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Journal of Chromatography A, 1187 (2008) 87–93

Hydrophilic interaction chromatography of nucleotidesand their pathway intermediates on titania

Ting Zhou, Charles A. Lucy ∗Department of Chemistry, University of Alberta, Gunning/Lemieux Chemistry Centre, Edmonton, Alberta T6G 2G2, Canada

Received 23 November 2007; received in revised form 26 January 2008; accepted 4 February 2008Available online 13 February 2008

bstract

Nucleotides and their pathway intermediates play important roles in all living species. They are essential cellular components in energy transfer,etabolic regulatory processes and biosynthesis. Titania (TiO2) has strong Lewis acid sites which have an affinity for the strongly electronegative

hosphonate group of nucleotides. Herein a bare titania column (150 mm × 4.6 mm I.D., 3 �m) with UV detection at 254 nm was used for theeparation of a set of nucleotides (AMP, ADP, ATP, UMP, UDP, UTP, GMP, GDP, GTP, CMP and CTP) and their intermediates (NAD, NADH,DP-Glu and UDP-GluNAc). Addition of phosphate to the eluent suppresses the ligand-exchange interactions with the titania surface such

hat hydrophilic interaction chromatography (HILIC) separations may be performed. Increasing the %ACN resulted in increasing retention and

fficiency (up to 13,000, 9500 and 4500 plates/m for AMP, ADP and ATP, respectively). The effects of pH, buffer concentration and other eluentnions (fluoride and acetate) were also studied. Fifteen nucleotides and their intermediates were separated in 26 min (Rminimum > 1.3) using anne-step gradient. 2008 Elsevier B.V. All rights reserved.

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eywords: Titania; Nucleotides; Hydrophilic interaction chromatography; Liga

. Introduction

Titania is attracting increasing interest as a chromatographicacking material. Titania has greater mechanical and pH stabilitypH 1–14) than silica [1,2]. It acts as an anion exchanger at lowH and a cation exchanger at high pH [3,4]. More importantly,imilar to zirconia [5], the unsaturated titania ions (IV) are strongewis acid sites which have an affinity for compounds donatinglectron pairs [5,6]. In contrast, silica only behaves as a cationxchanger and does not have any ligand-exchange properties.

The ligand-exchange behavior of titania may be treated sim-larly to transition metal coordination complexes:

i(OH)(H2O) + L− � Ti(H2O)L + OH− (1)

quilibrium (1) is particularly favored when the analyte lig-

nd L− is a Lewis base, especially fluoride and polyoxy anionsuch as borate, carboxylate and sulfate. In addition to the Lewisases mentioned above, the strongly electronegative phospho-

∗ Corresponding author. Tel.: +1 780 492 0315; fax: +1 780 492 8231.E-mail address: charles.lucy@ualberta.ca (C.A. Lucy).

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021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.02.027

change

ate group of organo-phosphates such as nucleotides also has atrong affinity for TiO2 [7].

Similar to zirconia [8], titania has been used extensivelyo trap organophosphates [9–13]. For instance, Ikeguchi et al.esigned a highly selective organophosphate analyzer by trap-ing phosphor–amino acids on a titania precolumn, followed byn anion exchange separation and postcolumn reaction detection10]. Pinkse et al. preconcentrated and isolated phosphopeptidesrom proteolytic digests using a titania preconcentration col-mn followed by two-dimensional (2D)-nanoLC–electrosprayonization (ESI)–MS/MS [12]. Hata et al. designed a 2D-LCystem for high-throughput phosphoproteome analysis using aitania column as the first dimension to trap organophosphatesnd a monolith silica column as the second separation dimension9].

In contrast to this trapping work, Miyazaki et al. recently useditania as a separation media for four nucleotides [7]. Moderatelyfficient (∼2600 plates/m) separations were achieved using an

luent containing 50 mM phosphate buffer in 60% acetonitrile.hile this mobile phase was effective, it was not clear why these

articular conditions were appropriate. In this paper we system-tically investigate the effect of the mobile phase composition

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8 T. Zhou, C.A. Lucy / J. Chr

n the retention and efficiency of the model organophosphonatenalytes AMP, ADP, ATP on titania. We then use our under-tanding of the retention mechanism to separate 15 nucleotidesnd their intermediates on a bare titania column with a minimumesolution of 1.3.

. Experimental

.1. Reagents

All solutions were prepared in nano-pure water (Barnstead,uburque, IA, USA). Sodium fluoride and HPLC–grade ace-

onitrile (ACN) were purchased from Fisher Scientific (Fairawn, NJ, USA). Sodium phosphate monobasic, sodium phos-hate dibasic, sodium acetate and acetic acid were from EMcience (Gibbstown, NJ, USA). Eleven nucleotides, adenosine′-monophosphate (AMP), adenosine 5′-diphosphate (ADP),denosine 5′-triphosphate (ATP), uridine 5′-monophosphateUMP), uridine 5′-diphosphate (UDP), uridine 5′-triphosphateUTP), guanosine 5′-monophosphate (GMP), guanosine 5′-iphosphate (GDP), guanosine 5′-triphosphate (GTP), citidine′-monophosphate (CMP), citidine 5′-triphosphate (CTP), andour pathway intermediates, nicotinamide adenine dinuclotideNAD), nicotinamide adenine dinucleotide reduced disodiumalt (NADH), uridine 5′-diphospho-d-glucose (UDP-Glu)isodium salt, uridine 5′-diphospho-N-acetyl-d-glucosamineUDP-GluNAc) were purchased from Sigma (St.Louis, MO,SA). All solutions were filtered through 0.22 �m Magna nylonembrane filters (GE Osmonic, Trevose, PA, USA) prior to use.he phosphate buffer was made from a concentrated sodiumhosphate solution. The buffer concentrations quoted in thisaper are that present after acetonitrile addition.

.2. Chromatographic conditions

Separations were performed on a Metrohm chromatogra-hy system (Metrohm, Herisau, Switzerland) consisting of

model 709 dual-piston pump operating at 1.0 mL/min.njections were made with a 6-port Cheminert CCP0140njection valve with a 20 �L loop (Valco Instruments, Hous-

on, TX, USA). A Lambda-Max Model 481 UV detectort 254 nm (Waters, Milford, MA, USA) was used. Dataas collected at 30 Hz using a Metrohm 762 data acquisi-

ion system with IC Net 2.1 software. A bare TiO2 column

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ig. 1. Effect of %ACN on (A) retention and (B) efficiency. Conditions: flow rate, 1.0CN; analyte, 0.1 mM AMP, ADP and ATP in the same %ACN as the eluent; UV de

gr. A 1187 (2008) 87–93

150 mm × 4.6 mm I.D., 3 �m) (Zirchrom, Anoka, MN, USA)as used. Sodium phosphate buffers with various percent-

ges of acetonitrile were used as the mobile phase. EluentH was adjusted using a Corning combination three-in-onelectrode (Corning, Big Flats, NY, USA) before adding ace-onitrile.

. Results

.1. %ACN

Fig. 1 shows the effect of %ACN on separations performedith eluents containing pH 7.0 sodium phosphate. The buffer

oncentration used in this paper was adjusted so that the finaloncentration of phosphate was 10 mM after the addition ofCN. The solubility of phosphate in ACN limited studies to0% ACN. Samples were prepared in the same %ACN as theobile phase for reasons discussed in Section 3.2. Under all

onditions, AMP was the least retained analyte and ATP washe most retained. As shown in Fig. 1A the retention of AMP,DP and ATP decreased as the %ACN was increased from 0

o 50%. Above 50% ACN retention increased with increasingACN. Fig. 1B shows that the efficiency gradually increasedith increased %ACN from 0 to 50%, and then dramatically

ncreased as the %ACN was increased above 50%. ATP showshe most dramatic change in retention and efficiency whenncreasing %ACN. For instance, between 65 and 70% ACN, thefficiency increased 350% and retention increased over 100%.ith 70% ACN and 10 mM phosphate (pH 7), efficiencies

re substantially higher than the ∼2600 plates/m observed byiyazaki et al. who used 50 mM phosphate buffer in 60% ACN

7]. Thus in all further studies %ACN higher than 50% wassed.

.2. %ACN in sample

Ideally samples in liquid chromatography should be dis-olved in the same solvent as the eluent. Differences in viscosityetween the sample and eluent can lead to peak distortion14,15], while differences in the solvent strength can lead to

and broadening [14,15]. The strong injection solvent preventshe analyte from absorbing at the head of the column. As the sol-ent bolus is diluted by the mobile phase, the analyte retentionradually approaches that characteristic of the mobile phase.

mL/min; eluent, 10 mM H2PO4−/HPO4

2− buffer (pH 7.0) in different % (v/v)tection at 254 nm.

T. Zhou, C.A. Lucy / J. Chromatogr. A 1187 (2008) 87–93 89

Fig. 2. Effect of %ACN in sample eluent on peak shape. Conditions: flow rate,1 − 2−A2

Ho

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Fig. 4. Effect of phosphate concentration on retention. Conditions: flow rate,1Ha

3

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Fa

.0 mL/min; eluent, 10 mM H2PO4 /HPO4 buffer (pH 7.0) in 70% (v/v)CN; analyte, 0.1 mM AMP and ADP in different %ACN; UV detection at54 nm.

owever, as the rate of dilution is finite, additional broadeningccurs.

The situation herein is more complex due to the bimodalluent behavior evident in Fig. 1A. Pure water is a weak elu-nt, while 50% ACN is a strong eluent, and higher %ACN aregain weak eluents. Further, the dramatically higher efficienciest high %ACN (Fig. 1B) make it highly desirable to performeparations under such conditions.

To investigate the effect of the analyte solvent, we prepared.1 mM AMP and ADP standards in 0, 30, 50 and 70% ACN.ig. 2 shows chromatograms for these solutions separated withmobile phase of 10 mM phosphate buffer (pH 7.0) in 70%CN. ADP is slightly more retained when injected in water,hich is consistent with the strong retention observed for water

n Fig. 1A. However, overall only minor changes in retentionere observed as the sample solvent was varied. Much moreramatic changes in efficiency with the sample solvent are evi-ent in Fig. 2. Peaks are very broad for both AMP and ADPissolved in water (N of 1700 and 1500 plates/m, respectively).he peaks sharpen dramatically as the %ACN in the sample

s increased, such that AMP and ADP dissolved in 70% ACN

xhibit efficiencies of 13,000 and 9500 plates/m, respectively.o avoid artifacts due to injection, all further studies wereonducted with samples dissolved in the same %ACN as theluent.

rpTp

ig. 3. Effect of pH of phosphate buffer on (A) retention and (B) efficiency. Conditionnalyte, 0.1 mM AMP, ADP and ATP in 55%ACN; UV detection at 254 nm.

.0 mL/min; eluent, H2PO4−/HPO4

2− buffer (pH 6.0) in 75% (v/v) ACN,

2PO4−/HPO4

2− buffer (pH 7.0) in 70% (v/v) ACN; analyte, 0.1 mM AMPnd Br− in 70% or 75% ACN; UV detection at 254 nm.

.3. Eluent pH

Eluent pH plays an important role in the retention ofrganophosphates. Fig. 3 shows the effect of eluent pH onhe retention and efficiency of AMP, ADP and ATP. Asn Section 3.1, higher %ACN could not be used due tohe limited solubility of phosphate. Increasing the eluentH from 6 to 8 decreased retention and increased the effi-iency. As in Section 3.1, ATP experienced the most dramatichanges: a 3-fold decrease in retention and 19-fold increasen efficiency from pH 6 to 8. The retention of AMP onlyecreased 0.5-fold, but there was still a significant (6.5-fold)ncrease in efficiency from pH 6 to 8. For eluent pH > 8, thehanges in retention and efficiency for AMP, ADP and ATPere modest. This behavior is consistent with the observa-

ions Carr and co-workers [16,17] who found that the ligandxchange adsorption of fluoride on zirconia decreased as pHncreased.

.4. Eluent anion

Fig. 4 shows the effect of phosphate concentrations on AMPetention with 75% ACN (pH 6) and 70% ACN (pH 7) as elu-nts. 0.1 mM AMP and bromide were used as the analytes. The

etention of AMP using pH 7 buffer was lower than that usingH 6 buffer, consistent with our observations in Section 3.3.he retention of AMP decreased dramatically with increasinghosphate concentration, and became constant for phosphate

s: flow rate, 1.0 mL/min; eluent, 10 mM H2PO4−/HPO4

2− in 55% (v/v) ACN;

90 T. Zhou, C.A. Lucy / J. Chromato

Table 1Retention of AMP on titania using different anions as the eluent

Eluent anion Concentration (mM) Retention factor of AMP

Phosphate 10 0.76Fluoride 100 >14Acetate 100 >14Hydroxidea 100 >14

Carbonatea 10 >1450 8.9

100 2.3

Conditions: flow rate, 1.0 mL/min; eluent, 10 mM or 100 mM eluent solution(a

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bporobcbaat pH 6. Similarly, Table 2 shows a slight increase in bromideretention as the %ACN increases.

With regard to adsorption via a ligand exchange interaction,Carr and co-workers prepared an eluotropic series for zirco-

Table 2Retention change when changing %ACN

Analyte anions k

0% ACN 50% ACN 70% ACN

Bromide 0.01 0.02 0.03AMP 0.88 0.10 0.76

pH 7, except carbonate (pH 11) and hydroxide (pH 13)) in 70% (v/v) ACN;nalyte, 0.1 mM AMP in the same %ACN as the eluent; UV detection at 254 nm.a 55% ACN was added to the mobile phase because of the limited solubility.

oncentrations greater than 0.5 (pH 7) or 2 mM (pH 6). A num-er of other eluent anions were tested. As shown in Table 1 onlyigh concentrations of carbonate could elute AMP.

The interpretation of these results will be discussed below.

. Discussion: mechanism of retention

.1. Models of retention

In their excellent summary and re-evaluation of theydrophilic interaction chromatography (HILIC) literature,emstrom and Irgum demonstrated that great insight can be

chieved by examining retention behavior using simple modelsor retention [18]. For partitioning the linear solvent strengthodel was used [19]:

og k = log kW − Sϕ (2)

here k and kw are the retention factor in the eluent and pureater, respectively, ϕ is the volume fraction of the strong eluent

nd S is the empirical slope derived from a plot of log k versus ϕ.lots based on Eq. (2) are rectilinear over short ranges of eluentonditions in reversed phase chromatogram [19]. Eq. (2) is alsohe basis of the RPLC optimization software DryLab [19,20].

For adsorption chromatography, retention can be expressedy [21,22]:

og k = log kB − AS

nBlog NB (3)

here kB is the solute retention factor with pure B as eluent,S and nB are the cross-sectional areas occupied by the solutend solvent B on the surface, respectively, and NB is the moleraction of the stronger eluent (B) of the eluent.

In addition to the retention models considered by Hemstromnd Irgum, retention on titania may also be due to ion exchange.he expression for retention in ion exchange chromatography

23] is:

x y−

og kA = const −y

log[E ] (4)

here x and y are the charges of the analyte (A) and eluent (E)espectively. The constant is dependent on the column charac-eristics and the charge on the eluent and analyte.

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gr. A 1187 (2008) 87–93

.2. Adsorption behavior

The retention behavior of the organophosphates on titaniaxhibits two regimes (Figs. 1 and 4), each of which will beiscussed separately. In Fig. 4 there was a rapid decrease inMP retention as the phosphate concentration was increased

rom 0 to 2 mM for pH 6 buffer and from 0 to 0.5 mM for pHbuffer. Replotting this data (plots not shown) as a log k versus

phosphate] (Eq. (2), R2 = 0.75 and 0.86 for pH 6 and 7, respec-ively) and log k versus log [phosphate] (Eq. (3), R2 = 0.92 and.987 for pH 6 and 7, respectively) indicates that the retentionehavior in the presence of low concentrations of phosphates better described by the latter mathematical form. Howeveroth adsorption (Eq. (3)) and ion exchange (Eq. (4)) follow thiseneral relationship.

Both ligand-exchange [5,6] and ion exchange [3,4] have beeneported on titania. Titania has an isoelectric point of 5.0 [24,25].hus it can act as an anion exchanger at low pH or a cationxchanger at high pH [3,4]. The studies above were all performednder neutral to alkaline conditions. The high percentage ofCN used makes it difficult to state whether conditions are abover below the pI of titania. To determine whether ion exchange isperative, the retention of bromide was monitored. Bromide washosen as it is both UV absorbing and has been used in previoustudies of anion exchange [4,5] on TiO2 under low pH aqueousonditions. Also, bromide is very low on the eluotropic seriesor zirconia [26], which means that there is almost no ligand-xchange between bromide and zirconia. The retention behaviorf titania has been shown to be similar to that of zirconia [5,26].hus the only possible interaction which will affect the retentionf bromide on titania is ion exchange.

As shown in Fig. 4, negligible retention was observed forromide in the presence of 70 and 75% ACN, even when nohosphate was present in the eluent. Similarly, in the presencef 10 mM phosphate (pH 7), bromide was essentially unretainedegardless of whether the eluent was pure aqueous or stronglyrganic (Table 2). This is consistent with the lack of retention ofromide on titania observed by Tani et al. under pure aqueousonditions within this pH range [4,5]. The retention factor ofromide increased slightly from −0.17 (ion exclusion) to 0.04s the phosphate concentration was increased from 1 to 10 mM

DP 13.37 0.44 2.77TP >14 1.99 10.68

onditions: flow rate, 1.0 mL/min; eluent, 10 mM H2PO4−/HPO4

2− buffer (pH.0) in different % (v/v) ACN; analyte, 0.1 mM bromide, AMP, ADP and ATPn the same %ACN as the eluent; UV detection at 254 nm.

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T. Zhou, C.A. Lucy / J.

ia to rate the relative strength of various eluents and observedcetate � fluoride < phosphate [26]. Titania behaves through aimilar ligand-exchange mechanism as zirconia [5]. The reten-ion of AMP in the presence of various eluents is shown inable 1. Phosphate is the strongest ligand-exchanger in the elu-tropic series [26] and herein acts as the strongest eluent onitania. Fluoride is just below phosphate in the eluotropic series,nd yet even 100 mM F− failed to elute AMP. This is consistentith Carr and co-workers’ observation that for extremely well

etained analytes such as organophosphates a phosphate buffers a decidedly stronger eluent than fluoride [26]. Acetate didot elute AMP under any conditions, consistent with its weakigand-exchange character [26].

Carbonate and hydroxide were not included in the eluotropiceries [26], but were still studied here due to their compatibilityith suppressed conductivity detection. Neither proved to be

trong eluents, although 100 mM carbonate did elute AMP.Finally the low efficiencies observed under adsorptive con-

itions (less than 2000 plates/m when using low %ACN (Fig. 1)nd for eluents containing low concentrations of phosphatedata not shown)) are consistent with literature ligand-exchangeehavior [27–29]. Carr et al. observed low efficiencies forigand-exchange retention of proteins (Lewis base) on zirco-ia. Addition of the strong Lewis bases fluoride and phosphateo the eluent effectively blocked the ligand-exchange retentionramatically improving efficiencies.

To conclude, the retention behavior on titania at low con-entrations of phosphate is indicative of adsorption, and theehavior of various eluents and analytes is consistent withigand-exchange retention.

.3. HILIC behavior

The retention behavior from 50 to 70% ACN in Fig. 1 wasxamined with respect to the mathematical models for parti-ioning (Eq. (2)) and adsorption (Eq. (3)). The plots (not shown)ere more linear for the partitioning model (R2 = 0.996, 0.97,

nd 0.987) than for the adsorption model (R2 = 0.991, 0.96, and.977) for AMP, ADP and ATP, respectively.

Alpert [30] defined the term hydrophilic interaction chro-atography (HILIC) as a separation in which: (a) water is the

trong eluent: and (b) the retention mechanism is partitioning. Ashown in Fig. 1, water does act as the strong eluent in the rangef 50–70% ACN, and as demonstrated in the last paragraph theetention mechanism is best described by Eq. (2) for partitioning.hus in the presence of high concentrations of phosphate andCN, the separation of organophosphates on titania is governedy a HILIC mechanism.

In general HILIC theory, there is a change in the retentionode as the % organic modifier is changed: RPLC at low %

rganic modifier, and HILIC at higher % modifier. Consequently,plot of k versus %modifier yields a U-shaped curve. If the selec-

ivity of the two modes differs, the elution order will change, as

as been observed in HILIC on silica columns [30,31]. Fig. 1hows no elution order switching for AMP, ADP and ATP whenhanging the %ACN. This means that the selectivity does nothange in the two retention modes (ligand exchange at low

Hht(

atogr. A 1187 (2008) 87–93 91

ACN and HILIC at high %ACN). In the ligand-exchangeode, ATP with a tri-phosphate group binds titania stronger thanDP or AMP. In the HILIC mode, ATP with more hydrophilicity

lutes later than AMP and ADP.

. Application: separation of nucleotides and theirntermediates on titania

Nucleotides and their pathway intermediates play importantoles in all living species. They are essential components inNA and RNA and are involved in processes such as energy

ransfer, metabolic processes and biosynthesis [32]. The level ofucleotides and their intermediates have regulatory potentials33] and can be used to evaluate the degree of DNA and RNAynthesis [34,35].

A multitude of chromatographic and electrophoretic methodsave been reported to detect and separate nucleotides and theirntermediates such as ion exchange chromatography [36–39],eversed phase liquid chromatography [40–44] and capillarylectrophoresis [45–48]. However, nucleotides and their inter-ediates often coelute when using ion chromatography [36–38].lternately two or more detectors are needed to resolve the

o-elution problems. For example, Vogt et al. used a conduc-ivity detector together with an in-line UV detector to resolveucleotides and their metabolites [36]. Similarly, Ritter et al.sed dual wavelength UV detection (220 and 260 nm) with con-uctivity, but still could not resolve UMP and UDP-Glu [37].ther concerns with ion exchange separations include long anal-sis times (>50 min) [27–29] and poor long-term stability dueo the use of eluent with low or high pH [42].

Direct reversed phase chromatography cannot separate polarugar nucleotides and highly charged nucleotides [40]. Ion-pairhromatography is more effective at separating nucleotides [49]nd their intermediates [50–56]. However, one problem withon-pair chromatography is that the reversed phase column isommonly a silica-based column, which cannot stand high elu-nt pH. The pH values used in literatures are no higher than 7.249–56]. Thus this limits the separation of compounds within aarrow range of pKa.

As mentioned before, titania is stable up to pH 14. Also, itas strong Lewis acid sites which have strong affinity for thehosphonate groups of nucleotides. Thus titania columns showromise for the separation of nucleotides. However, there havenly been a few examples of direct separation of phosphate-ucleotides using titania [7,57]. Kimura et al. quantified ADPnd ATP to measure the MDR1 ATPase activity on a bare titaniaolumn using 50 mM phosphate buffer (pH 7) in 50% acetoni-rile as the eluent [57]. Miyazaki et al. separated adenosine

ono-, di- and tri-phosphate on a titania-coated monolithic sil-ca column using 50 mM phosphate buffer in high percentage ofcetonitrile (>50%) as the eluent [7].

Based on mobile phase effects above, the separation ofucleotides and their intermediates was performed on TiO2.

igh %ACN is used to maintain high efficiency (Fig. 1B). Also,igh pH and phosphate concentrations are used to minimizehe separation time (Fig. 3A and 4) and maximize efficiencyFig. 3B). As shown in the inset of Fig. 5, the eight early-eluted

92 T. Zhou, C.A. Lucy / J. Chromato

Fig. 5. Separation of nucleotides under different conditions. Conditions: flowrate, 1.0 mL/min; analyte, 0.1 mM nucleotide mixture in 75%ACN; eluent(A) 10 mM H2PO4

−/HPO42− buffer (pH 6.0) in 75% ACN for 6 min, then

1 − 2−HH

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0 mM H2PO4 /HPO4 buffer (pH 8.0) in 65% ACN and (B) 10 mM

2PO4−/HPO4

2− buffer (pH 6.0) in 75% ACN for 6 min, then 20 mM

2PO4−/HPO4

2− buffer (pH 8.0) in 60% ACN; UV detection at 254 nm.

eaks are well separated using 10 mM phosphate buffer (pH.0) in 75% ACN as the eluent. Higher buffer pH or concen-ration resulted in poorer resolution of these early eluting peaksRs min < 0.1 for pH 7 phosphate and 20 mM phosphate). The elu-ion order for the nucleotides is adenosine, uridine, guanosinend cytidine, which is distinctly different from that observedsing ion-exchange column (cytidine, uridine, adenosine anduanosine) [58] and reversed phase ion-pair column (cytidine,ridine, guanosine and adenosine) [49–51,54,55]. This againndicates that the main retention mechanism on TiO2 is not ionxchange.

Under the initial eluent conditions the retention time for tri-hosphates was more than 60 min (data not shown). Thus ane-step gradient was used. Based on the results above, highACN, high phosphate concentration and high pH are desirable

onditions as they yield the greatest efficiency while reducingetention. Of these maintaining the %ACN above 60% is mostmportant. Fig. 5A shows the separation of 15 nucleotides andheir intermediates using a step gradient where the eluent pH isncreased from pH 6.0 to 8.0. Due to the limited solubility ofhosphate in ACN at high pH the concentration of ACN wasowered to 65% in the second step. The minimum resolution inig. 5A is 1.3, but the separation time is long (40 min). Similarly

he use of a step gradient in phosphate (10–20 mM) at a constantH of 6.0 did not significantly reduce the separation time.

Herein both high phosphate concentration (20 mM) and highuffer pH (pH 8) were used for the gradient separation. Fig. 5Bhows the step gradient separation of the 15 nucleotides and

heir intermediates in which the concentration of phosphate isncreased to 20 mM together with a high pH. From Fig. 5B,he tri-phosphate peaks are much sharper and the di- andri-phosphates are baseline resolved using 10 mM phosphate

[[[[

gr. A 1187 (2008) 87–93

uffer (pH 8.0) in 65% ACN as the eluent. Also, the separa-ion time is decreased to 26 min. The efficiency for AMP isround 15,000 plates/m. Miyazaki et al. observed 2800 plates/mor AMP on a titania-coated monolithic silica column using0 mM phosphate buffer (pH 6.0) in 60% ACN [7]. Kimurat al. observed 3000 plates/m for AMP on a bare titania col-mn using 50 mM phosphate buffer (pH 7) in 50% ACNs the eluent [57]. The higher %ACN used in Fig. 5B isesponsible for the significantly higher efficiencies observederein.

. Conclusions

The effect of mobile phase composition on retention and sep-ration on a bare titania column was discussed. It is shownhat two retention regimes: ligand-exchange and HILIC existepending on the mobile phase conditions. Higher %ACN favorshe HILIC mechanism, yielding better separation and efficiency.lso, higher eluent pH and higher phosphate concentration in

he eluent were helpful for shortening the separation time. Fif-een nucleotides and their intermediates were well separated onbare titania column in 26 min with a minimum resolution of

.3.

cknowledgements

This work was supported by the Natural Sciences andngineering Research Council of Canada (NSERC) and theniversity of Alberta.

eferences

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