Applicability of alkyl-bonded ultra-pure silica stationary phases for gradient reversed-phase HPLC...

J. Sep. Sci. 2006, 29, 889 – 904 J. D. M. Patring et al. 889

Johan D. M. PatringSvetlana A. LaninaJelena A. Jastrebova

Department of Food Science,Division of Food Chemistry,Swedish University ofAgricultural Sciences, Uppsala,Sweden

Original Paper

Applicability of alkyl-bonded ultra-pure silicastationary phases for gradient reversed-phaseHPLC of folates with conventional and volatilebuffers under highly aqueous conditions

Applicability of several alkyl-bonded silica stationary phases was tested for gradientRP-HPLC of folates under highly aqueous conditions. High retention of folates wasachieved on alternative phases with enhanced polarity and classical phases withhigher carbon content. Phases exhibiting polar secondary interactions were foundto provide better selectivity for late-eluting folates, whereas selectivity for early-elut-ing folates was mostly dependent on hydrophobic interactions. Best selectivity inphosphate buffered mobile phase was achieved on polar-endcapped silica phases(Aquasil C18 and HyPurity Aquastar) followed by alternative Atlantis dC18. Classicalphases exhibited poorer separation of 10-formyl-folic acid and 5-formyl-tetrahydro-folate, but it could be considerably improved by increasing the buffer pH. Strongsecondary interactions of ion-exchange character on polar-embedded phases resul-ted in marked peak deterioration, loss of recovery and dramatic changes in reten-tion behaviour for early- and late-eluting folates when changing the mobile phasecomposition and pH. Therefore, polar-embedded phases such as HyPurity Advancewere found to be unsuitable for separating folates. Stationary phases exhibited peakdeterioration when using volatile buffer of low ionic strength. Better results wereobtained with classical phases, whereas alternative phases showed not only peakdeterioration but also a decrease in recovery and poorer selectivity due to increasedsecondary interactions in volatile buffer.

Keywords: Polar-endcapped / Retention mechanism / Reversed-phase HPLC / Stationary phases /Volatile buffer /

Received: December 1, 2005; accepted: January 17, 2006

DOI 10.1002/jssc.200500481

1 Introduction

RP-HPLC using silica-based stationary phases is often themethod of choice for determination of biologically activenatural compounds such as vitamins, antioxidants,amino acids, peptides and proteins in complex biologicalmatrices. Many of these compounds are polar and con-tain various functional groups which make their chro-matographic behaviour complicated and difficult to pre-dict [1, 2]. Development of new stationary phases suitablefor chromatographic determination of polar ionisablecompounds with diversity of functionalities is therefore

of great interest. Furthermore, rapid development of MSapplications in analyses of biologically active naturalcompounds imposes new, more stringent requirementson the chromatographic performance of stationaryphases. These requirements include compatibility withvolatile buffers of low ionic strength and improved reten-tion of water-soluble polar compounds without excessiveretention of hydrophobic compounds.

Recently, considerable attention has been focussed onnovel silica-based stationary phases specially designedfor analyses of polar analytes under highly aqueous con-ditions. Due to the use of polar endcapping, polar embed-ding (modifying alkyl chains with polar functionalgroup) or mixed ligands, these alternative phases provideadditional retention mechanisms that often result inincreased retention of polar compounds and differentselectivity compared to more traditional alkyl-bondedphases [3–6]. Unfortunately, the information about bond-

Correspondence: Dr. Jelena A. Jastrebova, Department of FoodScience, Division of Food Chemistry, Swedish University of Agri-cultural Sciences, P.O. Box 7051, SE-750 07 Uppsala, Sweden.E-mail: [email protected]: +46-18-672995.

Abbreviations: ACN, acetonitrile; DAD, diode array detector;HAc, acetic acid; PhB, potassium phosphate buffer

i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

890 J. D. M. Patring et al. J. Sep. Sci. 2006, 29, 889 –904

ing technology and nature of functional groups used forpolar endcapping or embedding is usually classified asproprietary by manufacturers, which makes it difficultto predict the applicability of these novel phases for chro-matographic determination of highly polar multifunc-tional compounds. Moreover, literature data regardingcharacterisation of stationary phases are generally basedon standard test procedures using hydrophobic andmoderately polar model analytes and phosphate buf-fered mobile phases containing 30–80% of organic modi-fier (most commonly methanol) [7–13]. However, chro-matographic information obtained by these evaluationmethods does not always reflect specific column proper-ties in respect to retention and separation of polar ionisa-ble compounds in highly aqueous conditions. Recently,Euerby and Petersson [5, 11] have shown the necessity foradditional test procedures including several polar com-pounds and mobile phases with lower content of organicmodifier and lower ion strength in order to discriminatebetween different stationary phases.

In the present study, we have extended our previousinvestigation of ten alternative and conventional silica-based phases (alkyl-, phenyl- and polar-bonded) for separ-ating folates [14] to ten alkyl-bonded phases (both alter-native and classical). Folates, a group of derivatives offolic acid (water-soluble B-vitamin), have received muchattention in recent years due to their health-promotingeffects [15–18], and therefore development of sensitiveand selective methods for folate analysis is of greatimportance. Moreover, the study of the chromatographicbehaviour of folates can be of interest for a better under-standing of retention mechanisms for polar compoundscontaining various functional groups with widely differ-ent properties. As we have shown [14], alkyl-bondedphases, especially polar-endcapped Aquasil C18, were themost promising for the separation of folates, whereasphenyl-bonded phases lacked selectivity when separating10-formylfolic acid and 5-formyltetrahydrofolate, andpolar-bonded phases exhibited poorer peak shape. How-ever, a few phases tested made it difficult to explain thedifferences between phases and to give some recommen-dations for selection of columns most suitable for folateanalysis.

The main objective of this work was to study the separa-tion of folates under gradient conditions on alternativeand classical alkyl-bonded silica phases differing in bond-ing technology, ligand concentration, particle size, porediameter and type of endcapping. To understand the roleof organic modifier in folate retention, elution withdifferent percentages of ACN at the start of the gradientwas tested. To better understand the retention mechan-isms of different folate derivatives, mobile phases con-taining phosphate buffer at pH 2.3 and 3.5 and differentbuffer concentrations were compared. To find phases

most compatible with MS detection volatile acetate buf-fer (pH 3.5) of low ion strength was also tested.

2 Experimental

2.1 Materials

ACN was of isocratic grade for HPLC; the other chemicalswere of analytical quality. If not otherwise stated, thechemicals were purchased from Merck (Darmstadt, Ger-many). 2,3-Dimercapto-1-propanol (BAL) was obtainedfrom Sigma-Aldrich (St. Louis, USA). Water was purifiedusing a Milli-Q system (Millipore, USA).

Folic acid, (6S)-5,6,7,8-tetrahydrofolate, sodium salt(H4folate), (6S)-5-HCO-5,6,7,8-tetrahydrofolate, sodiumsalt (5-HCO-H4folate) and (6S)-5-CH3-5,6,7,8-tetrahydrofo-late, sodium salt (5-CH3-H4folate) were all a kind gift fromMerck Eprova, Schaffhausen, Switzerland. 10-Formylfolicacid, sodium salt (10-HCO-folic acid) was obtained fromDr. Schirck’s Laboratories (Jona, Switzerland). The folatestandards were stored at –808C until use. The purity ofall standards was checked according to the procedure ofvan den Berg et al. [19] using molar extinction coefficientsreported by Eitenmiller and Landen [15], but for 10-CHO-folic acid, the molar extinction coefficient reported byBaggott and Johanning [20] was used. The stock solutionsof folates of 200 lg/mL (purity corrected) were preparedunder subdued light in 0.1 M phosphate buffer pH 6.1containing 1% sodium ascorbate w/v and 0.1% mercap-toethanol v/v. Aliquots of the stock solutions were placedin separate tubes, flushed with nitrogen and storedbelow –808C for at most 3 months. The work solutions(100 ng/mL for H4 folate and 5-CH3–H4folate; 600 ng/mLfor 10-HCO-folic acid, 5-HCO–H4folate and folic acid)were prepared by dilution of the stock solution with acet-ate buffer, commonly used as elution buffer for SPE offolates from food extracts (0.1 M sodium acetate contain-ing 10% w/v sodium chloride, 1% w/v sodium ascorbateand 0.1% v/v BAL) [21, 22].

2.2 Instrumentation

Analyses were performed using an HPLC system (Agilent1100) consisting of a gradient quaternary pump, a ther-mostated autosampler, a thermostated column compart-ment, a diode array detector (DAD) and a fluorescencedetector. The HPLC system was controlled by a PC run-ning Agilent Chemstation software. The separation offolates was performed at 238C. The flow rate was 0.4 mL/min; the injection volume 20 lL; the temperature in thethermostated autosampler 88C. For the detection andquantification of H4 folate and 5-CH3–H4folate a fluores-cence detector was used (excitation at 290 nm and emis-sion at 360 nm) and for 10-HCO-folic acid and folic acid aDAD detector was used (the DAD channel was set at


J. Sep. Sci. 2006, 29, 889 – 904 Applicability of alkyl-bonded silica phases for RP-HPLC of folates 891

290 nm). For 5-HCO–H4folate both fluorescence (290 nmex/360 nm em) and DAD (290 nm) detectors were used.

2.3 Columns

The chromatographic separation of the five folate deriv-atives was tried on ten alkyl-bonded columns all basedon ultra-pure silica. Some data from our previous study[14] for five further alkyl-bonded columns were alsoincluded for comparative purposes. The characteristicsof columns are shown in Table 1. Columns were equili-brated with at least 30 column volumes before chromato-graphic data were collected.

2.4 HPLC analysis

The mobile phase was a binary gradient mixture of aque-ous buffer and ACN. Different gradient programmeswere used in the study (Fig. 1). The main differencebetween them was the initial concentration of ACNwhich ranged from 3 to 10%. The lag phase (5 min) aswell as the linear gradient raise (19% in 20 min) was thesame for all programmes. After 25 min run-time the ACN

concentration was kept constant for 2 min and there-after linearly decreased during 1 min to its initial con-centration that was applied for 14 min to re-equilibratethe column.

For all ten columns studied here, two buffers were usedfor preparation of mobile phases: 30 mM potassium


Figure 1. Description of the different ACN gradient pro-grammes used in the study.

Table 1. Survey of the columns used in this studya)

Abbreviation Column name Pore size (�) Particle size(lm)

Surface area(m2/g)

Total carboncontent (%)

pH range Comments

Columns with novel alkyl-bonded phases suitable for use in highly aqueous mobile phases (compatible with 100% water)Atl Atlantis dC18 (Waters) 100 3 340 12.5 2–8 C18 difunctionally bonded phase,

endcapped (proprietary)AqC18b) Aquasil C18 (Thermo Electron

Corporation)100 3 310 12 1.8–8 C18-bonded phase, hydrophilic

endcapping (proprietary)HyAqS HyPurity Aquastar C18 (Thermo

Electron Corporation)190 3 10 1.8–8 C18-bonded phase, polar end-

capped (proprietary)HyPAc) HyPURITY Advance (Thermo

Electron Corporation)190 5 200 10 1.8–8 C8-bonded phase, amide polar-

embedded group, endcappedZoAqc) Zorbax SB Aq (Agilent) 80 5 180 N/A 1.8–8 Unspecified, hydrophilic surface,

sterically protected

Columns with classical alkyl-bonded phasesZoC8ZoC8-5b)

Zorbax SB C8 (Agilent) 80 3.55

180 5.5 1.8–8 C8-bonded phase (diisopropyloc-tysilan), sterically protected

ZoC18 Zorbax SB C18 (Agilent) 80 3.5 180 10 1.8–8 C18-bonded phase (diisobutylocta-decylsilan), sterically protected

ZoRx Zorbax Rx C18 (Agilent) 80 5 180 12 1.8–8 C18-bonded phase (dimethylocta-decylsilan), nonendcapped

SyC12c) Synergi Max (Phenomenex) 80 4 475 17 1.5–10 C12-bonded phase, endcapped(trimethylsilane)

GeC18c) Genesis C18 (Jones Chromato-graphy)

120 4 300 18 2–8 C18-bonded phase, endcapped(proprietary)

Ace Ace C18 (ACT) 100 3 300 15.5 2–8 C18-bonded phase, endcapped(proprietary)

XBr XBridge C18 (Waters) 130 3.5 180 17.4 1–12 C18-bonded phase (trifunctionalbonding), based on polyethoxysi-lane, endcapped

Gold Hypersil Gold C18 (ThermoElectron Corporation)

175 3 220 10 2–8 C18-bonded phase, endcapped(proprietary)

SunF SunFire C18 (Waters) 90 3.5 340 15.8 2–8 C18-bonded phase (proprietarybonding), endcapped (propriet-ary)

a) All columns were 150 mm64.6 mm and based on ultra-pure silica.b) This column was tested in our previous study, but also included in the present study.c) These columns were tested in our previous study. Test results are included only partially in the present study.N/A – Not available.


phosphate buffer pH 2.3 (PhB) and 5 mM acetic acidpH 3.5 (HAc). For columns HyAqS, XBr and Ace, threeadditional buffers were used: 30 mM PhB pH 3.5, 5 mMPhB pH 3.5 and 6 mM H3PO4 pH 2.3. For five columnsstudied previously [14], some data obtained with 30 mMPhB pH 2.3 were included. For HyPA studied previously[14], data obtained with 30 mM HAc pH 3.1 were used.PhB (30 or 5 mM) were prepared by mixing either 30 mMKH2PO4 with 30 mM H3PO4 or 5 mM KH2PO4 with 5 mMH3PO4 until the desired pH was achieved.

All results were the mean of duplicate injections. Toavoid possible errors due to incomplete re-equilibrationof the column the check for repeatability of the measure-ment was always performed before data collection (RSDfor retention time, peak area and peak height of threesuccessive injections of the same work solution). Therepeatability of the measurement was better than 1% forretention time and better than 4% for peak area andpeak height for all folate derivatives.

2.5 Column performance calculations

Retention times (tR) of five folate derivatives were used tocompare column retention performance under highlyaqueous gradient conditions. Peak area was used for esti-mation of recovery. Peak widths (w0.5) were calculated athalf-heights. Peak asymmetry (As) was estimated at 10% ofthe peak height from the ratio of the widths of the rearand front sides of the peak. To compare the selectivity ofdifferent columns, the apparent resolution factor (Rs) fortwo adjacent peaks with retention times t2 > t1 was calcu-lated according to the formula commonly used for iso-cratic elution

Rs ¼ 1:186t2 � t1

w2 þ w1;

where w1, w2 are peak widths at half-height.

To compare the efficiency of some selected columns, theapparent plate number (N) was calculated from the peakwidth at half-height (w0.5) according to the formula com-monly used for isocratic elution

N ¼ 5:546�

tR

w0:5

�2

2.6 Check for applicability of general testprocedures for selection of columns forfolate analysis

To check the applicability of standard test procedures forcolumn selection we compared retention times of folatesand column characterisation parameters obtained pre-viously by Euerby and Petersson [5, 11] by using theTanaka protocol [23]. For this comparison we used chro-

matographic data for five folate derivatives obtained inphosphate buffered (pH 2.3) mobile phase with gradientprogramme ,Start 6%’. The following 11 alkyl-bonded col-umns from both our present and previous study wereincluded: ZoC8, ZoC18, ZoRx, ZoAq, HyPA, HyAqS, SyC12,GeC18, Ace, Atl and AqC18. Other alkyl-bonded columns(SunF, XBr and Gold) could not be included because ofthe lack of available characterisation data. The retentiontimes of folates on different columns were plottedagainst five main chromatographic parameters:

1. Hydrophobic selectivity (aCH2 ) – retention factor ratiobetween n-pentylbenzene and n-butylbenzene (MeOH-H2O (8:2 v/v), 1.0 mL/min, 408C);

2. shape selectivity (a(T/O)) – retention factor ratio betweentriphenylene (T) and o-terphenyl (O) (MeOH-H2O (8:2v/v), 1.0 mL/min, 408C);

3. hydrogen bonding capacity (a(C/P)) – retention factorratio between caffeine (C) and phenol (P) – (MeOH-H2O(3:7 v/v), 1.0 mL/min, 408C);

4. total cation-exchange capacity (aB/P pH 7.6) – the retentionfactor ratio between benzylamine (B) and phenol (P) atpH 7.6 (MeOH-H2O (3:7 v/v), 20 mM KH2PO4 pH 7.6,1.0 mL/min, 408C); and

5. acidic cation-exchange capacity (aB/P pH 2.7) – the reten-tion factor ratio between benzylamine (B) and phenol(P) at pH 2.7 (MeOH-H2O (3:7 v/v), 1.0 mL/min, 408C,pH 2.7 KH2PO4 buffer).

3 Results and discussion

3.1 Chromatographic behaviour of folates

In the present study we have investigated the RP separa-tion of the five most abundant folate derivatives, natu-rally occurring H4folate, 5-CH3–H4 folate, 5-HCO–H4folateand 10-HCO-folic acid as well as folic acid – syntheticfolate form commonly used for food fortification pur-poses. Folates are water-soluble multifunctional com-pounds possessing both acidic and basic properties. InRP-HPLC, folates are commonly separated at low pH [15,24–26] which is necessary to suppress the ionisation oftwo carboxyl groups in the glutamate part of the folatemolecule (Fig. 2). As seen from Table 2, a- and c-COOHgroups have pKa-values around 3.1–3.5 and 4.6–4.8,respectively [18, 27–33]. In other words, pH a3 is neededto completely suppress the ionisation of the a-COOHgroup. However, the use of low pH leads to protonationof the basic amide group (pKa = 7.7–10.5) and weaklybasic nitrogen atoms such as –NH2, N1, N5 and N8 in thepteridine ring moiety (Fig. 2, Table 2). As such, folates arepositively charged in mobile phase with pH a3 and pos-sess both positive and negative charges in mobile phasewith pH >3.



The degree of protonation depends considerably on sub-stituents in the folate molecule and the structure of thepteridine moiety. In folic acid and 10-HCO-folic acid thepteridine moieties are fully oxidised, that is, the pteri-dine exists as a fully double-bonded conjugated p-systemand nitrogen atoms N1, N5 and N8 have almost no basicproperties (Fig. 2, Table 2). In tetrahydrofolates two dou-ble bonds of the pteridine ring are reduced. This causes amarked increase of the basic strength of N5 of tetrahydro-folates containing –H, –CH3, –CH2– or –CH+– at N5 due toloss of aromaticity. Moreover, it may increase the basicstrength of the N8 atom for all tetrahydrofolates. On theother hand, introduction of an electron-withdrawing–CHO group on N5 completely abolishes the basic proper-ties of the N5 atom in 5-HCO–H4folate [34]. It may there-fore be concluded that 5-HCO–H4folate, folic acid and 10-HCO–folic acid are much less protonated at low pH thanH4folate and 5-CH3–H4folate. These differences in proto-nation and polarity determine to a large extent the elu-tion order of folate derivatives in RP-HPLC at pH a3:H4folate f 5-CH3–H4folate a10-HCO-folic acid f 5-HCO–H4folate a folic acid [14]; that is, the more protonatedH4folate and 5-CH3–H4folate elute first (early-elutingfolates) whereas less protonated 10-HCO-folic acid, 5-HCO-H4folate and folic acid elute much later (late-elutingfolates). The presence of substituents capable of hydro-phobic or polar interactions with the stationary phasecan influence the elution order as well. For example, the

higher retention of 5-CH3–H4folate compared withH4folate can be attributed to stronger hydrophobic inter-actions between alkyl chains of the stationary phase andthe CH3 group in the N5 position of 5-CH3–H4folate. Thedifferences in degree of planarity of molecules (e. g.,between reduced and oxidised pyrazine ring in 5-HCO–H4folate and 10-HCO-folic acid) may also influence theretention of folates.

At higher pH (pH = 3–4) the chromatographic behaviourof folates becomes more complicated because of partialionisation of the a-COOH group with the resulting forma-tion of bipolar folate ions carrying negative charge onthe glutamate part and positive charge on the pteridinering. In this case negative charge can become prevalentin less protonated late-eluting folates whereas positivecharge can still remain predominant in the early-elutingfolates due to protonation of N5 in the pteridine ring. Inthis connection, early- and late-eluting folates may exhi-bit widely different chromatographic behaviour atpH >3 when using phases with polar ionisable groups onthe silica surface.

3.2 Overview of stationary phases regardingretention of folates at pH 2.3

Retention times of five folate derivatives on 14 alkyl-bonded columns in mobile phase buffered with 30 mMPhB pH 2.3 and ACN gradient starting at 6% are pre-


Figure 2. Chemical structure for the various folate derivatives.

Table 2. pKa-values for different forms of folates

Folate Amide N1 N5 N8 N10 a-COOH c-COOH References

H4folate 10.5 1.24 4.82 ND – 1.25 3.5 4.8 [27]5-CH3-H4folate 10.8 1.24 5.2 ND ND 3.5 4.8 [33]5,10-CH+-H4folate 8.95 ND 4.5 ND – 1.2 ND ND [18, 28]H2folate 9.54 1.38 3.84 ND 0.28 ND ND [29]5-HCO-H4folate 10.4 ND ND ND ND 3.1 4.8 [30]Folic acid 8.38 2.35 – 1.5 1.5 0.20 3.5 4.8 [29, 31, 32]10-CH3-folic acid 7.68 2.40 – 1.5 ND 0.36 ND 4.62 [29]

ND = not detected.


sented in Fig. 3. Columns are placed according to theretention time for the last-eluting folic acid. Dependingon retention, columns could be roughly divided into thefollowing groups: high retentive – AqC18, ZoAq, HyAqSand Atl; moderate retentive – GeC18, ZoC8, SyC12, SunFand Ace; low retentive – ZoC18, Gold, XBr and ZoRx aswell as mixed retentive – HyPA. The high retentive groupinvolved two polar-endcapped columns (AqC18 andHyAqS) and two columns with enhanced polarity (ZoAqand Atl), which all are compatible with 100% water.These columns showed good retention of all five folates,whereas mixed retentive polar-embedded HyPA exhib-ited good retention of late-eluting folates but dramati-cally decreased retention of early-eluting H4folate and 5-CH3–H4folate. Moderate and low retentive groupsinvolved classical columns. Interestingly, the low reten-tive group comprised mostly columns with low carboncontent (10–12% for ZoC18, Gold and ZoRx). Only one col-umn in this group, XBr, has a higher carbon content(17.4%), but this column is prepared by using trifunc-tional bonding which usually gives much higher bond-ing density and a somewhat different surface chemistry[35]. Moderate retentive group, on the other hand, com-prised mostly columns with higher carbon content(15.5–18% for GeC18, SyC12, SunF and Ace). Only ZoC8 inthis group has a lower carbon content (5.5%) due tomuch shorter alkyl chain. By this means classical col-umns with higher carbon content and higher surfacecoverage may provide better retention of folates.

An important prerequisite for retention of polar ionisa-ble compounds in highly aqueous conditions is also goodpore accessibility of stationary phases. Apart from poresize and mobile phase composition it depends in greatpart on wettability of the silica surface. Higher wettabil-ity of silica surface can be achieved by increasing thepolarity of the silica surface (introducing polar groupsinto ,alkyl layer’ at silica surface) or by decreasing thehydrophobicity of the silica surface (using shorter alkyl

chains and monofunctional bonding) [3, 5, 6]. Accordingto our results columns with increased polarity (AqC18,ZoAq, HyAqS and Atl) provided higher retention offolates which can be explained by better wettability ofthe silica surface. However, improved wettability due toincreased polarity can result in loss of retention becauseof repulsive interactions of like-charged analytes andpolar groups on the silica surface [3, 5]. Such ion-exclu-sion effects might be responsible for very low retentionof H4folate and 5-CH3–H4folate at pH 2.3 on polar-embedded HyPA containing amide groups (Fig. 3). At thispH, H4folate and 5-CH3–H4folate are more positivelycharged than late-eluting 10-HCO-folic acid, 5-HCO–H4folate and folic acid (see Section 3.1 for explanations).Therefore, the embedded amide groups with positivecharge at low pH could repulse H4folate and 5-CH3–H4folate from pores, whereas repulsive interactions withthe less protonated 10-HCO-folic acid, 5-HCO–H4folateand folic acid seemed to have marginal effects.

3.3 Effect of the composition of the mobile phaseon chromatographic performance

The pore accessibility of stationary phase depends notonly on wettability of the silica surface but also onmobile phase composition. In the present work, we stud-ied the influence of ACN content in the mobile phase buf-fered with 30 mM PhB pH 2.3 on retention and otherchromatographic parameters of folates on ten alkyl-bonded columns. ACN content from 3 to 10% at the startof gradient was tested. As seen from Fig. 4A, a lower con-tent of ACN (3% instead of 6%) at the start of the gradientreduced markedly the differences between columns,whereas higher percent of ACN (7–8%) revealed consider-able variations in folate retention on different columns(Fig. 4B and C). It is interesting to note that all ten col-umns provided good retention and peak shape for folatesas well as excellent reproducibility at 3% ACN at the startof the gradient.


Figure 3. Retention times for five folate derivatives separated on different columns in 30 mM PhB pH 2.3 with 6% ACN at thestart of the gradient. Columns are placed according to descending of retention time for last-eluting folic acid.


Depending on chromatographic behaviour of folateswith increasing ACN content in the mobile phase, wecould roughly divide all ten columns into three groups:Group I (AqC18, HyAqS, Atl, ZoC8 and Gold): On these col-umns all five folate derivatives exhibited stable chroma-tographic behaviour over the range of 3–10% ACN withgradually reduced retention and progressive increase ofpeak width with minor changes of peak symmetry whenincreasing the ACN content. Group II (Ace, XBr, ZoC18and ZoRx): On these columns early-eluting H4folate and5-CH3–H4folate demonstrated a more dramatic decreaseof retention followed by marked deterioration of peakshape (both As and w0.5) when increasing the ACN content

in the mobile phase, whereas late-eluting folates exhib-ited chromatographic behaviour similar to that on col-umns in Group I. Group III (SunF): On this columnH4folate and 5-CH3–H4folate demonstrated a dramaticdecrease of retention followed by marked deteriorationof peak shape (both As and w0.5) at 7% ACN and loss ofretention at 8% ACN, whereas late-eluting folates showeda chromatographic behaviour similar to that on columnsin Group I.

Typical patterns for columns HyAqS and Ace belongingto Group I and Group II, respectively, are shown in Fig. 5.The observed distinctions between these two groups of


Figure 4. A–C. Retention times for five folate derivatives separated on different columns in 30 mM PhB pH 2.3 with 3% ACN atthe start of the gradient (A), 7% ACN at the start of the gradient (B) and 8% ACN at the start of the gradient (C). Columns areplaced according to descending of retention time for last-eluting folic acid.


columns may be attributable to differences in pore acces-sibility. Increasing the ACN content in the mobile phasemay decrease the ,effective’ pore size due to higher solva-tion of the alkyl layer at the silica surface by ACN. It mayalso lead to poorer desolvation of organic molecules andconsequently increasing size of solvated molecules [36].One would expect that phases with more hydrophobicsilica surface may demonstrate more pronounced solva-tion effects of the alkyl layer than phases with enhancedpolarity. Similarly, one would expect that phases withlonger alkyl chains may demonstrate more pronouncedsolvation effects than phases with shorter alkyl chains.Comparison of columns from Group I and Group II addsconsiderable support for this assumption. All columnsinvolved in Group II – moderate retentive Ace and lowretentive XBr, ZoC18 and ZoRx – are classical C18-bondedcolumns with at least moderate hydrophobicity and mayexhibit solvation effects of the alkyl layer when increas-ing the ACN content in the mobile phase. ZoC18, ZoRxand Ace have also small pores (80–100 �). XBr has largerpores (130 �), but it is based on more hydrophobic poly-ethoxysilane and is prepared by trifunctional bondingwhich usually leads to a more bulky alkyl layer on thesilica surface. Therefore, dramatic changes in chromato-graphic behaviour of early-eluting H4folate and 5-CH3–H4folate on these columns (Fig. 5) with increased ACN inthe mobile phase may be explained by size-exclusioneffects related to poorer pore accessibility. The fact thatlate-eluting folates did not exhibit similar changes inchromatographic behaviour may depend on differencesin solvation between early- and late-eluting folates,caused by differences in molecule geometry and polarity.

In contrast to Group II, the columns from Group I exhib-ited no noticeable size-exclusion effects for folates; thechromatographic behaviour of folates was stable over theACN range tested. Group I involved three columns compa-tible with 100% water (AqC18, HyAqS and Atl) and twoclassical columns (ZoC8 and Gold). The first three col-umns have enhanced polarity; therefore, the absence ofpronounced solvation effects of the alkyl layer withincreased ACN content and hence better pore accessibilitycould be expected. For classical ZoC8 better pore accessi-bility could also be expected due to much shorter alkylchains and consequently fewer solvation effects of thealkyl layer. The pore size has a role in pore accessibility aswell. Two columns from Group I (classical Gold and polar-endcapped HyAqS) have rather large pore size (175 and190 �, respectively) compared with other columns tested.

To better understand the differences in chromatographicperformance between columns from Group I and II wehave studied the influence of buffer concentration onretention of folates on columns HyAqS (Group I) and Aceand XBr (Group II). The mobile phase buffered with30 mM PhB pH 2.3 was compared with the mobile phase


Figure 5. Column characteristics (tR, peak width and peakasymmetry) for five folate derivatives separated on columnsHyAqS and Ace by using different ACN gradient pro-grammes ( ,Start 3–10%’, see Fig. 1 for description of gradi-ent programmes) in 30 mM PhB pH 2.3.


buffered with 6 mM H3PO4 pH 2.3. The use of 6 mM H3PO4

with much lower ion strength and absence of K+-cationsmay lead to enhanced size-exclusion effects (due toincreased solvation of the alkyl layer on the silica surfaceand increased solvation of folate molecules by ACN)resulting in decreased retention of folates. It may alsolead to revealing the possible ionic interactions of folateswith polar groups on the silica surface, which are usuallysuppressed in buffers with higher concentrations due tocompetitive interactions with buffer ions [37]. As seenfrom Fig. 6, the retention of H4folate and 5-CH3-H4folateon Ace and XBr was noticeably decreased when using6 mM H3PO4 instead of 30 mM PhB, which indicatedsome enhancement of the size-exclusion effects. The useof 6 mM H3PO4 also influenced the peak shape of folateson these columns resulting in a slight increase in peakwidth. In contrast to Ace and XBr, HyAqS exhibited onlymarginal changes of folate retention and peak shape,which indicated the absence of the size-exclusion effectsand strong ionic interactions at pH 2.3. Our results con-firmed, therefore, our assumptions about differences inpore accessibility between columns from Group I and II.

Classical SunF (C18-bonded with proprietary bonding andendcapping) exhibited widely different properties com-pared with the other nine columns. Its chromatographicperformance was similar to that of polar-embeddedamide-linked HyPA, tested in previous study [14]. Both col-umns showed loss of retention of H4folate and 5-CH3–H4folate when using higher ACN content in the mobilephase. SunF exhibited loss of retention at 8% ACN at thestart of gradient and HyPA even at 6% ACN, but retentionof H4folate and 5-CH3–H4folate could be improved whenusing a lower ACN content in the mobile phase. SunF pro-vided good retention of early-eluting folates with 3–6%ACN at the start of the gradient (Figs. 3, 4A) and HyPA with3–5% ACN [14]. This improvement of retention might beattributed to suppression of ion-repulsive interactions

due to better shielding of polar groups on the silica sur-face by buffer ions and water molecules when using alower ACN content in the mobile phase.

3.4 Comparison of selectivity of stationary phases

The selectivity of 14 alkyl-bonded columns for separationof folates in mobile phase buffered with 30 mM PhBpH 2.3 and ACN gradient starting at 6% is presented inFig. 7A. The apparent resolution factor (Rs) for separating


Figure 6. Differences in retention times (DtR) obtained in6 mM H3PO4 pH 2.3 and 30 mM PhB pH 2.3 (DtR = tR(6 mM) –tR(30 mM)) for five folate derivatives on columns Ace, XBr andHyAqS. ACN (6%) at the start of the gradient was used.

Figure 7. Apparent Rs for five folate derivatives separatedon different columns by using different ACN gradient pro-grammes with 30 mM PhB pH 2.3; A = 6%, B = 8% andC = 3% (see Fig. 1 for description of gradient programmes).


H4folate/5-CH3–H4folate, 10-HCO-folic acid/5-HCO–H4-folate and 5-HCO–H4folate/folic acid was used to charac-terise the differences between columns.

As seen from Fig. 7A, all columns except HyPA showedexcellent selectivity for H4folate and 5-CH3–H4folate, butvaried greatly in selectivity for late-eluting folates. Theseparation of 10-HCO-folic acid and 5-HCO–H4folateappeared to be a great challenge. Only three alternativecolumns (polar-endcapped HyAqS and AqC18 and polar-embedded HyPA) provided baseline separation (Rs F 1.5)of 10-HCO-folic acid and 5-HCO–H4folate under the condi-tions tested. This indicated that secondary interactionswith polar groups on the silica surface might be impor-tant for separation of 10-HCO-folic acid and 5-HCO–H4folate. By contrast, selectivity for H4folate and 5-CH3–H4folate appeared to be similar for both alternative andclassical columns (except HyPA) independently of differ-ences in endcapping and secondary interactions, whichmight reflect the predominant role of hydrophobic inter-actions. In a similar way, both alternative and classicalcolumns could provide baseline separation of 5-HCO–H4folate and folic acid (alternative HyAqS, AqC18, HyPAand Atl and classical ZoRx, Ace, ZoC18 and XBr). How-ever, several columns (alternative ZoAq and classicalGeC18, SunF, Gold, SyC12 and ZoC8) exhibited poorselectivity for all three late-eluting folates. The length ofthe alkyl chain seemed to be important for separation of5-HCO–H4folate and folic acid: columns with shorteralkyl chains (ZoC8 and SyC12) showed much poorer selec-tivity for 5-HCO–H4folate and folic acid than C18-bondedcolumns. This may reflect the importance of hydropho-bic interactions with alkyl chains in retention of thesetwo folate derivatives.

The use of a higher ACN content at the start of the gradi-ent (8% ACN, Fig. 7B) improved slightly the separation oflate-eluting folates, whereas the separation of early-elut-ing H4folate and 5-CH3–H4folate varied greatly, but stillremained very good (RsF2) for all columns except SunF.The use of lower ACN content at the start of the gradient(3% ACN, Fig. 7C) resulted in poorer selectivity for late-eluting folates and reduced the variations between col-umns regarding separation of early-eluting folates(Rs = 3.1–3.8). The poorer selectivity at 3% ACN may beassociated with higher suppression of secondary ionicinteractions due to better shielding of polar groups (bothon silica surface and in folate molecules) by buffer ionsand water molecules.

Among all columns tested, only two columns (polar-embedded HyPA and classical SunF with proprietarybonding and endcapping) exhibited a lack of selectivityfor H4folate and 5-CH3–H4folate at higher ACN content inthe mobile phase (for HyPA F6% ACN and for SunF F8%ACN) due to loss of retention. As discussed in Section 3.3,these effects might be related to ion-repulsive interac-tions. Typical chromatograms for separation of folateson different columns are presented in Figs. 8, 9.

3.5 Influence of pH on retention and separation offolates

The separation of late-eluting folates could be consider-ably improved by using phosphate buffer of higher pH.As evident from Fig. 9, the increase of pH from 2.3 to 3.5provided baseline separation of late-eluting folates onclassical Ace and XBr as well as further improved the sep-aration of 5-HCO–H4folate and folic acid on HyAqS.


Figure 8. Chromatograms for the separation of five folate derivatives on Atl, ZoRx and Gold by use of 6% ACN at the start of thegradient programme with 30 mM PhB pH 2.3. Peaks: (1) H4folate, (2) 5-CH3–H4folate, (3) 5-HCO–H4folate, (4) 10-HCO-folic acidand (5) folic acid.



Figure 9. Chromatograms for the separation of five folate derivatives on HyAqS, Ace and XBr by use of 6% ACN at the start ofthe gradient programme. Different buffers were applied – 30 mM PhB pH 2.3, 30 mM PhB pH 3.5, 5 mM PhB pH 3.5 and 5 mMHAc pH 3.5. Peaks: (1) H4folate, (2) 5-CH3–H4folate, (3) 5-HCO–H4folate, (4) 10-HCO-folic acid and (5) folic acid.


It is interesting to note that the effects of pH on folateretention were quite different for classical Ace and XBrand polar-endcapped HyAqS. Ace and XBr showed a pro-nounced decrease of retention, especially for H4folate,with increasing pH from 2.3 to 3.5. The peak shape ofH4folate was also adversely affected on these columns.These effects appeared to be independent of buffer ionicstrength. As illustrated in Fig. 9, the use of PhB of muchlower ionic strength (5 mM PhB pH 3.5) instead of 30 mMPhB pH 3.5 gave very similar results. The observedchanges in folate retention on Ace and XBr might beattributable to decreased hydrophobic interactions ofthe glutamate part of the folate molecule with the alkylchains on the silica surface when increasing pH. AtpH 2.3 the glutamate part of the molecule is protonatedand neutral, which favours the hydrophobic interactionswith the alkyl chains on the silica surface. However, atpH 3.5 the glutamate part of the molecule becomes nega-tively charged due to ionisation of the a-COOH group,which leads to shielding of this part by surroundingcounter-ions and water molecules. This in turn may con-siderably suppress the hydrophobic interactions withthe alkyl chains and result in lower retention of folates.However, the decrease of retention may vary for differentfolate derivatives depending on their hydrophobic prop-erties. For instance, one would expect that the decreasein retention will be more pronounced for H4folate ratherthan 5-CH3–H4folate containing the hydrophobic –CH3

group, and such is indeed the case (see Fig. 9). In contrastto Ace and XBr, the retention of folates on HyAqS wasaffected only slightly by increasing pH (Fig. 9), whichindicated considerable differences in folate retentionmechanisms on classical and polar-endcapped columns.The ionisation of the glutamate part of the molecule atpH 3.5 seemed to have no noticeable effect on folateretention on HyAqS, suggesting that interactions ofother parts of the folate molecule (possibly pteridinering) with polar groups on the silica surface could playan important role in the retention mechanism on polar-endcapped columns.

3.6 Influence of volatile buffer onchromatographic performance

Because of growing popularity of MS as highly selectiveand sensitive detection technique in HPLC, a search forstationary phases compatible with volatile buffers of lowionic strength is gaining in importance [36, 38, 39]. Inour previous study we have tested the compatibility often stationary phases with mobile phase buffered with30 mM HAc pH 3.1. We have found that 30 mM HAc hada pronounced adverse effect on the peak shape of folateson eight stationary phases compared with 30 mM PhBpH 2.3. Only two phases (ZoC8 and ZoAq) showed minorchanges of the peak shape when using 30 mM HAc, but

their selectivity was not sufficient for baseline separationof five folate derivatives. In the present study, we havetested 5 mM HAc pH 3.5 as volatile buffer because bufferconcentrations a10 mM are most typical for LC-MS appli-cations [36, 38].

An increase in ionic interactions between analyte mole-cules and polar groups on the silica surface is commonlythe basic challenge when using buffers of low ionicstrength [36]. It is associated with reduced shielding ofpolar groups on the silica surface because of decreasedcompetition from buffer ions. The impact of these inter-actions can be widely different depending on ion charge.If analyte and polar groups on the silica surface have theopposite charges, their interactions may result in anincrease of retention followed by peak deterioration.Such effects are usually observed when separating proto-nated bases on columns containing residual silanols onthe silica surface [37]. If analyte and polar groups on thesilica surface are like-charged, their interactions mayresult in decrease or loss of retention due to ion-exclu-sion effects. Another problem related to volatile bufferssuch as HAc or formic acid is the higher pH of these buf-fers compared with conventional phosphate buffers,which may lead to partial ionisation of residual polargroups of acidic nature on the silica surface. This canlead to an increase of ionic interactions with protonatedbases and thus increased retention. Conversely, reten-tion of acidic analytes may decrease due to ionisation ofacidic groups and decrease of hydrophobic interactionsas well as possible ion-repulsive interactions with ionisedacidic groups on the silica surface. A further difficultywith volatile buffers is possible size-exclusion effects dueto poorer desolvation of analyte molecules [36]. This alsoleads to a decrease of retention and is often attendedwith peak deterioration.

For folates all these effects described above may takeplace. As discussed in Section 3.5, the hydrophobic inter-actions of the glutamate part of the folate molecule withthe alkyl chains on the silica surface may decrease whenincreasing pH. Such effects were observed on two classi-cal columns (Ace and XBr) when using 5 and 30 mM PhBpH 3.5 (Fig. 9). However, these two columns exhibitedslightly different properties when 5 mM HAc pH 3.5 wasused. On Ace the retention of all folate derivatives wasincreased in 5 mM HAc compared with 5 mM PhB of thesame pH (Fig. 9). For H4folate and 5-CH3–H4folate (predo-minantly protonated at pH 3.5) this might indicate theincrease of ionic interactions with slightly ionised resid-ual silanols when using HAc with much lower ionicstrength. As seen from the chromatogram, this was fol-lowed by deterioration of the peak shape, especially forH4folate. The increased retention for late-eluting folates(less protonated and predominantly negatively chargedat pH 3.5) might be related to the increase in hydropho-



bic interactions of the ionised glutamate part of thefolate molecule with the alkyl chains on the silica due toless shielding of the carboxyl group by cations in HAc.Similar effects for late-eluting folates were also observedon XBr, whereas early-eluting folates exhibited adecrease of retention followed by peak deterioration,which might be attributable to size-exclusion effects. Thecomparison of chromatograms for Ace and XBr in 5 mMHAc showed that size-exclusion effects might have amore pronounced adverse effect on peak shape thanslight ionic interactions.

In contrast to Ace, polar-endcapped HyAqS showed muchgreater increase of retention of H4folate and 5-CH3–H4folate followed by marked peak deterioration (Fig. 9),suggesting that ionic interactions between early-elutingfolates (positively charged) and polar groups on the silicasurface were stronger on HyAqS than on Ace when using5 mM HAc as buffer. The late-eluting folates (negativelycharged) exhibited a decrease of retention in 5 mM HAccompared to 5 mM PhB pH 3.5, which might be attributa-ble to anion-repulsive interactions. The obtained resultssuggested that HyAqS might contain polar groups ofacidic nature on the silica surface. Due to considerablepeak deterioration and coelution of 5-CH3–H4folate and5-HCO–H4folate this polar-endcapped column appearedto be less suitable for separation of folates in volatile buf-fers compared with phosphate buffer.

The changes in folate retention, when using 5 mM HAcpH 3.5 instead of 30 mM PhB pH 2.3, are presented inFig. 10 for ten columns. Polar-endcapped AqC18 showedsimilar patterns as polar-endcapped HyAqS, i. e. pro-nounced increase of retention of positively charged

early-eluting folates, but decreased retention of nega-tively charged late-eluting folates, which indicated thatAqC18 also might contain polar ionisable groups ofacidic nature on the silica surface. Analogously to HyAqS,the marked changes in retention on this column resultedin coelution of H4folate/10-HCO-folic acid and 5-HCO–H4folate/5-CH3–H4folate, together with a change of elu-tion order: H4 folate a 10-HCO-folic acid a 5-HCO–H4 folatea 5-CH3–H4folate a folic acid. Compared with HyAqS,AqC18 exhibited less peak deterioration, which showedthat this column might be compatible with volatile buf-fers, but its selectivity for folates needs to be optimised.

In contrast to HyAqS and AqC18, Atl exhibited oppositepatterns in 5 mM HAc pH 3.5 (Fig. 10): pronouncedincrease of retention of negatively charged late-elutingfolates (possible attractive ionic interactions), butdecreased retention of positively charged early-elutingfolates followed by a marked decrease of recovery (possi-ble cation-exclusion effects). These effects might be attri-butable to ionic interactions of folates with polar ionisa-ble groups of weakly basic nature on the silica surface ofAtl. Moreover, this column exhibited loss of selectivityfor 10-HCO-folic acid and 5-HCO–H4folate when using5 mM HAc. Therefore, Atl was found to be unsuitable forseparation of folates in 5 mM HAc.

SunF exhibited excessive retention of negatively chargedlate-eluting folates (Fig. 10) followed by total loss of recov-ery (>90%) and marked peak deterioration when using5 mM HAc. This column required a special prolonged gra-dient programme (after the gradient raise, 25% ACN waskept 12 min instead of 2 min) to provide reasonableretention times for late-eluting folates. Marked peak


Figure 10. A–B. Differences in retention times(DtR) obtained in 5 mM HAc pH 3.5 and 30 mMPhB pH 2.3 (DtR = tR (HAc) – tR (PhB)) for five folatederivatives by use of 6% ACN at the start of thegradient programme.


deterioration and loss of recovery (>90%) were alsoobserved for early-eluting folates, which might be asso-ciated with cation-exclusion effects. These effects weresimilar to those observed on HyPA containing embeddedamide groups [14]. They might be attributable to strongion-exchange interactions. Because of these strong ion-exchange interactions resulting in widely differenteffects for early- and late-eluting folates these two col-umns seemed to be unsuitable for both volatile and con-ventional buffers.

As seen from Fig. 10, classical ZoC8, ZoRx, ZoC18 andGold showed changes in folate retention similar to Aceand XBr (Fig. 9), discussed above. On these columns, thechanges in retention might be attributable to bothhydrophobic and slight ionic interactions. The compari-son of these columns showed great differences in peakshape for early-eluting folates in 5 mM HAc. Gold andZoC18 showed marked peak deterioration for H4 folate(both As and w0.5), whereas ZoC8 and ZoRx exhibited lesspeak deterioration (increase of As and w0.5 less than 20%).In terms of peak shape, classical Ace, ZoC8 and ZoRxappeared to be more suitable for volatile buffers than theother columns tested. However, further optimisation ofthe chromatographic conditions for these columns isnecessary to minimise the adverse effects of volatile buf-fers and to improve selectivity.

3.7 Applicability of general columncharacterisation tests for selection of columnsfor folate analysis under highly aqueousconditions

The selection of a suitable column to separate com-pounds of interest is an important step in method devel-opment. The possibility to predict the chromatographic

performance of different columns on the basis of columncharacterisation tests is therefore of great value. In thepresent work, we studied relationships between reten-tion times of folates and major columns characteristicsfor two groups of columns: (1) 11 alkyl-bonded columnsfrom different manufacturers (ZoC8, ZoC18, ZoRx, ZoAq,HyPA, HyAqS, SyC12, GeC18, Ace, Atl and AqC18) and (2)four alkyl-bonded columns from the same manufacturer(ZoC8, ZoC18, ZoRx and ZoAq). The general characteris-tics such as hydrophobic selectivity (aCH2 ), shape selectiv-ity (a(T/O)), hydrogen bonding capacity (a(C/P)), total cation-exchange capacity (aB/P pH 7.6) and acidic cation-exchangecapacity (aB/P pH 2.7), all obtained by Euerby and Petersson[5, 11] according to the Tanaka protocol [23] were usedfor this comparison. The correlation coefficients and theslopes of linear regression lines are presented in Table 3.When comparing columns from different manufac-turers, no correlations were found between folate reten-tion and column general characteristics. Conversely, forcolumns from the same manufacturer (four Zorbax col-umns) a strong correlation was found between folateretention and two column characteristics: hydrophobicselectivity and hydrogen bonding capacity. The retentiontimes of five folate derivatives appeared to be directlyproportional to the hydrogen bonding capacity(r2 = 0.88–0.97) and inversely proportional to the hydro-phobic selectivity (r2 = 0.91–0.95). The general columncharacteristics can therefore be useful when comparingcolumns from the same manufacturer.

3.8 Particle size and chromatographicperformance

The use of columns with smaller silica particles is com-monly recommended as a simple way to improve peak


Table 3. Values of r2 and slope for correlation between retention times of folates and characterisation parameters for differentstationary phases

Folate form tRa) = f (aCH2 ) tR = f (aT/O) tR = f (aC/P) tR = f (aB/P pH 7.6) tR = f (aB/P pH 2.7)

r2 (a)b) r2 (a) r2 (a) r2 (a) r2 (a)

n = 11c) n = 4 n = 11 n = 4 n = 11 n = 4 n = 11 n = 4 n = 11 n = 4

H4folate 0.28(13.0)

0.94( – 22.9)

3610 – 3

(0.5)0.58( – 8.9)

0.08(1.5)

0.88(2.8)

0.11(0.5)

0.30(3.6)

0.01(20.4)

0.55(212)

5-CH3-H4folate 0.39(15.9)

0.93( – 18.6)

2610 – 3

(0.4)0.49( – 6.8)

0.07(1.5)

0.93(2.4)

0.09(1.5)

0.26(2.8)

6610 – 3

(14.1)0.49(165)

5-HCO–H4folate 0.06( – 2.4)

0.95( – 23.8)

0.01(0.5)

0.46( – 8.2)

0.17(1.2)

0.95(3.0)

0.20(1.1)

0.17(2.9)

0.14(35.3)

0.38(185)

10-HCO-folic acid 6610 – 5

(0.1)0.95( – 25.1)

2610 – 5

(0.02)0.46( – 8.7)

0.24(1.5)

0.95(3.2)

0.14(1.0)

0.19(3.1)

0.07(27.8)

0.4(199)

Folic acid 0.09( – 3.8)

0.91( – 21.8)

0.04(0.8)

0.40( – 7.2)

0.13(1.3)

0.97(2.9)

0.24(1.2)

0.19(2.8)

0.18(40.0)

0.39(175)

a) tR = ax + b, where x = aCH2 , aT/O, aC/P, aB/P pH 7.6 and aB/P pH 2.7 which were obtained by using Tanaka protocol [23]; (see Section 2.6for details).

b) r2 is correlation coefficient estimated by using linear regression; (a) is the slope of the line given in brackets.c) n – number of stationary phases.n = 11 (stationary phases: ZoC8, ZoC18, ZoRx, ZoAq, HyPA, HyAqS, SyC12, GeC18, Ace, Atl, AqC18); n = 4 (stationary phases:ZoC8, ZoC18, ZoRx, ZoAq).


resolution with only minor changes in column selectiv-ity. In the present work, the chromatographic param-eters (tR, Rs and N) obtained for five folate derivatives,when using mobile phase buffered with 30 mM PhBpH 2.3 and ACN gradient with start at 6%, were com-pared for two ZoC8 columns with particle size of 3.5 and5 lm. The use of smaller particles resulted in only slight(7–10%) improvement of peak efficiency for H4folate and10-HCO-folic acid, whereas peak efficiency for 5-CH3–H4folate, 5-HCO–H4folate and folic acid was decreased.The use of smaller particles led also to a marked changeof selectivity: Rs for separation of 10-HCO-folic acid and 5-HCO–H4folate increased from 0.75 to 0.95, whereas Rs forseparation of 5-HCO–H4folate and folic acid considerablydecreased from 0.84 to 0.16, which indicated that parti-cle size might influence the bonding procedure and con-sequently the surface chemistry. This marked change inselectivity when using smaller particles showed thatreplacing the column with standard particle size such as5 lm by a column with smaller particles may lead tounexpected changes in selectivity and should be donewith caution.

4 Concluding remarksApplicability of several alkyl-bonded silica stationaryphases (both classical and alternative) was tested for gra-dient RP-HPLC of folates with conventional and volatilebuffers under highly aqueous conditions. It was foundthat folates exhibited a mixed retention mechanism onsilica phases including both hydrophobic and secondarypolar interactions. These interactions were influenced bymobile phase composition, pH, buffer nature and ionicstrength. The wettability of the silica surface as well aspore accessibility were also found to be important forfolate retention. High retention of folates was achievedon alternative phases with enhanced polarity (AqC18,ZoAq, HyAqS and Atl) and classical phases with highercarbon content (GeC18, ZoC8, SyC12 and Ace).

The nature of polar groups used for endcapping orembedding in stationary phases appeared to markedlyinfluence the retention mechanisms of folates. Phasesexhibiting slight polar secondary interactions werefound to provide better selectivity for late-eluting folates,especially for 10-HCO-folic acid and 5-HCO-H4folate,whereas selectivity for early-eluting H4folate and 5-CH3–H4folate was mostly dependent on hydrophobic interac-tions. Best selectivity in phosphate buffered mobilephase was achieved on polar-endcapped silica phases(AqC18 and HyAqS) followed by alternative Atl, compati-ble with 100% water. Classical phases exhibited poorerseparation of 10-HCO-folic acid and 5-HCO–H4folate, butit could be considerably improved by increasing the pHof buffer.

Strong secondary interactions of ion-exchange characteron polar-embedded phases resulted in marked peak dete-rioration, loss of recovery and dramatic changes in reten-tion behaviour for early- and late-eluting folates whenchanging the mobile phase composition and pH. There-fore, polar-embedded phases such as amide-linked HyPAwere found to be unsuitable for separation of folates. Itshould be pointed out that some classical alkyl-bondedphases with unspecified bonding and endcapping (e. g.SunF) may exhibit properties more similar to those ofalternative phases with enhanced polarity than conven-tional phases.

All ten stationary phases exhibited peak deteriorationwhen using an LC-MS compatible volatile buffer of lowionic strength. Better results were obtained with classicalphases Ace, ZoC8 and ZoRx, whereas alternative phasesshowed not only peak deterioration but also a decreasein recovery and poorer selectivity due to increased sec-ondary interactions when using 5 mM HAc.

We thank Professor Margaretha J�gerstad for her encouragementand interest in this study and Dr. Vladimir Nikiforov (St. Peters-burg State University, Russia) for valuable discussions on themanuscript. Anders Grahn (ChromTech, Sweden) and Tony Wik-lund (Waters, Sweden) are gratefully acknowledged for helpfulsupport during this work. We are also grateful to ChromTech andWaters for kindly providing us with columns. This study was sup-ported by the Swedish Agency for Innovation Systems (Vinnova).The financial sponsorship of Dr. Svetlana Lanina by the SwedishInstitute is also gratefully acknowledged. The folate standardswere a kind gift from Merck Eprova AG.

5 References[1] Kowalska, S., Krupczynska, K., Buszewski, B., J. Sep. Sci.

2005, 28, 1502–1511.

[2] Buszewski, B., Kowalska, S., Krupczynska, K., Crit. Rev.Anal. Chem. 2005, 35, 89 –116.

[3] Layne, J., J. Chromatogr. A 2002, 957, 149 –164.

[4] Wilson, N. S., Gilroy, J., Dolan, J. W., Snyder, L. R., J. Chro-matogr. A 2004, 1026, 91 –100.

[5] Euerby, M. R., Petersson, P., J. Chromatogr. A 2005, 1088,1–15.

[6] Vervoort, R. J. M., Debets, A. J. J., Claessens, H. A., Cra-mers, C. A., de Jong, G. J., J. Chromatogr. A 2000, 897, 1 –22.

[7] Claessens, H. A., van Straten, M. A., Cramers, C. A.,Jezierska, M., Buszewski, B., J. Chromatogr. A 1998, 826,135 –156.

[8] Snyder, L. R., Dolan, J. W., Carr, P. W., J. Chromatogr. A2004, 1060, 77–116.

[9] Barrett, D. A., Brown, V. A., Davies, M. C., Shaw, P. N.,Anal. Chem. 1996, 68, 2170 –2178.

[10] Gilroy, J. J., Dolan, J. W., Snyder, L. R., J. Chromatogr. A2003, 1000, 757 –778.



[11] Euerby, M., Petersson, P., J. Chromatogr. A 2003, 994, 13 –36.

[12] Vervoort, R. J. M., Derksen, M. W. J., Debets, A. J. J., J. Chro-matogr. A 1997, 765, 157 –168.

[13] Vervoort, R. J. M., Ruyter, E., Debets, A. J. J., Claessens, H.A. et al., J. Chromatogr. A 2001, 931, 67–79.

[14] Johansson, M., Jastrebova, J., Grahn, A., Jagerstad, M.,Chromatographia 2005, 62, 33 –40.

[15] Eitenmiller, R. R., Landen, W. O., in: Eitenmiller, R. R.,Landen, W. O. (Eds.), Vitamin Analysis for the Health andFood Sciences, CRC Press, Boca Raton, Florida 1999, pp.411 –466.

[16] Scott, J., R�beille, F., Fletcher, J., J. Sci. Food Agric. 2000,80, 795 –824.

[17] Berry, R. J., Li, Z., Erickson, D., Li, S. et al., New Engl. J. Med.1999, 341, 1485–1490.

[18] Lucock, M., Mol. Genet. Metab. 2000, 71, 121 –138.

[19] van den Berg, H., Finglas, P. M., Bates, C., Int. J. Vitam.Nutr. Res. 1994, 64, 288 –293.

[20] Baggott, J. E., Johanning, G. L., J. Nutr. 1999, 129, 1315 –1318.

[21] Patring, J. D. M., Johansson, M., Yazynina, E., Jastrebova,J., Anal. Chim. Acta 2005, 553, 36 –42.

[22] Nilsson, C., Johansson, M., Yazynina, E., Stralsjo, L., Jas-trebova, J., Eur. Food Res. Technol. 2004, 219, 199 –204.

[23] Kimata, K., Iwaguchi, K., Onishi, S., Jinno, K. et al., J. Chro-matogr. Sci. 1989, 27, 721 –728.

[24] Vahteristo, L., Finglas, P. M., in: De Leenheer, A. P., Lam-bert, W. E., Van Bocxlaer, J. F. (Eds.), Modern Chromato-graphic Analysis of Vitamins, Marcel Dekker, New York2000, pp. 301 –323.

[25] Bagley, P. J., Selhub, J., Clin. Chem. 2000, 46, 404 –411.

[26] Konings, E. J. M., J. AOAC Int. 1999, 82, 119 –125.

[27] Kallen, R. G., Jencks, W. P., J. Biol. Chem. 1966, 241, 5845 –5850.

[28] Robinson, D. R., Jenks, W. P., J. Am. Chem. 1967, 89,7088–7097.

[29] Poe, M., J. Biol. Chem. 1977, 255, 3724–3728.

[30] Polhand, A., Flynn, E. H., Jones, R. G., Shive, W., J. Am.Chem. Soc. 1951, 73, 3247 –3252.

[31] The Merck Index of Chemical and Drugs, Merck, Rahway,New Jersey 1989.

[32] Pfleiderer, W., Comprehensive Heterocyclic Chemistry – TheStructure, Reactions, Synthesis and Uses of Heterocyclic Com-pounds, Pergamon Press, New York 1984.

[33] Horne, D. W., Biochim. Biophys. Acta 1990, 1023, 47–55.

[34] Blakley, R. L., The Biochemistry of Folic Acid and Related Pteri-dines, North Holland Publication, Amsterdam 1969.

[35] Gritti, F., Guiochon, G., J. Chromatogr. A 2005, 1099, 1 –42.

[36] McCalley, D. V., J. Chromatogr. A 2003, 987, 17 –28.

[37] Nawrocki, J., J. Chromatogr. A 1997, 779, 29 –71.

[38] Vervoort, R. J. M., Debets, A. J. J., Lamers, R. J., Claessens,H. A. et al., J. Pharm. Biomed. Anal. 1999, 21, 273 –289.

[39] Needham, S. R., Brown, P. R., Duff, K., Bell, D., J. Chroma-togr. A 2000, 869, 159 –170.


Applicability of alkyl-bonded ultra-pure silica stationary phases for gradient reversed-phase HPLC...

Documents

Transcript of Applicability of alkyl-bonded ultra-pure silica stationary phases for gradient reversed-phase HPLC...