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Journal of Chromatography A, 1216 (2009) 1140–1146
Contents lists available at ScienceDirect
Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma
Aqueous normal-phase retention of nucleotides on silica hydride columns
Joseph J. Pesek a,∗ , Maria T. Matyska a , Milton T.W. Hearn b , Reinhard I. Boysen b
a
b
Department of Chemistry, San Jose State University, San Jose, CA 95112, USA
Australian Research Council Special Research, Centre for Green Chemistry, Monash University, Clayton, Victoria 3800, Australia
a r t i c l e
i n f o
Article history:
Received 16 October 2008
Received in revised form 15 December 2008
Accepted 17 December 2008
Available online 25 December 2008
Keywords:
HPLC
Polar compound retention
Nucleotides
a b s t r a c t
The use of silica hydride-based stationary phases for the retention and analysis of nucleotides has been
investigated. Both reversed-phase columns with a hydride surface underneath as well as those with
an unmodified or a minimally modified hydride material were tested. With these systems, an aqueous
normal-phase mode was used with high organic content mobile phases in combination with an additive
to control pH for the retention of the hydrophilic nucleotides. Isocratic and gradient elution formats
have been used to optimize separations for mixtures containing up to seven components. All conditions
developed are suitable for methods that utilize mass spectrometry detection.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Nucleotides are important phosphate containing compounds
that are found in living cells and are associated with a vast array
of metabolic and biological processes. They play key roles in the
synthesis of DNA and RNA, are involved in signal transduction pathways, function as coenzymes in biosynthetic processes and serve
as energy reservoirs in biological systems [1–6]. Thus improved
methods of analysis for these compounds are of continued interest,
especially techniques which can distinguish among the nucleotides
based on their degree of phosphorylation.
Typically nucleotides are often separated by ion-exchange chromatography [7–9] or in some instances by reversed-phased HPLC
methods [7,10–14]. However, due to the high polarity of most
nucleotides retention is generally low with most C18 stationary phases. Thus, ion-exchange methods are more amenable to
the hydrophilic nature of these compounds. Samples of biological importance isolated from complex matrices are now frequently
analyzed utilizing mass spectrometry as the means of detection. The anion-exchange methods developed for the separation
of nucleotides are generally not suitable for MS detection due to
the high concentrations of buffers and salts which are usually not
volatile. Thus newer approaches which are compatible with either
optical or MS detection are desirable.
Silica hydride-based HPLC stationary phases were first proposed
as an alternative to other types of silica materials more than 10 years
ago. A large number of studies have confirmed that they possess
∗ Corresponding author.
E-mail address: [email protected] (J.J. Pesek).
0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.12.046
both high stability and a broad range of chromatographic properties
that are unique when compared to other separation media [15–18].
One of the desirable features that has been identified for hydride
separation materials is their ability to be used over a wide range of
mobile phase compositions from 100% aqueous to pure nonpolar
organic solvents. Therefore, these stationary phases can function in
high water (reversed-phase), high organic with some water present
(aqueous normal-phase) and pure organic (organic normal-phase)
mobile phases. The reversed-phase (RP) and aqueous normal-phase
(ANP) modes are highly complementary because water is the common component in the mobile phase. With silica hydride-based
stationary phases it is possible to rapidly change from RP to ANP due
to the rapid equilibration of these separation materials and in some
cases both mechanisms operate simultaneously thus retaining both
hydrophobic (by reversed-phase) and hydrophilic (by aqueous normal phase) compounds in a single isocratic run [17]. There have
been a number of silica hydride-based stationary phases studied
so it is now known that the relative amount of reversed-phase and
aqueous normal-phase capabilities can be adjusted by varying the
surface composition [17]. The surface composition that determines
the retention properties is a combination of the base silica hydride
and the organic moiety attached to it. This investigation has utilized several types of silica hydride separation materials in order
to determine the type and extent of organic moiety surface coverage that would provide the best ANP retention capabilities for
nucleotides. The designation of ANP for the retention of polar compounds, such as nucleotides, is used in this report to distinguish
silica hydride materials from HILIC stationary phases. While silica
hydride phases can make the transition from ANP to RP by the addition of water to the mobile phase HILIC materials do not have this
capability and thus can only retain polar compounds. In addition,
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J.J. Pesek et al. / J. Chromatogr. A 1216 (2009) 1140–1146
the experimental conditions in the methods reported are suitable
for detection by either optical or mass spectroscopic techniques.
2. Experimental
2.1. Materials
The undecenoic acid (UDA) stationary phase was synthesized
in house and packed into a 150 mm × 4.6 mm column. The other
silica hydride stationary phases used in this study were the
Cogent Type-C columns in 150 mm × 1.0 mm, 100 mm × 2.1 mm and
75 mm × 4.6 mm I.D. from MicroSolv Technology (Eatontown, NJ,
USA). The Diamond Hydride (DH) phase contains a small amount
of an organic moiety (∼2% carbon as reported by the manufacturer) on a silica hydride surface. All hydride columns were made
with 4.0 ␮m silica (Astrosil, Stellar Phases, Langehorn, PA, USA).
A common commercial C18 column (Agilent Eclipse, Wilmington,
DE, USA, 4.6 mm × 150 mm I.D.) was included in initial screening tests to provide a comparison for the reversed-phase hydride
materials. The nucleotides adenosine-3 ,5 -cyclic monophosphate; adenosine-5 -monophosphate; adenosine-5 -triphosphate;
thymidine-5 -triphosphate; uridine-5 -triphosphate; cytosine-5 triphosphate; guanosine-5 -triphosphate were purchased from
Sigma–Aldrich (Milwaukee, WI, USA). Samples for UV detection
were made at 100 ␮g/mL and for MS detection at 10 ␮g/mL. Solvents and buffer components for the mobile phase were obtained
in the highest purity available.
2.2. Instrumentation
All HPLC investigations utilized an Agilent (Little Falls, DE,
USA) 1100 Series LC system, including degasser, binary pump,
temperature-controlled autosampler and temperature-controlled
column compartment. The UV detector was a diode array (DAD)
system (Agilent Model DAD SL G1315C). For LC–MS, the mass spectrometer was an Agilent (Santa Clara, CA, USA) Model 6210 MSD
time-of-flight (TOF) with a dual sprayer electrospray source (ESI).
2.3. Methods
The undecenoic stationary phase was synthesized using a process that has been previously reported [19,20]. The column was
packed into a 75 mm × 4.6 mm I.D. column using a 90:10 carbon
tetrachloride/methanol slurry with methanol as the driving solvent.
Stock solutions of the nucleotides were made in deionized (DI)
water in the range of 0.2–0.7 mg/mL. Sample solutions were made
by diluting the stock in 50:50 acetonitrile/water containing 0.1%
ammonium formate. The mobile phase organic solvent was composed of 0.1% ammonium formate in acetonitrile. Water containing
0.1% ammonium formate made up the difference. The column flow
rate was 1.0 mL/min. The column temperature was 20 ◦ C. The gradients used in this study are designated in each figure caption.
For gradient experiments the flow rate was 1.0 mL/min with UV
detection and 0.4 mL/min for MS operation. The equilibration time
between successive runs was 5 min. For repeatability studies, the
number of replicates performed under each condition tested was
noted.
3. Results and discussion
3.1. Reversed-phase sorbents under ANP conditions
It has been demonstrated that a hydride-based column
designed for reversed-phase through the bonding of a hydrophobic moiety such as C18 can also function in the ANP mode
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[17]. Most columns based on ordinary silica do not have this
capability. In order to test this concept, a mixture of seven
nucleotides (adenosine-3 ,5 -cyclic monophosphate; adenosine-5 monophosphate;
adenosine-5 -triphosphate;
thymidine-5 triphosphate; uridine-5 -triphosphate; cytosine-5 -triphosphate;
guanosine-5 -triphosphate) was analyzed on several columns with
typical reversed-phase properties. The first examined was a commercial C18 based on organosilane bonding of octadecyl groups to
silica. Fig. 1A shows the result of testing this column using a type of
ANP gradient that was successful in the retention and separation
of other polar compounds [21]. As seen in the figure, all of the
components of this mixture co-elute at essentially the void volume
of the column. Using the same gradient on a shorter hydride-based
C18 column produced some retention of the components as seen
(Fig. 1B) as a separation between the baseline fluctuations at the
void volume and the two peaks for the nucleotides. While some
retention was achieved on the hydride-based C18, the selectivity
was only partially sufficient to resolve some of the components in
the mixture under these mobile phase conditions. A third column,
a hydride-based cholesterol-bonded phase, with significant RP
properties was also tested using a slightly longer gradient profile.
Under these conditions more retention of the nucleotides (Fig. 1C)
and somewhat better resolution of the four components were
achieved. The longer gradient on the standard silica-based C18
column showed neither improved retention nor any resolution of
the components while the hydride-based C18 had longer retention
but no significant improvement of resolution. For nucleotides,
the hydride-based stationary phases with hydrophobic groups
produce retention under ANP conditions, but the selectivity was
not high, at least under the conditions tested in this study.
3.2. Diamond Hydride column
The Diamond Hydride stationary phase is essentially a silica
hydride surface with a low coverage of organic moiety attached. A
previous study has demonstrated that this sorbent can be used for
the separation of metabolites such as amino acids, carbohydrates
and small organic acids in the ANP mode [21]. Thus, it is a good
choice for evaluation with nucleotides. A number of monophosphate and triphosphate nucleotides were analyzed under isocractic
conditions using acetonitrile/water mobile phases at high organic
content to achieve ANP conditions. In addition, various mobile
phase additives were evaluated including acetic acid, formic acid,
ammonium formate and ammonium acetate. Ammonium formate
at 0.1% (w/v) consistently gave the best peak shape but in order to
reduce the analysis time, and to produce better peak symmetry and
efficiency it was necessary to employ gradient elution conditions.
Fig. 2 shows the separation of a mixture of two monophosphate and
two triphosphate nucleotides using a simple linear gradient from
95% to 70% acetonitrile over 10 min. Under these conditions the
total analysis time was less than 10 min and the peak shape is good.
Two small impurities, possibly other triphosphate nucleotides, are
also seen in the chromatogram. These peaks could be due to either
contamination or degradation but no attempt was made to identify
them.
As shown in Fig. 2, the most strongly retained compounds were
the triphoshates (as predicted by the ANP mechanism) and some
tailing can occur depending on the mobile phase additive and gradient selected. Fig. 3A shows an example of the separation of the three
adenosine analytes using a gradient that was only slightly different
to the one employed to achieve the results shown in Fig. 2. The gradient starts at 90% acetonitrile in the mobile phase but also goes to
70% over 10 min thus making it not as steep as the gradient used
for the chromatogram in Fig. 2. The last component, adenosine 5 triphosphate, displays noticeable tailing in comparison to the first
two compounds. An improvement in peak shape can be achieved
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Fig. 2. Separation of nucleotide mixture on Cogent Diamond Hydride column.
Dimensions: 2.1 mm × 100 mm. Flow rate = 0.3 mL/min Detection at 254 nm. Gradient: 0.0 min 95% B, 0.0–10.0 min to 70% B. Mobile phase conditions are the same
as in Fig. 1. Solutes: 1 = adenosine-3 ,5 -cyclic monophosphate; 2 = adenosine-5 monophosphate; 3 = uridine-5 -triphosphate; 4 = cytosine-5 -triphosphate.
by adding a small amount of ammonia to the sample (Fig. 3B). The
amount used here (5 ␮L of 12% ammonia/mL) does not appreciably affect the retention times. The improvement of peak shape by
the addition of ammonia is consistent with previous results for
various organic acids and amino acids where efficiency and peak
symmetry are dependent upon the acidity of the solution [21]. In
particular, peak shape improved considerably for organic acids such
as fumaric, maleic and citric as the pH was raised by changing
the mobile phase from formic acid to ammonium formate and by
including NH4 OOCH in the sample solvent. Under more acidic conditions it is likely that a mixed retention mechanism takes places as
both protonated and unprotonated acid solute species are present.
These results suggest that mass transfer of the solute between the
mobile phase and the stationary phase is improved as the solution
conditions favor the greatest amount of unprotonated species.
3.3. Undecenoic acid column
The retention capabilities of the UDA column are the result of
both hydrophilic (hydride surface and carboxylic acid functionality) and hydrophobic (alkyl chain of bonded acid) properties.
With its greater hydrophobic nature, retention and selectivity for
nucleotides on the UDA column should be different than the Diamond Hydride in the ANP mode. These differences are illustrated
in Fig. 4 for the separation of seven nucleotides on the UDA column
under isocratic conditions. At 80% ACN in the mobile phase (Fig. 4A),
good retention was obtained with one pair of analytes (thymidine triphosphate and adenosine triphosphate) not resolved. If the
organic content of the mobile phase was raised to 85%, then retention became longer and the two previously unresolved components
were separated. This result is in contrast to the data obtained with
the Diamond Hydride column where complete resolution of this
mixture was not successful under isocratic conditions and required
Fig. 1. Chromatograms for gradient elution of a seven-component nucleotide
mixture on reversed-phased columns. (A), Agilent Eclipse, (B), Cogent Bidentate
C18 and (C) Cogent UDC Cholesterol. Detection at 254 nm. Gradient for A and B:
0.0 min 90% B, 0.0–10.0 min to 70% B. Gradient for C: 0.0–0.5 min 95% B, 0.5–20 min
to 30% B, 20.0–25.0 min hold 30% B. Mobile phase: A, DI water + 0.1% ammonium
formate; B, 90% acetonitrile/10% DI water + 0.1% ammonium formate. Solutes:
1 = adenosine-3 ,5 -cyclic
monophosphate;
2 = adenosine-5 -monophosphate;
3 = adenosine-5 -triphosphate;
4 = thymidine-5 -triphosphate;
5 = uridine-5 triphosphate; 6 = cytosine-5 -triphosphate; 7 = guanosine-5 -triphosphate.
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Fig. 3. Separation of three adenosine compounds on the Cogent Diamond Hydride
column. (A), Sample without ammonia and (B), sample with ammonia. Column and conditions same as Fig. 2. Gradient conditions are the same as
in Fig. 1A. Solutes: 1 = adenosine-3 ,5 -cyclic monophosphate; 2 = adenosine-5 monophosphate; 3 = adenosine-5 -triphosphate.
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Fig. 4. Isocratic separation of seven-component mixture of nucleotides on the undecenoic acid column. (A), 80:20 90% acetonitrile/10% DI water + 0.1% ammonium
formate/DI water + 0.1% ammonium formate and (B), 85:15 90% acetonitrile/10%
DI water + 0.1% ammonium formate/DI water + 0.1% ammonium formate. Column:
4.6 mm × 75 mm. Flow rate 1 mL/min. Detection at 254 nm. Solutes same as Fig. 1.
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Fig. 5. Gradient separation of seven-component mixture of nucleotides on the undecenoic acid column. Column and solutes are the same as given in the legend to Fig. 4,
gradient same as Fig. 1A.
a gradient. The improved resolution is most likely due to the
enhanced interaction of the nitrogen sites on the nucleotides with
the stationary phase provided by the carboxylic acid functionality.
The analysis time with the UDA column can be reduced but with
some loss in resolution by using the same gradient as in Fig. 1A.
The small shoulder on peak 5 is likely the small peak (identity not
determined) in Fig. 4. The results under these experimental conditions are shown in Fig. 5. While this may not be a practical solution
when using UV detection as shown, if quantitative determinations
are necessary, such resolution is certainly practical for MS detection. Adequate retention of all the compounds was obtained and
the m/z values for the (M−H)− ions of each of these compounds,
generated by ESI under these mobile phase conditions, were sufficiently different so that unambiguous identification can be made
via the extracted ion chromatogram (EIC). The closest values in m/z
for the compounds in this mixture differ by 2 amu (480 and 482)
which is more than sufficient for even a single quadrupole MS to
resolve at this mass/charge ratio.
The presence of ammonia in the sample solution also has a positive effect on peak shape for the UDA column in a manner similar
to that observed for the DH column described above (Fig. 3B). Fig. 6
shows a comparison of the separation of five nucleotides using
the same gradient where the only difference was that the sample
solution was without (Fig. 6A) or with (Fig. 6B) ammonia added. A
distinct improvement in peak shape and resolution was obtained
for the three triphosphates which were poorly resolved when there
was no ammonia in the sample solution. In this case retention
increased slightly due to the fact that a higher amount (twice the
amount used in Fig. 3B) shifts the sample further to the more unprotonated state and may also have resulted in greater ionization of
the carboxylic acid functionality of the bonded moiety. The process
of ANP retention on the hydride-based phases is not fully understood but more detailed investigations are currently underway to
elucidate the exact mechanism.
A comparison was also made between the UDA and DH columns
using flow rate conditions that gave approximately equivalent
retention times. All other conditions, gradient, mobile phase components and sample solvent, were held the same for both columns.
This comparison is shown in Fig. 7. As can be seen resolution and
efficiency are better on the UDA column (Fig. 7B) than on the DH
column (Fig. 7A). This result does not imply that the UDA is superior
under all conditions or that the DH separation of this mixture could
not be improved by further method optimization.
Fig. 6. Gradient separation of five-component mixture of nucleotides on the
undecenoic acid column. (A), Sample without ammonia and (B), sample with ammonia. Gradient same as Fig. 1A. Other conditions are the same as given in the
legend to Fig. 4. Solutes: 1 = adenosine-3 ,5 -cyclic monophosphate; 2 = adenosine5 -monophosphate; 3 = adenosine-5 -triphosphate; 4 = thymidine-5 -triphosphate;
5 = uridine-5 -triphosphate.
A final test on the UDA column involved the repeatability of
consecutive sample injections which is shown in Fig. 8. Five representative runs of the seven-component nucleotide sample run
under isocratic conditions on the UDA column are shown. As can be
seen, the chromatograms are highly repeatable and thus the column
provided stable operation for consecutive analyses under the same
experimental conditions. If a gradient method was used, consecutive runs also resulted in chromatograms with retention times for
the same peak with RSD values <0.5%. Similar repeatability results
were also obtained on the DH for various nucleotide samples using
both isocratic and gradient methods.
3.4. LC–MS applications
Many samples, particularly of physiological origin, are complex
and contain many constituents in low concentration. Thus for many
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Fig. 8. Repeatability of isocratic separation of seven-component mixture of
nucleotides on the undecenoic acid column. Conditions and solutes same as Fig. 4B.
Fig. 7. Gradient separation of five-component mixture of nucleotides on: (A), Cogent
Diamond Hydride column (4.6 mm × 75 mm) and (B), the undecenoic acid column (1.0 mm × 100 mm). Gradient same as Fig. 1A. Flow rates A = 0.3 mL/min and
B = 1.0 mL/min. Solutes are the same as given in the legend to Fig. 6.
analyses LC–MS is a logical choice to facilitate both identification
of the individual components in the sample and operation at low
detection limits. The total ion chromatogram (TIC) for simultaneous
detection of all species is often of limited use with these samples
since with a large number of analyte peaks significant overlapping and co-elution will usually occur. A better approach under
these conditions is to use either the EIC at a specific m/z or a tandem MS/MS spectrometer system for detection. If a TOF or other
high accuracy mass analyzer is used, the number of peaks in each
EIC is limited thus improving the chances for positive identification of each analyte. In an MS/MS system, the use of parent and
daughter ion masses for each compound also enhances the probability of correctly identifying a particular solute. For nucleotides
where good retention can be obtained using either the DH or UDA
columns, the main challenge when utilizing MS for detection is
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Fig. 9. LC–MS EIC for isobaric nucleotide species on the DH column: (A), m/z = 506 solutes: 1 = adenosine triphosphate and 2 = deoxycytidine-5 -monophosphate; (B), m/z = 346
solutes: 1 = adenosine-3 -monophosphate and 2 = adenosine-5 -monophosphate. Column: 2.1 × 150 mm. Flow rate 0.4 mL/min. Mobile Phase: A, DI water + 15 mM ammonium
acetate and B, 90% acetonitrile + 10% water + 15 mM ammonium acetate. Gradient: 0.00 min 95% B; 0.00–1.00 min to 90% B; 1.00–3.00 to 80% B; 3.00–4.00 min hold 80% B;
4.00–5.00 min to 50% B; 5.00–6.00 min hold 50% B; 6.00–7.00 min to 20% B.
the separation of isobaric compounds. Fig. 9 provides examples
of two separations with each involving a pair of analytes having the same m/z on a DH column. The EIC for each analysis was
obtained with a TOF mass analyzer in the negative ion mode and
the chromatographic gradient used was the same in each separation. Fig. 9A shows the separation of adenosine triphosphate
and deoxycytidine-5 -monophosphate (m/z for (M−H)− = 506). The
identity of each peak was established by injection of each compound individually although the TOF system provides sufficient
mass accuracy to distinguish the two compounds by their exact
m/z. In Fig. 9B, the more challenging pair of solutes, adenosine-3 monophosphate and adenosine-5 -monophosphate, with an m/z of
346 for the (M−H)− ion, are separated. The identity of each species
was confirmed by injection of the individual compounds which was
essential since the molecular masses of these two analytes are identical and thus indistinguishable using a TOF analyzer. The necessity
of having a suitable chromatographic method is also illustrated in
these examples since positive identification is facilitated by their
separation. These chromatograms also demonstrate another advantage of MS detection: the flat baseline which was obtained in the
EIC for a gradient elution. The experimental conditions used in the
LC–MS analyses described above result in both good peak shape and
high efficiency (∼200,000 plates/m). In addition, the ANP method
provides high sensitivity since a better S/N ratio is obtained for high
percentages of organic in the mobile phase [21].
4. Conclusions
In this study, it has been demonstrated that silica hydride-based
stationary phases with either a carboxylic acid functionality bonded
to the surface or a small amount of a hydrophobic moiety (2%) on the
silica hydride surface leads to good retention and separation capabilities for nucleotides. By adjusting the pH and ionic strength with
ammonium formate as the mobile phase additive, the nucleotide
polarity is sufficiently high (negatively charged) leading to ANP
retention on these columns. In addition the presence of ammonium formate is compatible with detection by mass spectroscopy.
Both isocratic and gradient elution can be used depending on the
constituents in the sample. Repeatability in both gradient and iso-
cratic modes is excellent. The experimental conditions described
here have also been shown to be applicable to LC–MS operation.
Acknowledgements
The financial support of the National Science Foundation (CHE
0724218) is gratefully acknowledged. J.J.P. would like to acknowledge the support of the Camille and Henry Dreyfus Foundation
through a Scholar Award. The authors would like to thank MicroSolv
Technology Corporation for donation of the columns used in this
study and Steve Fischer from Agilent Technologies in Santa Clara, CA
for his valuable contributions to the LC–MS experiments. The financial support of the Australian Research Council is also gratefully
acknowledged.
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