RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY JUNE 2003 2

Advances in the HPLC Column Packing Design

Even though HPLC column technology is considered to be
somewhat mature, new developments continue. Improvements
in packing-material design, bonded-phase chemistry, column
construction and formats have occurred. Users now have a
better understanding of the advantages and limitations of silica-
based materials and do not attempt to use them under
conditions that may shorten their lifetime or decrease their
performance. In addition, new phases have extended operating
pH ranges (high and low) providing more versatility. In this
article, I will update developments in packing morphology and
particle design. Instead of trying to cover the entire domain of
HPLC column development, I will focus on a few key areas.
Improvements in Porous Packings
Porous packings have been in favour throughout the history of
HPLC. The transition from large porous particles and pellicular
materials to small porous particles occurred in the early 1970s
when microparticulate silica gel (10 m d
p
) came on the
scene and appropriate packing methods were developed.
Irregularly shaped microparticulate packings were in vogue
throughout the 1970s until spherical materials were developed
and perfected. The spherical packings could be packed more
homogeneously than their irregular predecessors, gave better
efficiencies and could be manufactured in higher purity.
Indeed, the so-called Type B silica that was low in trace-metal
content became the standard in the early 1990s and now most
commercial silica-based analytical HPLC packing materials are
of this higher level of purity. Trace metals in silica gel cause
interactions with certain compounds and can affect the acidity
of residual silanols.
1
One aim of HPLC packings early in the game was to achieve
the best efficiency possible and consequently better
chromatographic resolution. To better understand the various
approaches employed to improve column efficiency, lets briefly
discuss the morphology of a porous packing material such as
silica gel or alumina.
Diffusive pores dominate a typical porous packing (Figure 1(a))
and the major surface area of the particle is contained within
these pores. A reduction in particle size improves both
interparticle and intraparticle mass transfer. In a porous particle,
solutes transfer from the moving mobile phase outside of the
particles into the stagnant mobile phase within the pores to
interact with the stationary phase. Following this interaction,
the solute molecules must diffuse out of the particle and
continue their journey down the column. Such a mass transfer
occurs many thousands or even millions of times as the
differential separation process proceeds and the solute is eluted
from the column. While the solute spends its time in the diffusive
pores, the mobile phase in which it was located originally moves
down the column ahead of the solute. This slow rate of mass
transfer into and out of the porous particles is a major source of
band broadening in HPLC. The use of smaller particles shortens
the path length of this diffusion process, improves mass transfer
and provides better efficiency. Manufacturers can now produce
small diameter particles with fairly narrow particle-size
distributions down to 1.5 m average diameter, although
33.5 m and 5 m particles are still the norm.
Advances in
HPLC Column Packing Design
Ronald E. Majors, Agilent Technologies, Wilmington, Delaware, USA.
Throughpore
(a)
(c) (d)
1.5 um
0.25 m
5 m
(b)
Diffusive pore
Thin porous
layer
Figure 1: Flow characteristics and design of packing particles
in HPLC. (a) totally porous particle; (b) perfusion packing;
(c) non-porous silica (NPS) or non-porous resin (NPR);
(d) Poroshell particle.
3 www.lcgceurope.com
Majors
However, congruent with this improvement in efficiency was a
decrease in column permeability; that is, an increase in column
backpressure. The increase in pressure is proportional to the
inverse of the particle diameter squared. Thus, halving particle
diameter will increase the column head pressure by a factor of
four. Although pumps can provide the necessary increase in
pressure output at normal flow-rates (e.g., 13 mL/min) with
commonly used solvents (e.g., water, methanol, acetonitrile,
hexane, etc.), users found that columns were not particularly
stable when run near to pump pressure limits as high as 450 bar.
Column efficiency, H or HETP (height equivalent to a
theoretical plate), is proportional to d
p
x
, where x is
approximately 1.61.9. Resolution is proportional to N
1/2
.
Thus, if one uses smaller particles packed into shorter columns
of the same internal diameter, the loss in resolution does not
fall off as rapidly as the efficiency improves. A current trend in
HPLC for high-throughput separations is to use shorter
columns with smaller particles (3 or 3.5 m particles in
4.6 mm i.d. by 2050 mm length), rather than longer
columns with larger particles (5 m particles in 4.6 mm i.d.
150250 mm lengths). Because separation time is proportional
to length, shortening the column results in faster separations.
Figure 2 provides an example of the time saved when using
smaller particles (d
p
5, 3.3, 1.8 m) in shorter columns
(L 250, 100 and 30 mm, respectively), compared with more
traditional HPLC analytical columns. The flow-rate on all
columns is the same except for Figure 2(d) in which the flow is
increased to 2 mL/min to illustrate a possible further decrease
the separation time by increasing the flow-rate. Compared with
the separation on a conventional 4.6 mm 250 mm column,
the separation time was reduced 15-fold (a little over 2 min).
Although short columns with small particles provide rapid
separations, the column plate number (efficiency) is not
increased. Thus, complex, multicomponent samples cannot
be separated on these columns. To increase plate count over
100 000 requires particles in long columns, but at the
expense of greatly increased column pressure. The first
demonstration of ultrahigh-pressure HPLC separations was
by Bidlingmeyer and co-workers
2,3
in 1969 with submicron
particles packed into long, thick columns. However, the
quality of the packings was not equivalent to todays
materials, and more recent studies by Jorgenson and
coworkers
4,5
from the University of North Carolina involved
small particles (down to 1 m) with ultra high pressure.
Conventional pumps cannot handle these columns so special
high-pressure pumps capable of pressures in excess of 5000
bar (75 000 psi) are required. However, such small-particle
columns have the capability of generating a quarter of a
million plates in less than an hour! The ultrahigh-pressure
chromatograph can also be used for gradient elution. To
direct users on how to employ these systems routinely, Milton
Lee and coworkers
6
from Brigham Young University in
Provo, Utah, USA have devoted their attention to solving
some of the practical concerns of ultrahigh-pressure systems.
Particular attention has been paid to the designs of injection
valves with respect to injection reproducibility, injection time,
maximum operating pressure, sample amount injected and
the valves impact on system efficiency. These workers have
also used supercritical carbon dioxide as a packing solvent.
Until commercial HPLC systems are available that can handle
these ultrahigh-pressure columns, they will be mostly used in
academic research laboratories.
Perfusion Packings
Perfusion packings were developed by Afeyan and
co-workers
79
and commercialized by Perseptive Biosystems
(Cambridge, Massachusetts, USA, now part of Applied
Biosystems) in the late 1980s that gave improved
chromatographic performance, particularly for larger
molecules. A simplified pictorial representation of a perfusion
packing is shown in Figure 1(b). Compared with the porous
packings, the perfusion packings consists of two different types
of pores: diffusive pore and through pores. The diffusive pores
are the same type present in the porous particles and provide
the sorption capacity. The through-pores allow mobile phase to
pass through the packing itself thereby increasing the rate of
mass transfer in the mobile phase. Instead of predominantly
flowing around the particles, a portion of the mobile phase
flows through the particle allowing the solute to spend less
time undergoing the mass transfer process and giving narrower
peaks. The process is actually a combination of diffusion and
convection.
Commercial perfusion packings are polymeric particles larger
than those typically used in HPLC packings, with the smallest
average particle size being ~12 m. However, when compared
Peaks: 1 estradiol, 2 ethylestradiol, 3 dienestrol,
4 norethindrone.
0 5 10 15 20 25 30
Time (min)
0 5 10 15 20 25 30
0.5 1
1
2
3
4
2.5 2 1.5
Rs (1,2) 3.1 4.6 30 mm, 1.8 m
2 mL/min
2.09
Rs (1,2) 3.7 4.6 100 mm, 3.5 m
12.71
1
2
3
4
0 5 10 15 20 25 30
Rs (1,2) 3.3 4.6 100 mm, 1.8 m
1 mL/min
4.15
1
2
3
4
0 5 10 15 20 25 30
Rs (1,2) 4.8 4.6 250 mm, 5 m
29.65
1
2
3
4
Figure 2: Effect of shortening column length and decreasing
particle diameter on chromatographic separation. Columns:
Zorbax SB-C18; mobile phase: 50% 20 mM NaH
2
PO
4
, pH 2.8:
50% ACN; flow-rate 1 mL/min; temperature: RT; detection: UV
230 nm. (Courtesy of Agilent Technologies, Wilmington,
Delaware, USA.)
RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY JUNE 2003 4
Majors
with a porous packing of the same particle and pore size, the
perfusion packings give better efficiency for large molecules.
9
In addition, compared with the older soft, organic porous
packings used for biomolecules such as fast-flow agarose or
polydextrans, which tend to collapse at higher linear velocities,
the perfusion packings may be used at higher flow-rates. At
these higher flow-rates they maintain their sample capacity
making them useful for preparative separations and
purifications.
Non-Porous and Superficially Porous Packings
The use of non-porous packings represents another approach
to improve the rates of mass transfer. There are two types of
non-porous packings: non-porous silica (NPS) and non-porous
resin (NPR). As depicted in Figure 1(c), the non-porous
packings are very reminiscent of the older pellicular or porous-
layer beads (PLBs) used in the early days of HPLC, but these
materials are of much smaller particle sizes, typically in the
1.52.5 m range.
10
The thin porous layer allows much faster
rates of mass transfer and separations of only a few minutes can
be achieved for both large and small molecules. Unfortunately,
the thin layer of stationary phase also limits the capacity of the
packing making NPS and NPR unsuitable for preparative
separations. In addition, because of their small particle size, the
backpressures from NPS columns are generally much greater
than those experienced with microparticulate HPLC porous
packings of popular particle sizes (i.e., 5 and 3 m). For more
information on the use and advantages of NPS packings,
consult reference 10. Such particles are finding less use in
todays chromatography laboratory.
Superficially porous packings (Figure 1(d)) are similar to
NPS particles described above but the particle diameter is
larger (~ 5 m), providing a much lower pressure drop. These
Poroshell particles (Agilent Technologies, Wilmington,
Delaware, USA) are recommended for larger biomolecules that
diffuse slowly into porous packings. When flow-rates are
increased with porous packings, the biomolecule peaks broaden
because of slow diffusion into and out of the pores. The thin
layer of stationary phase is derivatized with alkyl bonded
moieties such as C3, C8 and C18 providing rapid separations
of proteins by reversed-phase chromatography. Poroshell-type
packings combine the advantages of rapid mass transfer (i.e.,
improved efficiency), a decent sample capacity and good
recovery of biomolecules. Figure 3 shows the rapid gradient
separation of several protein standards in less than 1 min on
such a column.
Monoliths
Monoliths are columns that are cast as continuous
homogeneous phases (just like concrete in a mould) rather
than packed as individual particles. These types of columns
have been reviewed earlier in LCGC
11
and in this present
volume.
12
There are several types of monolithic column:
agglomerates of polyacrylamide particles
polymethyacrylate block
agglomeration of micron-size silica beads
polystyrene-divinylbenzene block
silica rods
membranes of various types (made by many manufacturers).
Monolithic columns have great potential in offering a stable,
easily replaced column for both analytical and preparative
separations. Both silica-based and polymer-based monoliths
have been extensively studied. The silica-based materials were
developed by Tanaka and coworkers in Japan,
13
introduced by
Cabrera and coworkers
14
at HPLC 98 in St. Louis, Missouri,
USA, and commercialized by E. Merck (Darmstadt, Germany)
as its SilRod column. These columns are solid rods of silica
monolith. Similar to the perfusion packings, they have both
flow-through pores with macroporosity (12 m in width) and
diffusive pores (called mesopores). The silica rods can be
modified using the same derivatization chemistries that are
used for regular HPLC packings (e.g., C18 bonded phase).
The SilRods, now encapsulated in PEEK, have been introduced
as Chromolith.
There are two important characteristics for current silica
monolith columns: they have the efficiency equivalent to a 35
m silica particle and their pressure drop is approximately
3040% lower than a 5 m silica particle. Thus, columns can be
coupled in a serial manner thereby generating higher plate
counts for more difficult separations.
The polymeric monolith columns have also made their
mark on separation science. These columns consist of a
continuous crosslinked, porous monolithic polymer usually
polymethacrylates or methyacrylate copolymerizates. They
can be fabricated into discs and tubes in convenient housings
for easy connection to an HPLC system. Some examples of
commercial products are the UNO from BioRad Laboratories
(Richmond, California, USA), the CIM copolymers from BIA
Separations (Ljubljana, Slovenia
11
), and the Swift
polystyrene-divinylbenzene monoliths from Isco (Lincoln,
Nebraska, USA). To illustrate the use of a polymeric
monolith, Figure 4 provides a 1 min separation of
oligodeoxynucleotides (8- to 16-mers) on a CIM DEAE disc
with a 3 mm thickness and a 16 mm diameter. This particular
run was a fast gradient elution using 6 mL/min flow-rate that
permits the use of a relatively thin disc of
poly(glycidylmethacrylate-ethyleneglycol) dimethacrylate
copolymer. There are several polymeric monoliths
Peaks: 1 angiotensin II, 2 neurotensin, 3 RNase,
4 insulin, 5 lysozyme, 6 myoglobin,
7 carbonic anhydrase, 8 ovalbumin
Time (s)
0 15 30 45 60
200
250
0
100
50
150
m
A
U
1
2
3
4
5
6
7
8
Figure 3: Fast high resolution separation of peptides and
proteins with Poroshell column. Column: Poroshell 300SBC18,
2.1 75 mm; mobile phase: (A) 0.1% TFA (trifluoroacetic acid);
(B) 0.07% TFA in AcN (aceonitrile); gradient: 5100% B in
1.0 min; flow-rate: 3.0 mL/min; temperature: 70 C; pressure:
260 bar; detection: UV, 215 nm.
5 www.lcgceurope.com
Majors
commercially available with ion-exchange, hydrophobic-
interaction, reversed-phase and affinity chromatography
capability. Note that the functionalized membranes that have
long been used in the isolation of biomolecules are also a
form of monolith columns.
InorganicOrganic Hybrids
Waters (Milford, Massachusetts, USA) has developed a unique
approach for making a hybrid packing, especially useful for
high-pH applications, in which silica gel has been less useful.
Traditionally, when high-pH conditions were required to
achieve greater retention of basic compounds or for compound
stability reasons, polymeric packings, coated zirconia, alumina
particles, or graphitized carbon materials were usually
considered. For various reasons such as lower efficiency,
swelling/shrinking problems, strong adsorption sites and other
undesirable features, these materials have never achieved the
popularity of silica gel as a base material. Waters has attempted
to combine the advantages of silica with those of organic
polymers.
Most modern silica gels used in HPLC are produced by the
polymerization of tetrachloro- or tetraethoxy-silane monomers
eventually resulting in a silica gel polymer with siloxane bonds
(Si-O-Si) and various types of terminal silanols (-Si-OH) at
their surface. In its synthesis process, Waters starts with a silane
monomer that contains both a methyl group and three ethoxy
groups thereby incorporating a methyl group into the silica-
based final packing material. The column constructed from this
material (called XTerra) has proved to be more stable in
alkaline conditions than its typical silica-based packings.
15
The
company has also used ethylene-linked triethoxysilane
(RO)
3
SiCH
2
CH
2
Si(OR)
3
instead of MeSi(OR)
3
to form a
sol-gel. At high-pH conditions, the particle from this latter
method has a 30% longer lifetime than the particle from the
sol-gel of MeSi(OR)
3.
16
Another approach to make more
alkaline stable silica bonded phases was used by Kirkland and
coworkers
17
in which a special bidentate bonded phase
anchored at two adjacent silanols combined with a high degree
of endcapping protected the underlying silica backbone from
attack by hydroxide ions.
Sol-Gel Silica
As silica gel is the most widely used base material for bonded
phases in HPLC, there is an interest in expanding the pH
operating range both on the acid and the basic sides of the
pH scale. There are two types of silica particles used in
commercial HPLC columns: sil-gel and sol-gel. Sil-gel
particles, usually made by gelling soluble silicates or
coalescing fumed silica, are characterized by higher porosities
and irregular pore shapes with variable wall thicknesses.
Sol-gel particles, which are made by aggregating silica sol
particles, have lower porosities and more-regular pores with
thicker walls defined by the surrounding solid silica-sol
particles. Sol-gels are generally more mechanically stable than
sil-gels. Both silica gels types can withstand typical mobile
phase buffers that are used on the acidic end of the scale. At
low-pH values, the unprotected siloxane bonded phases are
the more vulnerable part of these bonded packings and may
be subject to catalysed hydrolysis by the hydronium ion.
18
Pertinent to the discussion of intermediate-to-high pH
stability, because of their thinner pore walls sil-gel particles
appear to dissolve more quickly than sol-gel particles.
19,20
Accordingly, researchers anticipate developing more long-
term stable methods at intermediate-to-high pH using
columns made with sol-gel supports such as Hypersil
(Thermo Hypersil Keystone, Bellefont, Pennsylvania, USA),
Zorbax (Agilent Technologies) and Spherisorb (Waters)
columns.
Other Packing Materials
Other non-silicabased packings have been introduced in the
last few years; among them have been polystyrene-divinyl
benzene (PS-DVB) co-polymers, other organic polymers,
zirconia, graphitized carbon and hydroxyapatite. As graphitized
carbon was recently the subject of a review,
21
I will not
elaborate on this packing material.
The PS-DVB polymers have been around for a long time.
Users often turned to them when they needed a high-pH,
reversed-phase alternative to bonded silica gel. These polymeric
materials have a wide pH range, have high crosslinking, are
pressure and temperature stable, and have no silanols to
interact with basic compounds. However, they suffer from
poorer efficiency than silica gel. Typically, a 5 m polymeric
column may exhibit about a third of the theoretical plates that
Time (s)
0 10 20 30 40 50 60 70 80 90
0
10
20
30
40
50
60
90
190
140
10
40
R
e
l
a
t
i
v
e

a
b
s
o
r
b
a
n
c
e

(
2
6
0

n
m
)
%

b
u
f
f
e
r

A
8 mer
10 mer
12 mer
14 mer
15 mer
16 mer
solvent peak
Figure 4: Separation of Oligodeoxynucleotides on polymeric
monolith column. Column: CIM DEAE (Anion Exchange) Disc,
diameter: 16 mm thickenss: 3 mm; bed volume: 0.34 mL;
instrumentation: gradient HPLC System with extra low dead
volume mixing chamber; mobile phase: Buffer A: 20 mM
tris-HC1, pH 7.4, Buffer (B): Buffer (A) + 1 M NaC1; gradient:
shown on figure; flow-rate: 6 mL/min; detection: UV at
260 nm; temperature: ambient; injection size: 20 L. (Courtesy
of BIA Separations.
29
)
Number of Bases 5'3' Sequence Short Name
8 CCA TGT CT 8 mer
10 CTC CAT GTC T 10 mer
12 AGG TCC ATG TCT 12 mer
14 CGA CGT CCA TGT CT 14 mer
15 CCG AGG TCC ATG TCT 15 mer
16 GCCG AGG TCC ATG TCT 16 mer
RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY JUNE 2003 6
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a comparable silica gel bonded phase column. The rate of mass
transfer in the bulk stationary phase appears to be slower than
for a silica gel packing. Also, polymeric materials with a lower
degree of crosslinking may swell and shrink as the mobile-phase
composition is changed, as with gradient elution.
In the area of ion-exchange/ion chromatography and size
exclusion chromatography (SEC), polymers have gained
widespread acceptance. Silica gelbased ion exchangers do not
display the ruggedness that polymers display, especially in
strong buffers and with higher pH mobile phases. For ion
chromatography, special polymers have been developed by
Dionex (Sunnyvale, California, USA)
22
and applied for
separations of various anionic and cationic species. The
company's approach is to use a polymer bead consisting of
ethylvinylbenzene 55% crosslinked with DVB as a base material
and then to functionalize or graft it with various ionogenic
groups. In some instances, the surface of the bead is pellicular-
like and therefore the efficiency is better than the totally porous
polymer materials.
Polymeric materials are generally recognized as the standard
in gel permeation chromatography (GPC). Small particle (5
m) GPC packings, compatible with organic or aqueous
mobile phases display good efficiency and with various pore
sizes available can separate small and large organic molecules.
Silica gel packings with different pore sizes are available but
have limited acceptance in GPC, presumably because of their
surface activity. Silica gel packings with special deactivation
have found use in the size separation of proteins.
Zirconia (ZrO
2
) has many properties that make it attractive
as an HPLC packing, particularly its stability. It can be
produced in monodisperse, porous spherical particles and, for
many separations, it can provide the efficiency of silica gel.
Zirconia has excellent pH properties and can be used up to
pH 14; it also has high-pressure and high-temperature stability.
Unlike silica, the surface has no silanols for amine interactions.
However, its surface does possess hard Lewis acid sites and has
a strong affinity for hard Lewis bases (e.g., hydroxyl,
phosphate, fluoride and carboxylate). Therefore, one must add
competing anions (40 mM concentration) for analytes
containing these functional groups. Most successful separations
on zirconia-based columns have been those achieved on
polymer-coated phases. The most popular coated phase has
been the polybutadiene (PBD). The column can be used for
reversed-phase separations using wateracetonitrile or
watermethanol mobile phases; its selectivity is different than
C18-bonded silicas. Zirconia can also be covered with a thin
film of pyrolytic carbon resulting in a surface that, in addition
to possessing reversed-phase properties, has interesting
selectivity for the separation of diastereomeric compounds and
stereoisomers because of its high isomeric selectivity and for
highly polar solutes for which retention on bonded phases is
too low to be useful. The high-temperature capabilities of
coated zirconia have spawned several applications using
temperature (greater than 100 C) as a variable in the
optimization of LC separations. For a selection of applications
of zirconia-based HPLC packings, consult reference.
23
In its low-pressure application, hydroxyapatite (a calcium
phosphate material) has long been used for purification of a
wide range of biomolecules including proteins and monoclonal
antibodies. However, because of their fragility, these earlier
materials were unsuitable for the higher pressure operation
required in HPLC. A newer generation of materials, however,
is more structurally rigid and show good recoveries (typically
90100%) and provide high activity of sensitive biomolecules.
Because hydroxyapatite is a pure material, there is no bonded
organic phase that can strip to contaminate collected analytes.
Columns show high capacity; a 1.0 10 cm column can
handle 20.4 mg of lysozyme. A modern hydroxyapatite packing
is depicted in Figure 5. As can be seen in the scanning electron
micrograph, it has a unique porous structure. A typical gradient
elution application of the separation of nucleotides showed a
rapid separation and good pressure stability at a variety of flow-
rates.
24
Future Directions in Packing Development
Silica gel with chemically bonded phases will be around for a
long time. Even though many new materials have surfaced,
silica-based packings retain their dominance in most
laboratories. Their excellent efficiency, rigidity, lower cost than
alternatives, and ability to be functionalized will ensure their
continued success. The trend towards the use of smaller
particles (3 m) packed into short columns will continue and,
more than likely, a 3 m packed column of 7.515 cm length
by 0.46 cm internal diameter will replace the 5 m column
25 cm length by 0.46 cm column that is the standard today.
These shorter columns with smaller particles can provide the
same resolution as a longer column with larger particles. The
driving forces will be higher throughput requirements
quality assurance/quality control, LC/MS and LC/MSMS,
and combinatorial chemistry needs with solvent savings and
increased sensitivity as secondary benefits.
It remains to be seen if particles in the 12 m range become
mainstream. For optimized results with short columns (50
mm), extracolumn effects, dwell volumes, injection and
detection volumes must match the narrow peak widths so that
band spreading does not occur. For these same sized particles
packed into long columns (2550 cm), tremendous column
Figure 5: Scanning electron micrographs of Hydroxyapatite
particles (Courtesy of Wilhelmy Fine Particles, W. Leechburg,
Pennsylvania, USA).
7 www.lcgceurope.com
Majors
efficiency can be realized for very difficult separations but
ultrahigh-pressure instruments must become available to
permit their use.
Columns of smaller dimensions with internal diameters of
less than 100 m with small particle packings have become
interesting to those studying proteomics. The small samples
and low concentrations of analyte strongly favour these
miniature columns with small particle-sized packings.
Detection by MS, especially tandem MS, provides increased
sensitivity and structural information for tiny amounts of
peptides in tryptic digests.
Monolithic columns should become further commercialized
and cheape. The silica monoliths, currently only available in
4.6 mm i.d., require higher flow-rates than are desirable for LC-
electrospray. A 2.1 mm column would have optimized flow-rates
in a desirable range but these are not yet commercially available.
Recently, a 100 m i.d. monolith column prepared in situ has
been reported.
24
Although bonded silica columns are now
supplied with PEEK cladding, the silica rod cannot be easily
replaced because the cladding is integral to the column. If a
bonded silica monolith or silica rod column could be placed into
a holder or housing and provide the efficiency and lifetime that
clad columns have shown, they would be an ideal and easily
replaced column. A dead column could be removed easily and a
new rod slid into place without having to use any special
configurations or tools. Both analytical- and preparative-size
polymeric monoliths are already provided in housings and, in
some instances, the bulk monolith can be removed and replaced.
New types of particles constructed from newer materials will
undoubtedly be developed as chromatographers and
manufacturers look for the ideal packing that will provide high
recovery, excellent efficiency, low cost and extraordinary
stability. For example, titania is being studied as a base material
for bonded phases and as a bare material for ion-exchange
separations.
25
New co-polymers that provide unique surface
properties will continue to be developed, hopefully with better
efficiency. One example is the hydrophilic polymers provided as
SEC packings that can be used with either organic or aqueous
mobile phases and converted from one solvent system to
another. Similarly, chiral columns that are useful in both
aqueous and organic solvent systems have also become
available. These two types of columns now provide a lower cost
alternative to chromatographers who do samples in both types
of solvent systems because they only have to buy a single size
exclusion or chiral column. Bimodal SEC phases in which
multiple pore sizes are available in one bead or in one column
may give polymer chemists a packed bed that can cover a wide
molecular weight range in one column. Hybrid
inorganicorganic materials may prove to offer a better
compromise to solve stability problems for observed silica-
based materials at higher pH and more of them will be studied.
The current studies on lab-on-a-chip will result in packing
materials that will be fabricated (rather than packed) on the
inner walls of the tiny capillaries. The movement of liquids
through these tiny columns may be by electro-osmotic flow
rather than conventional hydraulic means although pumping
systems and nanovalves may be integrated onto the chip.
Methacrylate monoliths have been prepared inside capillary
tubes
26,27
for capillary electrochromatography (CEC) and
solid-phase extraction. Sol-gel silicas have also been prepared in
situ in chip channels
28
and used for the separation and
amplification of DNA. Many laboratories are investigating these
technologies. In fact, open-tubular liquid chromatography, the
ultimate in column performance, may become a reality if taken
to the dimensions of microchip flow channels.
References
1 R.K Iler, Chemistry of Silica Gel, John Wiley and Son, New York, 1979,
671.
2 B.A. Bidlingmeyer et al., Separation Science 4, 439-446 (1969).
3 B.A. Bidlingmeyer and L.B. Rogers, Separation Science 7, 131157
(1972).
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Ronald E. Majors is the business development manager,
consumables and accessories business unit, Agilent Technologies,
Wilmington, Delaware, USA. He is also the column editor of
Column Watch and Sample Preparation Perspectives and a
member of the LCGC Europe Editorial Advisory Board.