Separation of purine and pyrimidine bases and nucleosides by hydrophilic interaction chromatography

Research Article

Separation of purine and pyrimidine basesand nucleosides by hydrophilic interactionchromatography

The separation of 12 model compounds chosen among purine and pyrimidine bases and

nucleosides was studied by using hydrophilic interaction chromatography (HILIC). The

compounds investigated were small molecules with relevant properties for biomedical and

pharmaceutical studies. The mixture of pyrimidines and purines was applied on a ZICs-

HILIC 150� 2.1 mm, 5 mm, and two TSKgel Amide-80 150� 2.0 mm, 5 mm and 3 mm

particle size columns. The retention of the analytes was studied by varying ACN%,

ammonium formate concentration, pH, and column temperature. The results obtained

confirmed the elution order of nucleobases, nucleosides, and nucleotides based on their

hydrophobicity. The retention mechanism of the columns was studied considering the

models used for describing partitioning and surface adsorption. The influence on reten-

tion of chromatographic conditions (ACN%, salt concentration, pH, and temperature) was

described and discussed for both columns. The optimization of the conditions studied

allowed to assess a gradient method for the separation of the 12 analytes. The developed

method is a valuable alternative to existing methods for the separation of the compounds

concerned.

Keywords: HPLC / Hydrophilic interaction chromatography / Nucleosides /Purines / PyrimidinesDOI 10.1002/jssc.200900672

1 Introduction

In the last 30 years, there has been increasing request of

methods for the separation of nucleic acid constituents,

nucleotides, nucleosides, and bases, as well as their

degradation products, metabolites, and modified synthetic

analogs [1]. The main research areas concerned have been

genomics [2], pharmacology and toxicology [3], clinical

chemistry [4], forensic [5, 6] and evolutionary genetics [7],

and pharmaceutical chemistry [8–10]. A number of pyrimi-

dine and purine analogs, originating from mutations have

been studied in biological specimens as disease biomarkers

[11], and for evidencing changes of the structure of nucleic

acids [12, 13]. In pharmaceutical research, nucleotides are

used as diet supplements [14], and modified purines and

pyrimidines are expected to result in new drugs [15].

Separation methods have been implemented thus for

quality control and metabolism studies on this class of very

polar and hydrophilic molecules. For this purpose, capillary

electrophoresis [16], RP HPLC, ion-pairing HPLC, and,

more recently, hydrophilic interaction chromatography

(HILIC) have been used for years [2, 17].

HILIC stationary phases reported for separations of

nucleobases, nucleosides, and nucleotides include unbon-

ded silica for the separation of uracil, cytosine, and guanine

together with their chemotherapic derivatives 5-fluoro-

uracil, 5-fluoro-cytosine, and acyclovir [9], or for modeling

and predicting retention times of thymine, uracil, uridine,

cytidine, adenosine, adenine, cytosine, and guanine [18] (on

eight HILIC columns not including those studied here). The

same test compounds were studied on ethylene-bridged

hybrid HILIC columns, hydroxyl- and b-cyclodextrin-

columns [18], while for selected nucleosides diol [19], and

amino [20] columns equivalent to those used in normal-

phase HPLC were reported. Amide [20], polyhydroxyethyl-

and polysulphoethyl-aspartamide [21–23], and sulfobetaine

[20] stationary phases, more specifically designed for HILIC

were also used. Recent comprehensive reviews, however,

show that none of these studies neither used small diameter

columns together with gradient elution which allow lower

solvent consumption, better sensitivity, and easier method

transfer on LC-MS systems, nor presented separations of the

same set of analytes investigated here [17, 24].

Given target analytes and specific applications, several

HILIC columns can appear suitable a priori. However, it is

Giorgio MarrubiniBolivar Enrique CastilloMendozaGabriella Massolini

Department of PharmaceuticalChemistry, University of Pavia,Pavia, Italy

Received October 15, 2009Revised January 12, 2010Accepted January 13, 2010

Abbreviations: HILIC, hydrophilic interaction chromato-graphy; TSM, test standard mixture

Correspondence: Dr. Giorgio Marrubini, Department of Pharma-ceutical Chemistry, University of Pavia, Via Taramelli 12, 27100Pavia, ItalyE-mail: [email protected]: 139-0382-422975

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2010, 33, 803–816 803

not easy to select the more appropriate column because the

HILIC mechanism of retention appears as yet not fully

understood [23, 25]. Additionally, no general purpose

stationary phase in HILIC has emerged, and this forces

researchers willing to try a HILIC column to focus on the

mechanism of retention, as well as on the chemistry invol-

ving analyte, mobile phase, and stationary phase.

The primary retention mechanism of HILIC stationary

phases is to bind water onto their surface [17, 26], although

research on bare silica phases suggested that the estimations

of water content would be low [27]. Within the column, the

analytes retention is related to a complex mechanism, which

involves partition of the solutes between the organic and the

aqueous phases, as well as adsorption of the analytes on the

stationary phase [17, 23, 28–30]. HILIC retention seems to

depend also on secondary interactions (dipole–dipole and

ionic attractive/repulsive between solute and stationary phase

active groups), and therefore most HILIC mobile phases are

prepared including significant concentrations of salts.

Ammonium formate or acetate are mostly the salts of choice

because of their better solubility in highly organic solvents,

and both are suitable if method transfer to LC-MS may be

foreseen. However, other salts are also used despite their

lower solubility in aqueous-ACN eluents, including ammo-

nium chloride or bicarbonate [20], triethylammonium phos-

phate or phosphonate [23], trifluoroacetic acid buffers [25],

and sodium perchlorate [31]. HILIC stationary phases are

influenced by the ions present in the mobile phase; although

they may appear neutral (uncharged), the results on all

sorbents witness that ion-exchange interactions may contri-

bute to the overall retention observed [17, 23, 24, 28, 32]. The

salt type thus is also important in sample preparation, and

counter-ions should be the same in the mobile phase and in

sample solvent to avoid poor peak shape [23].

The aim of this study was to describe the behavior of 12

pyrimidines and purines on commercial HILIC columns,

namely the ZICs-HILIC and the TSKgel Amide-80. The

retention of the analytes was systematically investigated by

varying the chromatographic conditions, and the optimal

conditions for their separation were applied in gradient

elution in order to evidence the applicability potential of

these columns in biomedical and pharmaceutical applica-

tions regarding bases, nucleosides, and nucleotides.

2 Experimental procedures

2.1 Material and methods

TSKgel Amide-80, 150� 2.0 mm, 5 mm and 3 mm columns

were obtained from Tosoh Bioscience (Stuttgart, Germany).

The ZICs-HILIC 150� 2.1 mm, 5 mm column was supplied

by SeQuant (Umea, Sweden). Adenine, adenosine, cytidine,

cytosine, guanine, guanosine, hypoxanthine, inosine, thymi-

dine, thymine, uracil, and uridine of the highest purity

commercially available (minimum 98% purity) were

purchased from Sigma-Aldrich (Milan, Italy). HPLC-grade

water was produced by a Millipore Direct-Q system

(Millipore, Milan, Italy). HPLC-grade ACN and formic acid

(99% purity) were from Carlo Erba Reagenti (Milan, Italy).

Ammonium formate (molecular weight 63.06 g/mol, purity

Z99%) was purchased from Fluka (Sigma-Aldrich, Milan,

Italy).

2.2 Preparation of test solutions

Stock solutions of all analytes except thymine were prepared

by dissolving each compound in aqueous formic acid (1%,

v/v) at levels of 0.1–0.05 mg/mL depending on the analyte

solubility (Table 1). Thymine stock solution was prepared at

1 mg/mL level in methanol.

All stock solutions of the analytes were diluted 20-fold

with mobile phase before every working session in order to

prepare the corresponding working solutions at concentra-

tions ranging from 2.5 to 5 mg/mL. Thymine stock solution

was diluted 200-fold with mobile phase in order to obtain a

5 mg/mL final concentration.

The dilutions were chosen in order to have a solvent

composition of the sample as close as possible to the mobile-

phase composition.

A single stock solution containing the 12 analytes

considered in the study was prepared by weighing each

compound (10–20 mg) in a 200-mL calibrated flask and

dissolving the mixture in aqueous formic acid (1%, v/v).

This solution was diluted 20-fold with mobile phase just

before analyses, and named test standard mixture (TSM)

solution. The TSM solution was used for comparing the

columns ability of separating all peaks of the mixture while

avoiding effects due to sample composition or overload.

All aqueous formic acid (1%, v/v) stock solutions of the

analytes were stable for 1 wk stored at 41C in the dark,

except for thymine, which was stable for about 48 h.

Thymine stock solution in methanol at �201C was stable for

several weeks.

2.3 Apparatus

All experiments were conducted on Agilent HPLC series

1100 and 1200 systems, equipped with mobile-phase

degasser, quaternary pump, autosampler, thermostated

column compartment, and diode-array detector. For data

acquisition and analysis, the ChemStation software version

Rev. A.10.01 was used on the Agilent HPLC series 1100 in

Microsoft Windows XP environment. The ChemStation

version Rev. B.01.01 was used on the series 1200 system, in

Microsoft Windows XP environment.

2.4 Mobile-phase preparation

Stock solutions of 100 mM ammonium formate were

prepared in water and adjusted to each required pH by

J. Sep. Sci. 2010, 33, 803–816804 G. Marrubini et al.


adding formic acid. The pH was measured before the

addition of the organic solvent (wwpH) and after the addition

of the organic solvent (swpH) with a pH-meter calibrated in

aqueous buffer at room temperature (22–231C) in agree-

ment with previously published procedures [25, 33].

The swpH values reported here should be regarded

as indicative, and for this reason they have been

quoted in parenthesis accompanied by the correspond-

ing wwpH measurement as performed in previous

studies [25].

Table 1. Selected purine and pyrimidine compounds and some of their chemical physical data

Name Chemical

structure

pKa Log P(octanol/water) SWater (g/L) SOther (g/L)

Uracil 9.5 [59, 60] �0.70770.286 [61] Soluble [59] Almost insoluble in alcohol, ether [59]

Cytosine 4.68, 12.16 [59] �2.28970.75 [61], �1.730 [62] �7 [59] Alcohols, slightly soluble [59]

Thymine 9.94 [59] �0.11770.287 [61] 4 [59] Methanol soluble [59]

Adenine 4.15, 9.8 [63] �0.09 [63, 64] 0.5 [59] Alcohols, slightly soluble [59]

Guanine 9.92 [59], 3.3, 9.2 [63] �0.96 [63], �0.91 [64], �1.27 [65] Almost insoluble [59] Diluted acids (HCl 0.1N) and KOH

solutions [59]

Hypoxanthine 8.7 [59], 8.51 [63] �1.110 [62], 0.41 [65] 0.7 [59] Diluted acids and alkalies [59]

Adenosine 3.7 [66] �1.23 [64] Soluble [59] Insoluble in alcohol [59]

Cytidine 4.22, 12.5 [59] �2.510 [62] Soluble [59] Sparingly soluble in alcohols [59]

Guanosine 2.1, 9.2 [67] �1.85 [64] �1 [59] No data

Uridine 8.5�[66] �1.710 [62] Soluble [59] Somewhat soluble in alcohols, DMSO,

DMF, acetone, dioxane, ACN [68]

Inosine 8.8–9 [66] �1.4 [69] 16 [59] No data

Thymidine 9.72 [68] �1.11170.488 [61],

�1.25 [70]

Soluble [59] Soluble in methanol, acetone, ethyl

acetate, chloroform [59]

J. Sep. Sci. 2010, 33, 803–816 Liquid Chromatography 805


A stock solution of 500 mL of 100 mM ammonium

formate was prepared by weighing 3.15 g of salt and

dissolving it with 450 mL of water in an Erlenmeyer flask.

To this solution, 0.075 mL formic acid was added and the

solution was brought to the final volume of 500 mL in a

volumetric flask. The final wwpH resulted 5.0.

A second stock solution of 500 mL of 100 mM ammo-

nium formate was prepared by weighing 3.15 g of salt and




volumetric flask. The final wwpH resulted 4.0.

A third stock solution of 500 mL of 100 mM ammonium

formate was prepared by weighing 3.15 g of salt and




volumetric flask. The final wwpH was 3.0.

The mobile phases used were obtained by mixing the

salt 100 mM stock solution directly with ACN or diluting it

with water before mixing it with ACN. The salt concentra-

tion realized on-column can be deduced by the volume ratio

of the solvents. Therefore, for example 10 mL of 100 mM

aqueous salt solution adjusted at wwpH 4.0 were mixed with

190 mL ACN in order to obtain 200 mL of mobile phase

consisting of ACN/5 mM HCOONH4, wwpH 4.2 (95:5, v/v;

swpH �6.8) and about 75 mL/L formic acid (0.0075%, v/v).

The mobile phase ACN/1 mM HCOONH4, (85:15, v/v)

was prepared by diluting 1 mL of the 100 mM salt solution

adjusted to required pH (e.g. wwpH 3.0) to 15 mL with water.

After measuring the wwpH (in the case of initial w

wpH 3.0 this

was 3.2 after dilution), 85 mL of ACN were added (swpH

�5.2, total formic acid 145 mL/L, or 0.0145%, v/v).

The columns were equilibrated with at least 20-column

volumes of mobile phase at the beginning of every analytical

session. At the end of each session, the TSKgel Amide-80

columns were washed with at least 20-column volumes of

ACN/water (60:40, v/v) in reverse flow at 0.075 mL/min and

stocked with this wetting medium. The ZICs-HILIC was

washed with at least 20-column volumes of ACN/4 mM

HCOONH4, wwpH 5.0 (60:40, v/v and 6 mL/L formic acid,

swpH �7.2) at 0.5 mL/min, and the same mobile phase was

left as wetting medium when the column was not in use.

2.5 Methods

The experimental conditions were studied one-at-the-time,

and no experimental design was considered.

The effect of ACN% was considered in three series of

measurements at 85, 90, and 95% ACN on each compound

and on the TSM. The ACN% in the mobile phase was varied

while ammonium formate concentration on-column was

kept constant at 0.5, 1, and 5 mM at wwpH 4.0 and 201C.

The effect of salt was studied in three series of experi-

ments comparing the retention of each single analyte and of

the TMS using ammonium formate adjusted at wwpH 4.0 at

on-column concentrations of 0.5, 0.75, 1, 1.5, 3, 5, and

15 mM at 85% ACN (swpH �6.2), at 0.5, 1, 2, 5, and 10 mM

at 90% ACN (swpH �6.4), and at 0.25, 0.5, 1, and 5 mM at

95% ACN (swpH �6.8).

The effect of pH in the mobile phase was studied atwwpH 3.0, 4.0, and 5.0 (corresponding to s

wpH �5.0–5.6,

�6.0–6.8, and �7.0–7.6 depending on ACN%) using two on-

column salt concentrations for each ACN%, namely 1.5 and

15 mM at 85% ACN, 1.0 and 10 mM at 90% ACN, and 0.5

and 5 mM at 95% ACN.

The effect of temperature was studied at 20, 40, 60, and

801C on the 5 mm TSKgel Amide-80 column, and at 20, 30,

40, 60, and 801C on the ZICs-HILIC column using a

mobile phase consisting of ACN/1 mM HCOONH4wwpH 4.0

(90:10, v/v; swpH �6.4 measured at 231C).

Under all conditions tested, retention factor (k),

peak width at half height (W1/2), and symmetry were

recorded.

3 Results and discussion

3.1 Choice of analytes and columns

The compounds investigated are small molecules having

properties relevant for biomedical [2, 4–7, 11–13] and

pharmaceutical studies [8–10, 14] (Table 1). In particular,

five bases and the corresponding nucleosides are constitu-

ents of DNA and RNA. Hypoxanthine was added because it

is obtained from adenine by a metabolic route, which

involves oxidative deamination (like uracil which can

originate from oxidative deamination of cytosine [6]), and

inosine because it is a hypoxanthine-corresponding nucleo-

side.

Different and less numerous sets of compounds were

studied previously by adopting HILIC [17, 24], but the set of

analytes investigated here was neither investigated before,

nor studied using small diameter columns together with

gradient elution.

The two HILIC columns used, the TSKgel Amide-80

and ZICs-HILIC, were chosen based on the difference

between their stationary phases, popularity, and published

data on separations of bases, nucleosides, and nucleotides.

The Amide-80 is designed to operate both in normal

phase and in HILIC [24]. In the latter mode, it has been

reported for the analysis of saccharides, glycans, oligo-

saccharides [34–36], peptides [29], glycoproteins, and very

polar compounds isolated from biological extracts [37, 38].

For charged solutes, this column showed retention incre-

ments due to ionic interactions at pH about 4 [20, 23].

The ZICs-HILIC column contains a packing in which

spherical particles of silica are functionalized with a zwit-

terionic ligand which overall confers to the column high

capability of binding water and ion-exchange properties with

low contribution of silanophilic interactions [17]. The ZICs-

HILIC column has been reported in applications including

the separation of glycopeptides [39], proteins [24, 40],



glucosinolates [31], and studies devoted to the under-

standing of HILIC retention mechanism [9, 10, 20, 41, 42].

The columns were tested changing ACN%, salt

concentration, pH, and temperature to evaluate the effect of

these parameters on the retention of each compound and of

TSM. The optimal conditions were then selected for evalu-

ating the separation achievable for the components of the

TSM.

3.2 Mobile-phase strength

Most HILIC applications report the use of ACN/water

mobile phases although exceptions have been published

previously [30, 43, 44].

During method development here, the ACN/water

mobile phase yielded adequate peak shapes and therefore it

was further investigated. At ACN% lower than 85%,

regardless of the other method parameters (salt concentra-

tion, temperature, and pH), thymine, thymidine, and uracil

showed no retention on both 5 mm columns, and overall

peak resolution was poor. Thus ACN% was studied at 85,

90, and 95%.

On both columns, the TSM compounds’ retention

factor k increased with increasing ACN% as is typical of

HILIC experiments, with increments in k that were much

greater for late eluting analytes compared with the early

eluting ones. In particular, this was evident for guanine,

cytidine, guanosine, thymine, thymidine, and uracil that

eluted at the two extreme ends of the chromatograms of the

TSM. For the remaining analytes (adenine, adenosine,

hypoxanthine, uridine, cytosine, and inosine), intermediate

changes in k with ACN% were observed.

At levels of ACN lower than 95% on both columns, the

retention factors for the analytes ranged from about 1

(thymine, thymidine, and uracil) to less than 20 (guanosine,

being always the last eluted).

At 95% ACN, guanine, cytidine, and guanosine showed

k >20 on the 5 mm TSKgel Amide-80 column, whereas only

guanosine had an unacceptably high k on the ZICs-HILIC

column (k 5 47.8, Table 2).

The behavior of cytosine [20], aromatic amines, alcohols,

and amides [45] under different chromatographic conditions,

adopting wider ranges of ACN% (70–95%), and of cytosine

and acidic compounds at ACN% at levels higher than 90% on

ethylene-bridged hybrid and bare silica phases [30] have been

described. According to [30], the dependence of k appears

exponential. These reports are in agreement with the results

summarized here in the observation that despite the differ-

ences in columns, analytes, and chromatographic conditions

the retention factor variation with ACN% is smaller for less

polar compounds (less retained) compared with that of more

polar compounds (more retained) [20, 30, 45]. The explanation

given is that the more polar analytes are more retained due to

a complex mechanism involving partitioning, adsorption, and

secondary interactions on the stationary phase [20, 30, 45].

According to the literature on HILIC retention behavior of

bases, nucleosides, and nucleotides [19, 20, 23], at ACN% as

high as 85% in the mobile-phase retention is governed by

hydrophilicity, and retention factors increase with solute

polarity and molecular size. Thus, given a molecular frame for

the TSM compounds studied here, the order of elution

expected is from the least hydrophilic/polar compound, the

base (B, less retained, higher Log P, Table 1), to the nucleo-

side (B-Sugar, BS, more retained, Log P lower than the

corresponding B, Table 1), to the most hydrophilic/polar

Table 2. Retention factors, selectivity, and resolution values attained for the TSM compounds on the two 5 mm columns under isocratic

conditionsa)

TSKgel Amide-80 (2.0� 150 mm, 5 mm) ZICs-HILIC (2.1� 150 mm, 5 mm)

OE Name k a Rs OE Name k a Rs

1 Thymine 1.8 – 2.9 1 Thymine 0.9 – 4.1

2 Uracil 2.2 1.2 4.0 2 Thymidine 1.3 1.4 0.5

3 Thymidine 2.9 1.3 10.5 3 Uracil 1.3 1.0 8.3

4 Uridine 6.9 2.4 0.6 4 Adenine 3.2 2.5 1.3

5 Adenine 7.4 1.1 0.6 5 Adenosine 3.6 1.1 3.5

6 Hypoxanthine 7.7 1.0 1.1 6 Hypoxanthine 4.7 1.3 1.0

7 Adenosine 8.5 1.1 5.6 7 Uridine 5.0 1.1 7.8

8 Cytosine 13.3 1.6 2.4 8 Cytosine 8.3 1.7 6.1

9 Inosine 15.8 1.2 6.0 9 Inosine 12.1 1.5 6.1

10 Guanine 24.1 1.5 0.6 10 Guanine 17.7 1.5 0.6

11 Cytidine 25.2 1.0 6.8 11 Cytidine 18.5 1.0 25.7

12 Guanosine 44.9 1.8 – 12 Guanosine 47.8 2.6 –

a) Mobile phase: ACN/0.5 mM HCOONH4, wwpH 4.0 (95:5, v/v s

wpH �6.4). Flow rate, 0.15 mL/min; temperature, 20.01C, 260 nm. OE, Order of

elution. k: Retention factor; k 5 (tR�t0)/t0, where tR is the analyte retention time and t0 the retention time of toluene used as void volume

marker. a: Selectivity factor (relative retention), a ¼ ki11=ki, where ki is the i-th compound retention factor. Rs: Resolution factor,

Rs ¼ ðtRi11tRiÞ=2ðWi1Wi11), where tRi is the i-th compound retention time, and Wi the i-th peak width at half height.



compound, the nucleotide (base-sugar-phosphate, most

retained). In this study, it was verified that all series B, BS,

base-sugar-phosphate, sharing the same base B as nucleus of

the molecule, respected this order of elution. Data presented

here, however, report only on the retention of bases and

nucleosides which eluted in the first 150 min of run time

under isocratic conditions on either 5 mm column. Nucleo-

tides were so hydrophilic that they were not eluted at ACN%

higher than 85%, whereas at lower ACN% (i.e. 75%) they

showed severely tailing peaks, poorly resolved between them

at on-column total salt concentration of up to 15 mM. This

was considered a confirmation of the occurrence of ion

exchange effects (cation exchange/anion exclusion) as recently

reported [23]. Therefore, higher concentrations of salt and

higher water% in the mobile phase than those applicable for

the separation of bases and nucleosides resulted necessary to

attain acceptable peak shapes and separation of the available

nucleotides. For these reasons, nucleotides could not be

separated in a single run with the TSM compounds, and thus

they have been omitted here, and will not be mentioned

further.

As reviewed [17], the investigation over the mechanisms

of retention in HILIC can be conducted by studying plots of

ln(k) as a function of the volume fraction of the stronger

eluent in a binary mobile phase (lin–log plot), and of ln(k)

and the logarithm of the volume fraction of the same

stronger eluent (log–log plot), according to the equations

describing a partition model of retention or an adsorptive

one, respectively. From our data set, however, it is impos-

sible to draw conclusive evidence of a prevailing mechanism

of retention over the other, and therefore both mechanisms

(partition and adsorption) are believed to contribute to the

overall retention of the analytes on both columns under the

conditions applied.

It is interesting to observe that better separation was

obtained from organic solvent-rich condition, and that these

results are in agreement with published data for smaller sets

of molecules eluted on 5 mm, 250� 4.6 mm TSKgel Amide-

80 and ZICs-HILIC columns although under different

chromatographic conditions [18–20].

3.3 Effect of the salt concentration

Mixtures of plain solvents have been used in HILIC

[38], but for most applications ACN/aqueous saline

[29, 46] or buffered solutions are reported [10, 19–24,

28–32, 34, 39, 41, 43, 47, 48]. In unbuffered mobile

phases of ACN and water, cation exchange, anionic

repulsion, and dipole–dipole interactions hypothesized

for ZICs-HILIC, and TSKgel Amide-80 columns are

believed to occur at maximum strength [8, 17]. The

presence of an electrolyte seems preferred for the separa-

tions of molecules that undergo ionization in the mobile

phase [8].

Ammonium acetate concentrations of 5–20 mM were

reported to cause only modest increase in retention for

cytosine on 5 mm 250� 4.6 mm ZICs-HILIC and TSKgel

Amide-80 columns [20]. Most previous studies on a variety

of analytes and columns reported the use of 2.5–20 mM

ammonium acetate [24]. According to [23], on 250� 4.6 mm

columns salt concentrations lower than 20 mM are too low

to maintain a counter-ion layer that screens the underlying

stationary phase, and actually in some applications salt

(NaClO4) molarities as high as 250 mM were used in

gradient elution using a mobile phase in which the aqueous

fraction was 30% [49].

The TSM selected here is composed of heterocyclic

bases that under acidic pH form cations and polar hydro-

philic compounds by shifting charge densities within the

molecular frame (Table 1). The addition of salt in the mobile

phase was expected to provide more repeatable retention

times and sharper peaks due to better partitioning of the

analytes between mobile phase (most made of organic

solvent) and stationary phase onto which a layer of solvated

ions should attract the polar and ionized solvated analytes

[21–23]. Therefore, ammonium formate concentrations in

the mobile phase ranging from 0.25 to 15 mM at constant

ACN% and pH were compared with the absence of salt in

the mobile phase in order to verify the assumptions that

were published previously [8, 20–24].

In the three series of experiments carried out (85% ACN

and ammonium formate 0.5–15 mM, 90% ACN and

ammonium formate 0.5–10 mM, and 95% ACN and

ammonium formate 0.25–5 mM), differences were observed

in retention and peak shape when the mobile phase

contained ammonium formate compared with plain water,

but no differences in retention time precision measure-

ments were evident (RSD% o1% for all analytes after three

replicates in within-session repeatability testing).

The general trend observed was a reduction of retention

and an improvement in peak shape passing from water to a

‘‘critical’’ salt concentration, which was different between

the series of experiments. In particular, at 85% ACN the salt

concentration corresponding to minimum retention was

about 5 mM on both columns, at 90% ACN it was about

1 mM, and at 95% ACN it was about 0.25 mM. Salt

concentrations higher than these produced an increase in

retention accompanied by almost no increase in peak width

and with an improvement in peak symmetry, especially for

more retained analytes (viz. guanosine, cytidine, guanine,

and inosine). An example of the behavior observed at 95%

ACN is shown in Fig. 1.

Based on these data and on the previous studies [20, 23,

30], it is speculated that retention is strong on both columns

when no salt is present in the eluent due to the complex

interactions between the analytes, the mobile phase and the

sorbent (which results in hydrophilic partition), and the

analytes and the stationary phase (including polar and ionic

ligands and residual silanol groups which lead to retention

through adsorption). When salt is added in the mobile

phase, retention is decreased because ion exchange sites and

dipole–dipole interactions are shielded by the presence of

solvated ions supplied by the salt. When more salt is added



in the mobile phase, the reduced solubility of the solvated

ionized analytes in the ACN-rich mobile phase (which leads

to hydrophilic partitioning) becomes the driving force that

causes an additional increase in retention and optimal peak

shapes.

Other researchers explained analogous effects of

increased k with increasing salt concentration on home-

made neutral-bonded stationary phases and on a Luna

HILIC column. The increase in retention was related to the

increased concentration of solvated ions in the adsorbed

aqueous layer and thus yielding a thicker adsorbed water

layer and stronger retention [43].

3.4 Effect of pH

In this study, ammonium formate was preferred to

ammonium acetate, because of its lower buffering capacity

0

10

20

30

40

50

60

70

80

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5Salt concentration (mM)

Retention factor(k)

ThymineHypoxanthyneCytosine

UracilAdenosineGuanine

ThymidineAdenineCytidine

UridineInosineGuanosine

0

1

2

3

4

5

6

7

8

9

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Salt molarity (mM)

Peak width at half height

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Salt molarity (mM)

Peak Symmetry

A

0

10

20

30

40

50

60

70

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5Salt concentration (mM)

Retention factor(k)

0

1

2

3

4

5

6

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5Salt molarity (mM)

Peak Width at half height

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Salt concentration (mM)

Peak Symmetry

B

Figure 1. Effect of salt on retention, peak width, and symmetry of the TSM compounds. Chromatographic conditions. Mobile-phase ACN/HCOONH4, w

wpH 4 (95:5, v/v); temperature, 201C; flow rate, 0.15 mL/min; detector wavelength, 260 nm. The test compounds were injectedin mobile phase (5 mL injection volume) at concentrations from 2.5 to 5 mg/mL. The peak symmetry parameter used in this study is thatcalculated by the Agilent Chemstation software integrator, and it is based on the ratio between the leading and the trailing edges of thepeak. (A) The TSKgel Amide-80 column; (B) the ZICs-HILIC column.



range (viz. formic acid pKa is about 3.75, i.e. buffering rangewwpH �2.75–4.75) [50]. The mobile phase pH and analytes

pKas change with the composition of the mobile phase, and

this is recognized to influence the LogDs of the analytes

[50–52], as well as the overall ionization of the stationary

phase in the case of silica [26, 30, 33, 53].

In this study, the actual pH as well as the values of the

analytes pKas, and the LogDs, in the mobile phases realized

after adding ACN, have not been assessed. The data of wwpH

and swpH together with the aqueous pKas of the analytes

have been considered only for the practical aspect of method

development and basic understanding of the retention

observed.

On the ZICs-HILIC column, at 85% ACN and overall

salt concentrations on-column of 1.5 and 15 mM, and at

90% ACN and salt concentrations of 1.0 and 10 mM, pH did

not change significantly the analytes retention. At 95% ACN

in the mobile phase at salt concentrations of 0.5 and 5 mM,

the ZICs-HILIC column showed variations of the retention

factors with pH greater than the system precision but still

too close to these figures to allow safe conclusions (RSD%

ranging from about 0.5 to 8% depending on the magnitude

of the analyte retention factor, on six replicates in inter-

session repeatability testing). Data obtained at 95% ACN

(Table 3) indicate that molecules having a pKa1 close to thewwpH range investigated (adenine, adenosine, cytosine, cyti-

dine, guanine, and guanosine, Table 1) seem to have

decreased retention passing from wwpH 3 (s

wpH �5.6) to wwpH

5 (swpH �7.6). Similar data were evidenced for cytosine and

cytidine in one earlier report [20] and were related to the

acidity of these pyrimidines. From our data, it appears that

also purines having low aqueous pKas are subject to an

analogous effect, thus suggesting that this phenomenon is

related to cation exchange on the ZICs-HILIC column. The

limited variations of retention with pH are explained with

the fact that the ZICs-HILIC phase contains ligands always

charged that will not change ionization status with pH [54]

(http://viewer.zmags.com/publication/95db93bd/95db93bd/1).

Therefore, the net effect observed on retention can be rela-

ted only to the change in ionization that occurs on the

analytes.

On the TSKgel Amide-80 column, the effect of pH

change was more evident, and again it affected the retention

factors more at 95% ACN compared with lower ACN%. In

particular, at 95% ACN and 0.5–5 mM overall salt in the

mobile phase, guanine and guanosine showed an increase

of retention of 152 and 137% passing from wwpH 3 to 4 and

of 140 and 128% from wwpH 3 to 5, respectively (Table 3).

Analogous changes in retention, though less remarkable,

were observed for cytosine and cytidine, and for adenine and

adenosine (Table 3). Although less evident than at 95%

ACN, at ACN% of 90 and 85% for the same analytes the

same trend of increased retention with pH passing fromwwpH 3 to 4 seemed to occur, but again it was comparable in

magnitude with the measurements precision (which ranged

from about 1% to a maximum of 10% on six replicates in

different nonconsecutive working days).

Retention for the TSM compounds appeared not

strongly influenced by pH on the ZICs-HILIC column,

whereas on the TSKgel Amide-80 it reached a maximum atwwpH about 4. For this reason, w

wpH 4 was chosen for all

experiments, and for comparisons with the ZICs-HILIC

column.

One explanation of these results can be related to sila-

nophilic interactions. Ionic (cation exchange/anion exclu-

sion) and/or dipolar hydrogen bond-type silanophilic

interactions, as defined by Neue et al. [53], are well docu-

mented in RP-HPLC and depend mainly on pH and on the

solute chemistry under the elution conditions [33, 53].

Although high purity Type B silica is used, a certain amount

Table 3. Retention factor (k) at different pH valuesa)

ZICs-HILIC TSKgel Amide-80

wwpH 3.0 4.0 5.0 w

wpH 3.0 4.0 5.0

(swpH) (�5.6) (�6.8) (�7.5) (s

wpH) (�5.5) (�6.6) (�7.5)

Thymine 1.0 1.0 1.0 Thymine 1.1 1.2 1.2

Thymidine 1.5 1.5 1.5 Uracil 2.2 2.7 2.5

Uracil 1.7 1.7 1.7 Thymidine 2.7 3.4 3.2

Adenine 3.3 3.3 3.2 Uridine 5.3 6.6 6.3

Adenosine 4.7 4.6 4.4 Adenine 7.3 10.2 9.5

Hypoxanthine 6.7 6.7 6.4 Hypoxanthine 7.4 9.4 8.9

Uridine 8.5 7.6 7.5 Adenosine 7.8 10.6 9.8

Cytosine 11.3 11.4 10.7 Cytosine 12.4 17.0 15.9

Inosine 20.2 19.5 18.6 Inosine 18.2 27.0 24.5

Guanine 21.5 18.8 20.9 Guanine 21.6 32.8 30.1

Cytidine 24.9 23.1 22.5 Cytidine 31.9 45.5 39.9

Guanosine 45.3 48.6 48.7 Guanosine 53.8 73.6 68.8

a) The analytes are reported in order of elution at wwpH 3. Chromatographic conditions: ACN/5 mM HCOONH4 (5:95, v/v); temperature,

201C; flow rate, 0.15 mL/min. The test compounds were injected in mobile phase (5 mL injection volume) at concentrations ranging from

2.5 to 5 mg/mL.



of surface silanols remains available for interactions

between analytes, eluent agents, and stationary phase [19,

23]. These interactions may add adsorptive contributions

also to the overall HILIC retention [17, 19], and from the

results presented in the previous sections, it can be

supposed that adsorption occurred in experiments conduc-

ted here. In RP-HPLC stationary phases made of high-purity

silica, the degree of ionization of silanols increases with

increasing pH of the mobile phase [26, 33], and this was

observed in a bare silica phase with high ACN% too, at least

within a mobile-phase pH interval from 2.9 to 5.2 [55]. The

TSKgel Amide-80 column is based on silica, the silanols of

which are reported to provide some ion-exchange interac-

tion with charged analytes [20, 23, 41]. All solutes involved

in our study are protonated at least on one site according to

their pKas (Table 1), and this condition should cause

increase in retention from wwpH 3 to 5 due to the increase in

the number of ionized silanols. What is found is that all

analytes at 95% ACN and 5 mM salt molarity have a maxi-

mum increase in retention from wwpH 3 to 4, and a smaller

increase from wwpH 3 to 5. This is consistent with the

assumption that it occurs with an increase of the number of

ionized silanols with increasing pH, but this is counter-

balanced by a decrease in the ionization of the analytes,

especially those having a pKa close to the pH range

(adenine, adenosine, cytosine, cytidine, guanine, and

guanosine). Similar explanations were provided for the

behavior of amines on silica in RP-HPLC [56], and for

HILIC separations of basic analytes on bare silica columns

[25].

As far as the role of ACN at different pH values is

concerned, some authors provided an explanation of

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.8 2.9 3.0 3.1 3.2 3.3 3.4

2.8 2.9 3.0 3.1 3.2 3.3 3.4

2.8 2.9 3.0 3.1 3.2 3.3 3.4

2.8 2.9 3.0 3.1 3.2 3.3 3.4

1000x1/T (°K-1)1000x1/T (°K-1)

1000x1/T (°K-1)1000x1/T (°K-1)

ln(k

)

Cytosine Cytidine Thymine Thymidine Uracil Uridine Cytosine Cytidine Thymine Thymidine Uracil Uridine

0.0

0.5

1.0

1.5

2.0

2.5

ln(k

)

Adenine Adenosine Guanine Guanosine Hypoxanthine Inosine Adenine Adenosine Guanine Guanosine Hypoxanthine Inosine

| 80 | 60 | 40 | 30 | 20 (°C)

0.50

1.50

2.50

3.50

4.50

5.50

6.50

7.50

ln(k

)

1.50

2.50

3.50

4.50

5.50

6.50

7.50

8.50

9.50

ln(k

)

| 80 | 60 | 40 | 20 (°C)

A

B

C

D

Figure 2. The van’t Hoff plots for the TSM pyrimidine (A) and purine (B) compounds on the ZICs-HILIC column and for pyrimidines (C)and purines (D) on the TSKgel Amide-80 column. Retention factors at each temperature are the mean of the three measurements. Within-session repeatability expressed by the RSD% was never higher than 0.8%. Chromatographic conditions: Mobile-phase ACN/1 mMHCOONH4

wwpH 4.0 (90:10, v/v; s

wpH �6.4); flow rate, 0.15 mL/min; detector wavelength, 260 nm. The test compounds were injected inmobile phase (5 mL injection volume) at concentrations from 2.5 to 5 mg/mL after at least 1 h of column temperature equilibration.



analogous findings, although in a different application.

According to [55], by modulation of the mobile-phase pH,

the mechanism governing retention of the solutes can pass

from hydrophilic to the ion-exchange interaction. Such

interaction is strengthened by a low water% because the

ions present in the mobile phase have poor solubility in

ACN and thus are strongly retained into the thin aqueous

layer adsorbed onto the stationary phase surface. Therefore,

if by varying the mobile-phase pH cation exchange becomes

the dominant mechanism of retention, the aqueous pKa of

the analyte will provide information regarding the trend to

be expected for retention. The data collected in this study for

the TSKgel Amide-80 column support this interpretation. At

95% ACN and 5 mM salt concentration, all analytes showed

increased retention passing from wwpH 3 to 5 (Table 3).

3.5 Effect of temperature

The benefits of operating at high temperature on HILIC

columns have been recently illustrated [32], and it is

recognized that raising the column temperature will

generally result in shorter retention times for most analytes

[57]. The same condition applied here also as shown in

Fig. 2 in which van’t Hoff plots are illustrated for the

selected analytes. On both 5 mm columns, retention at 201C

was stronger generally than at 801C for most analytes.

Exceptions were represented by guanosine and guanine on

the ZICs-HILIC column (Figs. 2A and B). The ZICs-

HILIC column showed nonlinear van’t Hoff plots for all the

analytes concerned under the conditions applied, consis-

tently with the behavior of ionizable species, as reviewed

previously [58]. However, this result is not in agreement

with the previous data reported for cytosine [20]. Actually,

for cytosine a r2-value of 0.96 can be calculated in the linear

regression for the five points examined here (from 20 to

801C) and this determination coefficient corresponds with

that published, investigating similar temperature ranges,

but under different conditions (ACN/water 5 90:10, v/v,

10 mM ammonium acetate, pH not specified [20]) for

cytosine and cytidine on different columns and conditions

[19]. Nevertheless, in these reports nonlinearity was not

1

2

3

9

10

11

12

4

56

78

1

4

3

2

56 8

9

10

11

12

1

3+24

5 6

7 89

10

11

12

7

A

B

C

Figure 3. Separation of the test compounds on the ZICs-HILIC column at different temperatures. Mobile phase (A) 10 mM HCOONH4 (wwpH

4); (B) ACN. Chromatographic conditions. Linear gradient A/B 5 from 5:95 to 15:85% in 10 min, followed by an isocratic elution at 15:85%.Run time: 60 min. Test standard mixture of the 12 compounds was prepared as described in Section 2. Injection volume, 5 mL. Flow rate,0.15 mL/min; detector wavelength, 260 nm. Column temperature (A) 201C, (B) 501C, and (C) 801C. Peak identification: 1, thymine; 2,thymidine; 3, uracil; 4, adenine; 5, adenosine; 6, hypoxanthine; 7, uridine; 8, cytosine; 9, inosine; 10, guanine; 11, cytidine; 12, guanosine.



min0 5 10 15 20 25 30 35 40

mAU

-5

0

5

10

15

20

25

D AD 1 A, S ig=260,16 R ef=550,100 (C :\C H EM 32\1\D ATA\090930\M IX12000000.D )

1 2

3

4

5

6

7

8

9+10

11 12

min0 5 10 15 20 25 30 35 40

mAU

-5

0

5

10

15

20

25

D AD 1 A, S ig=260,16 R ef=550,100 (C :\C H EM 32\1\D ATA\091002\M IX12PH 4000000.D )

1

3

4

5

6 +7

8

9

10

12

2

Figure 4. Separation of theTSM compounds on the 5 mmTSKgel Amide-80 column.Chromatographic conditions:Mobile phase (A) HCOONH4

10 mM, (B) ACN, gradientelution A/B 5 from 5:95 to15:85% in 10 min, followedby isocratic elution atA/B 5 15:85%. Flow rate,0.15 mL/min; column tempera-ture, 201C; detector wave-length, 260 nm. Upperchromatogram eluent (A) atwwpH 3.0, lower chromatogrameluent (A) at w

wpH 4.0. Peakidentification: 1, thymine; 2,uracil; 3, thymidine; 4,adenine; 5, hypoxanthine; 6,adenosine; 7, uridine; 8, cyto-sine; 9, inosine; 10, guanine;11, cytidine; 12, guanosine.

12

34

5+6

78 9

10

11

12

Figure 5. Separation of the TSM solution on the TSKgel-Amide-80, 150� 2.0 mm, 3 mm particle size. Chromatographic conditions: Mobilephase (A) 10 mM HCOONH4

wwpH 4.0; (B) ACN. Isocratic elution with A/B 5 5:95% for 20 min, gradient to 10:90% in 20 min, and to 15:85% in

10 min. Flow rate, 0.1 mL/min; temperature, 20.01C. Peak identification: 1, thymine; 2, uracil; 3, thymidine; 4, uridine; 5, adenine; 6,hypoxanthine; 7, adenosine; 8, cytosine; 9, inosine; 10, guanine; 11, cytidine; 12, guanosine.



excluded, and in this study it is evidenced by the 12 plots of

pyrimidines and purines (Figs. 2A and B) that the

dependence of the ln(k) on temperature is not linear in

the case of the ZICs-HILIC column under the conditions

applied.

The TSKgel Amide-80 column showed linear van’t Hoff

plots with positive slopes for all analytes (Figs. 2C and D),

meaning that the retention mechanism does not change

with the temperature under the conditions applied, and that

the transfer of the solutes from the mobile to the amide

stationary phase is characterized by constant negative

enthalpies (retention process exothermic, favored by the

enthalpy contribution) [26, 58]. One additional observation

which is interesting for method development is that the

slopes of nucleosides are always greater than those of the

corresponding nucleobases, in agreement with the

published data for cytosine and cytidine found on diol

columns under different conditions [19].

As regards the selectivity, the ZICs-HILIC column

showed a relevant improvement in the separation of TSM

components passing from 20 to 501C (Fig. 3). On the

contrary, the 5 and 3 mm TSKgel Amide-80 columns showed

optimal resolution between most peaks at 201C compared

with runs conducted at higher temperatures (data not

shown).

3.6 Optimization of chromatographic conditions

The information collected allowed us to obtain a full

separation of the 12 compounds evaluated in the TSM,

which is of interest in bioanalytical studies [2–4, 11] and in

current research on the fidelity of PCR replication of DNA

[5–7, 12, 13].

From the first set of experiments, it appears that

isocratic elution is not viable for the separation of these 12

compounds due to long run times (Table 2).

Retention and peak shape were influenced by the

presence of ammonium formate at different concentrations.

At low salt concentrations, the behavior observed was a

reduction of retention to a minimum value, which resulted

different at different ACN%. An increase in retention with

increasing salt concentration was evidenced, accompanied

by an improvement in peak shape passing from water to

0.25–5 mM ammonium formate on both columns (e.g. at

95% ACN in Fig. 1). At salt concentrations higher than

those causing the minimum in retention (0.25 mM at 95%

ACN, 1 mM at 90%ACN, and 5 mM at 85% ACN), further

improvement in peak shape was appreciable for all analytes,

but only the more retained analytes (viz. guanosine,

cytidine, guanine, and inosine) had also a further increase

in retention.

The influence on retention of the mobile-phase pH in

the range from wwpH 3 to 5 was evident on TSKgel Amide-80

while it affected less the retention factors on the ZICs-

HILIC column (Table 3).

Column temperature affected retention and was used to

fine-tune the separation.

Gradient elution was necessary to achieve run times

shorter than those of isocratic elution at 95%. The gradient

starting conditions were of 95% ACN because it was the

parameter that caused maximum initial retention, initial

0.5 mM ammonium formate on-column, and wwpH 4.

Figures 3 and 4 show the chromatograms under

gradient elution, after applying the TSM solution on the two

5 mm columns. The ZICs-HILIC (Fig. 3, chromatogram B)

column provided, in about 60 min of run, the full resolution

of the 12 TSM compounds at 501C. The TSKgel Amide-80

columns had optimal selectivity at 201C and wwpH 4 but

neither the 5 mm nor the 3 mm particle size packings were

able to separate critical couples of peaks (Figs. 4 and 5). The

new 3 mm TSKgel Amide-80 column showed excellent peak

shapes for all analytes although at the expense of longer run

time (about 70 min) in comparison with the 5 mm column

(about 50 min run time). Based on the fundamental HPLC

theory [26] and recent reports [24], the 3 mm column can

allow the use of flow rates higher than those applied here,

maintaining at least the same column efficiency.

4 Concluding remarks

Different stationary phases are available on the market, but

the choice of the HILIC column is difficult due to the

complex mechanism that leads to retention on bonded and

bare silica.

This study provides practical data on the behavior of

bases and nucleosides, and information for method devel-

opment in applications involving nucleotide separations

also. This study confirms HILIC as an ideal tool

for the analysis of nucleobases and nucleosides. The results

on the effect of ACN%, salt concentration, pH, and

temperature are in agreement with the published data that

support the hypothesis of a complex mechanism of reten-

tion in HILIC, based on adsorption and partition both on

TSKgel Amide-80 and on ZICs-HILIC. The information

collected has been used to optimize the separation of 12

analytes. Only the ZICs-HILIC column provided full

resolution of the selected compounds in about 60 min.

The 5 and 3 mm TSKgel Amide-80 columns had demon-

strated great chemical and mechanical stability but were

unable to separate all the analytes under the conditions

applied.

The authors are grateful to Professor Raffaela Biesuz of theDepartment of Analytical Chemistry of the University of Paviafor invaluable discussions. The authors acknowledge the helpfulsuggestions of Andrew W. Alpert and the generous provision of anew 3 mm TSKgel Amide-80 column by Regina Roemling onbehalf of Tosoh Bioscience.

The authors have declared no conflict of interest.



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Separation of purine and pyrimidine bases and nucleosides by hydrophilic interaction chromatography

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