Page 267 - Multidimensional Chromatography

262 Multidimensional Chromatography

In its true sense, the term ‘multidimensional’ refers to LC systems in which there
is a distinct difference in retention mechanisms in the columns used. Still, column
switching without change in retention mode can be a relatively easy to implement
approach for separating complex mixtures. This is a practical substitute for linear-
gradient elution and is particularly useful for analyte enrichment and sample clean-
up, as has been extensively demonstrated by Hoogendoorn and co-workers (14, 20,
21) for environmental analysis. The concept is also useful in bioanalysis, as was
shown by Polettini et al. (22) who applied coupled-column RPLC using two identi-
cal ODS columns for the automated analysis of -agonists in urine samples. With
direct injection of large volumes (1.5 ml) of human urine and careful adjustment of
transfer volumes and mobile phase conditions, LC–LC with UV detection allowed
the determination of clenbuterol at the low-ng/ml level. In a subsequent study (23),
the RPLC–RPLC system was coupled to a tandem MS system in order to gain fur-
ther selectivity and sensitivity. With this set-up, -agonists could be analysed in
bovine urine with a LOQ of 0.1 ng/ml. For the determination of clenbuterol at the
1 ng/ml level, the inter-day reproducibility was 8.4 %. With similar hydrophobic
columns, still different retention modes can be established by using one in the ion-
pairing mode and the other in the RP mode. This approach has been used by
Yamashita and co-workers (34–38) for the analysis of basic and acidic drugs in bio-
logical fluids. For example, phenylpropanololamine was determined in human
plasma and urine by first pre-separating the basic drug from endogenous compounds
on an ODS column using a low-pH eluent containing butanesulfonate as the ion-pair
reagent. Subsequently, after column switching of the heart-cut containing the ana-
lyte, further separation is carried out on a second, identical column in the absence of
butanesulfonate. The selectivity of the method was such that short-wavelength UV
detection could be used, thus yielding a detection limit of 0.4 ng/ml in plasma. The
method was applied to the determination of phenylpropanololamine in the plasma of
human volunteers after oral administration of 25 mg of the drug.
The on-line coupling of achiral and chiral columns is now a proven concept for
the bioanalytical separation of enantiomers (40, 41). Table 11.1 summarizes a num-
ber of these LC–LC applications. Frequently, first an achiral column is used to sepa-
rate enantiomeric pairs from matrix components, and then the enantiomers are
transferred to a chiral column for selective separation (Figure 11.3). In this way, the
chiral separation is often enhanced by excluding interfering substances from the sec-
ond column. Van de Merbel et al. (43) used two-dimensional LC for the determina-
tion of D- and L-enantiomers of amino acids in biological samples. The amino acids
are first separated by ion-exchange chromatography and subsequent enantiosepara-
tion is achieved by injection of 3 l heart-cuts on to a second column with a chiral
crown ether stationary phase. The selectivity and sensitivity of the system was fur-
ther increased by using fluorescence detection after on-line post-column labelling of
the amino acids with o-phthalaldehyde. In this way, small quantities of D-enan-
tiomers could be determined in complex biological samples such as protein
hydrolysates, urine, bacterial cultures and yoghurt, with hardly any additional
pretreatment. Another typical example of achiral–chiral coupled LC is the