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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: giorgio.marrubini@unipv.itFax: 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.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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
dissolving it with 450 mL of water in an Erlenmeyer flask.
To this solution, 0.75 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 4.0.
A third 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, 7.25 mL formic acid was added and the
solution was brought to the final volume of 500 mL in a
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],
J. Sep. Sci. 2010, 33, 803–816806 G. Marrubini et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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.
J. Sep. Sci. 2010, 33, 803–816 Liquid Chromatography 807
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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
J. Sep. Sci. 2010, 33, 803–816808 G. Marrubini et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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.
J. Sep. Sci. 2010, 33, 803–816 Liquid Chromatography 809
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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.
J. Sep. Sci. 2010, 33, 803–816810 G. Marrubini et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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.
J. Sep. Sci. 2010, 33, 803–816 Liquid Chromatography 811
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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.
J. Sep. Sci. 2010, 33, 803–816812 G. Marrubini et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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.
J. Sep. Sci. 2010, 33, 803–816 Liquid Chromatography 813
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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.
J. Sep. Sci. 2010, 33, 803–816814 G. Marrubini et al.
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
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