2 column watch LC•GC Europe - December 2001

New Developments in
the Application of
Monolithic HPLC Columns
Dieter Lubda, Karin Cabrera, Wolfgang Kraas, Christian Schaefer and
Don Cunningham, Merck KGaA, Darmstadt, Germany,
Column Editor, Ronald E. Majors, Agilent Technologies, Wilmington, Delaware, USA.

This month’s “Column Watch” reviews recent developments in monolithic HPLC sorbent
technology and their possible benefits. The guest authors discuss polymer- and
silica-based monolithic sorbents and compare them with classic particulate sorbents.

Although many chromatographers feel high monolithic materials with large lower efficiency compared with silica-based
performance liquid chromatography (HPLC) throughpores, or channels, should cause columns. Most polymers, especially those
is a mature technique, chromatography higher column permeabilities. A with low degrees of cross-linking, swell or
research continues to make progress. hydrodynamic flow could speed shrink in organic solvents. This behaviour
Column-packing materials have changed separations. Alternatively, the surface area could have dramatic effects upon the
dramatically from irregular- to spherical- required for the chromatographic chromatographic performance of these
shaped particles and from large to small adsorption and desorption process could monolithic columns, and it frequently leads
particles. Developers have optimized the be obtained by creating mesopores on the to a lack of mechanical stability.
performance of HPLC columns filled with monolithic skeleton’s surface. Furthermore, the structure of porous
these small spherical particles to routinely Different research groups have studied polymers often contains micropores that
obtain reduced plate height values of 2 monolithic materials for use in HPLC. Based negatively affect column efficiency and
with commercial columns. upon the nature of their construction peak symmetry. Polymeric monolithic
In industrial applications, HPLC is materials, monolithic columns can be columns, however, offer excellent
routinely used for diverse applications such classified as organic polymer– or silica-based biocompatibility, a wide pH range, and the
as product quality control, monitoring columns. The first monolithic columns were ability to be cleaned with caustic mobile
analytes in biological matrices, and based upon organic polymers. Ross and phases. Polymeric monolith columns
analysing compound libraries synthesized Jefferson (2) first reported the preparation housed in convenient cartridge designs are
by combinatorial chemistry methods. In of monolithic GC columns from available commercially.
this last application, in particular, the polyurethane in 1970. Twenty years later, Taking into account some of these
number of samples that must be analysed Hjérten and co-workers (3) prepared drawbacks, porous inorganic monoliths
in a given day continues to increase. materials within a chromatographic tube could be the column filling materials of
Therefore, analysts need methods and based upon polyacrylamides. Those choice for conventional applications, just as
tools that can drastically reduce analysis columns were successfully applied to fast they are for microparticulate packed HPLC
time to achieve higher throughput. bioseparations. Frechet and colleagues (4) columns. In 1979, Pretorius and co-workers
described the preparation of either (5) reported two methods for forming
Development and Study polyacrylates or poly(styrene-co- open-pore silica foams suitable for the in
of Monolithic Materials divinylbenzene) in the presence of situ preparation of a new support. Later,
One approach to develop high-throughput porogens, which led to monolithic Nakanishi and colleagues (6) developed a
HPLC methods is using monolithic materials with a permanent macroporous new sol-gel process for preparing
columns. J.H. Knox and P.A. Bristow of structure. Their chromatographic suitability monolithic silica columns with a bimodal
Edinburgh University (Edinburgh, UK) was demonstrated by the separation of pore structure; that is, with throughpores
recognized the potential advantages of proteins. and mesopores. Their method was based
monolithic columns more than 30 years Monolithic polymers have also been upon the hydrolysis and polycondensation
ago (1). They suggested a preparation of prepared successfully within capillaries and of alkoxysilanes in the presence of water-
rigid foam inside a column. In their applied in HPLC and capillary soluble polymers. Tanaka and co-workers
proposal, they envisioned that electrochromatography (CEC). However, (7) and Cabrera and colleagues (8)
communicating pores would provide a using polymeric materials in HPLC has demonstrated that the method enabled
network through which the mobile phase several disadvantages. The general the preparation of chromatographic
would flow. The motivation for this drawback of these materials, as is typical columns with high efficiencies and low
approach came from their realization that for polymeric stationary phases, is their column back pressures.
LC•GC Europe - December 2001 column watch 3

“chromatographers synthesized each month. Each of these mobile phase would flow along the wall,
reaction mixtures must be separated for resulting in the so-called wall effect, and
can perform the component of interest. The standard column efficiency would be compromised.
separations at 15 or 20 min HPLC separation is too time-
consuming for high-throughput separation
Scientists in our research laboratory
developed a procedure to effectively shrink
higher flow-rates” and screening of so many samples. Likewise, wrap a polyetheretherketone (PEEK) plastic
the columns themselves must be very robust holder onto the silica rod. This procedure
because some laboratories must operate eliminates any possible wall effects. Both
Those improvements resulted from the 24 h/day to analyse these compounds. The silica-gel and bonded silica-gel materials
independent control of the sizes of the introduction of high-throughput HPLC can be encapsulated in the PEEK enclosure.
silica skeleton and throughpores. Moreover, instruments and the increasing automation The 2 µm macropores serve as flow-
the researchers demonstrated that the of separating processes using mass through pores and enable analytes to be
newly developed monolithic-type HPLC spectrometry (MS) detection systems have transported to the active surface for
column could be operated at high flow- resulted in a search for stationary phases subsequent chromatographic separation.
rates and provide high efficiencies. Following and column configurations that are The surface area created by these
this approach, Schulte and co-workers (9) suitable for use in these systems. mesopores is approximately 300 m2/g.
reported the application of 25 mm dp silica One separating material that is tailor- Because the total porosity of the
monoliths in preparative chromatography. made to fulfill the requirements of high- monolithic silica matrix is greater than
Tanaka and colleagues (10) and others throughput analysis is a monolithic silica 80%, users can perform chromatography
also reported the preparation of monolithic gel in rod form called Chromolith (Merck using a much lower back pressure than
materials, within the confines of fused- KGaA, Darmstadt, Germany). During they could with conventional packed
silica capillaries, that could be applied in LC manufacture, a silica-gel polymer is particles that have total porosities of
and CEC separations. This approach has formed, which, after aging, is dried into a approximately 65%. By optimizing the
the substantial practical advantage that no straight rod of highly porous silica with a ratio of flowthrough pores to total porosity
cladding procedure is needed. Researchers bimodal pore structure. The material has a and the silica-gel skeleton thickness,
have demonstrated the feasibility of these silica-gel skeleton that contains mesopores chromatographers can perform separations
microcolumns for practical applications, with diameters of approximately 13 nm at higher flow-rates. This capability is
but their general performance and long- and macropores with diameters of the result of accelerated adsorption and
term stability must be improved. approximately 2 µm (Figure 1). desorption kinetics of the substances
To connect the monolith column with an separated at the silica-gel surface.
Monolithic Silica Supports HPLC system, the silica rod must be The traditional van Deemter curve of
During the past several years, many contained tightly in a column sleeve that column plate height (H) versus mobile-
pharmaceutical companies have been ensures no void space between the solid rod phase linear velocity (u), which is often
involved with combinatorial chemistry. In and the column wall. If a void exists, the used as a measurement of HPLC column
their search for more effective drugs,
analysts must make massive arrays of
compounds and analyse them quickly. In
many instances, this exercise results in 5 µm
30
thousands of new compounds being

20
Plate height (µm)

1 µm 3.5 µm

10 Monolith

10 µm 0
0 2 4 6 8
Figure 1: Electron micrographs of the (a) Flow-rate (mL/min)
macroporous and (b) mesoporous structures
in a monolithic silica rod. Figure 2: H versus u curves for monolithic silica (Chromolith) and particulate HPLC columns.
4 column watch LC•GC Europe - December 2001

performance, demonstrates that these than 5 µm dp columns. The efficiency of separated the five substances within 5 min
monolithic silica columns provide these columns is equivalent to at a flow-rate of 1 mL/min. Under these
significantly better separation efficiency microparticulate columns filled with conditions, the total system back pressure
3.5 µm dp material. A clear advantage was 13 bar. We were able to obtain baseline
of the monolith column is that flow-rates separation of the five -blockers in 0.5 min
(a) 2 of as much as 9 mL/min can be used with a column back pressure of only 72 bar
(Figure 2). Knox and Bristow (1) proposed and a flow-rate of 9 mL/min (Figure 3).
using separation impedance (E) as a The low back pressure that results from
measure of total column performance. the enhanced permeability of monolithic
1 3 Because of the low pressure drop and versus particulate columns provides several
4 better efficiency of the monolith column, it additional benefits. One is the ability to use
5
is clear that monolithic silica columns flow gradients. Unlike conventional solvent
provide significantly better E values than gradients, for flow gradients, it is
the particulate HPLC columns studied. unnecessary to re-equilibrate a column at
Monolithic columns, which carry a C18 the end of each run. The flow-rate can
(b)
surface-modification and endcapping, are simply be adjusted to the initial conditions
comparable in selectivity to conventional before injecting the next sample (Figure 4).
reversed-phase columns. A comparison Because faster separations are of great
shows that although the selectivity of interest today, we have witnessed a
monolithic and particle columns are the pronounced trend toward the use of
same, the retention times achieved by the shorter columns filled with small particles
monolithic column are much shorter, (e.g., 3 µm dp). Complex separations,
therefore resulting in a faster separation. however, still require long column beds to
The equivalent selectivity enables easy provide the separation efficiency needed to
(c) transfer of existing methods from resolve all the compounds of interest.
particulate to monolithic columns. Typical examples of long-column
Because of the better mass transfer applications include the analysis of natural
properties of a monolithic skeleton over products, such as herbal medicines, and
distinct particles (i.e., flatter H versus u food and beverage samples. Using coupled
curves), high-speed separation is possible monolithic columns is one way to increase
without a noticeable effect on resolution. the column bed length and, therefore, the
For example, we separated five -blockers absolute plate number.
at high  values (  kSubstance2/kSubstance1, Figure 5 clearly demonstrates that
where kSubstance1 and kSubstance2 are the coupled silica monolith columns longer
(d) retention factors of two substances) and than 1 m can provide acceptable results
obtained excellent peak symmetry. We (11). These long columns are possible
because of the minimal back pressure
generated by monolithic HPLC columns.
(a) 1

2 45 6 6
3 5 7
7 8
9 10

3
(e) 0 1 2 3 4 5 6 7 8 9 10 11 12
2 4
(b)

0 1 2 3 4 5 6
Time (min) 0 30 60
0 1 2 3 4 5 6 Time (min)
Time (min) Figure 4: Separation of -blocking drugs
(a) without and (b) with a flow gradient. Figure 5: Separation achieved by coupling
Figure 3: Separation of five -blockers at Column: 100  4.6 mm Chromolith 14 100  4.6 mm Chromolith Performance
(a) 1, (b) 3, (c) 5, (d) 7 and (e) 9 mL/min Performance RP-18e; flow-rate: (a) 2 mL/min; RP-18e columns. Mobile phase: 80:20 (v/v)
flow-rates. Column: 50  4.6 mm (b) 2 mL/min for 2 min, 2–5 mL/min in 1 min, acetonitrile–water; column plate number:
Chromolith SpeedRod RP-18e; mobile phase: 5 mL/min for 3 min. Peaks: 1  fumaric acid, 108 000 plates; back pressure: 117 bar.
20:80 (v/v) acetonitrile–1% trifluoroacetic 2  pindolol, 3  nadolol, 4  pafenolol, Peaks: 1  thiourea, 2  benzene,
acid in water; detection wavelength: 222 nm. 5  metoprolol, 6  celiprolol, 3  toluene, 4  ethylbenzene,
Peaks: 1  atenolol, 2  pindolol, 7  carazolol, 8  bisoprolol, 9  alprenolol, 5  propylbenzene, 6  butylbenzene,
3  metoprolol, 4  celiprolol, 5  bisprolol. 10  propranolol. 7  pentylbenzene.
LC•GC Europe - December 2001 column watch 5

“Future developments will bring monolithic

silica products for preparative, semipreparative,
and micro and capillary liquid chromatography
applications and sample preparation.”
Materials for HPLC,” paper presented at the
Conclusion International Symposium on Chromatography,
Monolithic silica technology HPLC columns London, UK, October 2000.
represent a new and innovative type of
column for rapid chromatographic analysis.
In contrast to conventional HPLC columns, Dieter Lubda is laboratory leader for research
monolith columns are formed from a single and development of monolithic silica columns
piece of porous silica gel, giving them at Merck KGaA, R&D biochemistry and
greater porosity and permeability than separation, Frankfurter Strasse 250, D-64293
conventional particle columns. Silica Darmstadt, Germany. Karin Cabrera is head of
monoliths can also overcome the research, monolithic HPLC columns at Merck
drawbacks of polymer monoliths. They can KGaA. Wolfgang Kraas and Christian
be operated at higher flow-rates regardless Schaefer are international product managers
of back pressure. Because the selectivity is for chromatography consumables at Merck
comparable to microparticulate columns, KGaA. Don Cunningham is sales and
these columns allow chromatographic marketing manager for chromatography
analyses to be performed in a fraction of consumables at Merck KGaA.
the time previously required. Because of “Column Watch” editor Ronald E. Majors is
their better mass transfer properties, these business development manager, columns and
columns maintain high separation supplies at Agilent Technologies, Little Falls
efficiency, even at high linear flow-rates. As Site, Wilmington, Delaware, USA, and is a
a result, a typical laboratory can increase its member of the Editorial Advisory Board of
sample throughput. LC•GC Europe.
In addition to creating HPLC columns Direct correspondence about this column
with more than 100 000 theoretical plates, to “Column Watch,” LC•GC Europe,
future developments will bring monolithic Advanstar House, Park West, Sealand Road,
silica products for preparative, Chester CH1 4RN, UK,
semipreparative, and micro and capillary e-mail: dhills@advanstar.com
liquid chromatography applications and
sample preparation. These products will be
modified with a variety of stationary phases.
In response to the growing pressure to
improve laboratory efficiency and increase
sample throughput, more existing HPLC
applications using conventional particulate
columns could be replaced by those using
monolithic columns.

References
(1) J.H. Knox and P.A. Bristow, Chromatographia,
10, 279 (1977).
(2) W.D. Ross and R.T. Jefferson, J. Chromatogr.
Sci., 8, 386 (1970).
(3) Ch.M. Zeug et al., J. Chromatogr., 753,
227–234 (1997).
(4) S. Xie, F. Svec, and J.M.J. Fréchet,
J. Chromatogr., 775, 65–72 (1997).
(5) V. Pretorius, J.C. Davidtz and D.H. Desty,
HRC&CC, 2, 583 (1979).
(6) K. Nakanishi and N. Soga, J. Non-Cryst. Solids,
139, 1–13, 14–24 (1992).
(7) H. Minakuchi et al., Anal. Chem., 68,
3498–3501 (1996).
(8) K. Cabrera et al., J. High Resolut. Chromatogr.,
23, 99–104 (2000).
(9) M. Schulte, D. Lubda and J. Dingenen, J. High
Resolut. Chromatogr., 23, 100–105 (2000).
(10) N. Tanaka et al., J. High Resolut. Chromatogr.,
23, 111 (2000).
(11) Karin Cabrera, “Mololithic vs. Particulate Silica