Use of Hybrid Particles in HPLC Applications at Extreme pH
Thomas Walter, Waters Corporation, Milford, Massachusetts, USA
Hybrid Particle Technology has given rise to an
improved class of chromatographic packing materials
designed to overcome some of the limitations of
existing silica-based reversed-phase columns. The
recommended useable range of pH 1-12 is far greater
than the useable range of pH 2-8 for classical silicabased reversed-phase columns. The new Xterra™
columns from Waters Corporation feature Hybrid
particle Technology (patent pending, Waters
Corporation). These columns have been shown to
exhibit outstanding performance within the traditional
pH range, especially for basic compounds, but
additionally allow you to break out of these barriers to a
wider range of pH 1-12. These columns combine the
broad useable pH range of Polymer-based packings
with the speed, peak shape and high temperature
stability available in the best of the most recent crop of
silica-based reverse phase C8/C18 columns.
The Problem of Extreme pH
Applications in Silica-based Columns
Silica-based reversed-phase packing materials have
historically delivered the best combination of desired
chromatographic properties under the most common
range of conditions. However, silica-based reversedphase columns:

Have short lifetimes when the mobile phase pH is
below pH 2 (the bonded phase may be stripped off)

Have short lifetimes when the mobile phase pH is
above pH 8 (the underlying silica support begins to
dissolve)

Give broad, tailing peaks for many basic
compounds

Exhibit short column life at high temperatures
Waters' new Hybrid Particle Technology exhibits an
extended useful pH range and dramatically improved
performance for basic compounds, without sacrificing
the benefits of silica-based chromatographic supports.
Waters’ new Hybrid Particle Technology is now
available in XTerra™ columns.
Silica-based Packings
Traditional silica-based reversed-phase packings are
manufactured using silane reagents to derivatize the
silanol groups on the surface of silica particles. Because
the silanes are much larger than the silanols, not all
silanols are derivatized. In addition, the small pores in
many silica particles limits ability of bulky reagents
such as octadecylsilanes to reach the surface. Surface
derivatization is always incomplete, and may leave
large areas of residual silanols on the unbonded
surface3. Ion-exchange interaction with the acidic
residual silanols cause peak tailing for basic
compounds.
The technology of high-efficiency silica-based packings
has advanced dramatically since their introduction in
1973. Consistency of particle size, pore size, and
bonded-phase coverage of the particle were important
early advances. Recent advances include the use of
high-purity silicas such as the Symmetry® family of
packings (1) and the incorporation of a polar functional
group into the ligand as applied in the
SymmetryShield™ packings (2) to improve peak
shapes. Despite these advances, silica still deteriorates
at alkaline pH values.
Polymer-based packings
Packings made from organic polymers such as
poly(divinylbenzene) are stable over a wide pH range,
but suffer from low plate counts and often from
distorted peaks. Polymer-based packings swell and
shrink in the presence of some solvents, resulting in
poor mechanical stability.
Hybrid Particle based packings
Hybrid organic-inorganic technology provides a novel
solution to the problems created by the shortcomings of
classical silica-based and polymer-based packing
materials. The new XTerra™ packing material from
Waters Chromatography is a Hybrid Particle that
combines the broad pH tolerance of polymer-based
packings with the full range of benefits of silica-based
packings.
The Hybrid Particle is an organic/inorganic hybrid(3).
These materials contain both inorganic (e.g., silica) and
organic (e.g., organosiloxane) elements, and share the
advantages of both.
Equation 1 illustrates the process first described by
Unger et al(4), and now used for creating Hybrid
Particles. A mixture of two high-purity monomers is
used: one forms SiO2 units during the particle formation
process and the other forms RSiO1.5 (organosiloxane)
units. The resulting particles contain organosiloxane
groups incorporated throughout their internal and
surface structure. These particles are then surfacebonded to attach reversed-phase groups (e.g., C18, C8).
(RO)4Si + n (RO)3SiR* + (1.5 n + 2) H2O 
SiO2(R*SiO1.5)n + (3 n + 4) ROH
The final particle contains an alkyl group (R*) that is
incorporated into the matrix of the packing. This
approach yields a material that contains both inorganic
units (SiO2) and organic units (R*SiO1.5), combined at
the molecular level. In the hybrid XTerra™ packing
material, every third silicon atom in the matrix is
substituted with a methyl group. The carbon content of
the packing was determined to be about 7% carbon
before surface modifications.
1)
The packing is as hard as silica and does not swell or
shrink in the presence of organic solvents. The pH
stability of bonded phases based on these Hybrid
Particles exceeds that of silica-based bonded phases.
More Than a Surface Modification
The molecular structure of the entire Hybrid Particle is
a mixture of organic and inorganic groups. The result is
a fundamental improvement in the composition of the
underlying chromatographic particle. The
polymerization process is controlled to create a particle
with the right organic/inorganic balance. Too much
organic material, and the particle behaves like a
polymer – low mechanical strength, low efficiency. Too
much inorganic material and the particle behaves like a
silica – too many unbonded silanol groups that can
cause problems with peak shape for bases and poor
stability when used with aggressive mobile phases.
Hybrid Particles can be manufactured with pore sizes as
small as 2.5 µm, to allow extremely high speed
separations.
Performance of Hybrid Particle Packed
Columns
An important feature of the Hybrid Particle packings is
their significantly improved pH stability compared to
classical silica-based packings.
The stability of Hybrid Particle packings has been
investigated at both acidic and basic pH.
Performance at High Temperature and
Low pH
The improved stability of Hybrid Particles enables the
use of elevated temperatures for extended periods
without the degradation of column performance
exhibited by classical silica-based packings.
The low-pH stability of silica-based reversed-phase
columns is determined by two factors: the rate of
cleavage of the siloxane (Si-O-Si) bonds that attach the
bonded phase to the particle, and the rate of removal of
the products of this cleavage by the mobile phase. For a
surface bonded Hybrid Particle, the stability of the
bonded phase and the stability of the integral
methylsiloxane groups are both critical. In the Waters
Xterra Hybrid Particle, most of the methylsiloxane units
are attached by three siloxane bonds. These
methylsiloxane groups have exceptional low-pH
stability. Even after exposure to 1 M HCl at 100 °C for
16 hrs, no loss of methyl groups was detected. The
bonded phase in the XTerra MS C18 particle also
showed excellent stability at low pH, resulting from the
use of a trifunctional silane(5).
Waters XTerra MS C18 Hybrid Particle columns were
exposed to 1% trifluoroacetic acid at pH 1.2 at 50° C,
then tested the columns with a standardized test
condition. The Hybrid Particle packing worked as well
as or better than classical silica-based packings under
acidic conditions(6).
Performance for Basic Compounds
The peak shapes obtained for strongly basic compounds
in reversed-phase HPLC are dependent upon the
concentration and the acidity of the residual silanol
groups on the packing. Steric hindrance limits the extent
of derivatization for traditional surface bonded
packings(7). Because the new Hybrid Particles contain
methylsiloxane groups in place of roughly a third of the
siloxane units, the concentrations of residual silanols
are much reduced, enabling bonded phases based on
Hybrid Particles to deliver exceptional peak shape for
basic analytes.
High-pH Stability
Silica-based packings fail rapidly at very high pH
values. The lifetime of columns packed with 5 µm
Hybrid Particle was tested at pH 11.5 using a
pyrrolidine buffer. The column was run continuously at
30° C at 1 mL/min, and a mixture of basic analytes was
injected continuously. The column lasted for over 45
days without deterioration of plate count and peak
asymmetry or shifts in retention (other than those due to
changes in mobile phase composition)(8).
The high-pH stability of silica-based reversed-phase
columns is determined by the rate of dissolution of the
underlying silica particle (9). After dissolution has
proceeded to a critical point, the packed bed abruptly
collapses, causing voids and a catastrophic loss of
efficiency.
Because dissolution requires access of hydroxyl ions to
the silica surface, the rate of dissolution depends on the
amount of underivatized silica surface. Bonded phases
based on Hybrid Particles have an extremely low area
of underivatized silica surface because of the
methylsiloxane units incorporated throughout their
structure. Accordingly, Hybrid Particle columns
containing these particles show exceptional lifetimes in
high-pH mobile phases.
Walter et al compared the stability of a Hybrid Particle
Waters Xterra MS C18 column to a series of benchmark
silica-based columns. The test columns were heated to
50 °C and exposed to a mobile phase containing a pH
10 triethylamine (TEA) buffer. The columns were
periodically flushed with water and methanol and tested
for efficiency with acenaphthene. The XTerra MS C18
column lasted twice as long as the best benchmark
columns (6).
with 2.5 µm Hybrid Particles it is possible to maintain
maximum efficiency and achieve high throughput. The
result is significantly reduced gradient run times with
maximum resolution.
Conclusion
Hybrid Particle technology extends the advantages of
silica-based HPLC packings to cover the pH range from
pH 1 to pH 12 with no loss of efficiency or capacity.
The surface chemistry of Hybrid Particle packings is
identical to the surface chemistry of classical silicas.
This makes the use of the packing as simple as the use
of any other reversed-phase packing. The pH stability of
Hybrid Particle packings extends the pH range
accessible for reversed-phase packings. This opens up
new options in the manipulation of the selectivity of a
separation. The increased pH stability also results in
improved ruggedness of the packing under normal
operating conditions.
Exploiting the Extended pH range
A pH shift is a very powerful tool for separating
analytes of different charge. For example, ibuprofen and
toluamide overlap at alkaline pH, but a shift to a weakly
acidic pH results in a clean separation. Even for
compounds of the same charge, pH shifts can change
the elution order. At acidic pH, doxepin elutes before
imipramine and nortriptyline. Conversely, at alkaline
pH nortriptyline and doxepin elute very close to each
other, and imipramine elutes last. The best separation
between the three tricyclic antidepressants occurs
around pH 7 and pH 8.
While the recommended range for silica-based packings
is normally pH 2 to pH 8, Hybrid Particle packings can
be used from pH 1 to pH 12. This expands the range of
mobile phase conditions available to the
chromatographer. Now it is possible to run basic
analytes on a high-performance packing under nonionizing conditions. This provides new opportunities to
manipulate selectivity, as well as the reproducibility of
an assay. If one uses a packing at intermediate pH
values, the retention of ionic, especially basic, analytes
is highly susceptible to small changes in mobile phase
pH. This is not so when the analytes are either fully
ionized or not ionized at all, i.e. at low and high pH
values. Consequently, the reproducibility of an assay
improves at high and low pH.
Using standard silica particles, the inability to maintain
efficiency at high linear velocities with usable column
backpressure often limits the speed of analysis.
However, the stability of the Hybrid Particle at high
temperatures makes it possible to achieve high speeds.
At temperatures up to 80 °C, system backpressure is
reduced and column efficiency improved. In addition,
Hybrid Particle bonded phase packings
currently available
At the time of printing, four Hybrid Particle bonded
phases are commercially available, all from Waters
Chromatography, 34 Maple Street, Milford MA 01757,
(800) 252-4752.
The XTerra RP8 packing and the extended chain length
XTerra RP18 packing include a carbamate group
inserted into the chain. The best peak shapes and
wettability in 100% water are the key properties of the
XTerra RP packings.
The XTerra MS C8 and XTerra MS C18 packings are
made using a trifunctional silane. These packings are
designed for LC/MS applications, where very low bleed
is critical.
Both the XTerra RP packings and the XTerra MS
packings are fully endcapped.
References
1.
U. D. Neue, D. J. Phillips, T. H. Walter, M.
Capparella, B. Alden, R. P. Fisk, "The Relationship
between the Quality of a Reversed-Phase Column
and the Quality of a Pharmaceutical Analysis", LCGC, June 1994
2.
J. O'Gara, B. Alden, T. H. Walter, C. Niederländer,
U. D. Neue, " Simple Preparation of a C8 HPLC
Stationary Phase with an Internal Polar Functional
Group", Anal. Chem. 67 (1995), 67, 3809
3.
Laine, R.M., Sanchez, C., Brinker, C.J., Giannelis,
E., Organic/Inorganic Hybrid Materials, Materials
Research Society, Symposium Proceedings Vol.
519, 1998.
4.
Unger, K.K., Becker, N., Roumeliotis, P., J.
Chromatog. 125 (1976), 115.
5.
Neue, U.D., Walter, T.H., Alden, B.A., Jiang, Z.,
Fisk, R.P., Cook, J.T., Glose, K.H., Carmody, J.L.,
Grassi, J.M., Cheng, Y.F., Lu, Z., Crowley, R.J.
American Laboratory, in press
6.
the original Pharma article, in press.
7.
Berendsen, G.E., Pikaart, K.A., de Galan, L., J.
Liq. Chrom. 3 (1980), 1437.
8.
Neue, U.D., Walter, T.H., Alden, B.A., Jiang, Z.,
Fisk, R.P., Cook, J.T., Glose, K.H., Carmody, J.L.,
Grassi, J.M., Cheng, Y.F., Lu, Z., Crowley, R.J..,
American Lab,(31:22) 36-3 (1999) 99-0947
9.
Kirkland, J.J., van Straten, M.A., Claessens, H.A.,
J. Chromatog. A 691 (1995), 3.