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Talanta 76 (2008) 885–891

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Talanta

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ydrodistillation–adsorption method for the isolation of water-soluble,on-soluble and high volatile compounds from plant materials

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J. Mastelic ∗, I. Jerkovic, I. Blazevic, A. Radonic, L. KrDepartment of Organic Chemistry, Faculty of Chemistry and Technology, University of S

a r t i c l e i n f o

Article history:Received 26 November 2007Received in revised form 17 April 2008Accepted 23 April 2008Available online 2 May 2008

Keywords:AcidsTerpenes and isothiocyanatesHydrodistillation–adsorptionVolatiles isolationWater-soluble volatiles

a b s t r a c t

Proposed method of hydrwith solvent trap were copounds, such as acids, mon(Rosmarinus officinalis L.)The main advantages of HDand their simultaneous separticularly suitable for thcompounds. In distinctiondistillation water on activin the same apparatus.

1. Introduction

The plant volatiles are heterogeneous groups of organic com-pounds that include monoterpenes, sesquiterpenes, diterpenes

nd many other nonterpene compounds with different moleculareights and functional groups. Some essential oils contain glu-

osinolate degradation products, such as isothiocyanates, organicyanides, epithionitriles, oxazolidinethiones, and thiocyanates. Allhese classes of compounds exhibit different properties such asolatility, solubility in water and solvents or others that are essen-ial for their isolation and concentration from plant material as wellor the separation of their natural compound mixtures to differ-nt fractions. Hydrodistillation is one of the most frequently usedethods for volatiles isolation. Obtained isolates, called essential

ils, according to international definition are distillation productshat are not soluble in water and can be easily separated from dis-illation water. However, some compounds of the essential oils areater-soluble, especially at elevated temperature that can produce

osses and smaller oil yields during hydrodistillation. Many otherethods are used for volatiles isolation, such as extraction with

olvents and supercritical CO2 extraction [1–5].The differences in the obtained volatile yields and chemical

omposition from the same plant are well known due to the use ofifferent isolation methods. Namely, the most volatile compounds

∗ Corresponding author. Tel.: +385 21 329 436; fax: +385 21 329 461.E-mail address: josip.mastelic@ktf-split.hr (J. Mastelic).

039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2008.04.051

ovic. Tesle 10/V, HR-21000 Split, Croatia

llation–adsorption (HDA) on activated carbon and hydrodistillation (HD)ed for the isolation of water-soluble, non-soluble and high volatile com-enes, isothiocyanates and others from carob (Certonia siliqua L.), rosemary

ocket (Eruca sativa L.). Isolated volatiles were analyzed by GC and GC/MS.thod over ubiquitous HD method were higher yields of volatile compounds

tion in three fractions that enabled more detail analyses. This method isation and analysis of the plant volatiles with high amounts of water-soluble

previously published adsorption of remaining volatile compounds fromarbon, this method offers simultaneous hydrodistillation and adsorption

© 2008 Elsevier B.V. All rights reserved.

will the most rapidly distill and many of them can be lost by leak-ing through condenser, while less volatile compounds slowly andpartially distill. Furthermore, polar components of some essentialoils have high water solubility at boiling temperature and slowlyand incompletely distill. After condensation, the part of these com-pounds remains dissolved in distillation water that recovers indistillation flask, circuit during hydrodistillation and their isolation

is not complete. For the oils with large content of water-solublecompounds HD method gives lower yields due to the previous men-tioned losses. Therefore, we have developed a method of volatilesisolation by simultaneous hydrodistillation–adsorption (HDA). Sev-eral types of sorbents are available (polymeric adsorbents, zeolites,activated carbon, etc.). In this research, for adsorption of water-soluble and high volatile compounds activated carbon was used asadsorbent due to sorption capacity toward many compounds. Theprincipal binding mechanisms include hydrophobic interactions,charge-transfer complexation, hydrogen bonding, cation exchangeand another specific interactions [6]. The adsorption characteris-tics of activated carbon depend on the type of starting material,methods and conditions of preparation [7].

Suitable plant materials selected for this research were carob(Certonia siliqua L.), rosemary (Rosmarinus officinalis L.) and rocket(Eruca sativa L.). Carob belongs to Leguminosae family. It is ever-green tree which is cultivated in many parts of Mediterraneancoast and islands. MacLeod and Forcen [8] analyzed volatilecomponents from the carob beans from Spain by simultaneoushydrodistillation–extraction in Likens-Nickerson apparatus andidentified water-soluble isobutyric, valeric, isovaleric and butyric

lanta 76 (2008) 885–891

886 J. Mastelic et al. / Ta

acids as the main components (cca. 75%). Rosemary, the sec-ond selected sample, belongs to Lamiaceae family. This aromaticplant and its essential oil, isolated from leaves, are very famousand respected on world market and have been researched a lot[9,10]. Rosemary oil contains many volatile and non-polar monoter-pene hydrocarbons. Furthermore, the oil contains monoterpeneoxygen containing compounds, sesquiterpene hydrocarbons andothers. The third sample was rocket, member of Brassicaceaefamily that contains glucosinolates. It has been reported that4-methylthiobutyl isothiocyanate (erucin), that originates from glu-coerucin, was the major glucosinolate degradation product foundin the leaves oil of rocket [11].

Previous papers reported isolation of volatiles from aromaticplants using stream of nitrogen and their adsorption on acti-vated carbon column (purge-and-trap method) [12] as well as theadsorption of water-soluble compounds remained in the distilla-tion water on activated carbon [13,14]. The aim of this research wasto investigate and compare two isolation methods performed inthe same apparatus: hydrodistillation–adsorption (HDA) methodand its ability to diminish losses of water-soluble and high volatilecompounds and hydrodistillation method with solvent trap (HD).

2. Experimental

2.1. Plant materials

Ripe carob beans were collected on island Solta (near Split, Croa-tia) in September 2007. Carob beans have been de-seeded andgrinded in coffee mill to a particle size of cca. 1–2 mm. Rosemarywas collected near Split in June 2007. Fresh leaves were used forthis research. Rocket was, also, collected near Split in May 2007.Fresh flowers and leaves with green beans were used. The botani-cal identity of the plant materials was confirmed by a local botanistand the voucher specimens are deposited at Department of OrganicChemistry, Faculty of Chemistry and Technology, Split, Croatia.

2.2. Hydrodistillation–adsorption (HDA)

Clevenger type apparatus (Fig. 1) was used for proposed HDAmethod. Carbon (Kemika, Zagreb, Croatia, granules Ø = 0.3–0.5 mm)was activated in muffle furnace at a temperature of 800 ◦C for 2 hin reducing atmosphere. After cooling at room temperature, carbonwas added in water and deaerated under vacuum for 10 min. Acti-

vated carbon (5 g) was put in a separator (inner apparatus tube) forthe adsorption of water-soluble compounds, and in a small column(3 g) situated on the top of condenser for the adsorption of highvolatile compounds from gaseous phase.

The height of water layer above carbon column (Fig. 1, a) was5 cm. Plant material (200 g) and 600 mL water were put in 1000 mLdistillation flask. The distillate passed through the water layer:water non-soluble compounds were collected on the water layer(Fig. 1, b), and water-soluble compounds were adsorbed on car-bon column (Fig. 1, a). The most volatile compounds from gaseousphase were adsorbed on the carbon column on the top of condenser(Fig. 1, c). After hydrodistillation and adsorption (3 h), the layer ofwater non-soluble compounds (Fig. 1, b) was dissolved in pentaneand carefully separated from the water layer. This procedure wasrepeated twice and pentane extracts were combined (fraction b).After removing of water, through a pipe on the bottom of appa-ratus, the extraction of adsorbed compounds was performed inthe same apparatus using 50 mL diethyl ether for 8 h. The obtainedether extract was carefully concentrated by distillation to a smallervolume (fraction a). High volatile compounds were extracted fromupper column (Fig. 1, c) with ether, and the obtained extract was

Fig. 1. Apparatus for hydrodistillation–adsorption (HDA): a, column of activatedcarbon for the adsorption of water-soluble components; b, fraction of water non-soluble compounds; c, column of activated carbon for the adsorption of high volatilecompounds.

carefully concentrated (fraction c). All the extracts a, b and c weredried over anhydrous MgSO4. HDA method was performed in trip-licate for each sample. All the isolates were analyzed by GC and

GC–MS.

2.3. Hydrodistillation (HD)

Hydrodistillation (HD) of plant material (carob beans, rosemaryleaves, rocket flowers and rocket leaves with green beans) was per-formed in Clevenger type apparatus with solvent trap for 3 h. Amixture (3 mL) of pentane and diethyl ether (1:1, v/v) was used forvolatiles trapping. Prior HD, rocket was autolyzed for 24 h on 27 ◦Cto increase glucosinolate degradation by endogenous myrosinase[15]. All the extracts were dried over anhydrous MgSO4. HD methodwas performed in triplicate for each sample. All the isolates wereanalyzed by GC and GC–MS.

2.4. Analysis of isolated volatiles

2.4.1. Gas chromatography (GC-FID)Gas chromatography analysis was performed on a Hewlett-

Packard 5890 Series II gas chromatograph equipped with flameionization detector using HP-20M capillary column (Hewlett

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J. Mastelic et al. / Ta

Packard, polyethyleneglycol, 50 m × 0.2 mm i.d., film thickness0.2 �m). Chromatographic conditions were as follows: heliumas carrier gas, flow rate 1.0 mL/min; injector and detector tem-peratures, 250 and 300 ◦C. Oven temperature was isothermal at70 ◦C for 4 min, then increased to 180 ◦C, at a rate of 4 ◦C/minand held isothermal for 15 min. Volume injected 1 �L. Split ratio1:50.

2.4.2. Gas chromatography–mass spectrometry (GC–MS)Isolated volatiles were also analyzed by Hewlett Packard GC–MS

system (gas chromatograph model 5890 series II with mass selec-tive detector model 5971A). Polar capillary column, HP-20M, wasused (Hewlett Packard, polyethyleneglycol, 50 m × 0.2 mm i.d., filmthickness 0.2 �m). Oven temperature was programmed as fol-lows: isothermal at 70 ◦C for 4 min, then increased to 180 ◦C, ata rate of 4 ◦C/min and subsequently held isothermal for 15 min.Carrier gas was helium, flow rate 1.0 mL/min; injector tem-perature: 250 ◦C; volume injected: 1 �L; split ratio: 1:50. MSconditions: ionization voltage: 70 eV; ion source temperature:280 ◦C; mass range: 30–300 mass units, as in previous papers[16,17].

2.4.3. Qualitative and quantitative determinationsThe individual peaks were identified by comparison of their

retention indices to those of authentic samples, as well as by com-paring their mass spectra with the Wiley 275 library (Wiley, NewYork) and NIST98 (National Institute of Standards and Technology,Gaithersburg) mass spectral database and literature [18].

The percentage composition of compounds (relative quantity)in the essential oil and its fractions were computed from the GC-FID peak areas using the normalization method, without correctionfactors. Quantitative results were obtained as mean of data derivedfrom duplicate GC-FID analyses.

The yields of oils and fractions were determined by gravimet-ric method after complete solvent removal, by carefully distillationover Vigreaux column at low temperature. The yields of essen-tial oils were calculated as sum of the yields of all three fractions,and chemical composition was calculated as sum of chemicalcomposition of all the fractions. Absolute quantity [mg/kg] forparticular compound was calculated from average compound per-centage and average masses of each fraction (from triplicateexperiments).

3. Results and discussion

Volatiles of carob beans, rosemary leaves and rocket flowersand its leaves with green beans were isolated by two methods:hydrodistillation–adsorption (HDA) and hydrodistillation with sol-vent trap (HD). During the isolation by proposed HDA methodthe volatiles were separated in three fractions: water-soluble frac-tion a, water non-soluble fraction b and fraction c of high volatilecompounds. Water non-soluble compounds were isolated in theupper layer (above the water), water-soluble compounds and someemulsified compounds from distillation water were adsorbed oncarbon column in the separator, and high volatile componentswere adsorbed on carbon in the upper column, Fig. 1. Activatedcarbon has not the same adsorption power toward different com-pounds, but high number of theoretical plates in the columncorrects this fault. The identified structures of the main compo-nents are shown in Fig. 2, which indicates fractionation (a, b, c) ofisolated volatiles according to different water solubility and volatil-ity.

The yields and chemical compositions of volatile compoundfractions of carob beans isolated by two methods are givenin Table 1. The yield of volatiles obtained by HD method

76 (2008) 885–891 887

was 1450.0 mg/kg, and by HDA method 2728.3 mg/kg (88.2%yield increase). The yield of fraction b (water non-soluble com-pounds) was only 148.5 mg/kg with mass ratio 0.0544. Twenty-twocompounds were identified in this fraction. The main com-ponents were hydrophobic compounds such as hexadecanal(65.89 mg/kg), hexadecanoic acid (18.87 mg/kg), 2-tridecanone(15.17 mg/kg) and 3-dodecenyl acetate (15.82 mg/kg). The yieldof fraction a was 2567.4 mg/kg and of fraction c was only12.4 mg/kg. Fraction a was quantitatively the most significantwith mass ratio of 0.9410. Only seven water-soluble compoundswere identified in this fraction. The main components wereacids: 2-methylpropanoic acid (1853.98 mg/kg), hexanoic acid(513.08 mg/kg), butanoic acid (107.95 mg/kg), 2-methylbutanoicacid (48.49 mg/kg) and acetic acid (17.86 mg/kg). Fraction c con-tained eight high volatile compounds with mass ratio of 0.0045.Four of them were identified only in this fraction, Table 1.Volatiles isolated by HD method contained only ten compoundsin comparison with 25 volatiles isolated by HDA method. HDAmethod enabled higher overall yield and simultaneous fractiona-tion and, therefore, more complete analysis. In summary, the mainHDA components (a + b + c) of carob beans volatiles were water-soluble acids such as 2-methylpropanoic acid (1853.98 mg/kg),hexanoic acid (514.60 mg/kg) and butanoic acid (107.95 mg/kg).Octanoic, acetic and hexadecanoic acid were identified, but insmaller quantities. The volatiles also contained carbonyl com-pounds hexadecanal, 2-butenal, 5-methyl-2-hexanone, 2-octanonewith small quantity of esters, hydrocarbons and other com-pounds.

Rosemary leaves contain essential oil with relatively highamount of volatile compounds and small amount of water-solublecompounds. Monoterpene and sesquiterpene hydrocarbons andoxygen containing compounds were identified. The yields andchemical compositions of the isolated oil and its fractions forboth methods are given in Table 2. The yield of the volatilesobtained by HD method was 4972.70 mg/kg, and by HDA methodwas 5546.40 mg/kg (12% yield increase). The yield of fraction b(water non-soluble compounds) was 4234.50 mg/kg. Fraction bwas the main volatile fraction of rosemary leaves with 0.7634mass ratio in comparison with volatile fraction b of carob beans.Twenty-one compounds were identified in this fraction. Themain components were 1,8-cineole (2319.20 mg/kg), �-pinene(625.05 mg/kg), camphor (394.15 mg/kg), borneol (165.41 mg/kg),�-terpineol (119.76 mg/kg) and other hydrophobic compoundswith smaller contents. Polar compounds of this fraction were

also identified in fraction a such as 1,8-cineole, camphor,borneol, �-terpineol, terpinene-4-ol, linalool and other polarcompounds with small contents. The yield of fraction a was1254.70 mg/kg with mass ratio of 0.2261. Seven compounds wereidentified. Mass ratio of fraction c was 0.0105. High volatilecompounds were present. Two compounds (�-3-carene andmyrcene) were identified only in fraction c, while others werealso identified in fraction b. In summary, twenty compoundswere identified in rosemary volatiles isolated by HD methodand twenty-three compounds in the volatiles isolated by HDAmethod.

Volatiles from rocket flowers (I) and rocket leaves with greenbeans (II) were also isolated by HD and HDA methods. The yieldsand chemical compositions of the isolated volatiles and their frac-tions for both methods are given in Table 3. The yield of volatilesobtained from the flowers was: by HD method 152.32 mg/kg, andby HDA method 311.77 mg/kg (104.7% yield increase). Sixteencompounds were identified in the volatiles obtained by HD, andforty-eight by HDA. The yield of volatiles from the leaves withgreen beans was: by HD method 71.68 mg/kg, and by HDA method91.70 mg/kg (27.9% yield increase). Twenty-four compounds were

888 J. Mastelic et al. / Talanta 76 (2008) 885–891

Fig. 2. Chemical structures of the main volatile compounds in carob, rosemary and rock2-methylbutanoic acid (2); hexanoic acid (3) heptanoic acid (4); decanoic acid (5); (Z)-3-heisothiocyanate (9); 1,8-cinele (10); �-terpineol (11); camphor (12); borenol (13); undecan2-tridecanone (18); hexadecanal (19); 3-dodecenyl acetate* (20); methyllinolenate (21); �dimethyl sulfoxide (26); dimethylsulfone (27); cyclooctasulfur (28); *, correct isomer not

identified in the volatiles obtained by HD and thirty four by HDA.The yield of fraction b (water non-soluble compounds) for I was173.55 mg/kg with mass ratio 0.5567 including 21 compoundsand for II was 31.90 mg/kg with mass ratio 0.3479, including 12compounds. Depending of the used conditions [19], glucoerucindegradation can produce: 4-methylthiobutylisothiocyanate(erucin), 5-methylthiopentanenitrile (erucin nitrile) and sulfur.

et identified in fractions a, b and c. Compound names: 2-methylpropanoic acid (1);xen-1-ol (6); 2-phenylethanol (7); 3-butenyl isothiocyanate (8); 4-methylthiobutyl

oic acid (14); dodecanoic acid (15); tetradecanoic acid (16); hexadecanoic acid (17);-pinene (22); 2-butenal* (23); 5-methyl-2-hexanone (24); dimethyl disulfide (25);identified.

4-Methylthiobutylisothiocyanate was found mostly in fractiona (I: 29.52 mg/kg, II: 15.51 mg/kg). 5-Methylthiopentanenitrilewas detected only in fraction a (II: 0.31 mg/kg). In addition,water-soluble acids were found in this fraction, such as acetic acid(I: 6.73 mg/kg; II: 0.70 mg/kg), propionic acid (I: 0.14 mg/kg), 2-methylbutanoic acid (I: 17.57 mg/kg), pentanoic acid (I: 0.14 mg/kg),hexanoic acid (I: 2.61 mg/kg; II: 0.48 mg/kg), heptanoic acid (I:

J. Mastelic et al. / Talanta 76 (2008) 885–891 889

Table 1Identified constituents, composition and yields of volatile compounds of Ceratonia siliqua L. isolated by two methods: hydrodistillation with solvent trap (HD) andhydrodistillation–adsorption (HDA)

Compound RI HD [mg/kg] HDA [mg/kg]

a b c Calculated values (a + b + c)

2-Butenal* 1006 – – – 2.75 2.752-Butenal* 1010 – – – 5.49 5.495-Methyl-2-hexanone 1139 – – – 2.60 2.602-Octanone 1340 – – – 0.15 0.15Acetic acid 1380 – 17.86 – t 17.862-Methylpropanoic acid 1469 1077.04 1853.98 t t 1853.982-Nonanon 1545 0.26 – 1.01 0.07 1.08Butanoic acid 1549 36.35 107.95 – – 107.952-Methylbutanoic acid 1596 30.72 48.49 – 1.34 49.83Pentanoic acid 1657 0.15 – 0.59 – 0.592-Nonanol 1660 2.80 – 0.88 – 0.88Ethyl butanoate 1672 t – 0.44 – 0.442,6-Dihydro-2H-pyran-2-one 1743 – 4.35 – – 4.35Hexanoic acid 1770 290.00 513.08 1.52 – 514.602-Dodecanol 1859 – – 4.28 – 4.281-Hexadecene 1867 – – 3.35 – 3.352-Pentadecanone 1957 0.12 – 1.32 – 1.322-Tridecanone 1960 – – 15.17 – 15.17Octanoic acid 1966 12.56 21.69 – – 21.693-Dodecenyl acetate* 1967 – – 15.82 – 15.821-Octadecene 2054 – – 3.35 – 3.35Cyclooctadecene 2081 – – 6.09 – 6.092-Nonadecanone 2173 – – 0.49 – 0.49Hexadecanal 2197 – – 65.89 – 65.89Hexadecanoic acid >2200 – – 18.87 – 18.87

Yield [mg/kg] 1450.0 2567.4 148.5 12.4 2728.3Mass ratio of fractions 0.9410 0.0544 0.0045

HD, hydrodistillation with solvent trap; HAD, hydrodistillation–adsorption; a, fraction of water-soluble compounds; b, fraction of water non-soluble compounds; c, fractionof high volatile compounds; calculated values − sum [mg/kg] of fractions a, b and c; -, not detected; t, traces <0.01 mg/kg; *, correct isomer (E, Z) is not identified; RI, retentionindex on HP-20M column.

Table 2Identified constituents, composition and yields of volatile compounds of Rosmarinus officinalis L. isolated by two methods: hydrodistillation with solvent trap (HD) andhydrodistillation–adsorption (HDA)

Compound RI HD [mg/kg] HDA [mg/kg]

a b c Calculated values (a + b + c)

�-Pinene 1034 650.42 – 625.05 31.15 656.20Camphene 1071 80.28 – 90.85 4.85 95.70�-Pinene 1107 60.43 – 57.93 4.12 62.05�-3-Carene 1141 – – – 0.40 0.40Myrcene 1148 – – – 0.78 0.781,8-Cineole 1192 2755.57 558.20 2319.20 16.40 2893.80cis-�-Ocimene 1223 5.20 – 12.37 – 12.37�-Terpinene 1237 46.50 – 49.60 – 49.60p-Cymen 1253 51.65 – 62.21 0.50 62.71Terpinolene 1263 36.85 – 38.89 – 38.89Alloocimene 1353 t – 4.13 – 4.13Oct-1-en-3-ol 1412 t – 4.27 – 4.27Camphor 1482 501.51 282.92 394.15 – 676.07Pinocarvone 1506 25.89 – 34.75 – 34.75Isopinocamphone 1506 31.10 – 33.90 – 33.90Linalool 1507 30.95 4.78 60.10 – 64.88Bornyl acetate 1546 36.15 – 38.92 – 38.92Terpinene-4-ol 1555 – 13.50 24.78 – 38.28Trans-caryophyllene 1571 56.82 – 56.74 – 56.74�-Terpineol 1629 315.28 212.71 119.76 – 332.47�-Humulene 1638 20.71 – 20.80 – 20.80Borneol 1657 241.59 175.59 165.41 – 341.00Caryophyllene oxide 1912 25.80 3.53 20.69 – 3.47

Yield mg/kg 4972.70 1254.70 4234.50 58.20 5546.40Mass ratio of fractions 0.2261 0.7634 0.0105

HD, hydrodistillation with solvent trap; HDA, hydrodistillation–adsorption; a, fraction of water-soluble compounds; b, fraction of water non-soluble compounds; c, fractionof high volatile compounds; calculated values − sum [mg/kg] of fractions a, b and c; -, not detected; t, traces <0.01 mg/kg; *, correct isomer (E, Z) is not identified; RI, retentionindex on HP-20M column.

890 J. Mastelic et al. / Talanta 76 (2008) 885–891

Table 3Identified constituents, composition and yields of volatile compounds of Eruca sativa flowers (I) and leaves and green beans (II) isolated by two methods HD and HDA

Compound RI HD [mg/kg] HDA [mg/kg]

I II a b c Calculated values (a + b + c)

I II I II I II I II

�-Pinene 1034 – 0.25 – – – 0.10 – – – 0.10Dimethyldisulfide 1059 – – – – – – 0.04 0.08 0.04 0.08Tetrahydrothiophene 1107 – 1.60 0.14 0.35 0.21 – – – 0.35 0.35Heptanal 1157 – – – – – – 0.17 – 0.17 –(E)-2-hexenal 1180 – 2.36 0.69 0.87 – – 0.20 – 0.89 0.87Methylisothiocyanate 1228 – 0.25 – – – – – – – –(Z)-3-hexenyl formate 1230 – – 0.41 0.57 – – – – 0.41 0.572,4-Hexadiene 1280 t – – – – – 0.07 – 0.07 –(E)-3-hexen-1-ol 1322 – 1.77 1.10 1.18 – – 0.25 – 1.35 1.18(Z)-3-hexen-1-ol 1344 – 7.18 20.46 16.26 0.21 – 0.54 0.11 21.21 16.37Dimethyl trisulfide 1347 – – – – – – 0.07 – 0.07 –(E)-2-hexen-1-ol 1360 – 1.85 – 1.88 – – – – – 1.88(Z)-3-hexenyl isobutyrate 1361 – – 0.20 – – – – – 0.20 –Nonanal 1365 – – – – 0.43 – – – 0.43 –Acetic acid 1380 – – 6.73 0.70 – – 0.12 – 6.85 0.703-Butenyl isothiocyanate 1403 – – 1.10 0.35 – – 0.07 – 1.17 0.35(Z)-3-hexenyl-�-methylbutyrate 1440 – – 0.14 – – – – – 0.14 –Propionic acid 1473 – – 0.14 – – – – – 0.14 –4-Methylpentyl isothiocyanate 1487 – 0.76 0.14 0.22 0.21 0.20 0.06 0.27 0.41 0.69Dimethylsulfoxide 1522 – – 1.51 0.39 – – 1.04 1.57 2.55 1.96Hexyl isothiocyanate 1536 – – – – – – 0.10 – 0.10 –Methyl decanoate 1552 1.35 – 0.14 – 0.21 – 0.02 – 0.37 –�-Cyclocitral 1564 – – – – – – 0.02 – 0.02 –Phenylacetaldehyde 1596 – 0.59 – 0.20 – – – – – 0.202-Methylbutanoic acid 1596 0.84 0.74 17.57 – – – – 0.74 17.57 0.74(Z)-3-hexyl hexanoate 1614 – – 0.82 – – – – – 0.82 –Pentanoic acid 1657 – – 0.14 – – – – – 0.14 –�-Methylbenzyl alcohol 1737 – – 0.14 – – – – – 0.14 –Hexanoic acid 1770 – 0.17 2.61 0.48 – – – – 2.61 0.48Benzyl alcohol 1797 – – 0.27 – – – – – 0.27 –Dimethylsulfone 1815 – – – – – – 0.04 0.05 0.04 0.052-Phenylethyl alcohol 1835 – 0.26 2.88 0.14 – – – 0.26 2.88 0.404-Methylthiobutanenitrile 1851 – – 0.96 0.35 – – – 0.21 0.96 0.56�-Ionone* 1873 – 0.67 – 1.31 0.86 0.41 – – 0.86 1.72Heptanoic acid 1875 1.85 – 7.55 – – – – – 7.55 –

0.3–0.3

15.50.1––1.4–0.4–

5-Methylthiopentanenitrile 1892 – – –Nonadecane 1900 0.34 – –Octanoic acid 1966 0.84 0.34 1.244-Methylthiobutylisothiocyanate (erucin) 2041 32.18 28.63 29.52Nonanoic acid 2084 0.67 – 0.69Heneicosane 2100 – 1.94 –Methyl hexadecanoate 2182 0.51 0.17 –Decanoic acid 2192 56.11 1.60 25.85Ethyl hexadecanoate >2200 – – –Undecanoic acid >2200 2.70 0.76 1.65Dodecanoic acid >2200 6.57 0.76 1.78

Tricosane >2200 – 1.52 – –Tridecanoic acid >2200 – – – –Tetradecanoic acid >2200 2.36 1.09 – –Pentadecanoic acid >2200 0.51 – – –Methyl linolenate >2200 4.04 0.42 0.20 –Hexadecanoic acid >2200 41.45 16.00 0.27 –Cyclooctasulfur >2200 – – 0.27 –

Total mg/kg 152.32 71.68 127.31 43.4Mass ratio of fractions 0.4083 0.4

I, flowers; II, leaves and green beans; HD, hydrodistillation with solvent trap; HDA, hydrodnon-soluble compounds; c, fraction of light volatile compounds; calculated values from fris not identified; RI, retention index according to HP-20M column.

7.55 mg/kg), octanoic acid (I: 1.24 mg/kg; II: 0.35 mg/kg) andnonanoic acid (I: 0.69 mg/kg; II: 0.10 mg/kg). Decanoic acid wasfound in high amounts in fractions a (I: 25.85 mg/kg; II: 1.44 mg/kg)and b (I: 38.57 mg/kg). Acids with longer aliphatic chain, unde-canoic (I: 6.86 mg/kg), dodecanoic (I: 14.57 mg/kg; II: 0.31 mg/kg),tridecanoic (I: 0.64 mg/kg), tetradecanoic (I: 9.00 mg/kg; II:1.02 mg/kg), pentadecanoic (I: 2.14 mg/kg) and hexadecanoic acid(I: 72.65 mg/kg; II: 24.74 mg/kg) were found mostly in the fractionb. This is in agreement with the volatile analysis of carob beans,

1 – – – – – 0.310.86 0.25 – – 0.86 0.25

5 0.21 – 0.02 – 1.47 0.351 6.64 1.56 – – 36.16 17.070 – – – – 0.69 0.10

0.21 1.53 – – 0.21 1.532.14 0.51 – – 2.14 0.51

4 38.57 – – – 64.42 1.440.86 – – – 0.86 –

6 6.86 – – – 8.51 0.4614.57 0.31 – – 16.35 0.31

6.21 1.27 – – 6.21 1.270.64 t – – 0.64 t9.00 1.02 – – 9.00 1.022.14 – – – 2.14 –9.86 – – – 10.06 –72.65 24.74 – – 72.92 24.74– – 8.08 13.09 8.35 13.09

2 173.55 31.90 10.91 16.38 311.77 91.70735 0.5567 0.3479 0.0350 0.1786

istillation–adsorption; a, fraction of water-soluble compounds; b, fraction of wateractions and their mass ratios; -, not detected; t, traces <0.01 mg/kg; *correct isomer

which contain many of these compounds in the same fractions. Infraction c, nine compounds were identified, where cyclooctasulfur(I: 8.08 mg/kg; II: 13.09 mg/kg) was the major compound.

4. Conclusion

Proposed HDA isolation method is particularly suitable forplant samples whose volatiles contain larger amount of water-soluble and high volatile compounds regardless of their functional

lanta

J. Mastelic et al. / Ta

group. Mass ratios of the obtained fractions were dependent ontheir chemical composition, respectively, solubility in water andvolatility. This method offers simultaneous hydrodistillation andadsorption that is more practical. Distillation, adsorption, extrac-tion and concentration of volatiles are performed in only oneapparatus. The main advantages of HDA method over HD methodwere the higher yields, fractionation of volatiles in three frac-tions and elimination of peak overlaps on chromatograms thatproduced more complete volatile analysis. However, the extrac-tion of adsorbed compounds, performed with diethyl ether for 8 h,prolonged isolation of volatiles.

Acknowledgements

This work was supported by MZOS, Croatia, project “EssentialOils and Flavours - Biologically Active Compounds and their Mod-ifications” No. 011-098 2929-1329 in the framework of the MZOS“Spectroscopy and Modelling of Bioactive Molecules” No. 0982929.We acknowledge the botanical identification of the plant materialsby MSc Mirko Ruscic, Faculty of Philosophy, University of Split.

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