Ind ustria

J. sci. industr. Res, 401. 25 No. 7 Pp. 283-330

July 1966 - . .. .

~d by the ' uncil of Scientific 8 - - strial Research, T' r Delhi

.... .. . .. . . .. I . . . > . , .... 1 . 1 q . . . . . L b . s a , ;: . . ;

t. Sole Distrib@ors Outsid* India: ~ e r ~ i m o n Press

" . . . .. _ , %.< 0 e " . : O%feg ,Lendon Paris Frankfurt New York

< P . 1 '. - .. I . . . , I . . . . . . . . . , .

. - 4

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For example, the high performance 1755A Dual-channel Vertical Amplifier offers these sensitivities: I mv/cm at 20 mebandwidth,S mv/cm at 40 me or 10 mv/cm through 5 V/cm at SO me. Features include A·j B operation, differential A+ Buse for common mode rejection, a sync amplifier for triggering on channel B input.

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HZ

ISIR-JULY -<.

NUMBER 7

EDITORIAL BOARD

DR S. HUSAIN ZAHEER, Director­General, Scientific & I ndustrial Re­search (ex officio Chairman), New Delhi

DR VIKRAM A. SARABHAI, AtomicEnergy Establishment, Trombay,Bombay

DR K. VE~KATARAMAN, NationalChemical Laboratory, Poona

PROF. S. R. PALIT, Indian Associationfor the Cultivation of Science, Calcutta

Journal cbf& Industrial

VOLUME 25

ScientificResearch

JULY 1966

PROF. B. R. SESHACHAR, Delhi Uni­versity, Delhi

DR M. S. KRISHNAN, Osmania Uni·versity, Hyderabad CURRENT TOPICS

CONTENTS

PROF. N. R. KULOOR, Indian Instituteof Science, Bangalore

SHRI S. B. DEsHAPRABHu, ex officioSecretary

SHRI A. KRISHN AMURTHI, Editor

EDITORIAL STAFF

Editor: A. Krishnamurthi

Assistant Editors: R. N. Sharma, D. S.Sastry, S. S. Saksena, K. Satya­narayana & K S. Rangarajan

Technical Assistants: A. K. Sen,S. Arunachalam, R. K. Gupta, KuldipChand & R. P. Grover

Production Officer: S. B. Deshaprabhu

The lournal of Scientific & tndunrial Resurehis issued monthly.

The Council of Scientific & Industrial Researchassumes no responsibility (or the statementsand opinions advanced by contributors. TheEditorial Board in its work of examining papersreceived for publication is assisted. in an hono­rary capacity, by a large number of distinguishedscientists working in various parts of India.

Communicati~ns r-egarding contributions (orpublicuion in the Journal, books for review,subscriptions and advertisements sh~uld beaddressed to the Editor, Journal of Scientific &Industrial Research, Publications & InformuionDirectorate, Hillside Road, New Delhi 11.

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/' © 196; THE COUNCIL OF SCIENTIFIC &INDUSTRIAL RESEARCH, NEW DELHI

Sole Distributors Outside IndioPERGAMON PRESSp,..(ord London Pari' Frankfurt New York

. - .-.~

Conservation of Foreign Exchange: Contribution of the NationalLaboratories

Prof. P. Maheshwari, FRS

Summer School in Ferrous Metallurgy

B. K. SAXENA

Design of Cathodic Protection Installations

K. S. RAJAGOPALAN & N. SUBRAMANYAN

Adsorption & Other Related Phenomena in Paints

RUPENDRA KUMAR VERMA, MOHAMMAD YASEEN &]. S. AGGARWAL

Neural Control of Hormone Secretion

H. HELLER

Recent Advances in the Analysis of Lipids

U. K. MISRA

Reviews ...

The Theoretical Significance of Experimcntal Relativity; Advances in HeatTransfer: Vol. 2; AelOspace Ranges: Instrumentation, Principles of Guidedl\.tissile Design; Physical Properties of Magnetically Ordered Crystals; Osci1lo­scope Measuring Techniques; Applications of NMR Spectroscopy in OrganicChemistry: Illustrations fcorn the Steroid Field; Electroanalytical ;\Ietbods inBiochemistry; Modern Methods of Chemical Analysis; Tetracyclic Triterpenes;History Under the Sea; The Ideas of Biology; Scientific Societies in theUnitcd States

Notes & News

'Transit time' diodes - A new type of semiconductor for microwave powergeneration; Laboratory simulation and measuremEnt of lunar topography;New type of semiconductor; Induction and Illultisensitive end-product repres­sion; Antibody formation and the coding problem; Prostaglandins; Reacti­vities of histidine residues at the active site of ribonuclease; Fire Researcb inUK; Journal oj Combinatorial Theory; Dr Vikram A. Sarabhai; ForthcomingInternalional Scientific Conferences

For Inde.. to Advertisers, see page A21

283

283

285

287

292

298

303

319

324

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A22 .. IR-·)ULY 1966

Current

Conservation of Foreign Exchange:Contribution of the National

Laboratories

A I{EPO!{T* issued recently by the }{esearchSurvey and Planning Organization of the

Council of Scientiiic & Industrial l{esearch dealswith an important problem concerning the country'scurrent eCClnomic position, namely the contribu­tion of the national lahora tories in conservingforeign exchange. The determination of the extentof conservation of foreign exchange resulting fromresearch effort in exact quantitative terms is a verycomplex problem, since, in most cases, it is theconsequence of the combined impact of a varietyof factors. The isolation of the impact of researcheffort from that of other factors and linking it upwith proper quantum (monetary) of benefit (con­servation of foreign exchange) is thus a formidabletask. The present survey represents probablv thefir.st attempt in this direction. In fact, the purposeof the survey is more to ' build up a methodologywhich may serve as a working hypothesis for de­veloping the subject further', rather than to arriveat numerically'backed definite conclusions.

The survey covers the five-year period 1958-59to 1962-63 and the statistics provided are basedon data obtained from 20 national laboratories.The two main aspects of saving of foreign exchange,viz. (i) saving through import substitution, visibleas well as invisible (technical know-how and ,ervicesof experts, etc.) and (ii) earning of foreign exchangebv means of changing the quantum and structureof the commodities exported and/or rai,ing theprices per unit of export, have been taken intoconsideration. In the classification model adoptedthe contributions from the national laboratories havebeen grouped under these two broad heads. Underdirect sources of foreign exchange saving, contribu­tions from activities like designing and fabricationof equipment, production of import substitutes,release of processes, teaching and training facilities,and exchange of literature are listed. The indirectsources include consult~\I1cy services and practicaldemonstrations through extension services, testingfacilities and stam\;lrClization of proces,.;es andproducts. Contributions from facilities consideredto have been conducive to increa!-<ing the earningof foreign exchange bv intensifying export efforthave been grouped under: (i) sampling inspectionof cxport~ble goods; (ii) improvements in the methodsof preservation and packaging of exportable com-

*.-' study on the conser'i!ation of foreign exchange bythe Hational laboratories - Survey Report No.4, Research

_~ ~llrvcy and Planning Organiz;:,tion, (ollncil cd Scientific",J"'llistrial I{esearch, :-<ew Delhi, 1966. PI'. 15.

Topics

modities; and (iii) market research in foreigncountries and commodity studies within the country.

The gross conservation of foreign exchange duringthe period under survey due to the research effortof the national laboratories covered as well as theother contributing industrial factors has been esti­mated at Rs 259·02 million. The net contributionof the research effort works ont to Rs 76·91 million,i.e. about 30 per cent of the gross saving. The grosssaving figures break into Rs 234·44 million as thesaving through import substitution and Rs 14·58million as the exchange earning. The gross con­tribution from direct sources of foreign exchangesaving works out to Rs 183,83 million, the balance(Rs 75, 19 million) being the contribution fromindirect sources of saving. The correspondingfigures for net saving are Rs 1·86 million andI{s 75·05 million respectively. The contributionarising from the utilization of research results hasalso been broken up according to the frequency oftheir occurrence in the production processes, viz. ona continuous basis or only once. The contributionof the continuous component runs as high asH.s 149·27 million and that due to the use of researchresults only once, Rs 109·75 million.

Laboratory-wise, the largest contribution to thegross conservation of foreign exchange is from theCentral Food Technological Research Institute,Mysore (.Rs 128·17 million), followed by the NationalMetallurgical Laboratory, Jamshedpur (Rs 47·45million), the Central Fuel Research Institute, Jeal­gam (Rs 336 million), and the National PhysicalLaboratory, New Delhi (Rs 20,44 million). In termsof net conservation resulting from the research effortalone, the first po,ition is occupied by the National]\Ietallurgical Laboratory (Rs 45·0 million), followedbv the Central Fuel Research Institute (Rs 30,13),the Central Food Technological Research Institute(Rs 1·28 million), the Central Glass & CeramicResearch Institute, Calcutta (Rs 0129 million) andthe Central Mining Research Institute, Dhanbad(Rs 0·10 million).

The results of the present survey provide a basisfor condncting a more exhaustive and comprehensivesurvey covering not only the other national labo­ratoric, but also other centres of research in thecountry. Further surveys in this connection shouldcov('r those industries and others who have actnallvntilized the technical know-how. .

Prof. P. Maheshwari, FRS

WE record with deep regret the sad demise ofProf. P. Maheshwari, Professor and Head of

the Department of Botany, University of Delhi, on18 May 1966 after a brief illness. Prof. Maheshwari,a pioneer in the field of plant morphology in India,

283

J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

Prof. Maheshwari was elected Fellow of the RoyalSociety, London, in March 1965 for his significantcontribution in the field of botany, chiefly in themorphology and embryology of seed plants. Hewas elected Fellow of manv national and inter­national organizations. He' presided over mal1\'international congresses as Vice-President andPresident. In 1959 he was the recipien~f BirbalSalmi Memorial Medal of the Indian BotanicalSocietv and in 1964 of the Sunderlal Hora MemorialMeda(of the National Institnte of Sciences of India.He received the honorary doctorate of the McGillUniversitv, Montreal. .

Prof. ~1aheshwari travelled widely in Europeand USA. At the invitation of Vnesco he visitedIndonesia in 1952 and Egypt in 1954, and in 1958he paid a short visit to the USSR as a member ofa scientific delegation sponsored by the Governmentof India. In the summer of 1959 he was a visitingProfessor at the University of Illinois. In 1961he visited Germany and England, and in 1964 hegave lectures at many universities in USA. Hehad planned to visit some of the research centresin Japan, Hawaii, USA, England and Europe duringMay-June 1966, but fate prevented it.

Besides being an outstanding researcher he wasan excellent teacher and was responsible for traininga large number of students and research workersand establishing one of the best schools of botanyin the country. During the last 15 years about800 scientific papers have been published by thestaff and students of his department; 65 students,including one from Buenos Aires, were admittedto Ph.D. degree. Prof. Maheshwari was a tirelessworker. He expected from others the highestdegree of efficiency and devotion. He aimed atnothing but the very best and never tolerated poorstandards.

Prof. Maheshwari was associated with the Councilof Scientific & Industrial Research as a memberof the Governing Body, Chairman, Biological Re­search Committee and member of the ExecutiveCouncils of the Regional Research Laboratory,Jammu, and the Indian National Scientific Docu­mentation Centre, New Delhi. He was a memberof the Editorial Boards of the J oumal of Scientific& Industrial Research and the Indian Journal ofExperimental Biology.

has made significantcontributions to thestudy of embryo andits post-fertilizationdevelopment. In hisdeath India has lost aneminent botanist, heldin high esteem by bota­nists all over the world.

Born on 9 November1904, Prof. Maheshwarihad his education atthe Universitv of Allaha­bad, where' he laterworked as a researchscholar under late ProL P. Maheshwari, FRSDr Winfield Dudgeon.He obtained the D.Sc.degree of the University of Allahabad in 1931. Hebegan his teaching carrier as Lecturer in Botanyat the Ewing Christian College, Allahabad (1928-30),and later at Agra College (1930-37) and the Univer­sity of Allahabad (1937-39). In 1939 he wasappointed Reader in Botany and Head of the newlystarted Biology Department at the University ofDacca, and was later promoted to the professorship.In March 1949 he was invited by Sir Maurice Gwyer,the then Vice-Chancellor of the University of Delhi,to join the University of Delhi as Professor and Headof the Department of Botany which appointmenthe held till his death.

Prof. Maheshwari's important contributionsinclude studies on cryptogams, gymnosperms andangiosperms with special reference to taxonomy,anatomy, embryology and metabolism, on whichnearly 300 papers have been published by him andhis collaborators. He is the author of books en­titled Embryology of angiosperms, Gnetum (jointlywith his pupil Vimla Vasil) and Dictionary of eco­nomic plants of India (with his student Umrao Singh),and had in preparation a fourth publication onthe Morplwlogy of gymnosperms. He also editedProceedings of the summer school of botany: Darjee­ling; Plant tisSlte and organ culture: A symposium;and Recent advances i11 the embryology of angiosperms.He was Founder-Editor of Phytomorphology, officialorgan of the International Society of Plant Morpho­logists, of which he was the Founder-President.

28~

Summer School in Ferrous MetallurgyB. K. SAXENA

National Metallurgical Laboratory, Jamshedpur

T HE ;,npact of modern technological develop­ments in the metallurgy of iron and steelproduction has been fclt markedly in the

Indian iron and steel industrv. The prohl< ms thatconfront India are compkx, relating chiefly todifficulties of foreign exchange, raising of producti­vity potential, shortage of technically trainedpersonnel and insular shortages of indigenouscapital goods and maintenance spares. In spite ofvast reserves of good grade iron orcs, the vastexpansion of the steel industry has revealed somebasic metallurgical shortcomings in Indian rawmaterials for the steel industry. Unless thesedifficulties are overcome by the application of latestresearch results, the rationale of India's acceleratedsteel development would lose some of its force. Tofocus attention of ferrous metallurgists on the latesttechnological and research developments in themetallurgy of iron and steel and to provide a forumfor discussions on their rational utilization, a twoweeks' summer school on " Ferrous ;"letallurgy "was organized by the Ministry of Education,Government of India, at Shillong during 12-24 July1965, with Dr B. R. Nijhawan, Director, NationalYletallurgical Laboratory, as its convener.

Seventeen scientists and metallurgists fromdifferent research laboratories, technological insti­tutes and universities participated in the summerschool, which was inaugurated by Dr Taylor, Ex­Vice-Chancellor of Gauhati University. Whileinaugurating the summer school, Dr Taylor expressedthe hope that such a gathering of scientists andmetallurgists will offer stimulation of thought andprovide an ideal forum for exchange of technical, know-how' and 'know-why' of diverse aspectsof the complex subject of ' ferrous metallurgy'.

Technical SessionsTwenty papers covering various aspects of

research, production and planning in the field ofiron and steel were presented and discussed in thetechnical sessions held from 13 to 24 July relating to:(1) preparation and use of the Indian raw materials;(2) techniques and choice of iron and steelmakingprocesses; and (3) growth of iron and steel industryin India and abroad.

The technical sessions of the summer schoolstarted with a paper on 'Status of iron and steelindustry in India' by Dr B. R. Nijhawan, followedby five technical papers on the preparation and useof the Indian raw materials. Dr Nijhawan referredto the heavy capital investment in the integratediron and steel plants. He also discussed the dif­ferent possible sites for the location of the fifth andsixth steel plants vis-a.-vis the availability of rawmaterials in their vicinity, as well as the growthpattern of iron and steel industry in India. Hestressed that while considering proposals for theestablishment of coastal plants, such as at Goa,

the import of high grade metallurgical coking coal\or coke can be balanc(d against the e~port of ironore pellets made out of beneficiated iron are fines.It is a welcome feature of current Indian planningthat the establishment of new steel plants is dis­tinctly more 'raw materials oriented' rather than• equipment oriented'. He also illustrated thecost of production of O· 3 million tons/annum pig ironplant with a capital investment of Rs 2'5-3 crores.The total indigenous fabrication of such smallplants in India today is a definite possibility andwould encourage the growth of related engineeringindustries.

K. N. Gupta (NML) gave an appraisal of the rawmaterials for the blast furnace and about theinvestigations carried out at the NML on decrepi­tation and physical characteristics of some Indianiron ores, including porosity, abrasion and crushingstrength at room and elevated tempe I atures.

S. K. Gupta (lIT, Bombay) gave an account onthe interpretation of coke rates while varying chargebed heights during sintering fluxed charge. Alayerwise heat balance was drawn according to themethod suggested by A. A. Sigov. A similar heatbalance was drawn in the case of fluxed charge usingDurgapur iron ore. He also referred to the minera­logical structures of fluxed sinters from Rajharairon ores.

J. Goswami ( ML) in his paper on • Indian coalsand iron making' gave the main classifications ofIndian coals with their estimated reserves. Theabundantly available non-metallurgical coals canbe successfully used for the production of iron bysuitable alternate methods. He also discussed theresults of the use of non-metallurgical coalsin different forms in the low shaft furnace of theNML.

Dr B. R. Nijhawan (NML) gave an account of thelatest studies on the beneficiation of iron ores. Heemphasized that mechanical mining of high gradeiron ores liberates 30-40 per cent, _.~ in. fractioncontaining as high as 20 per cent Al 20 3 with a veryadverse Si02/AI20 3 ratio. He also mentioned thatthe cost of beneficiation will amount to aboutRs 12.00 per tonne inclusive of overheads. He indi­cated the difficulties of beneficiation of Indian ironores containing high clay contents.

S. L. Malhoha (Department of Metallurgy, BHU)discussed the various direct reduction processesand reviewed the researches on the developmentof suitable alternate proce~ses of iron are reductionin view of inadequate coking coal resources, deplet­ing resources of rich iron Oles, non-availability ofscrap, and the inherent high capital cost of the blastfurnaces. During the discussion Dr Nijhawandiscussed in detail the future prosp(cts cf the directrcduction pIOcesses in India ar.d pointed out thatthese are not suitable for Indian conditions, lar gelybecause of the non-availability of natural gas.

285

J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

B. K. Saxena (NML) gave an account of the recenttrends in the blast furnace practice. He discussedthe role of productivity and coke rate, the principalfactors in the economy of the blast furnace. Healso discussed the problems created by high aluminacontent and unfavourable AI20 3/Si02 ratio ofthe Indian iron ores, particularly in the orefines, aggravated by heavy monsoon rains, andthe high ash content of the metallurgical coals,etc.

Dr A. B. Chatterjea (NML) reviewed the techno­logical aspects of low shaft furnace process. Hementioned the various chemical and physicalcharacteristics of the burden, as well as the influenceof grain size on heat transfer. He also discussedthe furnace output, the rate of combustion of coke,the reduction of iron oxides by carbon monoxideand the temperature distribution in the low shaftfurnace.

A. 1. Djiakonov (Unesco Expert, lIT, Bombay)in his paper on electrical conductivity of moltenopen hearth slags and utilizing these data for con­trolling the process of steelm,aking described a devicefor estimating the electrical conductivity of moltenslag directly in the furnace and its correlation withdesulphurization. J. Mohan (NML) gave an accountof the different pneumatic steelmaking processesand their suitability to the Indian conditions. R. K.Dubey (NML) discussed the production of alloysteels, with special reference to the production ofstainless steel and high speed tool steels. He em­phasized that the development of oxygen lancingin the electric arc furnace for decarburizing themelt in the presence of high chromium has revolu­tionized the stainless steel melting practice. Healso discussed the melting of high speed steels andthe efficiency and economy achieved in differentstages of manufacturing tool and die steels.

V. S. Bhandari (NML) reviewed the recent develop­ments in cu pola practice, with particular referenceto the hot blast cupola. He also discussed theconversion of the cold blast cupola to the hot blastcupola along with its various advantages, withspecial reference to the hot blast cupola installedat the National Metallurgical Laboratory.

R. D. Gupta ( ML) in his paper on 'Electroslag melting' discussed the studies made on thechange in chemical composition, inclusion type andcontent and ingot structure of plain carbon, ball·bearing, and high speed steels.

P. K. Sen (lIT, Kharagpur) presented a paperon the decarburization of open hearth melts anddiscussed some features of the kinetics of the de­carburiilation process in the open hearth furnacesunder different conditions. He also discussed theapparent activation energy of the process of oxi­dation of carbon in Fe-C melts. The effect oftemperature on the rates of decarburization of Fe-Cmelts, subjected to two different oxidizing gaseousmedia, viz. still air and a mixture of 92 per cent argonand 8 per cent oxygen, have been studied inthe temperature range 1450-1650°C. S. K. Goel

286

(Department of Metallurgy, University of Roorkee)described the development of rotor process of steel­making. A. N. Kapoor (NML) in his paperon ' Some observations on surface protection' ga"ea description of various surface cleaning methodsprior to surface coating. He also described theadvantages of aluminium coating and the possibi­lity of replacing tinning by aluminium 01· chromiumplating, with special reference to the raw materialsavailable in India.

Dr S. S. Khanna (Heavy Engineering Corp.,Ranchi) in his paper on 'Role of Heavy Engineer­ing Corporation for the building of heavy capitalequipments' described the programme and thesalient features of the various projects under theCorporation.

A. 1. Djiakonov in his paper on ' Iron and steelindustry in USSR' dealt with the o;xygen enrich­ment of the flame in open hearth, decarburizingby roof lancing with oxygen and the use of 100 percent compressed air in steelmaking. He mentionedthat for increasing the output of a basic open hearthfurnace by 10 per cent, the use of compressed airwill be more economical, but for higher output,oxygen lancing was desirable. He also discussedthe expansion programme of the iron and steelindustry in USSR.

Dr A. B. Chatterjea, in his talk on ' A glimpse ofthe iron and steel industry in Japan' gave a pictureof the developments in the iron and steel industryof Japan, which imports both coal and ore and stillably competes' in the world market. He gave anaccount of the Japanese blast furnace working andpresented statistical data showing that Japan hasthe lowest coke rate.

Dr B. R. Nijhawan in his paper on ' Iron and steelindustry in developing countries' gave an accountof the growth and development of iron and steelindustry in India and other developing countries.He cited examples of Pakistan, Burma, Ceylon andsome African countries where no suitable rawmaterials are available for installing any iron andsteel industry; but even then they are setting upintegrated iron and steel plants, as it has becomea prestige issue for them. Japan is developingher iron and steel industry with a modern basemainly to balance their trade with food imports.It is interesting to know that ] apan is exporting10 million tons of steel to Canada, UK and othercountries from where they are purchasing the rawmaterials. He also discussed the iron and steelexpansion programme of the United Kingdom.

At the concluding session, the participantsexpressed general satisfaction at the way the summerschool was conducted. General appreciation wasexpressed of the role of the National MetallurgicalLaboratory in putting the metallurgical researchand developmental work in the country on a firmfooting.

While presenting a resume of the deliberations,Dr A. B. Chatterjea expressed appreciation for thevaluable contributions on various topics.

Design of Cathodic Protection InstallationsK. S. RAJAGOPALAN & N. SUBRAMANYAN

Central Electrochemical Research Institute, Karaikudi 3

.--:::~FIg: 1 - Cathodic protection installation [1, buried Or sub­merged structure made the cathode; 2, d.c. source; and

3, anode surrounded by anode bed]

I N an' earlier publication!, the criteria for cathodicprotectIOn were discussed l!1 terms of the elec­trode reactions taking place on the metal

surface under protection. I t was pointed out howknowledge of the fundamental parameters such asTafel ~Jopes and exchange currents of the electrcdereactions enables the calculation of the change inpotential leading to a specific degree of cathodicprotection. It was also mentioned that concentra­tion polarization caused by the diffusion of thespecies taking part in the cathodic reaction maybecome a dominant factor in neutral solutions. Inthis communication, the complimentary aspect ofthis subject of cathodic protection, namely theinfluence of extel'llal parameters, such as the resis­tance of the electrolyte, the variation of the poten­tial of the structure under cathodic protection,depending upon where the reference electrode isplaced, and the spread of cathodic protection arediscussed.

Electrolyte and Electrode Resistance

A cathodic protection installation (Fig. 1) isessentially a combination of an anode, the structureunder protection which is made the cathode, anda source of supply of d.c., which will flow from theanode to the cathode through the electrolyte. Whensacrificial anodes like zinc and magnesium are used,the current required for the installation is generatedby the difference in potential between the anodeand the cathode and no separate source of supplyof curren t is used. On examining this systemclosely, it is seen that the driving voltage, V, willdepend upon the current required as well as thetotal electrical resistance offered to the passageof current at each one of the component parts ofthe cathodic protection installation. The com­ponent parts are: (i) the anode, (ii) the anode-elec­trolyte interface, (iii) the electrolyte, (iv) the cathode-

Several general methods have been proposedfor tackling the problem2,3. The resistivity ofaqueous media can be readily measured using aKohlrausch bridge; ready-made instruments areavailable for the purpose. Since this cannot bedone in the case of soil resistivity, for this a soil

20.000,00020,0001000-5000100020-253000300,000

Resistivityohm-em.

Medium

Pure waterRain waterTap waterClaysSea waterChalkCoarse sand and gravel

electrolyte interface, (v) the cathode, and (vi) theexternal connections betwcen the cathode and theanode. The ohmic resistance of the anode maybe expected to be negligible. Oldinarily, theanode-electrolyte interface ~'oldd not contributesignificantly to the total resistance. However,this becomes significant if ancde passivation takesplace or resistive depo~;its are formed on the anode.Since the resistance of the electrolyte, R, is givenby R=p.l/A, where p is the specific resistance; t,-the length; and A, the cros,.-sectional area of theelectrolyte path, the geometry of the system hasto be taken into account. The presence of a non­metallic coating may ,.uggest that considerableresistance would be offered at the cathode-elec­trolyte interface. The resistance of the cathodeitself may become significant in spite of the veryhigh conductivity of the metal as compared to theelectrolyte, if current has to flow over very longsections, as may be obtained in the case of cathodicprotection of buried pipelines. The external co]]­nections are expected to contribute significantlyto the total resistance of the system only whencables have to be taken over large distances fromthe source of supply of current to the structure underprotection.

The electrolyte may be said to be homogeneouswhen protection is to be given in aqueous media.However, the electrolyte resistance may vary consi­derably from season to season in the case of estuarineand river water, while the resistance of sea watercan be expected to vary considerably with theprevailing temperature. Gress heterogeneity ofthe electrolyte is exemplified in the case of protec­tion underground. Large variations in resistivitymay be observed between the different soil strata,and the resistivity of any particular part of the soilitself may vary considerably depending upon itsmoisture content. Theoretical calculations of theresistance that would be offered by the electrolyte,therefore, becomes particularly difficult in the caseof underground structures. The resistivities ofdifferent media arc as follows:

3

IIIJ

\

\ \

\ \ \

\ \ \, \ \\ ,\\ \ \, \ ,\' ,

287

J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

box4 can be used. It is a cylindrical box made oflucite or perspex, in which four electrodes (usuallyof copper) are placed at equal distances from oneanother. A known current is passed through theouter electrodes and the potential difference betweenthe inner electrodes is measured. The resistivitycan then be calculated, applying Ohm's law, knowingthe cross-sectional area and the distance of separa­tion of the two central electrodes. This methodhas the serious drawback that it does not measurethe mean resistivi.ty of the soil through which currenthas to pass but only gives the resistance of an isolatedsample. For measuring soil resi.stivity in situ, anumber of methods have been proposed, of whichthe U"lOst widely used is the Wenner four·pin methods.In this case, four-point electrodes are introducedin the soil, such that these are equally spaced alonga straight line and the resistance measured in thesame way as in the case of the soil box. The resisti­vity, p, is given by

p=21ta.R ... (1)where a is the distance of separation of the pointelectrodes; and R, the measured resistance. Byadjusting the distance between the electrodes, thedepth of the soil, for which measurement has tobe made, can be varied. For example, if there aretwo strata of soil having resistivity PI (for the upper)and pz (for the lower), and if d is the depth of theupper stratum, then only PI will be measured so longas a is equal to or less than d. When a is very muchgreater than d, then only pz will be measured.

Resistance is given by the formula R=p.l/A.The geometry of the electrolyte, whose resistanceis being measured, is conditioned by the size andshape of the anode and the cathode and their rela­tive disposition. If this geometry is cylindrical,as for example, a vertical rod surrounded bya cylindrical cathode, it can be shown that theresistance R is given by

R=p/21tL.ln(D/a) ... (2)where D is the distance between the centre of therod and the surface of the outer cylindrical cathode;a, the radius of the rod; and L, the length of the rod.The manner in which the resistance of such a rodvaries with D /a is shown in Fig. 2. It is seen thatwhile the resistance values continue to increasewith D/a, the rate of increase is relatively small forvalues of Dja in excess of 300. On this basis,Eq. (2) can be simplified to

R=0·0324p/L ... (3)Eq. (3) has been tested in relation to lengths inexcess of 64 times the diameter and the agreement

r~E~,jo 100 2.00 '100 400 &GO

pIa.

Fig. 2 - Increase in resistance between a vertical rod ofradius a and ground with distance D from the rod

288

with measured values is exceedingly good 2• Theresistance of the anode to a certain point in theground has been considered because of its limitedarea. As compared to this, the resistance of thecathode to a similar point in the ground would benegligible because L will be much higher than forthe anode.

On the other hand, if the anode has asphericalshape the resistance R is given by

R=p/41t.(I/a-l/D) ... (4)Since D is very much greater than a, the expressionreduces to

R=p/41t.a ... (5)When the anode is close to the surface ofthe ground, only a hemisphere of the soil will betaking part in conducting the current. Hence

R=p/2u ... (6)The equation developed for vertical anodes is

also applicable to horizontal anodes, but the effectof proximity to ground surface has to be taken intoaccount. When a rod is longitudinally placed onthe ground, the soil through which CIIITent linesflow is a hemicylinder, and not a cylinder as in theearlier case and the resistance will not be like thatof a vertical rod. This situation will be alteredwith the depth of burial of the anode. At depthsgreater than 10 times the diameter, the resistancewill approximate to that given by Eq. (6), thougha correction factor may still have to be used. Butthen, the uncertain and variable character of resisti­vity (p) in the case of a heterogeneous medium likesoil is much greater than the small error which maybe introduced by an approximate formula.

It is not generally practicable to use a single anode.Anode bed, be it hori,zontal or vertical, usuallyconsists of several anodes connected in parallel.The theoretical formula for the resistance betweentwo anodes and a pipeline at infinity has beenpropounded by Dwight6 in terms of the capacityof the system. A practical test of the variationin resistance, as the number of anodes is increased,has been reported by Applegate'. The fact thatthe decrease in resistance with increase in the numberof anodes is much slower than what would beexpected from Ohm's law is illustrated in Fig. 3.It is also seen that the correction factor steeplyincreases with decrease in the spacing between therods (Fig. 4) (ref. 8).

The electrolyte resistance is further. modifiedby what is known as the 'backfill' at the anode

Fig. 3 - Relation between number of anodes and resistance(Soil resistivity, 200 Ohm-em.]

RA]AGOPALAN & SUBRAMANYAN: DES[G~ OF CATHODIC PROTECTlO:-l INSTALLATlOXS

Fig 5 -- Relative values of protective current required forthc' coated (I) and uncoated (Il) steel [C, cathode; and

A, anode]

oCUP-REo...

.0 10

CURREl'f,,,,

.J«r%..~..

~ \·4Fu..« 1·2«g 1·1L_~_ ___'__.l__~_ _I.__U

o f> 10 15 20 '2.5 30DIS"lAMCE. P>ETWull AIIODES,ft.

Fig. 4 - Relation l>ctween distance between anodes and thecorrection factor [No. of anodes, 5]

Ii '·8.---------------,~~ \·6

bed. The size of the anode bed is increased byplacing the metal anode in a bed.of coke-breezeor mixtures of gypsum and bentomte or commonsalt and bentonite. The ohmic resistance of sucha bed may he considered to be negligible, as com­pared to the soil resistance and hence the wholebed can be looked upon as an enlarged anode andthe resistance can be calculated taking into accountthe size and shape of the anode beel. Hdwever,care has to be taken to see that such an anode beddoes not get dried up by electro-osmosis, when theresistance of the backfill will increase steeply.

The resistance of the cathode-electrolyte interfaceis governed by the type of coating applied tothe structure under protection. It is, in fact,uneconomical to give cathodic protection to thestructure in the bare condition because of the largeconsumption of power, and the high cost of. main­tenance of such a system. Under mo~t cIrcum­stances, therefore, cathodic protection is combinedwith painting of the structure, the idea b~ing th.atthe area to be given cathodic protection will consistonly of the holidays that are invariably presentin almost all types of coatings. The current thatwould be required to bring steel coated with coal tarenamel to the protective potential as c.ompared ~o

a bare specimen of the same geometry IS shown mFig. 5. . . . .

It is eVident that the dlstnbutlon of current ona coated structure will be such that nearly all thecurrent would be going to the bare areas of the .coatedstructure. So the cathode-electrolyte resistancemay be considered to be .negligibl~. ~urther~ aspointed out earlier!, cathodic protectIOn IS obtamed_w~,-..."..- E~=E;-/(I./Ac)-I•.Rc ... (7)where E~ is the open circuit potential of the anod!csite; E;" the open circuit potential of the cathodIcsite; I., the applied current; Ac, the area of the catho­dic site' R the resistance of the cathode-electro­lyte int~rf~~e; and /(1./ Ac), the ~larization eff~ct,which is a function of the cathodIc current dens.lty,(I./A,). R, is given by p/21tL.ln D/fl. On a pIpe­line, L is very large and D/a IS ve.ry nearlyone. When the holidays are few, I. wlil also .besmall. Hence I.R, is negligible. So the coat~ng

-;:;''5:istance does not really figure in ~he calculatlo,nof the driving voltage. The econom~c~ of cathodIcprotection is governed by the dnvmg voltage,

because the power consumed is given by the prcd,!ctof the driving voltage and the current. By applymg

.a coating, the current is reduced, and, though thedriving voltage remains constant, the power con-sumption is reduced. . .

The resistance of the cathode becomes slgmficantonly in the case of long pipelines and cables. Insuch cases, there can be an appreciable voltage dropin the structure itself, so that the driving voltagesavailable at the drainage point and further alongthe pipeline become different. This would naturallyaffect the current distribution over the structure,This will be considered again under the section'Spread of Cathodic Protection '. Appreciablevoltage drops at the s~veral. connection~ should beavoided as far as possIble, smce otherWIse needlesspower loss would take place.

Potential of the Structure underCathodic Protection

The set-up for the measurement of the potentialof a structure under cathodic protection is shownin Fig. 6. The main problem is the location of thereference electrode for the measurement of thepotential of the structure. The str.ucture is b.roughtto a more negative potential to gIve cathodiC pro­tection. The potential of the structure measuredwill vary with the position of the reference electrode.To understand how this variation is brought about,it is necessary to first consider the potential of ametal specimen divid!ng .an elect~olyt~ throughwhich current is passmg m one dIrectIOn. Sucha condition is illustrated in Fig. 7 (inset). Thepotential is plotted against the distance of the metalin either direction in Fig. 7.

Fig. 6 - Set-up for the measurement of the potential ofstructure under cathodic protection [V.T.V.M., vacuum-tube

voltmeter]

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J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

Fig. 8 - Potential variation of two cylindrical electrodesmade of different metals [S, steel: and C, copper]

Fig. 9 - Corrosion cells developed in buried pipeline

J.

close to the anode, it will show the protectivepotential, before the cathode is polarized to thispotential. If it is placed close to the cathode, whenthe protective potential is reached, the anode poten­tial would have become more negative than theprotective potential. It is, therefore, essential tomake a reliable estimate of the half cell locationbefore using the potential criterion. It'should beplaced such that it is just at the boundary of thecorrosion cell, so that when the potential at thispoint becomes equal to the protection potential,no current flows in the corrosion cell and the poten­tial is the same both at the anode and the cathode.In other words, the appropriate location will dependupon the geometry of the corrosion cell, resistivefilms including coatings and the resistivity of theelectrolvte. If the size of the corrosion cell increaseseither due to an increase in the areas of the anodeand the cathode or due to an increase in the distanceseparating the two electrodes, the reference electrodemay be placed farther from the metal. If, however,the reference electrode is placed far away from theboundary of the corrosion cell, the IR drop wouldbe included and a more negative potential wouldbe measured before the cathode has been broughtto the anode potential.

Three types of corrosion cells are indicated inthe case of structures, e.g. buried pipeline: (i) cor­ro!3ion due to differences between adjacent points,(ii) corrosion at the bottom of the pipeline due tothe cell set up between the bottom and the top, and(iii) corrosion current flowing over long distancesbecause of differences in soil conditions in differentsections. These are shown in Fig. 9.

If the reference electrode is placed close to 1, forchecking the attainment of protective potential,all forms of corrosion will be prevented, though theanodic area at the distant point and at the bottommay become overprotected. It if is placed over

+6

__ it.-- 1

0

Fig. 7 - Variation of measured potential with relative positionof tll e reference electrode

The manner in which the potential varies withdistance will, however, not be as simple as shownin Fig. 7 in the usual case, because of the more com­plicated configuration of a structure under cathodicprotection and of the medium surrounding thestructure.

On a corroding structure, different potentialsmay be obtained in different regions, more negativepotentials being obtained on anodic areas and morepositive potentials being obtained on cathodicareas. If a reference electrode is brought insidethe boundary of the corrosion cell and brought closeto the anodic areas, it measures a certain potential.If it is moved to the cathodic areas, it measuresa different potential. Therefore, the potentialprofile will show considerable variation, when thereference electrode is moved very close to the metalsurface. However, if the reference electrode isplaced farther away, then it will scan both the anodicand cathodic areas and hence will measure a poten­tial, which is in between the two. When it is placedjust outside the boundary of the corrosion cell, itwill measure the compromise potential of the wholestructure as a result of current flowing betweenthe anodic and cathodic areas. This again em­phasizes the importance of the correct location ofthe reference electrode. A simplified approach tothis question may be made by a consideration ofthe potential variation of two cylindrical electrodesmade of different metals (say copper and steel),which have been short-circuited, thus resemblinga corrosion cell (Fig. 8).

The potential becomes more and more negativein the direction of N, because cathodic currentflows from the electrode C. Similarly, in the direc­tion of A, the potential becomes more positivebecause anodic current flows from A. The potentialvariation in between the two electrodes is also shownin Fig. 8. It is seen that somewhere in between,along BB' (outside the corrosion cell boundary)the compromise potential will be obtained. Whencathodic protection is given, the potential of thestructure is being changed in the negative direction.When the reference electrode is placed within theboundary of the corrosion cell, the potential willvary, depending upon whether it is close to the anodeor the cathode. If the reference electrode is placed

290

RAJAGOPALAN & SUBEAMANYAN; DESIG:-J OF CATHODIC PIWTECTIOX INSTALLATIONS

... (8)

Fig. 10 - Swing in potential in the case of cathodic prutectionof large pipelines

this pipe section of the soil surface, the overprotec­tion would be reduced, but the local cathode willnot be made sufficiently negative. If the half cellis placed far away, the distant anode will not beoverprotected, but the cathode potential will notbe sufficiently negative; and localized corrosionwill continue.

The location of the reference electrode becomesless critical, when the change in potential with dis­tance is less because of less current flowing to thestructure or the resistance of the electrolyte beingless. Coatings reduce current density and hencethe potential gradient in the electrolyte. Electro­lytes having a specific resistance less than 100ohm-cm. do not require accurate positioning.

In the case of large pipelines, the proximity ofthe pipeline to the surface of the soil has to be takeninto account. This is done by imagining a situation,where above the surface also there is soil, just asbelow, in which ca;;e, it is expected that an electricalimage of the pipeline will also be formed above thesoil. This is represented in Fig. 10. The resistanceof the soil can then be calculated in terms of thecapacity of the buried structure as well as of itsimage, and then, half of the capacity value will cor­respond to that of the buried structure alone. Inthis way, it has been shown9 that the potentialdifference between a point on the pipe and a pointx on the surface of the earth is given by

V~~ !2 [In(h2 +x2)-fl n lVl]2.. 2rh

where i is amp. per foot of the pipeline; p, the resisti­vity; x, the distance from the centre line onthe surface of the earth of the cylinders to the point

-~-O[l.~~, the distance from the centre of thepipe to earth's surface; and r, the radius of the pipeand the coating-resistance has been provided for,in the mathematically convenient form of p/21t In 111.On the vertical centre-line, the value of V will be

~iven by

V = i! [In(h-I.<)(h - s) +In ;:'1] ... (9)

where s is the depth at which the potentials aremeasured.

.' ~':fad of Cathodic Protection

For cathod~cally protecting long pipelines, theymay be consIdered as made up of several short

le~1gths ?f pipe and each section can be providedwIth. SUItable number of. anodes. In. general, aplpelme IS protected by a SIngle or a lImIted numberof anode installations. The number of anodeinstallations required depends on the spread ofprotection given by each installation. The spreadof protec!ion. is affected by the potentia! drop alongthe pIpelIne Itself, and also on the resistance of thecoating. Even with good coatings, it has beenfound that the coating is damaged by current inexcess of a few volts and so in practice, a shift inpotential of two volts is taken as the maximumtolerance at the drainage point. This sets a limitto the spread of protection that can be aimed at.Taking into account the metal resistance r and theconductance of the coating g it has been shownlothat the potential E A , at the drainage point (wherethe negative lead of the power source is connectedto the structure) is given by

EA =Em cosh.al ... (10)

where Em is the minimum shift of potential requiredto achieve protection, a= y'r.g and l is half of thelength of the pipeline getting protection. Sincethe change of voltage even at the dl ainage pointcannot be allowed to go beyond two volts and theminimum shift in the potential of the structurerequired for protection can be calculated from thepotential of the unprotected sh ucture, the lengthof the pipeline for which protection can be givenmay be estimated from Eq. (10).

Summary

Factors influencing the economic design ofa cathodic protection installation al e discussed.I t is shown that the geometI y of the system andthe size and shape of the anode have an importantpal t to play in determining the electrical resistanceof the cathodic plotection circuit and, thereby, thepower consumption. How the resistance of themetallic structure and the conductance of thecoating determine the length of the pipeline whichcan be protected with a single anode bed is discussed.The variation of pipe to soil potential withdIstance of the reference electrode from the pipeand its significance are discussed .

References

1. RAJAGOPALA:<, K S., j. sci. indllstr. Res., 20A (1961).27-33.

2. SHEPPARD, E. R. & GREISER, H. J., C"rrosio" , 6 (1950),630.

3. WELU:<GTON, J. R., CO/'1,""ion, 17 (1961), 550 t.4. MORGAN, J. H" Cathodic protcrtion (M~Graw-Hill Book

Co. Inc., New York). 1959, 54-.5. \VENNER, F .. cited in Underground coYrosion uy .M.

Romanoff, National Burean of Standards, Washington,Circnlar No. 579, April 1957.

6. DWIGHT, H. B., cited in Cathodic protection by J. H.Morgan [Leonard Hill (Books) Ltd, London], 1959.78.

7. ApPLEGATE, L. M., Cathodic protection (McGraw-Hili BookCo. In~., New York). 1960, 131.

8. ApPLEGATE, L. M.. Catlwdic pl'olection (Mc<;raw-J-lill BookCo. Inc., New York), 1960, 129.

9. MURGAN, J. H., Cathodic protectivl! (~lcGraw-HiIl BookCo. Inc., New York), 1959. 135.

10. MORGAN, J. H., Cathodic protection (McGraw-Hill BookCo. Inc.• New York), 1959, 142.

291

Adsorption & Other Related Phenomena in PaintsRUPENDRA KUMAR VERMA, MOHAMMAD YASEEN & J. S AGGARWAL

Regional Research Laboratory, Hyderabalj

THE phenomenon of adsorption at interfacesis of considerable importance not only inthe paint industry but also in the ink, plastics,

food and many other industries. The main aspectswhich concern the paint industry are (i) the effectof adsorption on the wetting and dispersion of pig­ments, (ii) the adhesion of paint films to the substrate,(iii) corrosion prevention of metallic substrates,(iv) the rheological properties of paint vehicles, and(v) the ageing and weathering properties of paintfilms.

Perhaps the most important factor in the adsorp­tion of molecules on solid surfaces is the heterogeneityof these surfaces. Bikerman' has suggested thatin surfaces consisting of more than one mineralogicalcomponent, as in the majority of alloys, the surfacehas a mosaic structure, since different compoundsare exposed at various points on the surface. Ifthe specimen is mineralogically uniform, its surfacecan still have various properties in different direc­tions due to the presence of different faces, edgesand corners. This heterogeneity accounts for thecatalytic and adsorption properties, and the varia­tion of thermionic and photoelectric work functionson the same crystal. Even one phase of the crystaldoes not have to be homogeneous as it normallycontains hill-and-valley structure2, kinks, jumpsand cracks. Harkins3 has pointed out that in allthe work on adsorption and on energy relationsfor the surface of solids, no account has been takenof the possible effects of different crystal faces, edges,corners, etc., the observed effect being a statisticalsummation of all these.

The heterogeneous nature of pigment surfaceswa, studied by Dintenfass4,5 by the adsorptionof polar organic molecules from organic solventson several industrial pigments and extenders suchas Ti02, talc, RaSO., PbCrO., etc. A selectivepolar adsorption pattern was observed which isdifferent from physical capillary condensation typeof adsorptio:l and from chemisorption. Apparently,the surface of pigments, extenders, etc., consistsof a number of distinct active areas, each of whichselectively adsorbs a specific type of polar group.An independent and simultaneous adsorption ofpolar compounds containing different polar groupscan take place, but the different polar compoundsdo not interfere with each others' adsorption.The active areas corresponding to various polarcompounds, i.e. acids, alcohols, phenols, soaps,esters, primary and secondary amines, have beeninvestigated.

The total surface area of a pigment appears tobe the sum of the individual active areas and insome cases of non-active areas. Every pigmentor solid surface can be characterized by the size,number and type of these individual areas. Thisgives a fingerprint of the pigment which apparentlydepends more on the origin and history of the

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pigment (or surface) than on the chemical structure.According to Dintenfass6 , in systems where com­pounds with identical polar groups and of differentchain length are present, there is no preferentialadsorption based on chain length; thus long chaincompounds can be displaced by short chain lengthcompounds with identical polar groups and viceversa. In each system, the ratio of amountsadsorbed on the surface is nearly the same as theratio of the molecular concentration of the twospecies in the solution.

The adsorption of linseed oil fatty acids at aninitial pH of 5·0-5·2 on magnetite during frothflotation was investigated by I{ajanne7• It wasfound that more umaturated fatty acids were pre­ferentially adsorbed at low collector concentration.An increase in the amount of collector is accom­panied by a decrease in the iodine value ofthe adsorbate. At low collector concentration,the amount adsorbed increases as the amount ofcollector is increased per unit weight of magnetite.A saturation point is reached and calculations ofthe specific surface show that the adsorbed layeris mul timolar. A rod structnre for the collectorlayer is proposed to accommodate the large numberof fatty acid moles, while the hydrophobic natureof the layer is retained during its formation.

Ultrasonic waves were found to increase theadsorption of methylene blueB• The adsorptionalso increased with the power and frequency of theultrasonic waves. The increase in adsorption wasaccounted for by the increase in the degree of fine­ness and change of electrochemical properties ofthe pigment. Studies on the adsorption of sodiumoleate on pigments like Ti02 and 2nO have revealedthat in low concentrations of sodium oleate there isthe usual increase in adsorption with concentration".Near the critical concentration of micelle forma­tion, especially at higher concentrations of sodiumoleate, several points of abnormal behaviour in theadsorption of sodium oleate were observed. Thecomparison of the adsorption isotherms with thoseobtained by other workers showed that the adsorp­tion of surface active agents on pigments dependsonly on the properties of the surface "tn'v<; "o·ol:." ..,in the solution and specific properties of the pig­ments have no effect.

Miller and Anderson10 made apparent adsorptionstudies of oleic acid from coefficient of frictionmeasurements. The coefficient of friction ofsolutions of oleic acid in paraffin oil (0-25 per centby weight) were measured on a friction pendulumover the temperature range 60-140°C. The frictionconcentration isotherms were amenable to treatmentby the kinetics of monolayer adsorption. As thetemperature was increased from 60°C. the frictiondecreased to a minimum at 120°C. and ':,~a-,

increased again. The decrease in friction 'withtemperature was ascribed to the formation of a

J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

The effect of physical properties of the pigmenton the adsorption of drier has been stressedby Bryson22 • According to him the shape of thepores and the nature of surface are more importantthan the porosity of the pigment. Generally, thepigments possessing a large specific surface inhibitdrying. A series of containers each containing thesame amount of the paint were taken, and increas­ing aliquots of drier were added to each and setaside for drying time tests at bimonthly intervals.A particular concentration of the drier, at whichno increase in drying time is observed for 3 or 4successive periods, corresponds to the correct amountto be added to the paint.

Wetting of PigmentsWhen a pigment comes in contact with a vehicle,

the physical properties of such a system canbe related to the extent and character of the inter­face between solid and liquid. The pigment-airinterface is replaced by a new pigment-vehicleinterface and the wetting of pigments follows.Wetting may be defined as the process by whicha liquid comes in contact with a solid to forma solid-liquid interface. The nature of the processwhich takes place at the pigment-vehicle interfacecontrols greatly the grinding and many otherproperties of the paints such as consistency, flow,levelling and gloss.

The wettability of a pigment by the vehicle hasgenerally been considered as the most importantcharacteristic associated with the stability of thepaint suspensions and this was first studied byHarkins and Dahlstrom23• The total free energyof immersion was taken as the measure of wettabi­lity. It was found that if dry clean pigment isimmersed in a liquid, heat is liberated, the quan­tity of which increases with increase of polarityof the liquid, or with increase of polarity of certaingroups in the liquid molecules.

Pigments are often difficult to wet with oil,because of the envelope of air or gas around theparticles that has been adsorbed by the pigmentsurfaces. Linseed oils of high acid value displaceabsorbed gases more readily than those of low acidvalue. If the pigments are first heated to removemoisture, they are wetted more easily. Carbonblack pigments are very difficult to wet with water,but if they are first heated or so treated as to removethe adsorbed film of hydrocarbons and air, theyare wetted more easily. The moisture adsorbedon the pigment may indeed be the main variableinfluencing the dispersion characteristic of com­mercial pigments. Pigment stocks are rarely givenan oven drying treatment immediately before use.Consequently, the pigment has generally anadsorbed film of moisture. The quantity of wateradsorbed is roughly equivalent to that necessaryto form a monomolecular film on the solid surface.

Eide and Depew24 reduced the reactivity of zincoxide in various vehicles by adding phosphoruspentoxide or other dehydrating oxides to the vehiclebefore dispersing the pigment. The plastic visco­sity of the finished dispersion is found to be reducedby this treatment and the paint is prevented fromthickening. Presumably, the reaction between

294

zinc oxide and fatty acids of the vehicle is consi­derably inhibited by dehydration of pigment beforedispersion. The use of hygroscopic organic liquidswith pigments in formulating lacquers has beenpostulated by Taylor and Chapman25, as a meansof preventing flocculation of pigments.

For wetting to occur, oil must be adsorbed onthe surface of the pigment particles. More vehicleis required to wet a unit weight of fine particlesthan is required for coarse particles, because theamount of oil required for pigment saturation orwetting is diiectly proportional to the specific sur­face area of the pigment mass. The oil absorptionfactor being relative to the surface condition of thepigment is possibly to some extent independentof its chemical composition or specific gravity. Asthe surface of a pigment is being wetted with oil,the pigment mass remains relatively dry andcrumbly, and will not smear when brought intocontact with a piece of glass. At the point, how­ever, where all pigment particles become wet, themass immediately forms a soft paste, which willthen smear when brought into contact with glass.The amount of vehicle required to wet a unit massof the pigment is known as its' oil absorption'.

The consistency of paints is affected to someextent by the oil absorption of the pigment. Ahigh oil absorbing pigment gives greater consistencythan a lower oil absorbing pigment, becauseit adsorbs more vehicle on the pigment surface,so that there is less free vehicle in the paint.Stieg2G has suggested that the oil absorption of anywhite pigment can be a factor in determining thespacing relationship and hence the hiding powerof the paint.

Freundlich27 utilized the contact angle relation­ship to derive an expression for adhesion tensionwhich is often taken as an indication of the degreeof wetting

'('.4 --Ysl.=AsL=I/.A cos 0

where YS A is the surface tension of the solid; 'YSL,

the interfacial tension of the solid and liquid; andlLA, the surface tension of the liquid. The termISA-1ST. is defined as the adhesion tension; ASLhas the same dimension as surface tension. Sincethere are three phases present, solid, liquid and gas,it becomes apparent that three separate interfacialtensions play a part in determining the contactangle. The liquid-gas interfacial tension has alwaysa positive value, and when the solid-gas interfacialtension is greater than the soJid-liquitl.... 1TIteTja.~;.:;,~tension, the value of cos () is positive which meansthat the contact angle is less than 900

• This is theresult when a liquid wets the solid. On the otherhand, when the solid-liquid interfacial tension isgreater than the solid-gas interfacial tension thevalue of cos 0 is negative and the contact angle liesbetween 90 0 and 1800

• This is the result whenthe liquid does not wet the solid. According toBancroft'S, a liquid is in actual contact with a solidonly if it is adsorbed on the surface of the solid.

The normal types of solvent-based media, suchas alkyd resins, are far from being pure liquids, .">rIllthey contain a wide range of molecular species.Apart from the differences in the size of the

VERMA et al.: ADSORPTION & OTHER RELATED PHENOMENA IN PAINTS

molecules, there is a range of polarity present inthem. In an alkyd, the free groups are mainlycarboxyl and hydroxyl, and the distribution of suchgroups is not uniform among the molecules ofdifferent degree of complexity, in fact, one wouldexpect the smaller molecules to be more polar byvirtue of having proportionately more end groups.

Calori.metric measurements of wettability ofpigments in various organic liquids29 confirm thebelief that polar-nonpolar molecules in the solventform a layer on the surface of the pigment with thepolar group towards the pigment ana the nonpolargroup towards the liquid. The polarity of theliquid in which the pigment is suspended determinesits degree of dispersion.

Dispersion of Pigments

First, the pigment must be wetted by the liquidused for dispersion. Secondly, mechanical workmust be performed on the system to effect disper­sion; in many cases the aggregates of the pigmentare hard and require a large amount of energy tobreak them up. Thirdly, the resulting dispersionmust be stabilized, i.e. the composition of the liquidphase must have been adjusted to provide condi­tions where there is no tendency for the pigmentto flocculate to an undesirable extent. Dintenfass4

considers that pigment dispersion occurs when amonolayer of sufficient thickness is adsorbed on thepigment to keep the particles at such a distance thatparticle-particle attraction is effectively negligible.

Every surface or interface has an energy asso­ciated with it. This can be expressed in ergs/cm.2;in the case of liquid, this may be approximated tothe surface tension in dynes/cm. With solids,however, the surface tension and the surface freeenergy are not necessarily equal as in creating afresh surface of the solid, the rearrangement of atomsto an equilibrium configuration is hindered by theimmobility of the surface region. Figures quotedfor the surface energy of solids vary widely, depend­ing on the method of measurement used, but anumber of inorganic surfaces, such as A1203' BaS04 ,

MgO, CaO, etc., appear to have surface energiesof the order of 1000 ergs/cm.2.

The surface energy of a powder will depend verylargely on the extent of the surface, and, when thesize of the individual crystals becomes very small,the edge energy factor, i.e. the energy associatedwith the junction of two planes of the crystallattice, becomes important.

~;,e ctii'lcive strength of solids can be influencedby adsorbed layers of surface active agent. Thepart played by surface active molecules in millingis thus quite complex. Almost all paint media assuch contain molecules which are themselves surface

'. active, i.e. they contain polar groups which caninteract with the solid surface in the same way asthe specific surface active agents. Certain solventslike chloroform, butanol, nitromethane and xyleneare effective in producing cracks, whereas aliphatichydrocarbon such as heptane and petroleum etherare ineffective. It was then noticed that the liquidswaich caused cracks to develop were all liquids witha fairly high cohesive energy density (CED), whilethose without any effect had low CED values. The

CED function is expressed as (/),H-RT)jV, i.e. thelatent heat of vaporization of the liquid at the tem­perature concerned minus the work done in expand­ing the vapour against surrounding pressureexpressed in terms of unit volume of liquid.

The amount of resin adsorbed from relativelydilute solutions increases with decreasing solventpower and with the oil length of the resin. Roth­stein30 made measurements from dilute resin solu­tions which allowed gravimetric study of adsorption,as well as separate study of bulk vi,cosity and ad­sorption on pigment dispersion. The value of thesaturation adsorption was found to increase almostlinearly with molecular weight of the resin. Thesame viscosity measurements that determined ad­sorbed resin thickness showed the effect of pigmentsurface coverage on the ease of dispersion of the,suspension. There are three basic interactionswhich govern the physical characteristics of asuspension made by dispersing a pigment into aresin solution. First is the resin chain\ segmentsolvent interaction which determines the configura­tion of the resin chain in solution. The secondinteraction is the adsorption of the resin onthe pigment surface, and the chain segment-pigmentsurface interaction which can determine the con­figuration of the chain on the pigment surface.These two interactions together determine theextent of the third interaction after initial dispersionand adsorption equilibrium has been reached, viz.the particle-particle interaction characterized bythe presence or absence of flocculation.

Stabilization of Dispersion

To stabilize dispersions, it is necessary to preventthe solid particles from approaching one anothertoo closely. There are various ways in which thiscan be brought about. In aqueous dispersionsespecially, electrical charges on the particles cancause a force of repulsion; this can also occur innonpolar liquids to some extent. When a solidis present in a liquid, a potential difference existsacross the boundary, the electrostatic potential.This potential exists whenever a solid surface isimmersed in a liquid, if the liquid is ionizing, ad­sorption of ions occurs to form what is describedas an electrical double layer. The principle is thatwith the charge on the surface of the solid particlesthere is associated a diffuse swarm of ions of oppositecharge surrounding the particles. These ions en­trap solvent molecules so that the effective sphereof action of the particles is greatly increased.

To investigate the state of pigments in oil-basedpaints when suspended in hydrocarbon medium,Khomikovskii and Revinder31 prepared submaddervarnish, pink varnish, yellow varnish and Prussianblue suspensions. Pigment dispersion in the sus­pension was measured with a vacuum sedimentater.The quantity of oleic acid adsorbed by the pigmentsand the change in the physical state of the pigmentsdue to the adsorption of oleic acid were determined.All the pigments were well stabilized with oleic acidin hydrocarbon medium; the stabilization limitwas reached when no more oleic acid could beadsorbed by the pigments. A simple relation wasfound to exist between the stabilization and wetting

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J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

of the pigment particles on the one hand and thequantity of stabilizer adsorbed on the other.

Vistosity of Pigment Dispersions

The viscosity of dispersion is affected by thequantity of pigment present and by the tendencyof the particles to aggregate and develop structureswhich immobilize the vehicle. In addition, thepigment is thought to adsorb a layer of vehicle im­mediately around the particles and this in effectincreases the pigment volume and reduces theamount of frec vehicle. The viscosity effect con­tributed by the vehicle is influenced by the tendencyof the molecule to coil and to form aggregates32•

The relationship log "I),=F/(I-F) between thesedimentation volume, F, and relative viscosity,"I)" holds for totally dispersed industrial pigmentsystems exhibiting Newtonian flow. F is a functionof the shape of the pigment particles, vehicle visco­sity and of the pigment phase (sum of the volumesof pigment and the adsorbed immobilized vehicle);and this relation is also true for thixotropic suspen­sions33• In concentrated pigment dispersiom, thesedimentation volume F increases with increasein viscosity of both the dispersed pigment phaseand the liquid phase. The F value of the floccu­lated pigment suspensions is not affected byternperature34 •

Thixotropy in Paints

Adsorption, flocculation and thixotropy innon-aqueous paint systems have been studied byDintenfass3s• The forces exhibited when a pigmentis immersed in a vehicle result in the phenomenaof adsorption, flocculation and thixotropy. Whenthe adhesion forces between the pigment and thevehicle are less than the attraction between pigmentparticles flocculation results. When the adhesiveforce is greater, the pigment is dispersed. Thixo­tropy is a reversible state resulting when the floccu­lating pigments form a network, which immobilizesthe vehicle. The degree of thixotropy dependsupon the pigment concentration, the degree of floccu­lation and the viscosity of vehicle. Factors affectingthe rate of dispersion and thixotropic build uphave been discussed by Dintenfass3s•

A thixotropic paint is one which possesses a highconsistency in the can, thus holding the pigmentsevenly dispersed. This high consistency, however;decreases under the action of mechanical treatmentwith the brush and after the application of paint,the consistency increases again. These anomalouschanges in the consistency with variation in appliedshearing stresses are termed together as the pheno­menon of thixotropy. Since the consistency dependsupon pigmentation of the vehicle, the optimumconsistency on the pigment content of the 'thixo­tropic' corresponds nearly to the critical pigmentvolume concentration.

Levelling characteristics of paint are determinedby proper formulations (pigmentation to the properdegree being an important parameter), so that thethixotropic rate of plasticity regain after applicationof the paint is fast enough to overcome the saggingtendency, but slow enough to allow the eliminationof brush marks before the paint sets. The following

296

relationship determines the levelling of the paintafter application: h= Wi/8Y) where It and d are thedepth and the breadth of the brw'h mark; i, theyield value; and Y, the surface tension of the sys­tem. For better levelling, paints should have ani value 2-8 dynes/sq. em.

Adhesion

The phenomenon of adhesion is due to forces ofvarious types36• The forces that hold a paint filmon a substrate include long-range body forces, sucha.s gravitation'al, magnetic and electrostatic forcesand intermolecular forces, loosely grouped under thetitle of van der Waals' forces and chemical bonds.The adhesion phenomenon must be differentiatedaccording to the nature of contact, i.e. whether itoccurs at points or over certain areas3? In thecase of paint coatings, adhesion over an areadepends on the structure of the metal surface andalso on the presence of dirt and greasy mater ialon the surface, which prevents the paint fromcoming into contact with and wetting the metalsurface. The fact that forces between substrateand film are exerted through layers, only afew molecules thick, explains why it is essentialto have a clean surface when applying paints tometals. If the oil material on the surface is fairlyfluid and easily soluble in the solvent of thepaint, all or most of this may be removed from thesurface before the solvent evaporates, and adhesionmay then be little affected311 •

Atoms on the surface of the solid have unsaturatedvalence electrons which are available to makebonds with reactive ingredients in the atmosphere,such as water vapour and oxygen. }{ober(39 haslisted the attractive forces that might exist betweenatoms as (1) chemical bonds including covalentbonds, (2) ionic bonds, (3) polymerization forcesbetween pairs of dipoles or between an ion anddipole, and (4) dispersion forces between inert atoms.Polymerization and dispersing forces are frequentlylumped together as van der Waals' forces, since theseare the only forces which are effective between mole­cules of gases and liquids at room temperature.Bullet40 suggested that the hydrogen bonds withenergies of the order of 5-10 kcal./mole are respons­ible for the strong adsorption of water moleculeson to the metal oxides but are unlikely to be themajor element in adhesion.

According to Dintenfass3S, the adhesion of paintfilms depends on the tensile strength of the filmand the total attractive forces between~tingand the substrate. Adhesion is affected by thenumber and type of the polar group available foradsorption. It may involve polar attraction,chemisorption involving electron exchange, andprecipitation when the metal ions on the surfaceform soaps or chelated compounds with the vehicle.The cohesion component of adhesion depends uponthe structure of the vehicle polymer.

According to Chatfield3M, as most surfaces areassociated with negative charge, greater adhesionwill be obtained by the use of an additive which inthe presence of moisture tends to confer a positivecharge by altering the zeta-potential. Such addi­tives are cationic surfactants in their various forms,

VERMA et al.: ADSORPTION & OTHER RELATED PHENOMENA IN PAINTS

which are sometimes so strongly adsorbed on certainsurfaces that more than a simple tendency toncutralize the charge is required to account for it,and actual ion exchange at the interface is assumed.Another instance in which additives are desirableis in the application of paint to rusty and corrodedmetal surfaces. The long chain amine salts, parti­cularly the acetates and oleates of primary amines,are used for this purpose, at about 0·5 per centconcentration on the paint weight. Provided theloosely adhering rust particles are removed bybrushing before the application of th~ paint, a paintwith such an additive can be applied direct to therusty surface when the remaining rust particleswill become incorporated into the paint in the samemanner as the pigment itself.

Some metallic structures are protected by paintcoatings and simultaneously by cathodic protection.Anderton4t observed loss of adhesion of paintcoatings on cathodically protected laboratory panelsnear standard coating breaks when the panels wereimmersed in sea water. He found that the loss ofadhesion occurs initially near the coating breaksand increases at a uniform rate depending on theapplied potential. The electrolyte solutions con­taining the alkali cations, potassium and sodiumhave been found to cause more loss of adhesionto coating on cathodically protected structures.Anderton suggested the mechanism that the diffu­sion of water into the film of wash primer at thecoating break dissolves the soluble componentsof the coating. Under the influence of the elec­trical potential there will be an inflow of cationswith large proportions of sodium ions, since sodiumis the predominant cation in sea water. The presenceof chromate ions and acid in aqueous solution nearthe coating break may produce soluble sodium orpotassium compounds with the binder which mayresult in leaching out of the film at the break. Theexcess of hydroxyl ions at the cathode will developan area of high alkalinity in the wash primer nearthe coating break which attacks the bond of theprimer to the steel. Cations diffusing into thewash primer film at the break will carry water withthem as hydrated ions and lead to loss of adhesionof the coating.

Summary

The various phenomena related to the adsorptionof paint vehicles at solid-liquid interfaces whichcauses the dispersion of pigments from the stand­point of fo, fnulation of paints are reviewed. Factorssuch as the heterogeneity of surfaces, nature of paintvehicles, fineness of pigments, presence of surfaceactive agents, driers, etc., which are closely asso­ciated with the phenomenon of adsorption andother factors which control the consistency, visco­sity, flow, levelling and gloss of paints are alsodiscussed. The wetting of pigments which mostlycontrols the grinding and stability of paints andfactors such as the adhesion tension, the degree ofwetting in terms of interfacial tension and thecontact angle which determine the degree of dis­persion and adhesion of the coating to the substratehave also been reviewed.

Acknowledgement

One of the authors (R.K.V.) wishes to expresshis thanks to the Council of Scientific & IndustrialResearch, New Delhi, for the award of a seniorresearch fellowship.

References

I. BIKERMAN, ]. ]., Surface chemistry (Academic Press Inc.,New York), 1948, 180.

2. HERRIKG, C., Phys. Rev., 82 (1951), 87.3. HARKL'S, K., The physical chemislry of swiace film

(Reinhold Publishing Corp., New York), 1952.4. DINTENFASS, L., Ko/loidzschr., ISS (1957), 121.5. DINTENFASS, L., J. Oil Col. Chem. Ass., 41 (1958), 125.6. DINTENFASS, L., Chem. &- Ind. (Rev,). (1957), 560.7. KAjANNE, P., Acta chem. Fenn., 31B (1958), 182.8. WITEKOWA, S. & KAMINSKI, W., ClIetn. Abstr., 61 (1964),

6429.9. SAKHAROVA, M. G. & SHUTOVA, A. I., LallVkrasoctmye

Ma!erialy iikh Prim."enie, (1954), 23.10. MILLER, A. & ANDERSO", A. A., J.ubric. Engng, 13 (1957).

553.11. PETIT, J., ] ACOV1C, M., HELMI, J. P. & BOSSHARD, G.,

C.R. Acad. Sci., Paris, 257 (25) (1963),3878.12. TARTAKOVSKAYA, V. E. & KHAZINE, Yu. G., CIte1n.

Abstr., 34 (1940). 6103.13. KOELMANS, II. & OVERBEEK, J. TH. G., Disc. Faraday

Soc., 18 (1954), 52.14. FISCHER, E. W., Kolloidzscilr., 160 (1958), 120.IS. DERJAGUIN, B. V. & KARASSEY, V. U., Proceedings,

Second illtm'national congYl'SS of surface activity, Part 3(Butterworlhs Scientific Publications, London), 1957,531.

16. LlvlNE, O. & ZISMAN, W. A., J. pilys. Cilem., 61 (1957),1068, 1188.

17. ZETTLEMOHR, A. C. & LOWER, G. W., J. Colloid Sci., 10(1955). 29.

18. ABRAM, J. C. & PARFITT, G. D., Chem. Abstr., 58 (1963).10670.

19. KOBAYASHI, T. & KITAHARA, A., Cile",. Abstr., 59 (1963),2194.

20. FINNE, W. rt al., Che",. Abstr., 31 (1937), 891.21. Montreal Paint & Varnish Production Club, Paint Oil

Chr",. Rev., 98 (1936). 114.22. BRYSON, H. C., Paint Manuf., 8 (1938). 115.23. HARKI"S, W. D. & DAHLSTROM, R., Ind1lsty. Engllg

GI..",., 22 (1930). 997.24. ElDE, A. C. & DEPEW, H. A., US Pat. 2,333,367 (to

American Zinc, Lead & Smelting Co.), 2 Nov. 1943;Ciletll. Abstr., 38 (1944), 2514.

25. TAYLOR, A. M. & CHAPMAN, A. R, US Pat. 1,824,177(to Atlas Powder Co.), 22 September 1931; Che",.Abstr., 26 (1932), 323.

26. STIEG, F. R., Off. Dig., 34 (1962), 1065.27. FREUNDLICH, H., Colloid and capillary chemistry (Methuen

& Co. Ltd, London), 1926, 157-60.28. BANCROFT, W. D., Applied colloid chemistry (McGraw-Hili

Book Co. Inc., New York), 1932, 74.29. RYAN, L. W., HARKINS, W. D. & GANS, D. M., Ind1lstr.

E1Igng Cilem., 24 (1932),1288.30. ROTHSTEIN, E. C., Off. Dig., 36 (1964). 1448.31. KHOMIKOVSKII, P. &: REVINDER, P., C.R. Acad. Sci,

URSS, 18 (1938), 575.32. DINTENFASS, L., Paint J. Aus!. N.Z., 2 (1957), 20, 25.33. DINTENFASS, L., Chem. &- Ind. (Rev.). (1957), 141.34. DINTENFASS, L., J(olloidzdschr., 162 (1959). 47.35. DINTENFASS, L., Paint J. A1Ist. N.Z., 1 (1957). 9, 11, 27.36. DR IN BERG, A. YA., GUREVICH, E. S. & TtKHOMIROV, A. V.,

Technology of non·metallic coatings (Pergamon Press Ltd,London), 1960,42.

37. DERYAGUIN, B. V. & KROI.OVA, N. A., Adhesion (Adgeziia)(USSR Academy of Science Press), 1949.

38. CHATFIELD, H. W., Tile science of surface coatings (ErnestBenn Ltd, London). 1962,443.

39. ROBERT, B. D., Off. Dig., 36 (1964). 664.40. BULLET, T. R, J. Oil Col. Cile",. Ass., 46 (1963),

441.41. ANDERTON, W. A., Off. Dig., 36 (1964), 1210.

297

Neural Control of Hormone Secretion*H. HELLER

Medical Research Council Group in Neurosecretion, Department of Pharmacolol(Y. University of Bristol, Bristol

TABLE 2 - RELEASE OF OXYTOCIN RESULTING FROM

ELECTRICAL STJMULATJON OF VARJOUS .PARTS OF THE

CENTRAL N ERVQUS SYSTEM

T~.BLE 1 - SlTl~S OF ELECTRIC ACTIVITY J:-J CENTRAL

NERVOUS SVSTEI\l EVOKED BY MAM~lARY OR VAGI:-lAL

STIMULATION

causes release of oxytocin, and increase in aterineactivity or the ejection of milk. Nauta2 andDenamur3 have recently outlined the neural path­ways which seem to be involved in the milk-ejectionand other nellro-endocrine reflexes. According tothese workers, the afferent pathways to the hypo­thalamus pass essentially through the brain-stemreticular formation and the non-specific thalamicnuclei which act as a first order analysing-integratingsystem. Two other similar systems, in parallel

Mesencephalic cerebral 64-68grey matter •

Midbrain reticular formationsMamillary and 5npramamil-

lary regionVentrolllc(lial hypothalamic

nucleiPerifornical areasL;mbic system including

eingulate gyrusAmygdaloid nucleus

Ref.

Ref.

57

60,61

62,63

61. 69, 70

12,58, 59

Mamillary a.nd supramamil­lary region

Ventromedial hypothalamicnuclei

Medial amygdaloid nucleiCingulate gyrus

Species Strudurc

Rat Lateral hypothalamusPerifornical arcasThalamusLateral preoptic areasHypothalamusMedial forebrain bundle

Rabbit HypothalamusThalamusLimbic systemSeptumHippocampusNeocortex

Cat

Species Structure

Rabbit

Goat MediallcmniscusMesencephalic rrticular

formationsAnterior hypothalamu,

Cat Supraoptic nucleiLateral preoptic areasMedial forebrain bundleCingulate gyrns

Afferent PathwaysMuch is known about sensory stimuli which

activate neuro-endocrine reflexes. In fact, it wouldseem that there is no type of external stimulus which,for example, may not give rise to the release ofone or the other of the hypophysial hormones.Enteroceptive, blood-borne releasing stimuli arealso known and they may be divided into two types:(i) hormonal feedback mechanisms by whichmessengers released from one of the 'peripheral'endocrine glands - say the gonads or the adrenalcortex - stimulate or depress some part of thecentral nervous system (CNS) functionally linked tothe pituitary, and (ii) changes in blood constituentsother than endocrines as, for example, a rise inplasma osmotic pressure which leads to the releaseof antidiuretic hormone from the neurohypophysis.

However, while the variety and efficacy of sti­muli have been widely investigated, relatively littleis known about that part of the reflex arc whichconnects the receptors with the fibres of the ulti­mate final path. that is to say, with the neurosecre­tory fibres which - by virtue of their secretory aswell as their neural function - must be regardedas part of the efferent system.

The complexity of the problems involved isperhaps best illustrated by discussing two neuro­endocrine reflex systems which have been intensivelyexplored. It is well known that suckling andvaginal stimulation lead to the release of oxytocinwhich contracts the mammary myoepithelium,thus causing the ejection of milk. Table 1 showsthat numerous parts of the CNS may be activatedby these stimuli. Table 2 shows the variety ofstructures ,of the CNS whose electrical stimulation

-Dedicated to the memorv of Heinrich Waclsch, lateProfessor of Biochemistry, Coiumbia University, New York,and Cbairman of the Department of Pharmacology, NewYork State Psychiatric Institute.

SOME neural mechanisms of hormonal control. are easily definable in Sherringtonian terms.

The pathways, for example, which servethe discharge of catecholamines from the adrenalmedulla are divisible into the classical triad receptor,conductor and effector, in close analogy to the reflexarcs for exocrine glands. Stimuli of various kindspursue 'private' pathways to reach internuncialneurones which, in turn, converge on another setof fibres providing the final common path. Thisultimate stretch may differ from the intermediatecommon pathway only in that 'it exhibits com­munism in a still higher degree' (Sherrington1),

but it may also contain neurones which elaboratespecialized 'neurosecretory' products. Reflex arcswhich contain such neurones (which, in vertebrates,are mainly situated in the hypothalamus) have inrecent years proved to be the main apparatus whichintegrates neuro-endocrine relationships.

298

HELLER: NEURAL CONTROL OF HORMONE SECRETION

and in series, seem also to be concerned. First,the limbic system-midbrain circuit of 1 auta2 towhich the supraoptic and paraventricular nucleiare directly connected (there arc also indirect con­nections bv way of the lateral hypothalamus) and,secondly, the thalamo-cortical apparatus whichcontains the sensory pathways.

The other neura-endocrine reflex I should liketo discuss is that concerned with the liberation ofvasopressin by osmotic stimuli. It differs from themilk-ejection reflex in not being initiated by sensorystimuli but by a blood-borne one. !iince, in somespecies at least, it could be shown that an increasein plasma osmotic pressure enhances vasopressinrelease and a decrease inhibits liberation we aredealing with a feedback type of neuro-endocrineregulation, not very different from others in whichthe concentration of a hormone from a peripheralendocrine gland influences neurones which regulatethe release of trophic hormones from the adenohypo­physis. Verney4 has demonstrated that in thewater-loaded dog, small rises of osmotic pressure(of the order of 2 per cent) in the carotid blood inhibitwater diuresis. He showed also that osmotic stimulifail to act after removal of the posterior pituitarylobe. Further observations on the effects of increasesin the osmotic pressure before and after ligation of theipsilateral internal carotid led to the conclusion thatthe osmoregulators lie in the vascular bed normallysupplied by that vessel. By evidence from blooddistribution or degenerative cell loss, Jewell andVerney5 restricted the site of osmoreception to adiscrete region of the diencephalon. Their work hasnow been supplemented by experiments6-8 in whichthe effect of hypertonic solutions on the electricalactivity of neurones in the anterior hypothalamuswere studied. It was found that the activity of theslJpraoptic neurones was always enhanced and thatof the paraventricular cells inhibited. These resultscombined with the evidence for the origin of vaso­pressin-secreting neurones in the supraoptic nucleus,and the fact that hormone secretion remains normalafter experimental isolation of the diencephalon9,

suggest strongly that the main site of the osmo­receptors is in - or very near to - the supraopticnucleus. A contribution of other osmosensitiveregions to the regulation of neurohypophysial activitycan, however, not be excluded.

There is a difficultv. The effect of intracarotitlhypertonic salt solutions on vasopressin release hasbeen demonstrated in the dog and almost certainlyalso in th~ ['oat, the rabbit and in man, but so farit has not been possible to show this mechanismunequivocally in the rat. It is difficult to believethat osmotic stimuli in this species do not operate intheir usual manner; the assumption that in the ratthe main osmoreceptors are not within the reachof carotid blood seems preferable.

The Efferent Arc

Whether relatively short or highly complex, theefferent portion of central neuro-endocrine pathwaysseems to contain neurosecretory neurones as itsessential constituent. The question may, therefore,be asked whether and in what manner this type ofneurone differs from ordinary nerve fibres.

Electroph)'siological Characteristics ofNeurosecretory Fibres

Neurosecretory cells show Nissl substance, bi­polarity, neurofibrils and dendrites like ordinarynerve cells. but their electric activity is relativelylittle explored. Where single-unit recordings arelikely to have succeeded (leech neurohaemal areas'O,

goldfish preoptic neurones", rat hypothalamic neu­rones!2, and others), action potentials of the usualkind have been observed. The action potentials ofneurosecretory neurones of fishes and of the leechwere of significantly longer duration than thoserecorded from nearby 'ordinary' neurones, butthose from the mammalian hypothalamus cannot beso distinguished. This statement must at presentbe made with the reservation that neurosecretoryneurones may not have been encountered in the latterrecordings.

·The postulate that neurosecretory fibres arefunctioning as both secretory and conductory unitis, however, difficult to avoid in the light of recenttheories on the mechanism of release of their hormonalproducts. Depolarization of the nerve endings in theisolated posterior lobe by a high concentration of K+in the presence of Ca2+ has been shown to causerelease of vasopressin and oxytocin!3,14. Suspendingthe isolated hormone-carrying granules in solutionat the same ion concentration did not lead to hormonerelease14• Acetylcholine releases hormones neitherfrom isolated granules nor from pituitary lobesin vitro but it does so from the whole isolatedhypothalamo-neurohypophysial complex l •• The bestfitting interpretation of these findings at presentwould seem to be that acetylcholine, liberated by theendings of the cholinergic fibres in synapse with theneurosecretory fibres, elicits an electric impulsein them which leads to changes in ion flux and in thepermeability of the axonal boundary and - in turn- to hormone release.

Morphological Criteria for Neurosecretory Neurones

Diagnosis of neurosecretory function by light­microscopy, based upon the occurrence of apparentsecretory inclusions in nerve fibres has proved to beunreliable. The classical staining method of Gomori­Bargmann and even the newer methods of stainingwith alcian blue or pseudocyanin are inadequateby themselves. For example, the neurones reachingthe medium eminence which serve as the source ofthe 'releaser' substances regulating the output ofadenohypophysial hormones have yet to be defined bylight-microscopical methods's.!?

Ultrastructural studies show that the perikarya andprocesses of neurone aggregations suspected of neuro­secretory function contain electron-dense granulesbetween 1000 and 3000 A. in diameter. Granules ofthese dimensions from the nerve endings in theposterior pituitary lobe have been isolated bydifferential centrifugation and the presence in themof vasopressin and oxytocin has been established!8-~o.

However, similar evidence for other specializedneurosecretory systems has, so far, not been provided.It seems likely that such granules are characteristic ofneurosecretory neurones, but in invertebrates at leastsimilar organelles are also encountered in fibreswhich are not known to be secretory. Moreover,

299

J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

catecholamines in peripheral nerves and acetylcholinein brain and nerves have also been shown to bestored in subcellular particles. The noradrenaline­containing granules isolated from beef splenic nerve,for example, have been described by von Euler21

as being 300-2000 A. in diameter. Some of themcontain electron-dense material which also appearsin units of about 200 A. Similar small submicro­scopic elements (diam. about 500 A.), called synapticvesicles, have been seen in cholinergic nerve endingsand have also been isolated22.

Vesicles, morphologically quite indistinguishablefrom these 'synaptic vesicles', occur abundantlyin the hypothalamo-neurohypophysial complex.They are characteristic for the median eminence ofhigher vertebrates" but occur also in the posteriorlobe either in the same fibre swelling as the large,hormone-carrying granules or, less frequently, on theirown24. The significance of these small vesicles isat present not established. Some workers2,,26 havesuggested that they contain transmitter substancesfacilitating neuronal hormone release, others27 ,26think that they may represent breakdown products ofthe hormone-carrying granules.

Topographicol Characteristics ofNeurosecretory Systems

It used to be thought that neurosecretory systemscould be characterized by their proximity to bloodvessels and there can be no doubt that many­or probably most - show this intimate relationship.Such ' neurohaemal organs '2' are the neurohypo­physis, the urophysis of fishes, the corpora cardiacaof insects, the crustacean sinus gland and others.In the neurohypophysis the nerve endings may lieagainst the pericapillary basement membrane directly(as, for example, in the teleost fish Lebisles30) or abuton the perivascular (collagen) space.

However, evidence has recently been accumulatingthat neurosecretory products may reach their targetcells not exclusively - or not primarily - throughthe blood. Neuronal processes of neurosecretory cellshave been described in amphibians31 ,32, mammals33and birds34 which extend towards and int<fthe liningof the third ventricle, suggesting that their productsmay be released into the cerebrospinal fluid.Lofgren33 has postulated that material secreted in thenuclear complexes of the tuber cinereum is carriedinto the infundibular cavity and from there to theprimary portal plexus and the anterior pituitary.Tinctorially (Gomori-negative, paraldehyde-fuchsin­positive) the neurosecretory material so considereddoes apparently not behave quite like that of neuro..hypophysial neurosecretion and may be susp~cted

to represent or contain' releaser' substances forthe adenohypophysial 'trophic' hormones. How­ever, the release of neurohypophysial hormonesinto the cerebrospinal fluid remains also a distinctpossibility.

A neurosecretory pathway which does not involvetransmission through blood has quite recently beenwell illustrated by Knowles3'. Fibres which areneurosecretory by all accepted criteria - they arestainable with Gomori's technique and containvesicles of about 1800 A. in diameter - have longbeen known36-38 to originate in the preoptic nucleus of

300

certain elasmobranch fishes and to enter the neuro­intermediate lobe. Knowles3; has now shown thatin the dogfish (ScylilJ,hinus steUaris) these fibresterminate on the secretory cells. Many such terminalswere found to make intimate contact with theregion of endoplasmic reticulum of the intrinsicsecretory cells in a manner which suggests thata neurosecretory substance affects these cellsdirectly.

There an~ several other arrangements, particularlyin invertebrates, by which the products of neuro­,secretory cells; reach their target tissue, but enoughhas been said to show that the older concept ofdefining neurosecretory neurones by their relationshipto blood vessels can no longer be sustained.

Chemical Characteristics of Neurosecretory Products

The suggestion has been made3s that there aretwo fundamentally different forms of neurosecretion,one concerned with the production of pepti(les andanother with the secretion of non-peptide substances,and that the two types ha\'e ultrastructural correlates.Present information ahout the chemical compositionof neurosecretory material, though scanty, is in favourof this concept. Only the products of one neuro­secretory system can so far be said to have beenintensively explored, namely those of the vertebratehypothalamo-neurohypophysi~ll complex. The bio­logically active products of these neurones seemto be a family of closely related octapeptides:The seven hormones whose chemical constitutionhas be~n ascertained differ from each other onlyby substitution of one or two amino acids in posi­t{ons 3, 4 or 8. Their distribution ranges fromthe primitive jawless fishes, the cyclostomI's, to themammals.

Another hypothalamic neurosecretory systemproduces the so-called' releasing factors' (RF), i;~.

substances which mediate the liberation of the, trophic' hormones from the adenohypophysis.Experimental evidence is extant for the existenceof a corticotrophin-releasing factor (CRF), a thyro­trophin-releasing factor (TRF), a luteinizing hor­mone-releasing factor (LH-RF), a follicle stimulat­ing hormone-releasing factor (FSH-RF)39 and agrowth hormone-releasing factor (GH-RF)40,41. Itseems that TRF42 LH-RF and FSH-Rp9 aresmall polypeptides ~vith molecular weights slightlygreater than those of the vasopressin or oxytocin.CRF presents a special problem since, according toGuillemin and Schally43, there are two such substances:<X-CRF which is chemically closely related to ct-MSM(13 amino acids) and ~·CRF, related to lysine vaso­pressin but with a somewhat larger molecule. Sincethe activity of LH-RF is not abolished by thio­glycollate44 , and TRF and <x-CRF, according to theirpublished structure, could not be inactivated either, •one would be tempted to assume that the releasingfactors belong to a family of peptides different fromthe neurohypophysial hormones which have adisulphide bridge and are hence all inactivated bythioglycollate. ~-CRF may be an exception: Guille­min and Schally43 have reported its inactivation bythioglycollate, but this finding was not confirmed byRamirez and McCann44. A further hypothalamicfactor, the prolactin-inhibiting factor (PIF),

HELLER: NEURAL CONTROL OF HORMONE SECRETION

acting directly on the anterior pituitary whereit inhibits prolactin secretion, has been describedby Talwalker et al:". Its chemical compositioni! unknown.

Relatively little is known about the chemical COlII­

position of the hormones produced by invertebrateneurosecretQry complexes. Only two groups ofhigher ihvertebrates have been studied.

Insects _. Groups of neurosecretory cells in theinsect brain discharge their products into the corporacardiaca and corpora allata. These ~eurusecretory

cells differ from ordinary neurones by the presenceof, usually, electron-dense granules. Moreover, theneurosecretory material stains with Gomori's tech­nique and with aldehyde-fuchsin. The neurosecretorycells seem to be akin to the intrinsic glandular cellsof the corpora cardiaca, organs which, in addition toproducing hormonal substances of their own, servealso as centres for the storage and release of theneurosecretory material of cerebral origin'·. A similararrangement probably obtains for the corporaallata.

The hormone of the brain cells has been claimedby Kobayashi" to be a lipid, and even cholesterol.However, other .J apanese workers'" have obtainedactive peptides from insect brain extracts, andGersch et al.''' have reported on four active substancesin the brain of the cockroach (Periplaneta americana),two of which seem also to be polypeptides. Whichof the many active substances found in extracts ofcorpora cardiaca arc neurosecretory products, stem­ming from the axons of the brain cells, has not beenestablished. Three, at least, of the active constituentsof the corpus canliaca of the cockroach have beenclearly defined as peptides'o, but a number ofpharmacologically active 'small molecules' ha\'ealso been found in extracts of this organ'I-'3.-Crustaceans --Most of the known endocrine systems

of crustaceans (sinus gland, postcommissural organs,pericardial organs) arc neurosecretory. Only thechemistry of the hormones of the pericardial organshas, to some degree, been investigated. Some of theseseem to be polypeptides" 56.

While clearly only a beginning, these findinl{ssuggest that the situation in invertebrates may not beso very different from the' chemical spectrum' ofvertebrate neurosecretory systems: if, for example,the activities in mammalian hypothalamic extractsare considered as a whole. one finds a similar mixtureof families of specific polypeptides and of smallmolecular substances of more general distribution(S-hydroxytryptamine, acetylcholine, etc.).Returnin~ now to the main theme, namely the

characteri~ation of neurosecretory systems, it wouldseem that anyone criterion is insufficient. At pres('ntthese systems may perhaps be best defined in thefollowiur; terms: (I) neurosecretory fibres formcomplexes which can be histologically recogni~ed;

(2) ultrastrncturalh', they contain membrane-boundsecretory granules; (3) they produce ~recilic sub­stances which lI1a\' he mainly (or perhaps e\'enexclusively) biologically active polypepticle~.

A sharper definition is difficult - the distinction,for example, between' true' neurosecretory systemsand cholinergic and adrenergic neurones is not anentirely clear one. However, there can be little doubt

that neurosecretory neurones are an essential linkin the control of endocrine secretions.

Summary

Neuro-endocrine reflex arcs and the significance ofneurosecretory neurones in such systems are dis­cussed. The complexities of the afferent part ofthese arcs are illustrated by enumerating the regionsof the central nervous system which may be concernedin the release of oxytocin by the stimulus of suckling.Another example given is the pathways leading to thesecretion of antidiuretic hormone after an increase ofblood oSll1oticity. The similarity between the lattermechanisms and the neuro-endocrine feedback of thehormones of the adenohypophysis is poin ted out.The neurosecretory fibres are defined as part of theefferent arc, but it is stressed that it is not easyto give an unequivocal characterization of these units:Their elcctrophysiological properties are probablythe same as those of' ordinary' neurones and whileultrastructural studies show that they usually containelectron-dense inclusions of 1000-3000 A. diameter,secretory granules (perhaps with morphologicallyslightly different characteristics) are also found inother parts of the nervous system. The topographicalrelationships of neurosecretory fibres are of some helpin defining them, but recent work has shown thatthe endings of neurosecretory fibres may also end in,or near, other structures than blood vessels. Arecent suggestion is then discussed, namely thatthere are two different forms of neurosecretion,one concerned with the production of peptides andone with smaller molecular substances, and that thesetwo types have ultrastructural correlates. A surveyof the chemical nature of neurosecretory products invertebrates and invertebrates is, on the whole, shownto favour this concept, although the important provisomust be made that the biochemistry of neurosecretoryproducts is as yet in its infancy.

References

1. SHERRINGTON, C. S., The integrative action oj the"ervous system (Cambridge University Press,Cambridge),1947.

2. X:\UTA, vv. ]. H., in Advances -in 'neuroendocrinology,edited by A. V. Nalbandov (University of Illinois Press,Urbana), 1963, 5.

3. DENAMUR, R, Dairy Sci. Abstr., 27 (1965), 193,263.4. VERNEY, E. B., Irish f. med. Sci., No. 345 (1954), 377.5. JEWELL, P. A. & VERNEY, E. B., Phil. T.ans., 240B

(1957),197.6. CROSS, B. A. & GREEN, J. D., J. Physiol., 148 (1959).

554.7. BROOKS, C. Me. C., USHIYAMA, J. & LANGE, c., Anter.

J. Physiol., 202 (1962), 487.8. KOlZUMl, K., ISHIKUM1, T. & BROOKS, C. Me. C., J.

Neurophysiol., (1964), 878.9. WOODS, J. W. & BARD, 1'., Acta elldocr., Copenhagen,

35 (1960), (Supp!. 51), 113.10. YAGI, K., BERN, H. A. & HAGADORN, J. R, Ge... compo

Endocri ... , 3 (1963). 490.11. K.\NDEL, E. R, J. ge... Physiol., 47 (1964), 691.12. llARRAeLOUGH, C. A. & CROSS, B. A., J. Endocrin., 26

(1963), 339.13. DOUGLAS, W. W., Nature, Lond., 197 (1963), 81.14. DAN.IEL, A. R & LEDERIS, K., Gen. compo Endoc.i... ,

3 (1963), 693.15. DANIEL, A. R. & LEDER!S, K., J. Endoc.i... , 34 (1966),

91.16. IlARKER-J13RGENSEN, C., Arch. Anal. micro Morph. exp.,

54 (1965), 261.

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17. BERN, B. A. & KNOWLES, F. C. W., in Neuroendocrino­logy, edited by L. Martini & W. F. Ganong (AcademicPress Inc., New York), 1966 (in press).

18. HELLER, H. & LEDERIS, K, in NCltrosecretio/!, editedby H. Heller & R. B. Clark (Academic Pres.' 1nc.,London), 1962, 35.

19. BARER, R., HELLER, H. & LEDER IS, K, Proc. roy. Soc.,158B (1963), 388.

20. WEINSTEIN, H., MALAMED, A. & SACHS, H., Biochem.biophys. Ac/a, 50 (1961). 386.

21. VON EULER, U. S.; Harvey Leel., Series 55, 1961,43.22. WHITTAKER, V. P., MICHAELSON, J. A. & KIRKLAND,

R. J. A., 13ioche1ll.]., 90 (1963), 293.23. OOTA, Y, 1- Fac. Sci. Tokyo Univ., 10 (1963), ISS.24. LWERIS, 1<., Z. Zellforsch., 65 (1965), 847.25. KOELLE, C. B.; Nat"re, Lond., 190 (1961). 208.26. DE ROBERTIS, E., ffistoph.ysiology of sYl1apses and 1UltrO­

secrelion (Pergamon Press, Oxford). 1964.27. HOLMES, R. L. & [(NOWLES, F. C. W., Nalure, T.olld.,

185 (1960). 710.28. LEDERIS, [(.,]. Endocrill., 27 (1963), 133.29. CARLISLE, D. 13. & KNOWLES, F. C. W., Nalure, Lond.,

172 (1953). 404.30. F'OLLENfUS, E. & PORTE, A., in Neurosecretioll, ('<1;ted

by I f. Heller & R. B. Clark (Academic Pre" I nr.,London), 1962, 51.

31. WILSON, L. D., WEINI1ERG, J. A. & BER", H. A.,J. comp.Neurol., 107 (1957). 253.

32. SMOLLER, C. G., Science, 147 (1965). 882.33. LOFGREN, F., Arln 1/Iorph. ·lIcc,.l. scand., 3 (1960),

55.34. NISHlOK.\, R. S .. BERN, H. A. & MEWALDT, L. R, G,n.

comp. EII</o(,riu., 4 (1964). 304.35. KNOWLES, F. C. W., Phil. 1'''''"5., 249B (1965), 435.36. SCHARRER, E., Z. Zellforsch., 37 (1952), 196.37. ~1AZZI, Y., 1Iiv. Bioi., 44 (1952), 429.38. BARGMANN, W., Z. Zellforsch., 42 (1955), 247.39. DH.\RIVAL, A. P. S., • 'ALLAR, R., BATT, M. & MCCANN,

S. 1-1., Endocrillolo/iY, 76 (1965), 290.40. FRANZ . .T., I{ASELBACH, C. H. & LmERT, 0., Acia. ,,,,Iorl'.,

Copenhagm, 41 (1962),336.41. DUEBEN, R. & MElTliS, J., Pror. SOf. e.rp. Riol., .V.Y.,

118 (1965), 409.42. SCHREIBER, Y., E.Tperien/ia, 18 (962), 338.43. (~UILLE:\IlN, R. & SCHALLY, i\. V., in Advanc?s ill neuYO­

cndocril1fi!ogy, edited ur A. V. Nalbanclov (llni\'ersit~· oflliinois Pres>, Urbana), 1963, 314.

44. RUIIREZ, V. I). & MCCA~N, S. ~1., ,4111er. 1. Physiol.,207 (1964), 441.

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-lS. TALWALKER, P. ],., HATNER, A. & MEITES, J., Amer. J,Physiol., 205 (1963), 213.

46. SCHARRER, B., Arch. Anal. Initr. Morph. exp., 54 (1965),331.

47. KOBAYASHI, M., Proc. XVI into congr. Zool., 4 (19(;3).226.

48. fSHIKAWA, M. & ISHlZAKI, H., Natllre, Lond., 198 (1963),226.

49. GERSCH, M., UNGER, H., FISCHER, F. & KAPITZA, \Y.,Z. Naillfr., 186 (1963), 587.

SO. BROW", fl. E., Cm. compo /i·"dorr;'/., 5 (1965), 387.51. GERSCH, M., FISCHER, F., lIN(;EI<, H. & KAPJTZA, \Y.,

Z. Na/urI, 166 (1961), 351.52. COLHOUN, H... H., Expel'ie"lia, 19 (1963). 9.53. n.\RTON-TlROWNE, 1.., DODSON, I .. F. & HODGSO", E. S.,

(;m. compo E"docrin., 1 (1962). 232.54. MWNARD, D. H. & WELSH, J. 1-1., J. Ph)'"iol., 149 (1959),

215.55. CHO\RNIAUX-COTTON, 1-1. & I,LEI"HOLZ, L. 1-1., in Th'

hormones, Vol. 4, edited hy (~. VinclIs, K. Y. Thimann& E. R. Astwood (Academic Pr('s~ Inc., Kc\\' York).1964, 135.

56. C.\RLlSLE. D, B., in Comparalh'l' 1U:t!"orltemistr)'. edited1)\' D. Hichter (Per~alllon 1'1'<',", Oxford), 1964, 323.

57. HOLLAND, R C. & CROSS, n. A., in O.1"1'loriu, editedhv R. Caldeyro- Barcia & H. Hl'il"r (P"rgamon Pr",s,Oxford), 1961, 24.

58. B.\RRACLOUGH, C A. & CROSS, Il. A., J. Neprod. [.'"./.,4 (1962), 213.

59. H,'RR."'·I.OlJGH, C'. A., AlIal. Htc., 136 (1960), 159.60. PORTER, C W., A1IIer. ./. Physiol., 189 (1957), 145.61. HEYER, F. C, ANGUIANO, I .. C & MENo\, J. F., Amer. J.

Phy"iol., 200 (1961), 625.62. ANDERSSON, B., .'leta l,h.1',,;..I. srand., 23 (1951), 1.63. '\NDERSSON, n. & Mct' A!'", S. M., Aria ph)'siol. sca"d.,

35 (1955), I'll.64. CROSS, n. A., in Oxvlorill. c·dih·d hy H. Caldc\TO-Barria

& H. ",,11,·1' (P,'rga'll1on I'r,·ss, OXf<>r<I), 1961.'24.65. CROSS, B. A. & SII.V"", f. :\.,./. E·mlorril1 .. 22 (1961),33.66. l'AWAKI'''', :vr., TERAS.\\\·A. E. & "A\\'ACHI, J., ./a1' . ./.

Physiol .. 14 (191)4), 102.67. SHl;lIZll, S., BAN, T. 8: )\{IROTS1I, E., jl1t'd . ./. f)'aka

U"iv., 7 (19.16), 79.68. l\:01KEC.Al\ll, II., Y.'\l\I:\J).\, T. & USl'l, 1\., Tulia. r.'yrMat.

·"e11.1·. jap., 8 11954\, 7.69. MEN,', F., .'\Nr;\II."o, J. C. & BEY"R, c., Tloln. JII,I.

Est/fd. mCd. hi ..l. Ulli,'. 11{/(. MeL. 19 (19611,119.70. SHEALEY, N. C. & I'''''''-E, I.. T.,). N'·/frnphvs;ol., 20

(1957),125.

Recent Advances in the Analysis of LipidsU. K. MISK ....

Department ')[ Radioisotopes & Biochemistry, Yallabhbhai Pah>1 Chest Institute, University of Delhi, Delhi 7

DURING ~.!Je last decade si~nificant progresshas been m'ade in understanding the chemistryand metabolism of lipids. These advances

have been mainly due to the ~reat emphasislaid on the etiology and the possible cure foratherosclerosis and other diseases connected \,ithlipid metabolism. In spite of the difficulties oftenencountered by workers in this field, a remarkableprogress has been made in developing improved pro­cedures for the isolation, fractionation and chemicalcharacteri7.ation of the biolof{ically occurring lipids.With the development of gas-liquid chromatography,followell by the application of silicic acid adsorbentsto column chromatography and later to thin layerchromatography, the biochemist has been able toseparate efficiently the many components of complexlipid mixtures. The present review will discussthe recen t methods employed in achieving theseparation and identification of biologically occurringlipids.

Extraction and Isolation of Lipidsfrom Tissues

The lipids in tissues exist largely bound to proteinsas lipoproteins. Therefore, the various extractionprocedures employed for isolating lipids from tissuesinvolve the usc of the solvent system, usually amixture of polar and non-polar solvents, whichrupture the proll'in to lipid bond and at the same timeextract the lipid completely. Various solvent mix­~ures have been used for extracting lipids fromtissues a])(1 body fluids, such as ethanol-ether,chloroform-methailOl, etc.1. These solvent mixtureshave inherent difficulties of achieving the desiredextraction. One solvent system, e.g. ethanol-ether,may be efficient for lipid extraction from plasma butnot from other tissues such as brain. The conditionsof lipid extraction may also vary. For example,heat is required in the case of fatty tissues.Chloroform-methanol mixtures in varying proportionsarc the solvent systems of choice for extracting lipidsfrom tissues and body fluids'. The extraction oftotal lipids is quantitative with this solvent mixture.The optimum solvent to tissue ratio for extraction oflipid may vary from tissue to tissue. Hanahan' hasreported that three parts of solvent to one part oftissue are quite adequate for complete extraction.The extraction of lipids at 4°C. will avoid the possibleactivation of certain lipid degrading enzymes.Exposure to atmospheric oxygen and light (whichinduces peroxide formation) during extraction shouldbe avoided as these will improve the quality ofthe final lipid extract. Sometimes a further checkshould be made for complete extraction by subjectingthe solvent extracted residue to acid or alkalinehydrolysis at reflux temperature. If no fatty acidsare recovered in the hydrolysates, it is reasonableto assume that the extracted material represent the'total lipids"-'.

Chloroform-methanol solvent mixture with certainmodification, e.g. addition of water, NaCI, IN HCI,prior treatment of the tissues with acetone, etc., hasalso been used for the extraction of proteolipids6- n ,phosphatidopeptidesI2, triphosphoinositidesI3, gang­liosides14-1H and sulphatides19 ' 24 .

Palmer and RossiterI3 have recently describeda method for the isolation of mono-, di- and tri­phosphoinositides from tissues. In this method mono­phosphoinositides are extracted with chloroform­methanol (2: I, vol./vol.) and di- and triphospho­inositides arc extracted when one part of conc.HCl is added to 300 parts of chloroform-methanol(2:1, voL/voL). The phosphoinositides are thenseparated chromatographically on formaldehydetreated paper.

Possible Alterations in Lipid Structureduring Extraction

The optimum conditions for the extraction of lipidsfrom tissues and body fluids vary from tissue to tissue.The nature of the sample, the purity of the solventsused, temperature, light and oxygen and the pro­cedures employed for extraction can significantlyinfluence the end results. Use of elevated tempera­ture may alter the lipid composition of the mixture.The elevated temperatures along with solvents mayactivate certain lipolytic enzymes, e.g. activation ofphosphalipases A, C and D by solvents'5-2H. Theimportant considerations to be kept in view whileattempting the isolation of lipids from biologicalsources are: (i) use of nitrogen atmosphere, (ii) use ofpurified and suitable solvents, (iii) rapid .removal ofthe tissues after sacrificing the experimental animalsto minimize enzymatic changes, (iv) quick macerationof the tissues, (v) use of proper solvent to tissue ratio,(vi) heat only when necessary, (vii) removal of non­lipid impurities without loss of lipid, and (viii) storageunder conditions that minimize alterations in lipids.

Removal of Non-Lipid Impuritiesfrom Lipid Extract

Lipids tend to solubilize non-lipid impuritiessuch as sugars, free amino acid, urea, etc. Thefollowing methods have been used to remove non-lipidimpurities from lipid extracts.

Partition method - In this method the lipidextract is partitioned with 0·2 volume of water orsalt solutions', viz. Na, K, Ca or Mg. The use ofsalt solutions avoids the interfacial fluffy layerand also decreases the loss of more acidic types ofphospholipids, at the interface or in the water phase.The lower phase (chloroform layer) contains the purelipids.

Dialysis - In this the lipid solution in petroleumether is dialysed through a rubber membrane for24 hr against petroleum ether. All the non-lipidcontaminants are dialysed out. A routine checkshould always be made on the dialysate for lipid

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J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

components. If they appear, the fractions arerecombined and dialysis repeated29.

Other effective methods for the removal of non-lipidmaterial from lipid extract have been reportedusing paper chromatography30, electrodialysis31 ,electrophoresis32 and sephadex columns33.

Storage of Lipid Extract

The conditions under which lipid extract is storedare quite important, for it has been reported thatnormal atmospheric conditions of storage andunfavourable temperatures significantly alter thecompositions of lipid mixtures3'. Lipid extracts, richin polyunsaturated fatty acids, are more susceptibleto these changes and may result in auto-oxidized andpolymerized products. Exposure of lipid extract tolight can modify its cholesterol content35. Thebrowning and formation of viscous solutions of aminophospholipids have been reported in cases whereadequate precautions for storage were not taken.In order to minimize these changes in lipid mixture,they should be stored in chloroform in a reasonablydilute solution at or under _8°C. in an atmo­sphere of nitrogen with minimum exposure tolight. .

Fractionation of Lipid Extract

In addition to the possible oxidative changes andenzymatic alterations occurring during the isolationof lipids from tissues, the presence of contaminantscomplicates the fractionation procedures. Severalschemes involving complex formation2-5, solventfractionationH , countercurrent distributionH ,36, saltfractionation2-5, column chromatography37-39, etc.,have been employed to fractionate lipid mixturesand these have been reviewed recently2-5. Whileno one procedure can be recommended as thebest, the ,column chromatographic techniques haveproved very satisfactory and these will be discussedhere.

Acetone Fractionation

Acetone has been widelv used for the initialfractionation of phospholipids from the lipid mixture2•

Phospholipids are insoluble in acetone whereasneutral lipids are soluble. Thus phospholipids of beefand rat liver have been obtained in 95 per cent yieldby acetone treatment of the total lipid preparationat room temperature. The fractionation can also be'carried out at 4°C., but in this case a significantamount of neutral lipids may be precipitated alongwith phospholipids. Acetone precipitates certainphospholipids adequately but in many instances itfails to precipitate all phospholipids. For example,phospholipids of erythrocytes and haddock flesh arenot completely precipitated even at _20' or -25°C.In order to overcome this difficulty MgClz, which aidsconsiderably in precipitating all phospholipids, isadded to acetone. The addition of MgCl2 may alterthe cation composition of some of the more acidicphospholipids found in many tissues (i.e. phospho­inositides and phosphatidylserine). Trichloraceticacid has also been used to precipitate phospholipids,but this precipitant may cause some hydrolysisof plasmalogenic phospholipids even under mildconditions'·.

304

Column Chromatography

Column chromatography is of immense value in theisolation and purification of lipids in the study oitheir metabolism. Development of chromatographicmethods has made most problems in lipid chemistryand biochemistry more practicable than. they werepreviously. The problem of resolv;;lg lipid extractsfrom natural sources on a preparative scale intoseparate, intact, molecular species has been investi­gated most su~.cessfully by column chromatographyon silicic acid"7,39. Therefore, it is important to stressthe necessity of complete separation of a lipidmixture. The technique of column chromatographyis dependent on many factors and the investigatorshould choose the conditions giving maximum degreeof separation of lipid mixture. Some of these factorsare discussed below.

Adsorbants - The adsorbants which have beenused in column chromatography for lipid separationare magnesium oxide38,41,42, alumina2,'3-'5, cellu­loseI2,:lS,46,47, silicic acid39 ,.s-67, florisiI6S-7:l, diethyl­aminoethyl cellulose3s, •• ,74 and methylated sephadexG_2575-7R. Among these, silicic acid has been mostwidely used. In most cases the selection of theadsorbant is the first problem. The capacity toadsorb the lipid strongly and allow the displacementwithout difficulty is a necessary requirement of anadsorbant. The adsorbant should have a preferentialaffinity for the lipid. The second important require­ment is the particle size of the adsorbant. Theparticles should be small enough to give a maximumadsorptive surface, yet not so small that they tightlybind the solute and give extremely low flow rateresulting in the involvement of more time and otherfactors. The next thing to be considered is theavailability and reproducibility of the adsorbant.This is particularly true of silicic acid.

Column - The selection of the column is governedto a great extent by the amount of adsorbant to beused. Long narrow glass columns usually give thebest separations. However, more narrow columnswith smaller particle size adsorbants give slower flowrates. A general practice is to keep the ratio of lengthto diameter of the column within a range of 5: 1 to15: 1, with the final choice made on the basis of theamount of adsorbant needed to hold and separate thesolute and the desired flow rate. Some preliminaryexperimentation will be necessary before a finalselection can be made.

Elution - In practice, solvent pairs arc often usedand elution carried out with the mixture of the two.A solvent pair usually includes a polar and non-polarmaterial, the selection of which is based on theirability to elute the most polar members of thelipid mixture to be resolved. The solvent pairswhich have been used to elute neutral lipids fromsilicic acid columns are mixtures of petroleum ether­benzene, diethylether-hexane and diethylether-pen­tane and benzene-hexane. For eluting phospholipids,chloroform-methanol mixture has been used38,39.When neutral lipids are desired to be separated fromphospholipids, they are eluted with chloroform andthen phospholipids with methanol. Once the selectionof solvent has been made, the lipids from the columnmay be eluted by (i) stepwise elution or (ii) gradient

MISRA: RECENT ADVANCES IN THE ANALY::iIS OF LIPlDS

elution79 • In stepwise elution, the column is elutedwith predetermined quantities of different specificmixtures of the solvent pairs. This type of elution isquite adequate when compounds of different polaritiesare to be separated. In gradient elution, the columnis developed by the addition of a continuouslychanging proportion of one solvent to the othermember of the Pilir. This method of elution is usefulwhen it is desiret} to separate as many individualcompounds as possible or to separate a mixture ofclosely related solutes on the basis of their polarity.In both stepwise and gradient elutitln, the usualpractice is to start with the least polar mixtureor a member of the pair and increase the polarityby the addition of the more polar material.

The elution of lipids from the column involvesdisplacement analysis. The relative degree of soluteand solvent affinity to adsorbant can be expressedin terms of the polarity, and this offers a convenientmode of expression of displacement development.If one considers a natural lipid mixture from thestandpoint of polarity, the individual componentsmay be rated with respect to degree of polaritybeginning with the least polar hydrocarbons andconcluding with the most polar phospholipids, i.e.lysolecithin.

Silicic Acid Column Chromato~raphy

The use of silicic acid for separating lipid mixturewas made by Trappe who separated glycerides fromcholesterol esters"s. Borgstrom's used silicic acidto separate neutral lipids from phospholipids. Sincethen, silicic acid has been used effectively to separatethe lipid mixtures from various animal tissues andbody fluids"-67 . Silicic acid has the advantage ofbeing commercially available in highly pure formand the results obtained with it are quite reproducible.:F-he elution pattern of neutral lipids from silicicacid columns is of the following order: hydrocarbon,cholesterol, triglyceride, unesterified fatty acid,free fatty acid, free cholesterol, diglyceride andmonoglyceride. In the class of phospholipids, moreacidic type phospholipids are eluted first followed byinositides and phosphatidyl choline, sphingomyelinand lastly lysolecithin (Table 1).

Silicic acid columns have certain limitations,though they are widely used for separating lipidmixtures. The main difficulties are tailing of peaksand non-reproducibility of results. These difficultiesarise because silicic acid is not an ideal adsorbantfor all classes of lipids, especially phospholipids,where various phospholipids such as cardiolipin,phosphatidyl serine, inositides, sulphatides andgangliosides overlap in the elution scheme80• Thedifference in the binding of sphingomyelin with silicicacid as compared to lecithin is not great enough forthe complete separation of these two substances38,81,82.

These difficulties can be overcome to a great extentif certain factors discussed below are taken into con­sideration in using silicic acid as the adsorbant forlipid separation.

Water content - Water content of silicic acid deter­mines its adsorption properties. The adsorptioncapacity decreases with increasing water content.For the separation of neutral lipids the water contentof silicic acid has to be nil, whereas the presence of

TABLE t - ORDER OF ELUTION OF LIPIDS FROM SILICICACID COLUMNS"

I Hydrocarbons

I Esters other than steryI esters and diglyccridesSteryl esters

~ Fatty aldehydes (1)Triglyceride,Long chain alcoholsFatty acidsOuinol1(,sSterolsDigl yceride,l\1onogl yccridesGlycolipidsLipoamino acidsBile acidsGlycerophosphatidic acidsInositol containing lipidsPhosphatidyl ethanolaminesLysophosphatidyl ethanolaminesl.ecithinsSph,ingomyelinsLysolecithinEthersMiscellaneous lipids

water in silicic acid favours the separation ofphospholipids.

Silicate content - Most silicic acids contain aconsiderable quantity of silicate which binds waterfirmly resulting in increased retention time of thelipid on the column. Therefore, the purity of silicicacid is of considerable importance in reproducibilityof results.

Particle size - Great care should be taken inchoosing the particle size of the silicic acid.Particle size should be such that there is enoughsurface for lipid binding and desirable flow rates areobtained with least overlapping of fractions.

Loading factor - The amount of lipid applied onthe column is of great importance. A convenientamount of lipid should be applied to achieve thebest results. The reasonable amount of lipid whichshould be applied on the column is in the range of6-10 mg.fg. silicic acid. Overloading of the columnmay result in the overlapping of certain fractions andpoor separation.

For a good fractionation of lipid mixture, freshbatches of suitably activated silicic acid should beused. This requirement is not so rigid in the case ofphospholipids where the same column can be reusedafter adequate washing of the column with methanolfollowed by chloroform. The use of cotton plugsfor supporting of column material should be avoidedas this can adsorb some of the phosphoinositides2•

Activation of silicic acid is achieved by heating it to110°C. for 24 hI' and sieved' to the desired particlesize. The silicic acid can also be treated withsolvents, e.g. diethylether and acetone and heated at1000 e. for 3 hI' and sieved to the desired particle size2•

The columns for separation are prepared by pouringa slurry of the activated silicic acid in suitable solventinto glass columns. After adequate washing of thecolumn with solvents, the lipid is applied at the topand elution started. The column can be operatedin a temperature controlled room, or fitted with a

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J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

Chart 1 - Ion exchange reaction of phosphatidyl ethanol­amine with silicic acid38

Substances eluted

Phosphatidyl serine andgangliosides

Cerebroside sulphate, ino­sitol phosphatides andcardiolipin

Lecithin, sphingomyelin,ccramidc, cerebrosides,cholesterol, lvsolecithin,sterol esters and glycerides

Phosphatidyl ethanolamine

Water-soluble non-lipids

Free fatty acids

Chloroform-methanol (7; 3)

Methanol

Chloroform-glacial aceticacid (3; 1) containingO'OOloM potassium acetate

Glacial acetic acid

Chloroform-methanol (4; 1)containing 10 mL/litreconc. aqueous ammonia

Eluting solvent (vol./vol.)

Chloroform-methanol (7; 1)

TABLE 2 - ELUTION SCHEME OF LIPIDS FROM DEAE­CELLULOSE (ACETATE FORM) COLUMNS"

Diethylaminoethyl (DEAE)-CelluloseColumn Chromatography

The major drawback of silicic acid is that theseparation of acidic and non-acidic lipids is not clea.~.

To overcome this difficulty cellulose ion exchangershave been used for fractionating lipid mixtures.Several ion exchange cellulose columns such ascarboxymethyl cellulose, disodium <-hosphocellulose,Ecteola triethylaminoethyl (T t:AE)-cellulose andDEAE-cellulose have been used for phospholIpidseparation""'S4~ These exchangers do not require thepresence of water for the rete.ntlOn of lIpids andreproducible results are obtamed under carefulconditions. Among these DEAE has been reportedto be the most promising for lipid separation. Ecteolabehaves essentially like DEAE and TEAE is similarto DEAE in many respects. The difference betweenDEAE and TEAE is that acetate and chloride formsof TEAE do not bind lipid, instead hydroxyl formbinds lipids readily. Another difference betweenDEAE and TEAE is that DEAE in acetate formreadily binds phosphatidyl ethanolamine in theabsence of water, whereas the hydroxy form of TEAEdoes so only in the presence of water. DEAE­cellulose has been used in the acetate form becausethis form does not liberate strong acid during ion ex­change reaction that would destroy acid labile lipids,e.g. plasmalogens. After a thorough prewashing withIN HCI followed by water and IN KOH, the DEAE­cellulose is put in the acetate form by treatmentwith glacial acetic acid. The aCid IS washed out withmethanol and the column is prepared in a suitablechloroform-methanol mixture, before application ofthe sample. Phosphatidyl ethanolamine is obtainedin pure form from DEAE-cellulose column andall the water-soluble non-lipid contaminants areeluted in a single fraction free of lipid (Table 2).The other fraction obtained, however, must :::separated into individual lipid classes by other means,e.g. silicic acid column chromatography. . .

The zwitterion lipids, sllch as leclthm andsphingomyelin, show very lit~le tendency to bind toDEAE-cellulose and are retamed only very slIghtlyby DEAE. They are eluted with non-ionic solvents

PE

o- II +O-P-O-(CH,),- NH,

dR PE ""\,.

t 0

I 1 + II-Si-O H,N-(CH,l,-O-P-OR

t I J)HI ~ }t/Jf PE silicale

-Si-OI-1 + H,N-(CH')'-i-OR

I OH

Silicic acid

I-Si-OH +

ISilicic acid

water jacket to maintain a constant temperatureduring elution. The columns should be protectedfrom light. The flow rate should be reasonable(10 ml./2-3 min.). If the flow rate IS sl0:-V, th: desIr­ed flow rates can be achieved by applymg mtrogenpressure. Neutral lipids are eluted with inc~easing

concentration of diethylether in hexane, or dlethyl­ether-petroleum ether (b. p. 40-60°C) or diet~}:'lether

in pentane, or benzene in hexane.. PhospholIpids a~e

eluted with increasing concentration of methanol 111

chloroform. The progress of elution of lipids from thecolumns can be followed either by weight assay orby chemical analysis of the particular lipid componentin effluent"a. For every unknown sample the amountof a particular solvent system to elute a particularcomponent has to be predetermined. The lipid com­ponents eluted from the column are pooled accordingto the size of their peaks and evaporated todryness in vacuo, preferably in a rotary. evaporatorunder nitrogen at 40-45°C. The dned reSidueis dissolved quantitatively in a known volume ofchloroform and stored under nitrogen at or under_8°C.

Silicic acid columns appear to function largelyby ion exchange and hyd~ogen bonding38.. The ionexchange reaction of SIlICIC aCid can be Illustratedby the behaviour of phosphatldyl :tha!10lamme onsilicic acid columns (Chart I). A Similar IOn exchangereaction occurs with lecithin, resulting in its strongerbinding on the column than the phosphatidylethanolamine.

The role of hydrogen bonding in silicic .acidcolumn chromatography is illustrated by the differ­ences in the elution characteristics of a number ofcompounds. Lysolecithin is eluted from silicic ~cid

c.olumns after lecithin. The ion exchange reactIOnsare presumably the same for both molecules, but thepresence of hydroxyl group in lecithin creates anadditional site for hydrogen bondmg resultmg m thestronger binding of lysolecithin on the column.The ion exchange reactIOns do not occur m the case ofcholesterol. A substance such as lysolecithin whichinteracts bv ion exchange and hydrogen bondingrequires methanol for elution. Cholesterol withone OH group is rapidly eluted with ether, whilecerebrosides containing several OH groups reqUIrechloroform-methanol mixture for elution.

306

:lIlSRA: RECENT ,\IJVANCES IN THE AN ..\LYSIS OF LIPIDS

1'1' hOllnd to DEAE

Chart 2 - Ion cxchallJ{t' rl'action of phosphaticlyl ethanol­amini' with 1)Ei\E-ceIl1lIos(~3M

such as chloroform-methanol. The non-ionic lipids(cholesterol and cerebrosides), which do not havestrong hydrogen bonding, pass through the ionexchange cellulose in the presence of polar solvents~ methanol. Phosphatidyl ethanolamine is a

proton donor in the zwitterion form and is retainedmore firmly by DEAE and is elutable from the columnwith a non-ionic solvent such as chloroform-methanol(7: 3, vol./vol.) mixture. This follows from the factthat DEAE is itself a proton donor. Escape from thepositively charged site on the adsurbant is possibleas the proton of the diethylaminoethyl group can betransferred to the phosphate group of phosphatidylethanolamine with the subsequent release of phos­phatidyl ethanolamine molecule from the adsorbant.A proton transfer from the phosphate to -NH2to give -NH,t would then restore the· phosphatidylethanolamine to its original zwitterion form (Chart 2).Acidic lipids in the salt form exchange ions withDEAE. Phosphate or other acidic groups interactwith the positively charged site of the adsorbant,with the production of potassium acetate, which iswashed through the column. Acidic lipids areretained very firmly and are eluted from DEAEwith acidic or basic solvents. The overloading ofDEAE-cellulose columns results in the elution ofacidic lipids with non-ionic solvents.

Cellulose Column Chromato~raphy

Cellulose columns have been used for removingnon-lipid materials from lipid mixtures'2,3s,~6,~7.

Svennerholm and Svennerholm's have used cellulosecolumns for preparing gangliosides from brain. Rouser

Lipid eluted

HydrocarbonsCholesterol estersTriglyceridesCholesterolDiglyceridesMonoglyceridcsFree fatty acids

Eluting soh-ent

Hexane·Ether (5 %) in hexaneEther (15%) in hexaneEther (25%) in hexaneEther (50%) in hexaneMethanol (2 %) in etherAcetic acid (4%) in ether

'Purified skellysolve B

TABLE 3 - ELUTION SCHEME. OF LIPIDS FROMFLOltISIL COLUM~87

et al.as have used cellulose columns for separatinglipid mixture. Whatman cellulose is washed withmethanol, followed by water and then dried. Thecolumn is prepared by pouring a slurry of cellulose inchloroform. The column is then washed with chloro­form-methanol-water (16: 4: I, vol./vol.) mixture.All the lipids are eluted first with the above solventmixture and then the gangliosides with chloroform­methanol-water (4:15 :1, vol./vol.) mixture. A goodseparation of gangliosides is achieved by cellulosecolumn chromatography"'.

Silicic Acid-Silicate-Water ColumnChromato~raphy

These types of columns have been successfully usedfor the fractionation of phosphatidyl ethanolamine(PE) from phosphatidyl serine (PS) from brains5,liver and tiles6 . These columns are convenientlyprepared by passing a mixture of chloroform­methanol-aqueous ammonia through a bed of silicicacid in a chromatographic column. When a mixtureof PE and PS is applied on the column, PE is elutedwith chloroform-methanol (4: 1, vol./vol.) mixtureand PS with methanol. Silicic acid-silicate-watercolumns appear to function in part as partitionchromatography columns. A pure silicate columnwith a monovalent ion (such as ammonium ion) isof limited value for the separation of lecithin andsphingomyelin, as such columns have a very limitedcapacity. A mixture of silicic acid and silicate isadvantageous because the ion exchange propertiesof silicic acid are retained and the system has ahigh capacity. Lipids are bound less firmly to thestationary partit ion phase and the peaks do not havethe long tailing portions characteristic of pureabsorption columns.

Ma~nesium Silicate (Florisil) ColumnChromato~raphy

Florisil has been used by various workers toseparate faecal lipids66, tocopherols69 , standard lipidmixtures", steroid hormones7o• cerebrosides71 andfattv acid esters". Recently, Carroll13 has presenteda scheme for the quantitative elution of lipids fromf10risil columns (Table 3). The order of elution ofneutral lipid classes from f10risil columns is the sameas that observed with silicic acid columns, but f10risilhas the advantage of being in a coarse mesh size.Florisil seems to have as much adsorptive surfaceas the commonly used fine mesh preparation ofsilicic acid and lipids up to 10 mg. or more per gram

oII -I-

H..-O-P-O· (C1I,l,-NH,

-6PE

oII

IIO-I'-Oll.

IoI

11,:\-(('11,\,

j-l-o

- IIO-P-OH J

Ia-I- I

II,N - (CII,),

I.j,

H,(/

/(l- I' = () -1- C11,. ,CO()(l

" .0- ((,11,),- :\11,

··-"~1

o-I- - II

R-O-(CH,),-!\-J-l O-C-CH"I .

(CH,-CH,),11EAE ac<'tate

-I­R-O-ICII,),-!\ -II

I(ell,-C1-I,,),

1­I

-l-R-O- (CII,),-:"(C1I,-CI-I,),

307

J. SCI. INDUSTR. RES., VOL. 25, JULY t966

of adsorbant may be readily separated withoutevidence of overloading. Although f10risil has provedvery satisfactory for the separation of neutrallipid class, it cannot be used to separate and recoverphospholipids satisfactorily. Free fatty acids likephospholipids arc adsorbed more strongly on f1orisil,but it is possible to recover free fatty acids quanti­tatively by including acetic acid in the elutionsolvent mixtures. This is a further advantage offlorisil over silicic acid, since the free fattv acidsand triglyceride fractions found to overlap onsilicic acid are easily separated on f1orisil. Recently,Carrol1'3 has reported that if .fiorisil is treated withcone. HCI, a separation of phospholipids similar tosilicic acid columns can be achieved.

Magnesium oxide columns have been used toseparate choline containing phospholipids fromnon-choline containing phospholipids, which arestrongly adsorbed on the columns38 ,41,42. The re­sults obtained with this adsorbant are not quitereproducible. Aluminium oxide columns have beenused for the preparation of lecithin of highpurity2,43-4s. But recently it has been reported thataluminium oxide degrades some lecithin to Iyso­lecithin88. On aluminium oxide columns non-cholinecontaining phospholipids are firmly retained, whereascholine containing phospholipids are eluted withchloroform and methanol mixtures. For a succes:;fulfractionation of phospholipids on these columns, thepH of alumina should be slightly on basic side.

Recently, methylated and acetylated sephadexcolumns have been used for the separation of lipidmixtures's-'8. The advantage of using methylatedsephadex in lipid separation is that phospholipids areeluted ahead of triglycerides, cholesterol esters,cholesterol and fatty acids. Methylated sephadexappears to be of considerable potential value in theseparation of lipid-soluble substances and mayserve as a complement to partition and adsorptionchromatographic procedures. The separation appearsto be based partly on the differences in size and/orshape of the molecules and partly on liquid-liquidpartition between a mobile phase and a stationarygel-solvent phase. By choosing different solven-tmixtures, liquid-liquid partition chromatography ofeither the reversed phase or the straight phase typecan be obtained. Recently, Hirsch89 has described thecolumn chromatographic separation of neutral lipidsusing factice, a hydrophobic polymer, as the stationaryphase and aqueous acetone as the moving phase.The neutral lipids, i.e. cholesterol esters, triglycerides,dlglycendes and monoglycerides, have thus beeneasily separated from each other. Separations ofcomponents with different fatty acid compositionwithin the same lipid class can also be achievedwith this liquid-gel system. Thus cholesterol esterswith fatty acid chain length from 2 to 18 carbons areeluted as individual peaks. Similar separations arepossible with glycerides or with mixtures of methylesters of fatty acids. However, with the exception ofcholesterol esters,lipids within a given class containingfatty acids of equivalent polarity are inseparable.E1.ution of lipids is performed with a single solventmixture and detection of lipid in the effluent streamcan be achieved by the use of automatic differentialrefractometry. Free fatty acids and phospholipids

308

are poorly separated and must be removed from thelipid mixture prior to chromatography.

From the foregoing discussion on the columnchromatographic separation of lipid mixtures, it j.

apparent that no adsorbant gives a clear cut andpure fractionation of lipid classes. Therefore, it isnecessary that the eluted fractions be examinedcarefully for their purity prior to)urther analysis.This could be achieved by .y"p~r and thin layerchromatographic techniques. Both these chromato­graphic techniques are rapid and sensitive means ofidentifying an'd separating lipids. A brief discussionof these techniques is presented below.

Paper Chromatography

The intact lipids can be chromatographed on filterpapers90-98 , formalin treated papers99-102, acetylatedpapers'03, tetralin impregnated papers104, silicic acidimpregnated paperslOS-10', phosphate impregnatedpaperslO', glass fibre papers'09-112 , and on thin layersof silica gel coated on glass plates"3, 114. Each ofthese methods for the separation of lipid mixtureinto various constituents has its own advantages anddisadvantages.

Silicic acid impregnated papers - RecentIy, excellentreviews have appeared on the preparation and use ofsilicic acid impregnated papers for the separation ofneutral and phospholipids11s,116. This method hasbeen successfully used for both qualitative andquantitative separation of neutral and phospholipids.After applying suitable amounts of lipids (about15 IJ.g. phospholipid phosphorus and equivalentamount of neutral lipid) the papers are developed insolvent mixtures. Various solvent mixtures havebeen used for developing the chromatograms, butdiisobutyl ketone-glacial acetic acid-water (40: 25: 5,vol./vol.; 40: 20: 5, vol./vol. at room temperatureand 40: 20: 3, vol./vol. for 0_5°) mixtures have prov!,..':1to be the best for phospholipid separation. Forneutral lipid separation solvent systems n-heptane­diisobutyl ketone (96 :6, vol./vol.), n-heptane-diisobutylketone-acetic acid (91: 9: 2, vol./vol.) have been used.The development of the papers should be carried outin a temperature controlled room. Recently, a methodhas been reported where aqueous systems such asplasma, serum, cytoplasm or finely dispersed particlescan be chromatographed directly without priorextraction with organic solvents"6.

Identification of Spots on Chromatograms

The lipid spots on developed chromatograms areidentified by their staining characteristics andmobility behaviour (R! values). This identification,however, can by no means be interpreted as conclusiveand should be further supported by chemical andphysical data. The various staining reagents usedare the following: Rhodamine-6-G (0'001 per centaqueous solution) for all lipids; ninhydrin (0,25per cent in acetone-Iutidine, 9:1, vol./vol.) for amino­lipids, iodine vapours for unsaturation, phospho­molybdic acid (1 per cent aqueous solution), stannouschloride (1 per cent in 3N HCI) or Dragendorfreagentl17 for choline containing lipids, and 2,4-di­nitrophenyl hydrazine (0,15 per cent in 3N HCI) orSchiff's reaction for plasmalogens. Other stainssuch as Tri-complex Brilliant"8,1l9, Biebrich Scarlet,

MISHi\: HECE:'\T ADVANCES IN THE ANALYSIS OF LIPIDS

Nile Blue, Rhodamine B, acid fast violet green andprotoporphyrinl20 have also been used for detection oflipid on the chromatograms. The labelled lipids~n be visualized by exposing the chromatogram to a

photographic emulsion in the conventional manner.The lipid spots on silicic acid impregnated paperscan be eluted with suitable solvents and quantitized.The quantitati'~9 analysis of phospholipids onchromatograms ciiL~I;;;) be done by neutron activa­tion of the phospholipid spotsI21.122. This methodhas limitations. The background ma~ be high, theneutron flux may not convert the same amount ofphosphorus in each spot to radioactive phosphorus,and the amount of activity may not be enough.

The separation of lipids has also been done onsilicic acid impregnated glass fibre papersI09-1l2.

After applying the lipids the papers are developed.in diisobutyl ketone-acetic acid-water-benzene(160: 50: 8: 7, vol./vol.) and the spots detected bycharring (spraying with 20 per cent H2S04), phospho-

• molybdic spray and 0·001 per cent aqueous solutionof 1(hodamine-6 G. The lipid spots can be elutedand quantitized.

The separation of lipids on silicic acid impregnatedpapers is affected by various factors such as theimpregnation procedure, humidity, temperature,the amount and way of application of lipid, exposureof the paper to volatile basic solvents and the,,·ay of storage. In order to get reproducibleresults, all these factors have to be borne in mind.Increased humidity in the atmosphere results inhigher mobilities of the lipids. In some cases,chromatography should be performed at a lowertemperature to minimize degradation of labilecompounds such as plasmalogens. Unless one iscareful, wrong conclusions arc drawn regarding theidentity of one compound from paper chromato-

'"'I\'l'aphic techniques. A single spot on a chromatogrammay not be from a single compound. Improper use ofcolour tests for location of lipid spots can give rise toconfusion. Auto-oxidation of lipids, especially ofphosphatidyl ethanolamine, phosphatidyl serine andlecithin, can give rise to streaking, extra spot orfailure to migrate. Small amounts of divalent cationsmay cause retention of phospholipids, particularlylecithin and sphingomyelin at the place of application.Several other artifacts may be introduced such asthe use of a strongly acidic silicic acid paper orstrongly acidic solvents for chromatography. Theapplication of labile plasmalogen forms of phospho­lipids (particularly of PEl to acidic silicic acid papermay result in appreciable hydrolysis of ()(,~-unsatu­

rated ether bond in the plasmalogen to give a lyso­phosphatide. Therefore, all the interpretations of apaper chromatogram must be confirmed by isolation,hydrolysis, infrared examination, etc.

Formalin treated paper has been reported to begood for the separation of inositolphosphatidesI3,99-102,and three types of diphosphoinositides, and tri­phosphoinositides13 have been separated employing it.Lecithin has been fractionated into five componentson tetraline impregnated paperslO4 . Papers impreg­nated with anionic and cationic resins, ZnC03, CaC03and RaS04 and cellulose phosphate, carboxymethylcellulose and DEAE-cellulose papers have also beenused for lipid separation115 ,116. The resolving power

of these papers is inferior to that of silicic acidimpregnated paper, but good separation is achievedof amino phosphatides. Paper chromatography hasalso been used for analysis of fatty acids of lipidmixtures.

Thin Layer Chromatography

Stahl has demonstrated the potential usefulness ofthin layer chromatography (TLC) in lipid separa­tionln Several excellent books and review articleshave a.ppeared on thin layer chromatography and itsapplication'14,123,124. The main advantage of TLC isits quickness for separation of the lipid mixtures.TLC has been applied for the separation of neutraland phospholipids from several biological sources.TLC can be used both for qualitative and quantitativeseparation of lipids. Certain important points such ascoating material, its thickness on plate, activation ofplate, solvent system, humidity and temperatureshould be kept in mind for achieving the best results.

Other methods such as nuclear magnetic reso­nancel25, mass spectrometryl26 and infraredl27 andnear infrared128 spectroscopy have also been usedfor the identification of isolated lipids.

Gas-Liquid Chromatography

Gas-liquid chromatography has lately attracted theinterest of the lipid chemists and is a techniqueundergoing rapid development. The method wasfirst introduced by James and Martinl?9 when theyseparated the normal saturated carboxylic acids up tolauric acid, and was later extended up to behenicacid130 and since then there has been a rapid expansionin the use of this method in the lipid field. Thegeneral theory and practice of the gas chromatographicmethods are extensivelv treated in recent text-booksand reviewsl31-138 and the problem of presenting theincreasing amount of data in a consistent wayhad led to the recommendation of the AmsterdamCommittee139-141 . Some developments in the field ofseparation of fatty acid methyl esters have beendescribed by Farquahar et aU'". The emphasis inrecent studies of GLC in lipid field has been inimproving separation by finding more suitablestationary phases and in improving the instrumenta­tion. It has thus been possible to extend theworking range for methyl esters of fatty acidsas far as 34 carbon atoms143 and to detect andestimate components below 0·05 per cent in theoriginal sample142.

During the past few years a number of instrumentsfor GLC have become available commercially.In order to reduce the working temperature, tospeed up the development of the chromatograI?sand to improve the separation, it is a common practiceto convert the substances to lower boiling derivatives.The fatty acids are usuaJly separated as methyl estersalthough sometimes higher esters are used. A numberof methods have been used for the preparation ofmethyl esters of fatty acidsI31-138 . The stationaryliquid phases used for fatty acid methyl esters andsimilar compounds are either of the non-polar typesuch as silicone products or of the polar type such aspolyesters. With non-polar phases, unsaturated andbranched components emerge before saturated ones,whereas with polar phases, unsaturated acids emerge

309

J. SCI. INDUSTR. HES. VOL. 25, JULY 1966

after their saturated analogues. Late-emerging com­ponents of one chain length group may be retainedin the column so long that they overlap with compo­nents from the following chain length groupJ44,

The efficiency of separation in a column is the resultof at least two factors: column efficiency and solventefficiency. Column efficiency is often expressedas the highest equivalent of a theoretical plate and ismeasured by the spread of a component when it passesthrough the column. This is related to many factorsassociated with the packing and design of the column.The solvent efficiency or sepatation factor dependson the distribution coefficients of the different solutesbetween the liquid and the gas phase. It is expressedby the ratio of the retention volumes of two peaks.The choice of stationary phase depends in each caseon the particular separation desired. The plate effi­ciency of polar column is usually less than that of anon-polar column '45-J47 . The capillary columns usedby GolayJ4' have height equivalent of a theoreticalplate of the same order as packed columns, but theygive better separation because of the extreme lengthof the column and the very small charge used'49-151.The carrier gas used for fatty acid esters have beenargon, helium, hydrogen all(lnitrogen. Argon is usedin instruments equipped with ~-ray argon ionizationdetectors, while hydrogen and nitrogen are used withthermal conductivity detectors. Hydrogen, alone ormixed with another gas, is used with flame andflame ionization detectors.

The practical identification of fatty acid methylesters on chromatograms is done by comparing theirretention time with suitable standards. Members in ahomologous group may be identified by plotting thelogarithms of retention times, or retention volumesversus chain length or degree of unsaturation.James146 has devised a method to determine thedegree of 'unsaturation of straight chain andsimpler branched chain compounds. By plotting thelogarithm of the retention time measured on onestationary phase against similar values from adifferent stationary phase, saturated components lieon one straight line and monoenes, dienes, trienes, etc.,lie each on a separate parallel line. The quantitativeaspect of GLC has been studied by several workers.A serious problem arises from the fact that thedetectors do not always give a response proportionalto concentration. The detectors most frequentlyused arc gas-density balance'52-J59 , the thermalconductivity cellsI60- 162, and ~-ray argon ionizationcell163,164. In spite of the difficulties in makingquantitative measurements, variation between thelipid compositions of similar biological samples isoften so great that the inaccuracies of the detectorare not apparent, and are small compared to theinaccuracies involved when biological comparisionsare made.

There are certain limitations of GLC analysis andthe quantitative interpretation of the chromatograms.One of them is the stability of the substances insidethe columnI6!>. The esters of polyunsaturated acidsare very stable up to 200°C. (ref. 166). Transesterifica­tion of fatty acid in polyester packed columns has beenreported'67• Esters of some unsaturated hydroxy andhydroperoxy acids undergo dehydration to morehighly unsaturated derivative and the conjugated

310

polyunsaturated esters undergo cis-trans isomeriza­tion'·". Another disadvantage is that some com­ponents of the samples may never emerge from thecolumn and, therefore, will go undetected. This maycause significant errors in calculating the compositionof the samples since this is often based on emergingcomponents only.

GLC technique has also been uselU\)r the separationof fatty alcohols'6lI, fatty -"aI1fchydes169- 17I , fattynitriles ' '', meJllO-, di- and triglyceride173 17\ glycerdethers17• and steroids177 . It is possible to collectmicrogram qU~lIItitiesl7R of substances from analyticalgas chromatograph, and also connect it to a radio­activity counter''''·''". For this specially designedcolumns ami detectors arc needed. Onf' seriou,;<lisadvantag" of preparative ~as liquid chromato­graphy is the possible contamination of productsdue to blcedin~ of the stationary phase and somesubsequent purification is often necessary.

From the foregoing discussion on the separation oflipids by column and impregnated paper chromato­graphy, it is apparent that the elution pattern of lipidsfrom the column and their mobilities on the papershould not be considered as conclusive proof of theiridentity. All the lipid components must be chemi­cally characterized before conclusions about theiridentity arc made. Some of analytical methods ofthese lipids arc discussed bclow. An excellentcompilation of these methods published in thelast twenty years is available"·.

Cholesterol

Numerous methods have been used for the e;ti­mation of total free and esterilied cholesterolm-,o".The most popular reaction for cholesterol estimationis the Lieberman-Burchard (L-B) method20.' • Thereaction mixture consists of a combination of H2S04

and acetic anhydride, which when added to a solutiohof cholesterol in chloroform ~ives blue colour.The fact that the colour stability is affected b~'

H2S04 concentration, solvent, temperature and lightand the question whether free or esterified form ofcholesterol is measured by this reaction have gi\'Cnrise to great number of modifications for the properuse of this reagent''''. The difference in reacti\·it~·

between free and esterified cholesterol is greaterat low temperature and low concentration of H50,.Higher temperatures and larger amounts of acid causesuch differences to become smaller and eventualh'disappear. L-B reaction is not specific for cholestero·1.Highly unsaturated compounds interfere with colourdevelopment. Since the colour formation depeJl(l,;on the combination of the sterol with a strong acid.many modifications using reagents other than H2S04 •

viz. p-toluencsulphonic acid. sulphosalicylic acid andchlorosulphonic acid, havc been used. The additionof an aldehyde, e.g. salicylaldehyde, has been reportedto intensify the colour, but these reagents give ared colour in the blank and also react with estrone,estradiol, anhydrosterone, testosterone and cortico­sterone'o,.

Tschugaeff reaction gives red colour when asolution of cholesterol in glacial acetic acid is treatedwith zinc chloride and acetyl chloride. In this methodboth free and esterified cholesterol give similarextinction coefficients on molar basis. Old solution

M1SR"-: ImCENT ADVANCES IN THE ANALYSIS OF LIPIDS

of zinc chloride and acetyl chloride do not yield(juantitative colours. Acetyl chloride also tends todistil away from the reaction mixture. Becauseof this other reagents such as benzoyl chloride,o-nitrobenzoyl chloride, phenylacetyl chloride andm-nitrobenzyl chloride have been used. Hanel andDam have mwlified the original Tschugaeff reagentsfor the cstimation of cholestero!'o". This method isvery simple and q;.[lck, but does not give quanti­tative values for esterified cholesterol. Choleste101,epicholestanol and cholic acid give • faint colour.Both cholesterol and lathosterol can be determinedsimultaneously with this method. This method hasalso been adopted on a ultramicroscale for theestimation of cholesteroP"".

One of the most popular methods of cholesterolestimation is that of Ziatkis ct at. IRS. In thismethod cholesterol is measured directly by addinga fixed volume of conc. H 2S04, glacial acetic

, acid and ferric chloride solution to serum or plasma.On equimolar basis free and esterified cholesterolgive equal colour intensity. The presence of bilirubin,proteins, tryptophan, haemoglobin, etc., interferein the analysis. Therefore, this method has beenmodified in various respects such as varying theconcentration of ferric chloride, preparation ofstock solution in phosphoric acid, use of extractionprocedures before colour development and use ofother metal halides20

'.

Other methods such as gravimetric204, titrimetric20l,turbidimetric20', fluorometric 20H,209, spectroscopic2!O,

polarographic211 - 213 , haemolysis red blood cells2!4 andthe monolayer film technique3 have also been usedd:lfor the estimation of cholesterol. These methodshave been critically reviewed by Kabara204

.

Total Lipids

Numerous methods using gravimetric, titrimetricand colorimetric techniques have been used forestimation of lipids on micro- and macroscales2!5-222.

In the commonly used gravimetric analysis one hasto be very careful for the moisture which couldeasily be adsorbed by the dried lipid during weighingprocedures. Quantitative estimation of lipids onmicroscales by GLC has also been reported223 ,224.

The method is based on the difference in volatilitybetween lipid and solvents.

Triglycerides

Triglycerides have been estimated by a number ofmethods225 2:.6 such as gravimetric, colorimetric andchromatographic. The most widely employed non­gravimetric methods have been (i) estimation bydifference either of cholesterol ester and phospholipidfatty acids from total titrable fatty acids or ofcholesterol and phospholipids from total lipids;(ii) determination of glyceride glycerol, after alkalinehydrolysis and periodate oxidation, by measurementat 570 m!L of the chromotropic acid-formaldehydecomplex formed; and (iii) determination of glyceridefatty acids by formation of their hydroxamic acids.Recently, infrared spectroscopic methods have beenused for the estimation of triglycerides after theirextraction and separation from other lipids237- 239 .

By this method simultaneous estimation of tri­glycerides and cholesterol esters has been achieved.

This method has been claimed to be simple andquicker than glycerol oxidation method.

Fatty AcidsA number of methods221 ,24D-259 have been used for

the estimation of fatty acids, viz. alkaline isomeriza­tion, iodine value, gas-liquid chromatography, esterdistillation, thiocyanogen value, quantitative paperand thin layer chromatography, titrimetry and colori­metry. The oxidative methods using potassiumdichromate have the disadvantage of being unspecific.The titrimetric methods of Gordon240 and Dole24!

for unesterified fatty acids have been widely usedfor their simplicity, quickness and reproducibility.In the Gordon method the recoveries of palmitic,stearic and oleic acids are quantitative and thetitration values are unaffected by acetoacetate,~-hydroxybutyrate,succinate, etc. The reproducibi­lity and accuracy of Dole's method are as good asthat of Gordon's. Values for plasma fatty acidstend to be somewhat higher by Dole's method,indicating that the exclusion of titratable compoundsother than unesterified fatty acids may not becomplete. These methods have been modifiedaccording to needs of the later investigators.Gas-liquid chromatography has been found to be thebest for fatty acid analysis.

Lipid PhosphorusNumerous methods are available for the estimation

of lipid phosphorus and have been critically reviewedby Lindberg and Ernster260• In the estimation oforganic phosphorus, advantage is taken of theproperty of orthophosphate to form a complex withmolybdic acid which on reduction gives a blue colour.All the reducing agents reduce molybdic acid in theabsence of phosphate. Phosphate accelerates therate of reduction and this acceleration occurs over awide acid range but decreases with increasingacidity. The rate of reduction of molybdic acid,the accelerating effect of phosphate and the end pointsreached in these reactions are all dependent on theconcentration of molybdate and the concentration andnature of reducing agent. Phosphate is converted intophosphomolybdic acid in O·5M H 2S04 and reducedwith l-amino-2-naphthol-4-sulphonic acid (Fiske­Subba I~ow's method). This method has beencritically reviewed by Furukawa et at.261 • A greatnumber of substitutes have been introduced in placeof amino-naphthol-sulphonic acid, viz. hydroquinone,2,4-diaminophenol, monomethyl-p-aminophenol, as­corbic acid, thiosulphate, stannous chloride andferrous sulphate. From the point of sensitivity andreproducibility, Fiske-Subba Row's method givesunsatisfactory results. The acidity remaining aftersample incineration affects the end results. To avoidthese effects perchloric acid (King's method) has beenused in place of sulphuric acid. Some modificationsof King's method have been suggested to increase itssensitivity262. The interference caused by acid labileorganophosphate compounds, e.g. creatine phosphate,in Fiske-Subba Row's method has been eliminatedby carrying out the formation and reduction ofphosphomolybdate at pH 4, in acetate buffer andascorbic acid as the reducing agent (Lowry andLopez's method). In all these methods non-phosphate

311

J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

compounds occurring in biological system interfere.These non-phosphate compounds have been dividedinto three groups: (i) those which alter the acidityof the medium, e.g. acids, alkali, buffers, etc.:(ii) those which form molybdenum complex, e.g.fluorides, citrates, oxalates, etc.. and (iii) those whichchange the concentration of the reducing agent,e.g. nitrates, hypochlorites, etc.' In later modifiedmethods of Lowry and Lopez, the phosphomolybdicacid formed in an aqueous solution of sulphuric acidis extracted with isobutanol (Berenblum-Chainmethod)'60. After separating the two phases, thecolour intensity is determined in the isobutanol.Isobutanol has been substituted with a mixture ofequal parts of isobutanol and benzene (Martin-Dotymethod). The reduction of phosphomolybdic acid isperformed in an aliquot of the organic layer withSnCI2• These modified methods are completelyindependent of interfering substances and the colourintensity is stable within a wide range of time. Thismethod can be employed even when small amounts ofproteins are present in the sample by including silico­tungstic acid in the water phase. The method hasbeen modified to determine 0·002 I-'-g. (ref. 263) phos­phorus. Some methods have been reported which arebased on a colorimetric determination of phosphorus,without involving a reduction of phosphomolybdicacid260. Another procedure is based on the changein a solution of dye quinaldine red in the presenceof phosphomolybdate'64. The determination ofphosphorus based upon Misson's reaction involvingthe determination of yellow phospho-vanadio­molybdate has also been reported'Go. Attemptshave been made to determine phosphorus by flamespectroscopy, but this method is too sensitive to dis­turbances'G•. Recently Bartiett'G6 has reported a heat­ing method for the estimation of lipid phosphorus.This method is quick, simple, sensitiveand reproducibleand the colour developed is stable for long periods.

Glycerol

The estimation of free glycerol in hydrolysatesinvolves the oxidation of glycerol with periodateto form 1 mole of formic acid and 2 moles of form­aldehyde. Formaldehyde is then determined colori­metrically by formation of a complex with chromo­tropic acid267 ,'68. Under the conditions of theperiodate oxidation there is essentially no interferencewith the common sources of formaldehyde such asglucose. Alpha monoglycerides are oxidized andcontribute partially to the glycerol values. To makethe procedure quantitative, a standard solution ofglycerol is similarly treated. During acid hydrolysiswith 2N HCI about 2 per cent of the glycerol is lost2G'.Other methods, such as enzymatic289··m , fluorimetricand chromatographic216, have also been used for theestimation of glycerol in plasma and tissues.Wheeldon et al.277 have described a method where asimultaneous analysis of glycerol, inositol, ethanol­amine and serine can be quantitatively determinedin a lipid sample.

Determination of Inositol, Ethanolamine andSerine in Lipids

Inositol- A number of chemical278-'87, micro­biological288-'99 and enzymatic methods288 (inositol

312

dehydrogenase and pig heart diaphorase) are availablefor the estimation of lipid inositol300 The chemicalmethods in use are nearly all based on oxidation withperiodic acid. Estimation of inositol is based on th~

amount of periodate consumed. The oxidationapparently does not result in the conversion ofall carbon atoms to formic acid. .T!lis procedurerequires complete liberation _ot.-Iipid inositol byhydrolysis since all hydroxy1groups must be freefor complete reaction with periodate. Further,inositol must ,be isolated from hydrolysate becauseglycerol, sugars, amino sugars, ethanolamine, serineand sphingosine, all react with periodate. Thehydrolysis is carried out with strong acid, i.e. 4Nor 6N HCI, in sealed tubes at elevated temperatures(HO-20°e.) for prolonged periods (20-40 hr). Theproducts of hydrolysis can be separated by paperchromatography and the individual componentsdetermined301.

Ethanolamine and serine - Several methods havebeen used for the estimation of lipid ethanolamine andserine, e.g. manometric measurements of nitrogen gasafter treatment with nitrous acid302-a04, manometricmeasurements of carbon dioxide after ninhydrin treat­ment305-308, determination of ammonia after periodateoxidation309-313 and colorimetric measurements usingfluorodinitrobenzene:1l4-31U, ninhydrin320-3ao and1,2-naphthoquinone-4-sulphonate30o. The methodsused for the estimation of serine, with the P .~o'Jtion

of periodic acid oxidation, are not specific anddetermine other amino acids arising from proteolipids,glycoproteolipids, etc. The manometric methodsmeasuring liberated nitrogen are excellent in theirreproducibility and have been used as the standardmethods for the evaluation of newer methods. Theprincipal disadvantage is the inconvenience and therather large amounts of lipid needed for estimation.Also, nitrous acid reacts with unsaturated fatty acid­and releases extra nitrogen giving higher valuesfor lipid nitrogen. The manometric method ofdetermining carbon dioxide liberated from amino acidsby ninhydrin has been widely used for the estimationof lipid serine. The main disadvantage of the methodis its lack of sensitivity. Ethanolamine and serinehave been estimated 'by periodic acid oxidationafter their hydrolysis and separation277• Dut thedrawback of this method is that ninhvdrin does notquantitatively react with amino acids and the

. colour is affected by metal impurities in t.he paper.The liberated formaldehyde is complexed withchromotropic acid. Periodic acid is more specificfor the determination of lipid ethanolamine andserine than the other amine reagents since itrequires a hydroxyl group vicinal to an aminogroup.

In colorimetric methods of estimation of ethanol­amine and serine the dinit.rophenylhydrazine methodis the most sensitive and accurate. Ammoniainterferes with this method. Ninhydrin has beenused to estimate amino nitrogen' and has theadvantage of being a stable reagent and the colouredproducts are the same for all primary amines. Thedisadvantage is that the colour of the reactionproducts is unstable. Lea and Rhodes have usedninhydrin reaction in buffered methyl cellosolveto estimate ethanolamine and serine in unhydrolysed

MISJLI\: RECENT ADVA"lCES IN THE ANALYSIS OF LIPID::;

phospholipids302,32G. The results with egg phospho­lipids showed considerable variation in molar colouryield while free ethanolamine and serine gave~ore consistent result with greater colour yield.A critical review of these methods has been given byMcKibbin30o.

Choline ---;-..Jhe quantitative estimation of cholinein tissoe extracts and hydrolysates is subject to thelimitations inherent' 111 the extraction proceduresand in the specificity and sensitivity of the methodsemployed33H38. The hydrolytic qrocedures ofphospholipids may influence the quantities of itsnitrogenous constituents. The colorimetric reineckatemethod is generally used for routine analysis becauseof its simplicity and speed. The specificity of themethod depends on the nature of the hydrolysis.A more sensitive method is based on the precipitationof choline as the periodide33fi • The method is quitesensitive, but is not very specific because manyother bases, proteins, peptones, purines and alkaloids

'give positive colours. Appleton et al. 33G havedeveloped a very sensitive method for the estimationof a choline-iodide complex. Choline in phospholipidextracts is precipitated as the iodine complex which isextracted with ethylene dichloride and its absorptionwas measured at 365 ml~. The excess of free iodinedoes not interfere and betaine, urea, creatine,creatinine, uric acid, cystine, histidin, guanidine,lys~...;t!1sl arginine are not precipitated by theiodi'ffe reitgent. As little as 5 ",g. of choline can bemeasured. The various methods for choline estima­tion have been critically reviewed by Engel et a[,339.

Fatty acid ester - A number of methods areavailable for the determination of fatty acid estersusing hydroxamic acid formation coupled withferric ion complex production340-35o. However, noneof the methods yields quantitative results forcholesterol esters of long chain fatty acids. Recently,Skidmore and Entenman349 have modified theabove methods to give quantitative estimation oflong chain esters of cholesterol.

Plasmalogens - The methods most widely usedfor the determination of free and plasmalogen boundaldehydes in lipid fractions are the fuchsin-sulphurousacid methods30H,342,3,;t-353, the p-nitrophenylhydrazinemethod254 ,3f,5 and iodometric titration356-358. Thefuchsin-sulphurous acid method has the advantageof being highly sensitive but is not very specific.The p-nitrophenylhydrazine method is very specificbut tends to give too low values owing to the low rateof reaction. The iodometric method is, on theone hand, too specific in that it determines only theunsaturated structures in which the aldehydes arebound (alkenyl ether structure); on the other hand,polyunsaturated acyl groups may give rise to too highvalues.

I dentijication of hydrolytic products of phospho­lipids - From the foregoing discussion on theseparation of phospholipids by column and impreg­nated paper chromatography, it is apparent thatelution pattern of phospholipid components from thecolumn and the mobilities on the paper should not beconsidered as the conclusive proof of their identifica­tion. As stated earlier, all the lipid componentsmust be chemically characterized before conclusionsabout their identity are made. This can be further

substantiated by total or partial hydrolysis of thephosphatides.

Total acid hydrolysis of phosPholiPids - Thephosphatides are hydrolysed with 2N HCI insealed tubes at 11 DoC. for 48 hr. This hydro­lysis will split phospholipids completely to yieldglycerol, serine, ethanolamine, choline, inositol,fatty acids and aldehydes. The hydrolysis productscan be estimated chemically. Recently, Wheeldonet al.m have reported a procedure of hydrolysiscoupled with enzymatic hydrolysis to estimateglycerol, inositol, ethanolamine and serine in thesame hydrolysate. It has been reported that 6N Helhydrolysis destroys some of the glycerol amino acids,ethanolamine and serine2G8,277,293. In certain casesphosphatidyl ethanolamine and phosphatidyl serineare not completely hydrolysed330. The hydrolysisproducts yield important information on the chemicalcomposition of phospholipid molecules.

Partial acid or alkaline hydrolysis - These reactionshave been widely used for the identification ofphospholipids. The phosphatides are hydrolysedunder specified mild conditions with methanolic­NaOH or with trichloracetic or acetic acid329. Ingeneral mild alkaline hydrolysis splits ester bondsand liberates fatty acids. In methanol, the fatty acidsare cleaved off as methyl esters. Acetic acid hydro­lysis cleaves the vinyl ether bond of plasmalogento yield fatty aldehydes. The resulting water-solublephosphate esters are isolated and then separatedby paper chromatography and/or ionophoresis. Thequantitative analysis is carried out by elution of thespots and determination of phosphorus in each spot.Some phospholipids resist alkaline hydrolysis329.

Mild alkaline hydrolysis should be carried outunder carefully controlled conditions. Uncontrolledmild alkaline hydrolysis gives rise to cyclization,acetylation and changes in the mobilities of phosphateestersl16,329,

Marinetti has used methanolic sodium for thehydrolysis of neutral lipids and phospholipids1J6.

The ester phosphatides are completely deacylated atroom temperature within 10 min. Sphingomyelin andcerebrosides are stable towards this reagent and theplasmalogens of monovinyl ether are converted totheir corresponding Iysoplasmalogens. The water­soluble phosphate esters formed by the methoxidehydrolysis can be placed directly on filter paper andseparated by Dawson method329. There is no needto neutralize the methoxide or to isolate the water­soluble phosphate esters before chromatographingthem. It is important to keep the reaction timebelow 30 min., otherwise the N-base of thephosphate esters will be split. Marinetti has usedthis hydrolytic procedure for the preparation ofIysophosphatides (35 per cent yield) and mono- anddiglycerides by carrying out the reaction at DoC.

Plasmalogens are estimated by hydrolysing with90 per cent acetic acid containing one drop ofsaturated mercuric chloride for 30 min. at 90°C.(ref. 359). Lysophosphatides are produced, and theycan be estimated chromatographically. Dawsonestimated plasmalogens by hydrolysing' lower phase'obtained from alkaline hydrolysis (see above) with1 per cent trichloracetic acid containing 5 millimolesof HgCl2for 30 min. at 37°C. The phosphate esters

313

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REVIEWS

THE THEORETICAL SIGNIFICANCE OF EXPEHIMENTAL

RELATlVIp! by R. H. Dicke (Blackie & Sons Ltd.London), 1964. Pl'. xii-j-153. Price 32s. 611.

The volume under review represents the notesincluding a number of reprinted papers for a course oflectures at the Les Houches Summer.school in July1963, given by one of the leading authorities on boththe theoretical and experimental aspects of relativity.The aim of the course was to present the rathermeagre experimental material available on thesubject and use it as a criterion for defining aclass of relativistic theories of gmvitation which iscompatible with it.

The various experiments arc classified into the)gllowing groups: (I) Null experiments of extreme

precision and of ordinary precision. Under thisheading the early experiment of Eotvos and thelater precision experiment of Dicke and his collabo­rators arc dealt with at length and their importancewhich rests on the fact that the null result obtainedis a necessary condition to be satisfied in order thatEinstein's general theory holds is pointed out.(2LNext, the experiment of Hughes et at. and Drever

- ."h,-.'J./' :'Ulonstrates the isotropy of space is discussed.Tni~ experiment eliminates the possibility of intro­ducing a second long-range tensor field (in additiollto the ordinary gravitational field tensor) into thetheory. A zero mass long-range scalar field is not,however, excluded. (3) The ether drift experiments,the prototype of which is the famous Michelson­Morley experiment, and the three tests of generalrelativity arc next discussed. The poor accuracy ofobservation on the gravitational deflection of light andon the perihelion rotation of mercury (data which couldbe used to exclude gravitational t~lCories incorporat­ing both scalar and tensor fields) is pointed out.

There are twelve appendices, reproducing someof the papers of the author and his collaborators.The subjects range from Mach's principle and ;1

ne\l· theory of gravitation based on a long-rangescalar interaction to cosmology and its implicationsfor terrestrial phenomena (such as the earth's expan­sion, the geomagnetic field and the surface tempera­ture of the earth), stellar and galactic evolutiollrates and dating of the galaxy by uranium decay.

The subject matter of the volume forms delectablereading; but this is marred by the numerous printingerrors (which indicate bad proof-reading). At Ol1('

place the symbol for the cosmological constantchanges from A to A and then to V within a few lines.At another place we are wafted to gastronomicalregions by a reference to a ' supper limit'! Theseand similar mistakes arc perhaps the price one hasto pay for emphasizing speed of publication.

K. VENI(ATESAN

AVVANCES IN HEAT THANSFEH: Vol. 2, edited byJ. P. Harnett & T. F. Irvine (Jr) (Academic PressInc., New York), 1965. Pl'. xi-t-465. Price $ 16.00

The first volume of Advances in heat transfer appearedin 1964 and coverecllsix topics, viz. (I) The interaction

of thermal radiation with conduction and convectionheat transfer by R. D. Cess, (2) Application ofintegral methods to transient non-linear heat transferby T. R. Goodman, (3) Heat and mass transfer incapillary-porous bodies by A. V. Luikov, (4) Boilingby G. Leppert and C. C. Pitts, (5) The influence ofelectric and magnetic fields on heat transfer toelectrically conducting fluids by M. F. Romig, and(6) Fluid mechanics and heat transfer of two-phaseannular-dispersed flow by M. Silvestri. H.esearchin the field of heat transfer continues vigorouslyas the work is connected with important currentfields of interest like atomic energy, aviation andastronautics. Because of advances in the field ofinstrumentation, high speed photography and highspeed computation, experimental work on complexproblems can now be tackled with great vigour.Because of the highly specialized nature of thedevelopments taking place, it is difficult for researchworkers in the broad field of heat transfer to knowwhat is happening in the field unless reviews areavailable as various topics compiled by specialistswho conld give adequate background informationand point out the implications of hundreds of paperson each topic appearing in journals.

The present volume covers the following fiveimportant topics in heat transfer and each topic isreviewed by an eminent specialist in the respectivefield: (1) Turbulent boundary layer heat transfer fromrapidly accelerating flow of rocket combustion gasesand of heated air by D. R. Bartz, (2) Chemical reactingnon-equilibrium boundary layers by P. M. Chung,(3) Low density heat transfer by F. M. Devienne,(4) Heat transfer in non-Newtonian fluids by A. J3.Metzner, and (5) Radiant heat transfer betweensurfaces by E. M. Sparrow.

The review on heat transfer from rapidly accelerat­ing flows by Bartz is a topic of great current interestand deals with high temperature, high pressure heattransfer connected with rocket engines on whichconsiderable research is going on. However, morework is still required before we can say that theproblem is solved. In this excellent review Bartz hasdiscussed at length the background of the problemsand the possible analytical approach for the solutionof the turbulent boundary layer development and heattransfer in rapidly accelerating flows. The reviewalso deals with air experiments and measurements inrocket thrust-chamber.

The next review on chemical reacting non-equi­librium boundary layer by Chung concerns with anold problem which classically received much attentionin connection with the combustion of solid andliquid fuels and also in catalytic reactors. During thelast 10 years the problem has received great attentionbecause of the advent of higher velocity vehicles inthe aerospace technology. The review deals withthe governing equations, chemical kinetics, boundarylayers with surface reactions and gas phase reactions.

The problem of low density heat transfer which isthe next review has received a spurt of interest

319

J. SCLINDUSTR. HES., VOL. 25, JULY 1966

because of the current problems of long-range missilesand satellites. F. M. Devienne has given an excellentreview of the advances in heat transfer in rarefiedgases stressing the results obtained recently andpointing out those problems not yet solved.Accommodation coefficient has received considerableattention in this review.

Prof. A. B. Metzner has written a review on heattransfer in non- Tewtonian fluids. Being a pro­minent worker in this field, Prof. Metzner's review isauthoritative and comprehensive. The whole field ofheat transfer in non-Newtonian fluids is a recent oneand most of the work done in the field was publishedduring the last 25 years. The review coversrheological and thermal properties of non-Newtonianfluids, heat transfer in steady ducted flow fields,boundary layer problems and miscellaneous heattransfer problems.

The last review is on radiation heat transferbetween surfaces by Prof. E. M. Sparrow. Thesubject of radiation heat transfer has received arenewed interest because of its importance inspace flights and high temperature energy sources.Most of the review is devoted to recently developedcomputational methods and results for radiant inter­change between surfaces which are separated bynon-participating media. The analysis and solutionof radiation problems involving participating mediahave been discussed by R. D. Cess in the first volumeof the book.

The book is extremelv valuable for research workersworking in the broad 'field of heat transfer whetherthey are physicists, mechanical engineers or chemicalengineers. To the non-specialist it serves to keep himup to date with what is happening in this rapidlyexpanding exciting field.

N. R. KULUOR

AEROSPACE I{ANGES: INSTRUMENTATION, PRINCIPLESOF GUIDED MISSILE DESIGN by Joseph J. Scavullo& Frederick J. Paul (D. Van Nostrand Co. Inc.,New York). 1965. Pp. xv+457. Price 15.75

A modern aerospace range accomplishes the missionsassigned to it with the help of range instruments.The mainline range instruments have to measureaccurately the trajectories and internal performanceof test vehicles, control the flight of targets andboosters, correlate the observations with range timeand process the data gathered. The book Aerospaceranges: 1nstrwneutat£on is one of a series entitledPrinciples of Guided Missile Design. It presents acomprehensive review of the instrumentation thatgoes into the make-up of an aerospace range, theirfunctions and their limitations. The book beginswith a chapter giving a general outline of the require­ments of a test range and problems associated withrange instrumentation. Subsequent five chapterstreat in some detail individual systems, such as'optical and photographic systems, telemetry and flighttest instrumentation, electronic instruments for trajec­tory measurements, data processing instrumentationand systems necessary for test communication andtime signal distribution. The concluding chapterdeals with the nature, magnitude and causes of themain inaccuracies of measurements by range instru­mentation, a necessary and useful chapter indeed.

320

The book is a source of information to studentsand to persons newly introduced to a test range.It provides answers to numerous questions that arisein their mind, answers that are not easy to obtainfrom men working on a busy range. The knowledgepresented in the book helps one to get a compre­hension of aerospace range instrumentation and theworking of a test range as a whole. The book,however, falls short of the needs of a serious engineerinterested in the design of systems and subsystemsfor a range., Detailed discussions concerning thedesign philosophy of systems, subsystems andinstrumented payloads have been avoided and refe­rences to such material are not presented for thereason that they are not accessible to all. This vitalinformation still eludes the engineer. I t is eitherclassified or remains the monopoly of the specialists.

The book is certainly of use to all connected"'ith a range.

PHYSICAL PI/OPERTIES OF MAGNETICALLY OIWEREDCRYSTALS by E. A. Turov (Academic Press Inc.,New York), 1965. Pp. xx+222. Price S 10.00

Some of the physical properties of magnetic crystalsdepend on the nature of the energy spectrum of theelementary excitations over the ground state of thesystem. While the available microscopic theoriesprovide a basic understanrling governing :'':..;',,:,,![-.r--=of excitations, it is difficult to derive quanti'tall\'einformations from them. It is in this context thatphenomenological approaches prove to be morefruitful. This book is an excellent example of theapplication of phenomenological methods to theexposition of physical phenomena in ferro- andanti ferromagnetic insulators. Since the major contri­bution in this direction has come from Russianworkers, the fact that the author of the bookbelongs to the same school is an additional virtue.One gets a first hand account of their work. Thedescription of the weak ferromagnetism is particularlyappealing.

This is indeed a valuable book and it will be ofgreat assistance to the workers in this field.

K. 1'. SINH,\

OSCILLOSCOPE MEASURING TECHNIQUES by J. Czech(Centrex Publishing Co., Eindhoven), 1'J65.Pp. xviii+620. Price Hs 84.00

This is another valuable addition to the PhilipsTechnical Library. It contains a wealth of informa­tion and is very useful as a reference book forsenior students as well as engineers who arc engagedin CRO design or its application and are seekingup-to-date knowledge on the subject.

The book is a revision of the book Cathude rayoscilloscope by the same author published in 1957and has resulted in a completely new book. Thebook is well written and A. Smith Hardy has donea fine job of almost flawless translation from (;erman.The book contains a fabulouslv rich and well-chosenbibliography, but unfortunately it contains a verylarge number of referenccs in German which arenot so easily available to English readers.

The book is divided into four parts. Part I de­scribes the various building blocks which make the

]. SCI. INDUSTR. RES., VOL. 25, JULY 1966

the authors have spared no pains to make the textall-embracing. Even so, there are a few omissions.For instance, there is no mention of moisturedetermination by the Karl Fischer reagent. A.C.polarography, colorimetry anel controlled potentialelectrolysis are not referred to. Nor does fluorescenceX-ray spectroscopy and the related electron probemicro-analysis find a place. The separation ofelectrolytes from non-electrolytes by ion-exclusionthrough ion exchange resin is also not included.

On the whole the book is well conceived andwell got-up and is a helpful guide to the aspiringstudent.

P. R. SVBBARAMAN

TETRACYCLIC TRITERPENES by Guy Ourisson, PierreCrabbe & Oscer R. Rodig (Holden Day Inc.,San Francisco), 1964. Pp. 237. Price $ 8.75

This book, which is an adaptation of the Frenchedition (1961) by G. Ourisson and P. Crabbe, presentsthe subject matter of tetracyclic triterpenes with arefreshingly original approach. The subject matterhas been divided into four chapters and a catalogue.The introductory Chapter 1 is followed by the mainchapter, which is entitled reactions of tetracyclictriterpenes. These reactions are discussed in terms offunctionalities ring-wise, and stereoelectronic factorsare highlighted. Chapter 3 discusses syntheses oftetracyclic triterpenes, while Chapter 4 gives someaspects of tetracyclic triterpene biochemistry. Thecatalogue gives an up-to-date coverage of variousnaturally occurring tetracyclic triterpenes and theirderivatives, in terms of physical constants, andultraviolet, infrared and rotatory data.

The authors are to be congratulated for presentingthe subject matter reaction-wise, which has resultedin a concise and lucid treatment of the subject.The book, which is very well produced, is recom­mended alike to students and the specialists.

SUKH DEV

HISTORY UNDER THE SEA bv Mendel Peterson(Smithsonian Institute, vVasbillgton), 1965. Pp. 108+56 plates. Price S 3.00

Man's interest in the exploration of the seas hasgrown con~iderably in recent years. He is devotinghis attention more and more towards developingtechnifJues of undenmter exploration so that hemay see for himself the various treasures that Naturehas strewn on the sea bed. Apart from his interestin the natural history of the ,ea bed as well as itsgeological history, he is also anxious to study whatwe may call the "man-made history that lies onthe sea bed ". In shert, man has hegun to carryhis archaeological studies from land to the sea iiiorder that he may' unearth' the vast submergedtreasures which the sea had claimed from the king­doms that once existed on the coasts of contineaband thereby reconstruct the past history.

This intere:;ting book is a practical guide for theundersea archaeologist. In the words of the authorhimself, "there has not been a single volume sofar to which a serious underwater explorer mayturn for instruction on exploration, recovery, pre­servation techniques and identification of artifacts ".This book fulfils this long-felt need to a great extent.

322

Beginning with the historiml a'ped.~ andnarration of the various under,;ca archaeologicalexploration in the Western Hemisphen', particularlythose undertaken dm ing the past ten years hy theSmithsonian Institution in the region of the Gulfof Mexico, the author goes on to discuss the variou'~

search techniques, the condition of underwatersite" surveying 11I1derwater sites, recovery lech­niqnes, and preservation of rC'coveredmaterial in sixchapters. The last chapter is devoted to the pro­blem of identi~cation of ship\\TCck sites, which apartfrom its archaeological aspect" has gre:lt significance[rom the naval point of view.

The presentation is simple and the various chaptersinclude considerable practical details. The chapteron the preservation of recovered material containsinformatiou of great value to a museum techno­logist. The bibliography, although described asselective, is fairly comprehensive. The 56 platesshowing photographs of underwa\{'r surveying ansLthe recovered material ado to the vahle of thepuhlication.

I{. JIY.'IRAMA:\

THE IDEAS OF BIOLOGY by John Tyler Bonner(Methuen <\: Co. Ltd, London), 1965. I'p. 240.Price ISs.

The book written as a supplementary reading withthe biology text also serves a good purp~e ir.· a.';...;..."intelligent layman to aCfJuaint himself with --TIleconcepts of biology.

As the title suggests the book deals more withideas thau facts. The book, spread over sixchapter,; entitled Cell, Evolution, Genetics, Develop­ment, Simple to complex, and Man, includes24 ligures which facilitate understanding th.deasset forth in the text.

A reading list and an index are provided at theend of the book. The book is worth possessing byall interested in biology despite the unusually highprice of this paperback edition.

KUI.ll!l' CHAI\Il

SCIENTIFIC SOCIETIES 1:-1 THE U:\ITEIl SLITES byRalph S. Bates (Pergamon Press Ltd, Oxford),Third Edition, 1965. I'p. 326. Price 63s.

A book of more t.han ordinary interest, the ScientificSocidies in the United Stat,~s presents the histor~' ofscience associations since t.he foundin/-( fathersestablished the American Philosophical Societ.y in1729. Prior to that date, the scientific societies ofEurope, particularly the Royal Society of Lonoon,fulfilled the needs of American scientists and themore prominent among them, Cotton Mather, RogerWilliams and Benjamin Franklin, for instance, weremembers of the Royal Society and contributed toits Transactions. The umbilical cord connectingAmerican science with European learned societieswas severed in the early years of the eighteenthcentury and from then on numerous regional andnational societies, academies, associations and coun­cils came into existence as research enterprise inAmerica /-(ained momentum and attained an im­pressive magnitude, and American scientists madeincreasingly important and significant contributionsto the pool of knowledge.

HEVIEWS

The book under review brings out vividly thedynamism of scientific societies in the UnitedStates established by scientists who firmly sub­scribed to the' principle of association' and believedin 'action by joint force'. Science is clearly theconcern of scientists; and by virtue of their know­ledge and understanding, scientists alone can speakfor science. As associations of scientists, the scien­tific societies of America have exerted considerableinfluence on the progress of science in the countryand on national science policies. Almost everyconceivable activity germane to the'developmentof science is covered by the societies. There aresocieties for the promotion of industrial and voca­tional education and for 'encouraging students onthe threshold of a scientific career, by makingscientific work seem more attractive to them'.Junior academies. have been established for highschool students and there are associations to fosterimprovements in teaching of science and to exchange;~:'as on teaching techniques. Societies have beenestablished for the dissemination of science, forgiving impetus to improved types of journals andfor promoting excellence in the preparation, editingand publication of scientific and technical docu­ments. The advent of the atomic and space agehas seen the rise of new societies and internationalcooperation in science has been greatly strengthened

_ in r"cent years.The Ar;,erican Scientific Societies have been a

potent force in the intellectual life of the peopleand at times of national crisis, like the Civil War,World War I anel World War 11, they respondedablv to the call for mobilizing the nation's scien­tific resources. The societies are the acceptedagencies for the publication of the scientific out­put of the nation and the publications issued bythem not only serve the needs of contemporaryscience, but also constitute the intellectual heritageof generations to come. Their intlnence in diffusingscientific information among the people and inleavening scientific education is unique and im­pressiVl'.

The fact that the book is in its third edition isan eloquent testimony to its popularity. It in­cludes a valuable bibliography and a chronology ofscience and technology in the United States. Thebook is not altogether free from errors. It is stated,on page 3, that the Royal Society began the publica­tion of Philosophical Transactions in 1756; the actualdate is 1665. The hydrogen isotope, deuterium, is

_....

made synonymous with 'heavy water' (p. 139last line and p. 140 first line); line 27, on page 184,should read 'collect mineral, botanical and zoo­logical specimens'; the date of establishing theSociety for Industrial Microbiology is given as 1949on page 208 and as 1948 on page 321. It is hopedthat all the errors would be corrected in the sub­sequent edition.

The book provides stimulating reading. It hasa message for scientists in every country to formassociations to examine objectively all issues whichconduce to the advancement of science. The bookshould be studied by the executives of all scientificsocieties in India with a view to ascertaining howbest the societies can be activated to higher levels ofachievement. It is warmly recommended to allcollege libraries and to individuals interested in thehistory of science.

B. N. SASTRI

PUBLICATIONS RECEIVED

WET COMBUSTION AND CATALYTIC METHODS INMICROANALYSrs: Vol. 2, edited by J. A. Kuck(Gordon & Breach Science Publishers, NewYork), 1965. Pp. xx+412. Price $ 21.00

INTRODUCTION TO ATOMIC AND NUCLEAR PHYSICSby Otto Oldenberg (McGraw-Hill Book Co. Inc.,New York), Third Edition, 1961. Pp. xiii+380

A BIBLIOGRAPHY OF INDOLOGY: Vol. 2 - INDIANBOTANY, Part II - K-Z, compiled by V.Narayanaswami (National Library, Calcutta),1965. Pp. xxx+412. Price Rs 8.25

COMPLEX ANALYSIS by Lars V. Ahlfors (McGraw­Hill Book Co. Inc., New York), Second Edition,1966. Pp. xiii+317. Price $ 8.95

SOUND CONTROL OF THERMAL INSULATION OF BUILD­INGS by Paul Dunham Close (Reinhold Publish­ing Corp., New York), 1966. Pp. vii+502.Price $ 17.00

RELATIVITY AND THE NEW ENERGY MECHANICS byJakob Mandelker (Philosophical Library, NewYork), 1966. Pp. xii+84. Price $ 4.00

BIOSYNTHETIC PATHWAYS IN HIGHER PLANTS byJ. R Priclhan (Academic Press Inc., New York),1965. Pp. xi+212. Price 75s.

THE ENCYCLOPEDIA OF PHYSICS edited by R. M.Besancon (Reinhold Publishing Corp., New York),1966. Pp. xii+832. Price $25.00

CONSTRUCTION, REGLAGE ET ESSAISDES INSTRUMENTSD'OPTIQUE by M. Lachenaud (Dunod, Paris),1966. Pp. xxvii+599

323

NOTES & NEWS

. Transit time' diodes-A new type of semiconductorfor microwave powergeneration

Three new types of semiconduc­tors called the 'transit time'diodes developed at the Bell Tele­phone Laboratories have potentia­lities for being developed as thefirst solid state devices that cangenerate sufficient power for usein microwave systems. Till nowthe only practical sources of sub­stantial microwave power are thevacuum tube devices which havethe disadvantages of being bulky,expensive and having a limitedlife. The solid state devicesthough free from these defectscould not produce the high powerlevels required in the microwavesystems. Even though none ofthe 'transit time' devices so fardeveloped has equalled the powerhandling capacity of vacuum tubes(i.e. above 1 kW.), still because oftheir promise of providing morepower output (or higher frequencyoperation with a given poweroutput) compared to transistorsor tunnel diodes, these will beuseful for the high gain, highpower stages that follow a lownoise preamplifier. However, be­cause of their high noise output,these devices are not likely tobe used in the sensitive, lownoise input stages of a receiver.

All the three types of 'transittime' devices developed, viz.(1) bulk gallium arsenide wafer,(2) silicon avalanche diodes and(3) Read avalanche diodes, operateat the room temperature as self­excited generators or oscillators.A 'transit time' device gets itsname from the fact that itsoperating frequency is determinedby the time it takes the electronsto move or ' transit' through thematerial. The details and mecha­nism of working of these devicesare as follows.

The bulk gallium arsenide hasno junctions but operates due tothe' Gunn effect' when a suitabled.c. field is applied across a waferof uniform gallium arsenide. The

324

frequency of microwaves generatedis a function of the time it takesfor the moving domains of highelectrical resistance accompanyhlgthe Gunn effect to travel throughthe semiconductor and is con­trolled by the length of thematerial along the applied electricfield. While operating conti­nuously, these devices amplifiedsignals in the 2-10 gigacycle rangewith power outputs exceeding60 mW.

The second type of transitdevice, viz. silicon avalanchediode, is made from a semi­conductor junction which isreverse biased to produce ava­lanche breakdown. As amplifiersthese devices achieve a gain of20 db. with a bandwidth of30 Mc/s. at power output ofseveral milliwatts.

The Read avalanche diode, acomplex electrical structure, isusually made of silicon with aPNIN structure whose impurityprofiles are controlled by phos­phorus and boron diffusion.Recently developed diodes of thistype have produced 19 mW. ofpower at 5·2 gigacycles with anefficiency of about 1·5 per cent.

Used as oscillators, some ofthese 'transit time' devices haveexhibited spectral purities betterthan any other self-excited micro­wave oscillator, generating fre­quencies that are relatively freeof random modulation and hencemay find wide use as generalpurpose microwave oscillators forreceivers or low power transmitters[Bell Lab. Rec., 43 (1965), 409].

Laboratory simulation andmeasurement of lunartopography

Characteristics of the moon'ssurface roughness have beendetermined at the Bell TelephoneLaboratories, New Jersey, bybouncing a laser beam off materialsprepared in the laboratory andcomparing these findings withmeasurements of microwavereflections from the moon's surface.The statistical values determined

by this method for the first timefor the dimensions of height,length and slope of lunar irregu­larities compare well with theestimates ba~ed on photographstaken by the Ranger 7 moon probe.The values for these dimensionsare: height of irregularities, 16 ±4in.; distance between irregularitit:s,9±2'5 it; and slope, 8±4 deg.This attempt, in addition tofurnishing a basis for determininglunar terrain, has provided amethod of deducing statisticalvalues for tbe surface irregularitiesof planets.

A surface having randomdistribution of irregularitiesrequired for the invEstigation ofbackscatter characteristics duri~

bombardment of the surface withlaser beams was obtained byblasting blocks of soft aluminiumwith different size alumina grits.These rough surfaces were electro­polished to remove burrs andsharp points since this processhas an effect similar to the pro­cesses believed to be iQvolved iT}<'­the shaping of the surface of themoon, specifically the smocthingof crater edges through bom­bardment by small meteorites.To study the statistical propertiesof the prepared surfaces, a styluswas run over the samples and thetracings showing the contour ofthe surfaces were statisticallyanalysed on a computer. Then,in controlled experiments withlaser light scattered from thesesurfaces, the energy distributionwas obtained as a function ofangle of incidence for each surface.The curve for one of the surfacesstudied was almost identical tothe curve showing the variationof microwave energy backscatteredfrom the moon. Hence, it wasconcluded that the particularaluminium sample has statisticalproperties similar to that of themoon. The dielectric constant ofthe lunar surface was also deter­mined from a comparison of theenergy level of backscattered laserradiations at normal incidence onthe aluminium sample with theenergy level backscattered fromthe moon. This value (1,9 ±0,3)was near the values of dielectricconstant for porous volcanic glassor loose sand (News from BellTelephone Laboratories. NewYork, Release dated 30 September1965).

New type of semiconductor

During the course of systematicstudies on thermistors, HisawFutaki of Hitachi Ltd reported anew type of semiconductor calledcritical temperature resistor(CTR): This semiconductor showsan abrupt resistance change of2-4 orders of magnitude at 68°C.with negative temperature co­efficient. CTR is prepared bysintering a mixture of V20, andan acidic oxide (B, Si or P oxide)or a basic oxide (W, Ca, Sr orBa oxide) in a suitable reducingatmosphere. Subsequent experi­ments showed that CTR preparedusing ternary systems of vana­dium oxide, the acidic oxide andthe basic oxide has better char­acteristics than that preparedfrom the binary system in suchproperties as the large magnitudeof the abrupt resist:lnce change,the large negative temperaturecoefficient of resistance, stabilityat higher temperature and the

- ease in its manufacture.Life of the samples obtained

under suitable conditions istheoretically estimated to beseveral tens of years in air at200°C. The large temperaturecoefficient of resistance of thisnewly developed semiconductor­30 times as large as that of theusual thermistor - offers mall\'interesting applications. Th-efollowing prospective applicationsof CTR are envisaged.

Based on the property of theresistance change by ambienttemperature, it can br used in(a) thermometers and as a tem­perature controller, having sensiti­vity of ±0·5°C.; (b) infrareddetector in which output signalis 30 times as large as in the usualthermistor; and (c) thermalswitches. By virtue of the largechange in thermal dissipationconstant it finds application inmanometer, gas analyser, anemo­meter, etc. The property ofthermal inertia enables it to beused in delay circuit of relay,low frequency oscillator. Theuse as thermal switch, automa­tic gain controller, high fre­quency oscillator and as logicelement in a flip-flop circuit areother examples in which theCTR holds promising applications[Japan j. appl. Phys., 4 (1) (1965).28).

NOTES & NEWS

Induction and multisensitiveend-product repression

In the classical example of'sequential induction' mandelateis degraded by the followingpathway:

Mandelate (SI) ~ benzoyl formateE E

(5,)~ benzaldehyde (5,) ~ benzoate

(S,l~ catechol (S,) ~ ~-oxoadipatc~ SlIccinyl CoA+ncetyl CoA

According to the OJ iginal hypo­thesis such inductions are sequen­tial, the product of each step actingas an inducer for the next enzymein the sequence. It is now knownthat such induction is not sequen­tial but is coordinated. E), E2and E3 are induced by S1> E4 bybenzoate, and E5 and subsequentenzymes by catechol.

The enzymes E1> E2 and E3 arerelated in function and are saidto be under the control of a single, regulon ' (regulon is distinguishedfrom the operon, in that it de­scribes a group of genes not neces­sarily closely linked). J. Mandel­stam and G. A. Jacoby [Biochem.1-, 94 (1965). 569) found that all'the three, benzoate, catechol andsuccinate, repress Ec E3 . Theysuggested three possible modesof action as due to (1) multi­sensitive repression, (2) repressionby a common end-product, and(3) sequential repression. UsingappropIiate mutants, they ruledout the last two possibilities,showing thereby that the mecha­nism (I) is the most likely one.

Degradation of p-hydroxy-mandelate also follows a similarpathway. The intermediates ofthis pathway, p-OH benzoate,pyrocatechol and succinate,also repressed the enzymes E1-E3,

which have been shown to becommon to both the pathways.Benzaldehyde and poOH benz­aldehyde repressed E1-E3 . Thisenigma of an enzyme beingvirtually completely repressed byits own substrate was solved whenan alternate enzyme, oxidizingaromatic aldehyde, was detected.Multisensitive repression systemswere also found in the secondregulon in each pathway.

This coordinate induction hasthe advantage of reducing the lagphase in the production of enzymes.The ' multisensitive ' controlmechanism also overcomes thedisadvantage in terms of protein

economy that might occur in'coordinate induction' in thatwhen the end-product of a groupof enzymes is present in excess,it represses the formation of allthe preceding enzymes in thedegradative pathway. It is alsoof interest that the points wherethe new regulons begin are thosewhere either the degradativepathways of different aromaticcompounds converge or wherethere is the likelihood of the con­cerned compounds to occur undernatural conditions. So an enzymeis not synthesized unless its sub­strate is present and even thenit is not synthesized if the end­products required for cell growthare already present in excess.The site of action of these re­pressors is, however, not known.­V. N. UMA

Antibody formation andthe coding problem

Antibodies are typical globulir.sand hence their formation isessentially biosynthesis of proteir.s.According to the current conc£ptof protein synthesis, plOteins areformed on the )ibowmes from apool of free amino acids with1IlRNA which is coded from thechromosomal DNA. The conforma­tion of the plOtein is believed tobe determined by its amino acidsequence and no genetic informa­tion is needed.

In spite of the numerous theoriesof antibody formation there is noagreement about the selectiverole of the antigen and its IOleas the template. If the antigenhas merely to select betweendifferent types of receptors, thenit is not necessary to postulateinterference of the antigen in theprocess of globulin formation orits modification. If the antigeninterferes in this process in sucha manner that antibodies areformed instead of normal glo­bulir.s, the question naturallyarises as to in which phase ofprotein synth£sis this interferenceoccurs and how the complementaryconformation of the combinationof the combining group of theantibody is accomplished?

The several possible ways inwhich a template molecule mightinterfere with any of the principalsteps in protein synthesis are:(i) the replication of DNA, (ii) the

325

J. SCI. INDUSTR. RES., VOL. 25, JULY 1966

transcription of the genetic codeinto the nucleotide sequence inRNA molecules, (iii) the aminoacid sequence, (iv) the folding ofthe peptide chain, and (v) in caseof the antibody the combinationof the two A-chains and the twoB-chains of the globulin to givethe antibody molecule of com­position A2B2•

Any interference in step (i),replication of DNA, could beexcluded as it would involye amutagenic action of the antigen.Evidence is against such a sup­position.

Modification of step (ii) or (iii)should lead to changes in thecomposition and sequence of aminoacids in the antibody molecule.Occurrence of such changes isindicated first by small but signi­ficant differences in the peptidemaps of antibodies against humanand horse serum albumins andagainst different types of pneumo­coccal polysaccharides. Since theantigens contain a number ofdifferent determinant groups, theantibodies formed are hetero­geneous mixtures directed againstdifferent gronps. It is difficultto resolve whether the differencesreflect differences in the anti­genic determinants or merelyreflect different distribution of theinjected antigens in the organismand antibody formation indifferent organs or cells. Thissituation would be simplified bythe isolation of hapten-specificantibodies and by comparison oftheir amino acid composition andsequence. Small but significantdifferences have been found inthe amino acid composition ofanti-AI'S (cationic diazobenzenearsonate) and anti-R(N (cationicdiazobenzene N-trimethyl ammo­nium) [Koshaland, M. E. & Engl­berger, M. F., PYoc. nat. Acad.Sci., Wash., 50 (1963), 61; Groff,1. L. & Haurowitz, F., bmnuno­chemistry, 1 (1964), 39]. Many ofthe peptide spots of the trypticdigests of anti-AI'S and anti,R(Nwere identical. The observeddifferences cannot be caused bygenetic differences, because theywere also found when the twoazoproteins were injected into thesame animal or when a doublelabelled azoprotein containing bothAI'S and R(N was injected [Gold,E. F., Knight, K. L. & Hauro­witz, F., Biochelll. biophys. Res.

326

COlll'llIU11., 18 (1965), 76]. Th('differences in the amino acidcomposition of hapten-specificantibodies seemed to indicatemodification of step (ii) or (iii)of the protein synthl'sis system.It has been postulated that iso­topically labelled antigen deter­minants are associated with sRNAor other R As, that antigenicdeterminants are present in t1Wvicinity of the sites of amino acidincorporation and hence till'modifying effect [Campbell, D. 1-1.& Garvey, j.S., Advanc. !m1ll1ttwl.,3 (1964). 262; Saha, A., Gan'cy,J. S. & Campbell, D. 1-1., Arch.Bioclle1ll. Biophys., 105 (1964),179].

The conformation of the com­bining site of the antibody isdetermined principally by thethree-dimensional shape of theantigenic determinant and muchless by its charge and polarity.The questions that arise are: Inwhich phase of the biosyntheticprocess is the information concern­ing the shape of the antigenicdeterminant transmitted to thesites of protein biosynthesis andhow does this occur ?

Since long-range forces do notexist in biological systems it mustbe postulated that the folding ofthe peptide chains, i.e. step (iv).occurs in the immediate vicinityof the antigenic determinant,although this seems to he incom­patible with the obsc'T\'ed dif­ferences in amino acid sequenceand also with the claim that theconformation of the peptide chainis determined by its amino acidsequence. It could be said thatconformation and amino acidsequence are interdependent andthat changes in conformationcaused by a template mightsecondarily lead to changes inamino acid sequence. This isquite contradictory to the originalcentral dogma. However, ex­ternal factors have been shownto modify the coding process[Commoner, B., Nature, Land.,203 (1964), 486]. The possibilityof deviation from the centraldogma in the biosynthesis of anti­bodies has been pointed out. F.Haurowitz [Nature, Land., 205(1965), 847), on the basis of theabove views, assumes that anti­genic determinants are bound tosome of the ribosomes or thelIlRNA molecnles and the deter-

minant group, which is usuallyrigid and polar, affects the bio­synthesis sy,tem in two ways.Firstly, dipole induction and otherintermolecular forces 01 iginatingfrolll the template induct' a smallportion of the newly fOl mulpeptide chain to fold onr theStll face of the antigenic dder­minant and thus to prcduct' acomplementary patteln. Second­lv, while most of the aminoacylr~sidues arc incorpol akd into till'peptide chains of th" antihodylI10lecule accortliug to mHNA,deviations [1'(.111 the cede occurduring the formation of thecombining site. Only thoseaminoacyl residues are incorpo­rated which allow the peptidechains to fold complementarilyover the antigenic determinant,whereas other aminoacyl residuesarc rejected. This rejection maybe considered as the inabilitv ofunsuitable aminoacyl-sHNA com­plex to displace the terminalsI~NA residue, as the incorpora­tion process has been i1fscribed •as displacement of the terminalsRNA residue of an sRNA peptideestc'r by tht' incoming aminoacyl­sHNA complex.

Antibody formation, accordingto this view, involves primarilydirect interference of the antigenictemplate in step (iv) of proteiusynthesis by rejection of unsuit­able aminoacyl residues, only byindirectly interfering in step (iii).The process most probably occursin the same cell in which the normalglobulins are formed and is con­trolled by the same geneticapparatus, the same types of DNAand ImA.

The last step (v) in plotciusynthesis (here the combinationof the two sub-units) could beentirely excluded by any int,"r­ference, as the four sub-units cancombine in vitro spontaneouslyeven in the absence of the antigento yield biologically active anti­body.- M. R. SAIRAM

Prostaglandins

von Euler and Goldhlattdemonstrated independently thepresence of blood pressure depress­ing and smooth muscle stimulatingmaterial in human seminal plasmaand sheep vesicular gland extracts.The factor was provisiC'J~ally

named as prostaglandin and was

OH

~'"11

8

H leOOH

o"d » ·15 -dihydroxy ) 9 - keto - prost ­

13.nolc acid (PGE,)

lipid soluble and had acidic pro­perties. For nearly three decadesthis smooth muscle stimulatingprinciple eluded isolation and aseries of compounds, PGE" PGE2,

PGE3, PGF" PGF2 and PGF3,

having a basic structural formula(I) have been isolated from sheepvesicular gland extracts, prostrateglands, human semen, etc. Thechemical structures of all thesecompounds have been elucidated;PGE, is 11tY.,15-dihydroxy-9-keto­prost-13-enoic acid; PGE. differsfrom PGE, by a !:;5 -double bondand PGE3 has a third double bondbetween C'7 and C'8' The PGFcompounds have a hydroxy groupat C9 instead of a keto group foundin the PG E compounds. Thedifferent prostaglandin compoundsarc present in many organs, thehighest amount being found inhuman seminal plasma and sheepvesicular glands. Semen of bull,boar, dog, rabbit, pig and horsedoes not have prostaglandins.Semen from men with documentednormal fertility contained moreprostaglandin than semen frominfertile men, where hardly anysmooth muscle activity is seen.

D. A. Dorp, R. K. Beerthuis,D. H. Nugteren and H. Vonkeman[Biochem. biophys. Acta, 90 (1964),204] and S. Bergstrom, D.Danie1sson, D. Klenberg and B.Samuelsson [j. bioi. Chem., 239(1964), 1135] suggest that prosta­glandins in sheep and humansemen are formed in the seminalvesicles and in the vesicular glandsrespectively. When Y-homolino­lenic acid, arachidonic acid or aneicosa pentaenoic acid was incu­bated at 37° with homogenizedvesicular gland from sheep, PGEt ,

PG E2 and PG E3 respectively wereformed.

All the prostaglandin compoundsas well as the crude prostaglandinextract from human semen andvesicular gland of sheep stronglystimulate all the smooth muscle

NOTES & NEWS

organs except the fallopian tubulesof rabbit and man where prosta­glandins and various PGE com­pounds inhibit the spontaneousmotility. Blood pressure of rabbit,cat, dog and man is lowered.Most pronounced blood pressurereducing activity is seen withPGE compounds. On humanmyometrium all PGE compounds.~1ave almost the same activity,indicating that the number ofdouble bonds appears to havelittle importance for their activityon this organ. This is not thecase, however, for other smoothmuscle organs (jejunum, duode­num and uterus of rabbits) whereusually PGE2 is the most potentand PG E3 the least active of thesesubstances. The keto group,however, seems to be essentialfor the inhibitory effect, sincePGF, and PGF2 have a tendencyto stimulate the myometrium.

With regard to the proposedrole of prostaglandin in normalfertility, pure prostaglandin com­pounds present in human semenwere examined for their effecton the motility of human myo­metrium [Rygdeman, M., AdaPhysiol. scand., 63 (1964), 242].Normally all PGE compounds andHSF-PG inhibit the spontaneousmotility of human myometrium.Contrary to PGE compounds PGF,and PGF2 have a tendency tostimulate the spontaneous motilityof human non-pregnant myo­metrium. An inhibitory effect onhuman myometrium of an auto­pharmacological active substanceis uncommon, the only othersubstance with such an effectin vitro is bradekynin. Thesensitivity of the myometrium toPGE and HSF-PG vades in acharacteristic way during thedifferent phases of menstrual cycle.The activity is pronounced aroundovulation time and less duringthe other phases of the cycle.

The factors which regulate thereactivity of prostaglandin duringthe menstrual cycle, however,stress the importance of ion grad­ients, especially the intra- andextracellular relationship betweencations, i.e. K, Na and Ca. Varia­tions in the extracellular ionconcentration profoundly changethe reactivity pattern of themyometrium. A high extra­cellular potassium concentration,for instance, decreases the mem-

brane potential, increases thefrequency of the contractionsand increases the sensitivity ofthe myometrium to differentstimuli.

At low extracellular potassiumconcentrations the effect of PGEis enhanced at the same time asthe stimulatory effect of PGF,is abolished. The change insensitivity of human myometriumto PGE compounds when potas­sium concentration is varied seemsto be specific. The effect ofpotassium concentration on spon­taneous motility and ion distri­bution indicates that the membranepotential of human myometriumis charged simultaneously. Withthis assumption, the effect ofprostaglandin is also dependentnot only on the extracellularpotassium concentration but alsoon the membrane potential. Alow extracellular potassium con­centration corresponds to ahyperpolarized membrane. Atthis time the sensitivity of myo­metrium to PGE, is enhancedand to PGF, depressed. A highextracellular potassium concen­tration and consequently a partlydepolarized membrane depressesthe effect of PGE" but increasesthe stimulatory effect of PGF,.A low extracellular calciumconcentration affects the sensiti­vity of myometrium to prosta­glandin, especially at ovulationtime.

The sensitivity of human myo­metrium to PGE compounds andvaried effects during differentphases of the menstrual cycle mayalso be due to the influence ofestrogen and progesterone. Theeffect of estradiol and progesteroneon the sensitivity of human myo­metrium to PGE, was also investi­gated. Hormones investigatedaffected the sensitivity of myo­metrium to PGE in vitro. Theeffect was most pronounced atovulation time. Here proges­terone and estradiol together withprogestrone decreased the sensiti­vity of the myometrium to PGE.During early and middle proli­ferative phases very confusingeffects were observed. No statis­tical differences were foundbetween the ion content, eitherduring the various phases of themenstrual cycle or under differenthormonal treatments.- T. S.ANANTHA SAMY

327

J. SCI. INDUSTR. RES., VOL. 25, JULY 1%6

Reactivities of histidineresidues at the active site ofribonuclease

There is considerable evidenceto indicate that the histidineresidues at positions 12 and 119in ribonuclease are at the activesite of the enzyme. It wasdemonstrated that inactivation ofRNase A by iodoacetate at pH5·5 and 25°C. was due to theintroduction of a single Cal boxy­methyl substituent on eitherimidazole nitrogen 1 of histidine119 or imidazole nitrogen 3 ofhistidine 12 under these conditions.The yield with substitution atN1 of 119 histidine was found tobe 8 times that of the product withsubstitution at Na of histidine 12.These two chIOmatographicallydistinct carboxymethylated der i­vatives were shown to be alternateproducts which are formed simul­taneously. In order to explainthese and other findings, includingthe behaviour of the aggregatesof RNase the following mechanismhas been suggested. In thesimplest hypothesis, histidine 12and 119 are both considered toform the active site and the pro­tonated form of the one bindsthe anionic alkylating agent whichthen alkylates the unprotonatedform of the other. In the othermechanism, the orientatio.n isprovided by lysine 41.

The difference in yields of thetwo alkylation products may bethe result of greater steric hind­rance at one site or the other.In order to test this hypothesisa study has been made of thereaction between ribonuclease anda series. of halo acids [Heinrikson,R. L., Stein, W. H., Crestfield,M. A. & Moore, S., J. bi91. Chem.,240 (1965), 2921]. The resultsof this study indicate that thereaction with a series of unbranchedoc- and ~-bromo acids ranging from3 to 6 carbon atoms also leadsto the alkylation at the same sites.Reaction may occur predomi­nantly either at histidine 12 orat histidine 119, depending uponthe chain length, the position ofhalogen atoms relative to thecarboxyl group and the opticalconfiguration of the oc-carbon of thereagent employed. These findingsconfirm the earlier contention thatboth 12 and 119 carboxymethy­lated derivatives of ribonuclease

328

arise from the same molecularspecies of the enzyme; the 12derivatives are not produced byside reactions involving some minorcontaminant of ribonuclease.

The most striking finding fromthe present study is the greatdifference in the reactivities andsite of reaction of the D- and L­antipodes of oc-bromopropionic acidand lX-bromo-n-butYlic acids. ThGD-antipodes of these acids formhistidine 12 derivatives in 70-78per cent yields. The L-anti­podes form histidine 119 deri­vatives in yields of 78 and 74per cent respectively. Moreover,the D-antipodes react at histidine12, 20 times more rapidly and athistidine 119, 2-3 times morerapidly tlian do the L-antipodes.

These effects must reflect thestereochemistry of the active siteitself, as such differences are notobserved in the model reactionswith histidine. Notable is thevariation in the site of reactionswith the nature of the alkylatingagent. Only D-IX-bromopropionicacid and D-IX-bromo-n-butyric acidsalkylate predominantly at histi­dine 12 while the others alkylate athistidine 119. With the IX-bromoacids the rate of reaction decreasesas the size of the substituent onthe IX-carbon atom increases.Part of this effect is intrinsic tothe reagent itself. The 300-folddecrease in the reactivity athistidine 119 in passing frombromoacetate to L-IX-bIOmoplO­pionate is significantly larger thanwould be expected on the basisof the model reactions and mustbe caused in part by ster ic effect.The decrease in the rate of reactionat histidine 12 as one proceedsfrom bromoacetate to D-IX-bromo­propionate (less than 5-fold) isless than 3D-fold that expectedfrom the histidine reaction andmay be a result of secondarynon-polar interactions between' themethyl group in the alkylatingagent and some group or groupsin the enzyme. Probably similarinteractions operate in the caseof D-IX-bromo-n-butyric acid sinceits rate of reaction is nearly iden­tical with that of the C3 acid.These results indicate that thespace into which these reagentsfit when they are oriented in sucha fashion as to permit alkylationat histidine 12 is big enough toaccommodate both the Ca and

C, oc-bromo acids easily and theC5 acid with difficulty. It is notlarge enough to accommodateIX-bromocaproate, however, forthis reagent does not alkylatehistidine 12.

One aspect that is not entirelyclear on steric grounds is why thehaloacetate, the least hindered ofthe reagents studied, reacts sopredominantly at histidine 119.In the reaction with iodoacetatethe formation of the 12 derivativeis somewhat favoured as thetemperature is raised. Thissuggests that there may be presentan interfe ring group, whose positionshifts slightly with change intemperature. It is likely thatthere are groups in the vicinityof imidazole nitrogen 3 of histidine12 which get in the way of thehydrocal bon side chains ofoc-bromovalerate and oc-bromo­caproate, when these reagents arebound at histidine 119. One ormore of these intefering groupsmust be arranged on one side ofthe axis between the histidineresidues to provide hindrance sothat reaction with L-enantiomcrphsof IX-bromopropionate and oc­bromobutyrate cannot readilyoccur at histidine 12. Conversely,the reaction at this position withthe D-isomers of Ca and C, acidsmay actually be facilitated some­what by a non-polar region betweenthese histidine nit! ogen atoms.

The differences in the ratesand sites of alkylation with thereagents of systematically varyingstructures are reflections of thestereochemistry and conformationof the active site. A final pointthat bears emphasis concer!. s thepcssible use of these reagents.Because of their selectivity interms of both rate of reactionand the site at which alkylationoccurs, these reagents provide apotential sensitive method ofdetecting conformational changesin the active site of ribonuclease.- A. S. ACHARYA

Progress Reports

Fire Research in UK

The annual report for 1964 ofthe UK Fire Research Board,besides covering the activities ofthe Board relating to preventionand control of fire hazards, designof building and choice of building

materials to minimIZe chances offire hazards, general characteristicsof flames, and processes of combus­tion, presents the results of astatistical study regarding fire out­breaks on various aspects, such ascost of damage, industries affected,causes of fires, etc.

Studies made on spontaneousheating and ignition in miscellane­ous materials have led to conclu­sions on methods of shippingcarbon safely and economically.An equipment for studying thethermal explosion of organic per­oxides in the paste form has beenconstructed and it has been foundthat a paste containing 65 percent peroxide undergoes thermalexplosion at a lower temperaturethan dry peroxide and in the caseof dry benzoyl peroxide rust andsome decomposition products ofthe peroxide added to the dryperoxide reduce the inductionperiod of explosion. Measurementshave been made on the speed ofrise of lower flammability limitflames in vertical tubes usingmethane-air and propane-air mix­tures. The results have beenexplained on the basis of thecontrolling effect of rising bubblesof hot gaseous products.

As a result of experiments onthe factors that affect the rate ofburning of 'cellulose materials therate of charring has been found tobe proportional to the density andthe permeability along the grain.An equation connecting the den­sities of the flame and surroundingair and the entrainment velocityof air by flames has been deve­loped and the experimentally deter­mined values of flame heightshave been found to agree with thevalues predicted on the basis ofthe equation. Studies made onradiative transfer of heat fromflames have shown that the radia­tive transfer from alcohol flamesis about five times that of convec­tive transfer at the centre of thetray containing the flame. Theburning rate of wood cribs isaffected by heat received fromsome other source as, for example,radiative transfer in the fuel bed.

A theoretical expression derivedon the basis of dimensional ana­lysis for the conditions of mergingof separate fuel bed flames toform a single flame has beentested by actual observation. Thetheory is found to give reasonable

NOTES & NEWS

estimates of flame heights at merg­ing. Another expression for thelength of an advancing flame hasbeen found to agree with anempirical formula derived for lowrates of burning. A thoroughstudy of the characteristics offires in compartments is in pro­gress and the observations madeso far agree with the deductionsmade from an assumed theoreticaln~odel. Several important deduc­tions regarning the effect of ceil­ing, ventilation, the corridor,etc., in the case of indoor fireshave been arrived at. The studieshave resulted in further deduc­tions regarding several aspectssuch as effect of ambient atmo­sphere on burning wood, productsof combustion with restrictedventilation, soot production indiffusion flames, etc. A theoreticalexpression for the conditions ofequilibrium of flow of hot gasesin vented single-storey buildingshas been developed. An ap­paratus for testing small concretebeams in flexure at high tem­peratures under well-controlledconditions has been developedand a number of preliminaryconclusions have been drawn as aresult of investigations made onthe spalling of concrete. Experi­ments have been made to compareunder identical conditions thebehaviour of ducts of variousmaterials forming part of a venti­lation system and the results willbe of interest to industry, especi­ally to the plastics industry.

A thorough study of fire resis­tance of different building mate­rials has been made and deduc­tions useful to the building industryhave been made. From studieson various aspects of the problemof keeping escape routes in abuilding, methods of dealing withthe smoke hazard have been de­veloped. Several types of heat­sensitive and smoke-sensitivedetectors have been developedand their sensitivity and operationcharacteristics are being studied.Studies have been made on theperformance of sprinkler systemsand the rates of application ofwater required to extinguish deve­loping fires in three types of woodcrib have been determined. Froma study of the new techniques inextinguishing fires, recommenda­tions have been made regardingsuch extinguishers as freezing point

depressants, inert gas and foamgenerator, vaporizing liquid ex­tinguishers and dry powders.

As a result of studies made ongas explosions and dust explosions,standards for keeping industrialequipment safe have been de­veloped. Special hazards such ascyclones, fires due to electriclamps, fires in brickwork impreg­nated with domestic fuel oil,incidence of fires on newsprint, etc.,have also been studied and re­commendations have been madefor reducing loss in such accidents.

New Periodical

Journal of Combinatorial Theory

This new journal, to be pub­lished quarterly commencing fromspring 1966, is devoted to thepublication of original mathemati­cal articles dealing with theoreticaland physical aspects of the studyof finite and discrete structures inall branches of science. Manu­scripts in English, French orGerman should be addressed tothe Editor-in-Chief, Journal ofCombinatorial Theory, AcademicPress Inc., New York. Annualsubscription for the journal is$ 15.00 for institutions and $ 9.00for individuals.

Dr Vikram A. Sarabhai

A worthy successor to the lateDr H. J. Bhabha for the chair­manship of the Atomic EnergyCommission has been found bythe Government of India inDr Vikram A. Sarabhai, whosepersonality represents a rare com­bination of the fine qualities of anoutstanding scientist, a capableorganizer and a connoisseur offine arts. He has also beenappointed Secretary to the Depart­ment of Atomic Energy, Govern­ment of India.

Born in August 1919 at Ahme­dabad, Dr Sarabhai studied at theuniversities of Bombay and Cam­bridge, taking his Natural ScienceTripos from the latter in 1939.After about six years of studyas a research scholar in the fieldof cosmic rays under Sir C. V.Raman, he continued his investiga­tions at the Cavendish Labora­tory, Cambridge, from wherehe got his doctorate degree.Dr Sarabhai has been an excellent

329

J. SCT. INDUSTR. RES., VOL. 25, JULY 1966

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organizer of scientific institutionsand the rapid and brilliant progressof such instit.utions as the PhysicalResearch Laboratory, Ahme­dabad, the Textile Industry'sResearch Association, Ahmedabad,and the Indian Institute ofManagement, Ahmedabad, withwhich he has associations, beartestimony to this aspect of hiscapacity.

In recognition of his scientificcontributions, Dr Sarabhai wasawarded the Shanti Swamp Bhat­nagar Memorial Award for 1962and he was honoured by theGovernment of India by the awardof Padma Bhushan early thisyear.

Dr Sarabhai is a fellow of severallearned societies, both Indian amiforeign. He has been an activemember of the Scientific AdvisoryCommittee to thc cabinct, theNational Planning Council ofthe Planning Commission, theGoverning Body of the Councilof Scientific & Industrial Researchand the Central Advisory Board ofEducation, Government of India.He is a member of the EditorialBoards of the]oltmal of Scientific0- I ndltstrial Research and theIndian ]oumal of Pitre & AppliedPhysics since its inception. Deep­ly interested in the problems ofdisarmament, Dr Sarabhai hasdevoted considerable time andeffort in propagating the ideasbehind the Pugwash movementand is the convener of the IndianPugwash Committee.

One of his major interests inresearch centres around astro­physical implications of cosmicray time variations and the in­vestigations made so far from hisschool of studv have led to adeeper understa'nding of the solaractivit.y with respect to cosmicrays. Space research is anotherfield of interest to Dr Sarabhaiand it was under his directionthat the Equatorial RocketLaunching Station at Thumba andthe Experimental Satellite Com­munication Earth Station atAhmedabad were set up.

330

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JOURNAL OF PALYNOLOGY, Vol. 1,1965 - Contains papers from Venkata Rao; B. K. Nayar &Surjit Kaur; R. K. Ibrahim; S. Chanda & G. Erdtman; P. K. K. Nair & Mithilesh Sharma; S. I. Saad &R. K. Ibrahim; K. F. Adams & H. A. Hyde; U. C. Banerjee, J. R. Rowley & Mary L. Alessio;D. N. Shivpuri &·M. P. Singh; V. S. Raman; L. R. Wilson; and Raj D. Bamzai & G. S. Randhawa

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PALYNOLOGICAL BULLETIN, Vol. II, 1966 - Contains some papers presented to the Sympo­sium on Palynology, held at the National Botanic Gardens, Lucknow

Revised Prepublication Price: Rs. 20, $ 6, £ 2

JOURNAL OF PALYNOLOGY, Vol. II, 1966, Special Number - Contains a monograph onthe" Pollen Morphology of Indian Monocotyledons" by Dr. (Miss) Mithilesh Sharma

Revised Prepublication Price: Rs. 20, $ 6, £ 2 for members of PSIand Rs 25, $ 7, £ 2 sh. 10 for institutions

JOURNAL OF PALYNOLOGY, Vol. II, 1966 - Contains papers from B., Gulvag; VimalaMenon; P. K. K. Nair; Mithilesh Sharma; S. S. Bir; S. 1. Saad; and H. A. Khan

Price not fixed

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