Professor Joseph J. Pesek Department of Chemistry San Jose State University

Transcription

Professor Joseph J. Pesek Department of Chemistry San Jose State University
A White Paper:
Why Everyone Should Have Silica Hydride Based HPLC Columns in Their Lab
Professor Joseph J. Pesek
Department of Chemistry
San Jose State University
San Jose, CA 95112
Dr. Maria T. Matyska
MicroSolv Technology Corporation
1 Industrial Way West, Bldg. E
Eatontown, NJ 07724
Abstract
The usefulness of silica hydride-based HPLC stationary phases, commercially
available today, is described and a number of unique features of these materials are
illustrated. These stationary phases can be used for a broad range of applications
including traditional reversed-phase (with unique selectivity) as well as both aqueous and
organic normal phase analyses. This suite of columns is becoming a very popular tool for
orthogonal data during method development. The ability to analyze hydrophobic and
hydrophilic compounds in the same run, to separate basic compounds at low pH and for
methods with very high organic mobile phases is especially interesting for use with mass
spectroscopy as a detector. Several types of phases are available so the correct choice of
separation material can be made depending on the degree of hydrophobic/hydrophilic
selectivity required.
Introduction
Silica-hydride based HPLC stationary phases have been investigated over the last
ten years and possess both high stability and a broad range of chromatographic properties
that are unmatched by any other single separation medium (1,2). A number of these
features are especially attractive when coupling HPLC to mass spectroscopy. These
separation materials span the range of mobile phase compositions from 100% aqueous to
pure non polar organic solvents. Thus they can be used at high water (reversed-phase),
high organic with some water present (aqueous normal phase) and pure organic (organic
normal phase). The reversed-phase and aqueous normal phase modes are highly
complementary and it is possible to rapidly move between them with the silica hydridebased stationary phases due to the rapid equilibration of these separation materials and in
some cases both mechanisms operate simultaneously thus retaining both hydrophobic and
hydrophilic compounds in a single isocratic run. Because there are several types of silica
hydride-based stationary phases currently available, it is possible to adjust the relative
amount of reversed-phase and aqueous normal phase capabilities by varying the surface
composition. The surface composition is a combination of the base silica-hydride and the
organic moiety attached to it. There are currently five different types of silica hydride
stationary phases to consider when assessing particular separation goals. The materials
are in order of increasing hydrophobicity: Silica-C™ (silica hydride only surface);
Diamond Hydride™ (a small amount of carbon on the silica hydride surface); UDCCholesterol™; Bidentate C8™(octyl); and Bidentate C18 (octadecyl) .
Experimental
Columns: All of the columns used in this study were obtained from MicroSolv
Technology (Eatontown, NJ) www.mtc-usa.com. Column sizes ranged from 75 to 150
mm all with a standard 4.6 mm id. for UV detection and with 2.1 mm i.d. for mass
spectroscopy detection.
Instrumentation: All LC experiments were conducted on either an Agilent 1050 HPLC
system equipped with a diode arrary detector or an Agilent 1050 HPLC system interfaced
to a Waters Micromass Platform LC mass spectrometer having ESI and APCI sources with
0
Masslynx 3.4 for data analysis. Temperature was maintained at 25 C for all experiments.
Results and Discussion
The Silica-C™ column has the greatest retention capabilities for hydrophilic
compounds since there are no organic groups attached to the hydride surface and thus is
most suitable for aqueous normal phase (ANP) operation. It also possesses good
selectivity for certain functional groups like hydroxyl, carbonyl, carboxylic acid, and
halides so it is also a suitable separation material for organic normal phase separations.
Amino acids are difficult to retain on typical reversed phase columns and generally elute
at or near the dead volume. The only choice is to derivatize these compounds and make
them more hydrophobic. This adds an additional step to the analysis and is a source of
error that can lead to non-reproducible results. With underivatized amino acids, HILIC
columns have been used for retention. However, HILIC depends on a uniform and
consistent water layer on the surface for reproducible results (3). This is often difficult to
achieve and is probably the main reason some laboratories report poor reproducibility on
HILIC columns for the analysis of many hydrophilic compounds from run to run. Figure
1 shows the separation of phenylglycine and phenylalanine on the Silica-C™ column.
Repeated injections of these and other amino acids give RSD values less than 0.3% in
both isocractic and gradient methods.
An even more versatile material for the analysis of hydrophilic compounds is the
new Cogent Diamond Hydride™. With a small amount of carbon engineered onto the
silica-hydride surface, the polar retention properties are diminished slightly with respect
to Silica-C™. An example of the retention capabilities of this stationary phase is shown
in Figure 2 for the separation of adenosine and guanine in the ANP mode. When this
mixture is injected ten consecutive times, the RSD of the retention for these two
compounds is less 0.1%. Another important feature of the Diamond Hydride™ is the
relatively low amount of TFA in the mobile phase needed to maintain excellent peak
shape; in this case only 0.001%. This extremely low concentration of TFA is particularly
important when using mass spectroscopy for detection since the amount typically used on
other phases is 0.1% which can significantly depress the MS signal and lower sensitivity.
The Diamond Hydride™ can be used for a wide variety of metabolites including amino
acids, small organic acids and carbohydrates. Both the Silica-C™ and the Diamond
Hydride™ do not have a strong affinity for water, which leads to the high reproducibility
and fast equilibration after gradients in the ANP mode in comparison to HILIC. The lack
of water affinity also accounts for the fact that both of these materials are excellent
choices for organic normal phase (ONP) methods. Water does not have to be added to
the mobile phase in trace amounts as is common practice with typical stationary phases
used in ONP methods (silica, amino, cyano and diol) and this feature also makes
gradients more feasible in this mode of operation.
The attachment of bonded organic groups to the hydride surface results in more
hydrophobic retention and a diminishing, but not elimination, of the ANP retention. An
example of ANP retention with a bonded organic group is shown in Figure 3 for the
retention of the drug Tobramycin™ on a silica hydride-based UDC-Cholesterol™ phase.
The drug is highly polar with five amine groups and five hydroxyls. The peak shape is
very symmetric and the retention, as on the Silica-C™ and Diamond Hydride™ phases is
quite reproducible. Attempts to run this compound on several types of typical reversedphase columns resulted in very low retention and significant tailing, even for end capped
materials. The interesting feature of silica hydride-based materials is that the addition of
organic groups on the surface result in the attainment of reversed-phase (RP) properties.
The extent of RP retention depends on the type of moiety bonded to the surface. In this
case the reversed-phase properties of the UDC-Cholesterol stationary phases are shown
in Figure 4 where a mixture of steroids is separated in a mobile phase consisting of 50:50
methanol/water. This is a typical RP mobile phase composition. Detection is by mass
spectroscopy using the APCI+ mode. In addition to its ANP and RP retention
capabilities, the UDC-Cholesterol™ bonded material also has good shape selectivity for
such compounds as polycyclic aromatic hydrocarbons and steroids. This feature is a
result of the liquid crystal properties of cholesterol, some of which are preserved even
when bonded to a silica surface.
Of the other commercially available silica hydride-based phases, the Bidentate
C8™ is slightly more hydrophobic than the UDC-Cholesterol™ phase and the Bidentate
C18™ is the most hydrophobic. In general, a longer alkyl chain with no functional
groups produces greater hydrophobic retention. However, the presence of the silica
hydride surface imparts different properties to the material and hence selectivity is often
different than most other reversed-phase materials. This can be advantage in at least two
circumstances. First, different selectivity may allow for the resolution of analytes that
cannot be separated on other stationary phases with the same bonded group. Second, it
can provide a second (orthogonal) means for identifying one or more components in a
mixture. This capability is especially useful for impurity profiling, assays, drug
metabolism and stability indicating methods. A totally different column can provide the
confirmation for an analysis that is often required in quality control and regulatory
situations. The properties of more than four hundred stationary phases have been
evaluated including a large number of octadecyl materials (4). Their properties have
been cataloged and an examination of the silica hydride based phase shows that the
parameters used for determining column equivalency are much different for the hydride
materials than many others thus making it possible for their use in the two situations
described above.
The versatility of the silica hydride-based C18 is shown in the next two figures.
In Figure 5 a mixture containing both hydrophilic and hydrophobic compounds is
separated on the Bidentate C18™ column. Under the mobile phase conditions used
(60:40 acetonitrile/water) the reversed-phase mechanism is dominant since the first three
compounds are hydrophilic and the last four are hydrophobic. When the amount of
acetonitrile is increased, the two groups switch position as the ANP retention becomes
stronger and the RP effects are diminished. The chromatogram shown represents the best
resolution of this mixture. However, in other samples using a mobile phase where ANP
retention dominates will produce the best resolution. The excellent RP capabilities of the
Bidentate C18™ silica hydride-based phase are shown in Figure 6 where a group of
carbohydrate isomers are separated in a 100% aqueous mobile phase. The excellent
reproducibility of this phase is illustrated by comparing the 1st and 10th injections, which
are essentially identical. The identity of the structural isomers is accomplished through
mass spectroscopy since each compound has unique fragment ions.
Another significant advantage of using mass spectroscopy for detection in
combination with the ANP mode is the expansion of solvent choices. For example, it has
been demonstrated that acetone is a very useful solvent for ANP applications. It has
different solvent properties than acetonitrile and hence different selectivity. With UV
detection acetone is not a suitable mobile phase component due to its own strong
absorption a lower wavelengths. Thus an expansion of separation capabilities is possible
when the detector is not sensitive to the mobile phase composition. Another example of
the same insensitivity to the solvent is the evaporative light scattering detector (ELSD).
From a practical point, the other common solvent in aqueous based mobile phases is
methanol. However, methanol being more polar than acetonitrile or acetone generally
produces less ANP retention than these two solvents. ANP behavior can be observed for
strongly polar (usually basic) compounds like tobramycin with methanol. The ANP
mode offers another significant advantage with respect to mass spectroscopy detection.
At higher amounts of organic in the mobile phase, sensitivity increases in MS due to
lower noise in the spectrum. This can sometimes result in up an order of magnitude
improvement in the lower limit of detection. Finally, ANP is a more instrument friendly
method for the analysis of basic compounds than reversed phase. In RP methods the pH
must be increased to the range of 8-10, which can lead to not only shorter column
lifetimes but more importantly an increase in the amount of instrument down time due to
replacement of valves, seals and other parts degraded by exposure to basic mobile phases.
Conclusions
Silica hydride-based stationary phases are emerging as a unique type of separation
material that provides analytical capabilities not available in a single type of ordinary
bonded silica phase, and in some instances not in any other commercial column. The
type and degree of selectivity can be controlled by starting with the base material (SilicaC) and increasing the amount of hydrophobicity on the surface by increasing the amount
of bonded material (either via higher surface coverage or using larger molecular weight
hydrocarbon chains). Thus the selectivity changes from strong retention for hydrophilic
compounds to increased retention for hydrophobic compounds when the surface has a
high amount of C18 bonded. However, the ANP mode does not disappear even for the
octadecyl phase so this stationary phase can provide significant retention for both
hydrophobic and hydrophilic compounds as the mobile phase composition spans the
range from 100% aqueous to essentially pure organic. For these reasons, and others,
many labs worldwide have adopted these columns to challenge every method they
develop, for complex mixtures, for compounds not typically retained in RP and as a way
to extend the range and reach of each and every HPLC method.
References
(1) J.J. Pesek and M.T. Matyska, J. Sep. Sci. 28, 1845–1854 (2005).
(2) J.J..Pesek and M.T. Matyska, J. Liq. Chromatogr & Rel. Technol. 29, 1105–1124
(2006).
(3) D.L. Roush, L.Y. Hwang and F.D. Anita, J. Chromatogr A 1098, 55–65 (2005).
(4) http://www.usp.org/USPNF/columns.html
Figure 1
Figure 1. Separation of phenylglycine (1) and phenylalanine (2) using a Silica-C
hydride based stationary phase. Mobile phase: 80:20 acetonitrile/DI water + 0.5%
formic acid. Detection at 254 nm.
Figure 2
Figure 2. Separation of adenosine (1) and guanine (2) using a Diamond Hydride™ silica
column. Mobile phase: 80:20 acetonitrile/DI water + 0.001% TFA. Detection at 232
nm.
Figure 3
Figure 3. Separation of uracil and tobramycin on a silica hydride-based cholesterol
column. Mobile phase: 70:30 acetonitrile/DI water + 0.5% formic acid. Detection by
mass spectrometry using the APCI+ ionization mode with single ion monitoring.
Figure 4
5.00
10.00 15.00
20.00 25.00 30.00 35.00 40.00 45.00 50.00 Time (min)
Figure 4. Separation of a six steroid component mixture on a silica hydride-based
cholesterol column. Mobile phase 50:50 methanol/DI water. Detection by mass
spectroscopy in the APCI+ ionization mode with single ion monitoring. Solutes: 13.29
min. = andrenosterone; 16.99 min = corticosterone; 27.46 min = 4 –androstene-3,17dione; 32.12 min = 11α-acetoxyprogesterone; 38.07 min. = estrone; and 48.89 min. =
estradiol
Figure 5
Figure 5. Separation of a seven-component mixture containing both polar and nonpolar
compounds on the Bidentate C18 silica hydride stationary phase. Mobile phase: 60:40
acetonitrile/water. Detection: 254 nm. Compounds 1-3 are polar and 4-7 are nonpolar.
Figure 6
Figure 6. Separation of carbohydrate isomers with MW = 505 on the Bidentate C18
silica hydride stationary phase using a 100% aqueous mobile phase. Detection by mass
spectroscopy in the APCI+ mode.