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Article 321548
Article 321548
com
Multidimensional and
Comprehensive Liquid
Chromatography
Column
Watch
M ultidimensional-chromatogra-
phy has been practiced for
many years, both in off-line
and on-line modes. Basically, the experi-
ured cleanly provided no other components
in the fraction happen to elute at the same
time on column 2. A simplified schematic
of a coupling experiment, a size-exclusion
In this month’s “Column ment involves transferring a fraction or column with an affinity separation column,
fractions from one chromatographic is depicted in Figure 1.
Watch,” Ron Majors
medium (usually a column) to a secondary When the demand requires the complete
discusses advances in (or additional) chromatographic medium characterization of complex mixtures, then
multidimensional (column or columns) for further separation. the term comprehensive chromatography is
chromatography and The technique can be used for further reso- used. The late Prof. Calvin Giddings of the
lution of complex mixtures that cannot be University of Utah, Salt Lake City, coined
comprehensive
separated entirely on a single medium, for the term comprehensive multidimensional
multidimensional sample cleanup by removing matrix or separation (10). Unlike multidimensional
chromatography. These interfering compounds, for increased sam- chromatography, in comprehensive chro-
techniques are well suited ple throughput, and for trace enrichment of matographic systems, every fraction from
minor compounds of interest. The most the primary column is subjected to the sec-
for the separation of
popular version of multidimensional chro- ond dimension. Normally, fractions are
complex samples. matography is two-dimensional (2-D) diverted from the primary column (10) to
Although GC GC is fairly chromatography. Table I provides examples the secondary column (20) at defined time
well developed, LC LC is of popular combined modes that have been intervals.
used. In a recent special issue of LCGC Proteomics is an area in which extremely
in its infancy. But with
Europe, several of these approaches were complex samples are encountered. All pro-
fast LC columns, based on reviewed in detail (8,9). In the column teins within a cell may be in need of char-
monoliths, superficially mode, 2-D chromatography also has been acterization to determine how they are
porous particles, and short referred to as column switching, multiphase affected in disease states. Because there are
chromatography, coupled column chro- potentially tens of thousands of proteins or
sub-2-m particle matography, boxcar chromatography, and more, a 1-D separation is inadequate to
columns, it has a bright sequential analysis. characterize this complex sample. However,
future, especially when In conventional 2-D chromatography, if the proteins (or tryptic peptides) are bro-
coupled with MS usually one or only a small number of com- ken into smaller fractions, which in turn
pounds are of interest. One example would are broken into even smaller fractions by a
techniques. Some of the be the high performance liquid chromatog- second (or sometimes even a third) separa-
latest information from raphy (HPLC) separation and determina- tion dimension, and if they are coupled to
HPLC 2005 provides tion of a drug in a biological fluid such as a powerful detection measurement tech-
examples of the potential urine. The matrix (for example, proteins, nique such as mass spectrometry (MS)
uric acids, and so forth) is of no interest yet (which is really an added dimension), then
of LC LC. may interfere with the analysis and identifi- it is conceivable that individual proteins (or
cation of the drug compound in a one- peptides) could be identified at these trace
dimensional (1-D) experiment. Other drug levels. Then, perhaps, the biochemists can
metabolites and small molecular weight measure those individual proteins that can
compounds might also be of little or no lead to new drugs or other treatments that
interest. By directing a fraction containing could block or modify their behavior.
the drug peak plus any possible overlapping Comprehensive 2-D gas chromatography
Ronald E. Majors contaminants from the primary column to (GC) (abbreviated GC GC) is a mature
Column Watch Editor a secondary column, the drug can be meas- technique with commercial products
www.chromatographyonline.com OCTOBER 2005 LCGC NORTH AMERICA VOLUME 23 NUMBER 10 1075
plex the samples that can be resolved. multidimensional GC, a cold trap might be
Mixture
When a sample is separated using two dis- used; in HPLC, a trapping column could
similar columns, the maximum peak capac- be employed to minimize this band disper-
ity max will be the product of the individ- sion. Another approach might be to use a
ual column’s peak capacity n (as depicted narrow bore or capillary column for the
in Equation 1). first dimension and a wider bore column
Size separation
for the second dimension.
max 1 2 [1] Obviously, if the speed of the first
dimension is faster than the speed of the
For example, if each separation mode second dimension, something must be
generates peak capacities of 100 and 200, done to halt or slow up the primary separa-
Affinity separation respectively, the theoretical peak capacity of tion that will affect the overall time. An
the 2-D experiment will be 20,000, a huge alternative approach could be to speed up
gain in separation space. To achieve this the separation in the second dimension by
gain, however, the two techniques should using a shorter column, faster gradient
be totally orthogonal, that is, based upon (LC), faster temperature program (GC),
Figure 1: Simplified schematic of 2-D multidi- completely different principles. For exam- and higher flow rate. Schoenmakers (13)
mensional chromatography LC separation. The ple, in HPLC, if the primary separation pointed out how Poppe plots (14) can be
first and second dimension separation modes
were size-exclusion and affinity chromatogra- column were based upon chirality while the used to predict the optimum resolution at
phy, respectively. second separation column separated on the reasonable separation times using currently
basis of hydrophobicity, the individual peak available pressures. Combinations of iso-
already on the market. The technique has capacity of the combined system would be cratic and gradient elution analyses for the
tremendous separation power, uses simple their multiplicand. two dimensions might also help to alleviate
robust hardware, and has similar analysis There are many practicalities to be real- the speed dilemma.
times to temperature-programmed high-res- ized when coupling two different chro- The compatibility issue is important for
olution capillary chromatography. On the matographic techniques. In a plenary lec- the practical coupling of the techniques
other hand, comprehensive 2-D LC (LC ture during the opening session at HPLC outlined in Table I. An example of the
LC) is still in its infancy and is more com- 2005, Peter Schoenmakers of the University incompatibility of the two orthogonal tech-
plex to perform. However, it is driven by of Amsterdam, The Netherlands, a member niques in HPLC would be the application
user needs, especially in proteomics, and of the Permanent Scientific Committee,dis- of normal-phase chromatography using
the topic is getting more attention. cussed the state-of-the-art in multidimen- nonpolar solvent such as hexane as the first
The purpose of this month’s installment sional chromatography and LC LC com- dimension. Solvent compatibility difficul-
of “Column Watch” is to discuss multidi- prehensive 2-D separations (13). He ties will be encountered if the second
mensional chromatography and compre- detailed some of the factors that govern dimension is reversed-phase chromatogra-
hensive 2-D chromatography. I will briefly multidimensional techniques. The most phy which uses a polar solvent such as
discuss GC GC, but because much has important factors are: water. In GC, if a packed column is used
been written on this approach (3,11), I will ● peak dispersion for the first dimension and a capillary col-
focus mainly on LC LC. I will confine ● relative speeds, and umn is used for the second dimension, the
my discussion to the use of 2-D techniques ● mode compatibility. sample capacities of the two columns may
in GC and HPLC. It is obvious that one Let’s examine these factors. As the sample not be mutually compatible. In coupling
can extend the experiment to “n” dimen- components move down the primary sepa- LC with GC, the HPLC mobile phase sol-
sions, but for the most part, many of them ration column, they become diluted, influ- vent can be detrimental to the GC station-
are difficult to put into practice. I will also encing the injection dispersion of the sec- ary phase, especially if a large volume is
summarize several papers presented on ond dimension. Ideally, their bandwidth injected.
multidimensional chromatography and should be very small so that they start out
comprehensive multidimensional HPLC as narrow band on column two. If not, the Off-Line Multidimensional
from the recent HPLC 2005 Symposium in sensitivity and resolution may be compro- Chromatography
Stockholm, summarized in last month’s mised. In many cases, an intermediate Off-line 2-D techniques are performed
“Column Watch” (12). “refocusing” step might be required. In every day in a normal laboratory operation.
Table I: 2-D chromatographic techniques
Background in Multidimensional
Chromatographic Separations Primary Technique Secondary Technique Off-Line On-Line Reference
By the combination of multiple chromato-
TLC (or HPTLC) TLC (or HPTLC) 1
graphic steps, there is one great advantage: Preparative TLC GC or LC X 1
an increase in peak capacity. Peak capacity GC GC 2,3
is, simply stated, the maximum number of LC LC 4
peaks that can be resolved in a given time- LC GC 5
frame. The more peaks that a combination SPE or SBSE LC or GC 6
SFC LC 7
of techniques can handle, the more com-
1076 LCGC NORTH AMERICA VOLUME 23 NUMBER 10 OCTOBER 2005 www.chromatographyonline.com
Absorbance (mAU)
small molecules such as drugs to diffuse 250
Absorbance (mAU)
a plasma sample from a LiChrospher RP-4 60
before a switching valve was diverted to the 0.2 0.4 0.6 0.8
second dimension column, a LiChrospher 40
Time (min)
overall performance.
Other multidimensional chromatography
applications at HPLC 2005 were reported. 10
15 20 25 30 35 40 45 50
Egidijus Machtejevas and colleagues, First dimension (min)
Johannes Gutenberg-University, Mainz,
Germany; Merck, Darmstadt; and Astra- Figure 4: (a) Comprehensive 2-D-LC Mode: normal-phase LC–reversed-phase LC-UV-MS. (Top)
First dimension normal-phase LC. Column: 250 mm 1 mm, 5-m dp Betasil diol (Thermo Electron
Zeneca, Molndal, Sweden (24) developed a
Corporation, Waltham, Massachusetts; mobile phase: 85:15 hexane–1-butanol 0.2%
novel sample cleanup column to study ethanolamine, isocratic; fl gradient: 25% B for 3 s, to 50% B in 3 s, to 100% B in 15 s, 100% B for
endogenous peptides in biofluids by direct 9 s, back to 25% B in 3 s; flow rate: 5 mL/min (170 bar); 30 °C; sample: pharmaceutical compounds
injection. The packing consisted of a RAM B-mix; response time: 0.1 s. (b) Comprehensive 2-D-LC-UV chromatogram.
with strong cation-exchange functionality.
By adjusting the pore size of the packing,
size exclusion could be used to determine compounds. Using bisphenol A as a model system with partially correlated selectivities,
the molecular weight fractionation range compound, trace levels were determined in allowing to predict the range of the struc-
and the selective ion exchange chromatog- serum combining column switching and tural units in a 2-D sample (according to
raphy could be performed within the pores. LC–MS. the Giddings’ notation) that can be
For added selectivity, Jan Haginaka and H. From a more theoretical side, Pavel Jan- resolved in a given time range for the two
Sambe of the Mukogawa Women’s Univer- dera and colleagues from the University of dimensions for not fully orthogonal sys-
sity, Nishinimiya, Japan (25) developed a Pardubice, Czech Republic (26) studied tems. The group also investigated the possi-
combined RAM and molecular imprinted structural effects on the separation selectiv- bility of phase transfer between a reversed-
polymer (MIP). MIPs are used for the very ity and performance optimization in 1-D phase system (1-D) and a normal-phase
selective extraction of target compounds, and 2-D LC systems. The goal of the work system (2-D). A hydrophilic interaction LC
while RAMs have been used for the direct was to characterize the degree of orthogo- (HILIC) system was proposed for the sec-
injection assays of drugs and metabolites in nality between the first and the second ond dimension in a reversed phase–normal
biological fluids. Although MIP template dimension systems in 2-D LC using the phase LC LC system. The HILIC system
leakage is still a problem, by the use of an correlation characterizing the similarity of was more resistant to aqueous solvents
isotopologue form of the template molecule selectivity in the two separation systems, transferred from the first dimension than
and MS detection, the template leakage will allowing more efficient use of the peak classical NP systems with nonaqueous
not interfere because MS can discriminate capacity theoretically available in 2-D mobile phases. This system was used for
between the labeled and unlabeled target HPLC. A model was derived for the 2-D comprehensive 2-D separation of ethylene
www.chromatographyonline.com OCTOBER 2005 LCGC NORTH AMERICA VOLUME 23 NUMBER 10 1081
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1082 LCGC NORTH AMERICA VOLUME 23 NUMBER 10 OCTOBER 2005 www.chromatographyonline.com
lary–nanocolumn–chip formats, superfi- the use of LC LC as well as the combi- dimension but with a different composi-
cially porous packings (28), and short, fast nation of supercritical fluid chromatogra- tion. A modulating device was constructed
sub-2-m columns, the ultrahigh-speed phy and HPLC (SFC LC) as applied to from a 10-port PC-controlled valve and
separations of small and large molecules, complex mixtures of natural products. In two 50-L sampling loops. The modula-
especially in the second dimension, should the latter technique, the use of supercritical tion time was only 1.0 min. Because of the
improve the situation. carbon dioxide as the mobile phase for the low flow rate in the first dimension and the
first dimension offers the advantage of easy rapid flow rate in the second dimension, it
Selected HPLC 2005 Applications transfer and excellent compatibility since was possible to transfer every fraction to
of LC LC the carbon dioxide can be evaporated dur- completely characterize the organic acid
A number of papers presented at HPLC ing the process. They also combined nor- content of the aerosol. The use of UV and
2005 showed that LC LC could be put mal phase chromatography (1o) with time-of-flight TOFMS aided in the identi-
to practical use. reversed-phase chromatography (2o) and fication of the separated acids.
In an oral lecture at HPLC 2005, Tanaka overcame the solvent compatibility problem
and colleagues of the Kyoto Institute of by using a 1-mm i.d. normal phase column Conclusion
Technology, Kyoto, Japan reported on their operating at 50-L/min coupled to a 4.6- Multidimensional chromatography has long
continued work with silica monoliths (29). mm reversed-phase column operated at a been used for resolving selective compo-
Their emphasis has now turned to compre- high flow rate to give a 1-min analysis. The nents from poorly resolved samples in both
hensive 2-D applications of monoliths,in small amount of nonpolar solvent from col- off-line and on-line applications. The
which columns of different dimensions are umn 1 could be accommodated by the advent of modern low-volume, high-pres-
coupled via a switching valve. Using 2-D larger volume of column 2 without detri- sure multiport switching valves conve-
contour plots, they looked at the types of mental effects. niently packaged that can be precisely con-
monolithic column and mobile phase con- Y. Zhao and colleagues (30) presented a trolled and programmed by computerized
ditions (isocratic and gradient elution and pharmaceutical application of LC LC instrumentation, on-line multidimensional
high flow rates) to maximize the time reso- combined with UV and MS detection. chromatography should receive more atten-
lution of multicomponent mixtures. Obvi- They assembled a fully automated multidi- tion as a way to cleanup difficult samples
ously, when both monolith columns were mensional LC system that could be used and improve analytical results. Comprehen-
of the same types such as C18, there were for LC–LC (heartcutting) and LC LC. sive multidimensional chromatography
many blank spaces in the contour plots, This initial system was used to ensure that should see even more widespread applica-
meaning that the resolution per unit time there were no coeluted compounds in their tion as more difficult samples that must be
was not optimal. By using different mobile formulations and to characterize impurities. fully characterized are encountered. GC
phase compositions, water-tetrahydrofuran Coupling normal phase chromatography GC has already proved itself in handling
in the first dimension and water–methanol (diol) as the first dimension using 1-mm difficult volatile samples such as hydrocar-
in the second dimension, they achieved a i.d. columns with a 4.6-mm i.d. C18 bons in petrochemical samples (32). In
semi-orthogonal separation that gave an monolith column as the second dimension, HPLC, with full characterization, require-
increased peak capacity. But, when they they were able to analyze a fraction every ment could dictate the use of longer
switched to a truly orthogonal set of 60 s. Figure 4a shows the separations columns with small particles to give
columns using a strong cation exchange achieved on the two columns for a propri- enhanced chromatographic resolution but
microparticulate column (1-D) coupled to etary drug mixture. Note the good orthog- at the expense of increased pressure. How-
a reversed-phase monolith (2-D), the peak onal separations achieved on the two differ- ever, because the increase in peak capacity
capacity was increased dramatically. By the ent systems. Selectivity was quite different will increase only with N1/2 with the longer
use of these orthogonal columns combined on the two columns. Finally, Figure 4b pro- column, such a system will have a difficult
with a gradient run in the first dimension vides a colored contour plot resembling a time competing with the significant
and isocratic elution in the second dimen- typical 2-D profile resulting from the LC increase of peak capacity with LC LC
sion, they achieved peak capacity of 700. LC UV coupling experiment. performed at more moderate pressures.
Tanaka then spelled out a set of ideal con- An environmental application of LC
ditions for the second dimension column. LC was reported by Riekkola and col- References
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