Multidimensional Chromatography

conformations and 19 form stable enantiomers due to the restricted rotation around the central carbon – carbon bond at ambient temperatures (14). There are at least nine of the conformationally stable chiral PCBs which are present in commercial formulations and these are expected to accumulate in the environment. In order to analyse them, an achiral column was coupled to a chiral column (14, 25). The gas chromatographic separation of most of the chiral PCB congeners was achieved on different cyclodextrin phases (14).

MDGC has been used for separating commercial formulations of PCBs (11, 12, 22, 23, 26) although it is not widely used on real samples. In some examples, MDGC has been applied to determine PCBs in sediment samples (13, 14, 27) and water samples (14, 24).

For PCB analysis, Glausch et al. used an achiral column coated with DB-5 and a chiral column coated with immobilized Chirasil-Dex (14). The column was switched with a pneumatically controlled six-port valve and peak broadening was minimized by cooling the first part of the second column with air precooled with liquid nitrogen, thus focussing the cut fraction. These authors determined the chiral polychlorinated biphenyls 95, 132 and 149 in river sediments, using microsimultaneous steam distillation – solvent extraction as the sample treatment technique. Figure 13.2 presents the MDGC-ECD chromatograms of PCB fractions from sediment samples, where it can be seen that the separation of the PCB enantiomers is good.

In many environmental extracts, the analytes of interest overlap with other analytes or matrix components. MDGC is therefore essential for improving accuracy when identifying non-target methods. In a typical example (20), contaminated water, clay and soil samples have been analysed. While water and clay extracts can be analysed by GC-IR–MS, soil samples, because of their greater complexity, need MDGC- IR–MS to identify the pollutants present in the sample. In such cases, an MDGC-IR–MS system with multiple parallel cryogenic traps and sample recycling is used (see Figure 13.3).

The chromatogram from the first column was divided into five areas of five heartcuts each. Some peaks identified in the chromatogram from the first column were used as heart-cut markers. This method has some limitations which mainly concern contamination of the system, and also with the determination of less volatile pollutants. However, such a system is able to detect and accurately identify about 40 pollutants.

Another interesting application of MDGC is in the rapid determination of isoprene (the most reactive hydrocarbon species) and dimethyl sulfide (DMS) (the major source of sulfur in the marine troposphere and a precursor to cloud formation) in the atmosphere (16). The detection limits were 5 and 25 ng l 1, respectively.

A programmed temperature-vaporization (PTV) injector (with a sorbent-packed liner) was used to preconcentrate and inject the sample. Thermal desorption was performed and the analytes were passed to a primary column (16 m 0.32 mm i.d., film thickness 5 m, 100% methyl polysiloxane) and separated according to analyte vapour pressure. Selected heart-cuts were transferred to a second column (15 m 0.53 mm i.d., Al2O3 /Na2SO4 layer, open tubular column with 10 m stationary phase) where final separation was performed according to chemical functionality.

Figure 13.2 MDGC-ECD chromatograms of PCB fractions from sediment samples, demonstrating the separation of the enantiomers of (a) PCB 95, (b) PCB 132, and (c) PCB 149; nonlabelled peaks were not identified. Reprinted from Journal of Chromatography, A 723, A. Glausch et al., ‘Enantioselective analysis of chiral polychlorinated biphenyls in sediment samples by multidimensional gas chromatography–electron-capture detection after steam distillation – solvent extraction and sulfur removal’, pp. 399 – 404, copyright 1996, with permission from Elsevier Science.

Figure 13.4 shows the standard chromatogram obtained by using this method. In this sample, isoprene and major interference compounds, i.e. 2-methyl pentane and hexane, were spiked at 1 g l 1, plus DMS at 10 g l 1.

When using one-dimensional GC (1D-GC), the analysis took about 60 min (shown in the inset to Figure 13.4), while with the use of two-dimensional (2D-GC) it took only about 12 min.

Figure 13.3 Schematic diagram of the parallel cryogenic trap MDGC-IR–MS system: A, splitless injection port; B, Rtx-5 non-polar first-stage separation column; C, HP 5970B MSD; D, HP 5965B IRD; E, four-port two-way valve (300 °C maximum temperature); F, external auxiliary carrier gas; G, six-port selection valve (300 °C maximum temperature); H, stainless-steel cryogenic traps; I, three-port twoway valve (300 °C maximum temperature); J, Rtx-5 intermediate polarity column. Reprinted from Journal of Chromatography A, 726, K. A. Krock and C. L. Wilkins, ‘Qualitative analysis of contaminated environmental extracts by multidimensional gas chromatography with infrared and mass spectral detection (MDGC – IR – MS)’, pp. 167 – 178, copyright 1996, with permission from Elsevier Science.

13.3MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY

13.3.1INTRODUCTION

Liquid chromatography (LC) is a good alternative to GC for polar or thermolabile compounds. While polar compounds need to be derivatized for GC analysis, this is therefore not necessary for LC analysis.

When environmental samples are analysed by reverse-phase liquid chromatography, the most widely used technique, polar interferences usually appear (ions, plus humic and fulvic acids). This makes it difficult to determine more polar compounds that elute in the first part of the chromatogram. This is specially important when detection is not selective, e.g. UV detection, which is one of the most common techniques in routine analysis. In such cases, multidimensional chromatography plays an important role.

The application range of coupled-column technology is determined by the separation power of the first column. In general, it can be said that low resolution favours multiresidue methods (MRMs), while high resolution leads to methods for a single analyte or for a group of analytes with similar properties.

Multidimensional LC – LC, using two high-resolution columns with orthogonal separation mechanisms, has only a few applications in environmental analysis. The limitations that such a multidimensional system has with regard to selectivity must

be taken into account. For example, the differences in the physico-chemical bases of the separation processes involved may lead to poorly compatible mobile phase systems, thus requiring complex interfaces. Moreover, the separation obtained in the first column can, at least partly, be decreased in the second column.

In spite of such limitations, some examples can be found in literature. For example, a reversed-phase C18 column has also been coupled to a weak ion-exchange column to determine gluphosinate, glyphosate and aminomethylphosphonic acid (AMPA) in environmental water (28). This method will be described further below.

Zebühr et al. (29) developed an automated system for determining PAHs, PCBs and PCDD/Fs by using an aminopropyl silica column coupled to a porous graphitic carbon column. This method gives five fractions, i.e. aliphatic and monoaromatic hydrocarbons, polycyclic aromatic hydrocarbons, PCBs with two or more ortho- chlorines, mono-ortho PCBs, and non-ortho PCBs and PCDD/Fs. This method employed five switching valves and was successfully used with extracts of sediments, biological samples and electrostatic filter precipitates.

As mentioned above, the most commonly used liquid chromatographic technique is reverse-phase liquid chromatography (RPLC), which is also the most often used coupled technique. When two RPLC systems are coupled to analyse aqueous samples, there is an additional advantage because large sample volumes can be injected without causing extensive band broadening. This means that there is on-line enrichment. In general, therefore, the less polar the analyte, then the more the sample volume can be enriched without causing band broadening or breakthrough. However, when highly polar analytes have to be determined, the enrichment and clean-up needed to eliminate the interference become more limited. However, results have been good for some analytes, as we will see later.

Most work on LC–LC in environmental analysis has been developed by the Van Zoonen group (30, 31) and the Hernández group (32 – 34).

A commonly used system in environmental analysis is the heart-cutting technique which uses the separation power of the first column to obtain a higher selectivity than with the previously described precolumn enrichment. The two columns are coupled via a switching valve, as shown in Figure 13.5.

Separation in column 1 (C-1) removes early-eluting interference compounds, and so considerably increases the selectivity. The fraction of interest separated in C-1 is then transferred to column 2 (C-2) where the analytes of the fraction are separated. These transfers can be carried out either in forward mode or backflush mode. The forward mode is preferred because the backflush mode has two disadvantages for polar to moderately polar analytes. For most polar compounds, it leads to additional band broadening, while for more retained analytes there is a decrease in the separation obtained earlier in the process (31).

An important parameter in LC – LC is the transfer volume, i.e. the time that C-1 is coupled to C-2, since the selectivity is highly dependent on this. In environmental samples, it is important to remove early-eluting interference in order to ensure selective analysis. A short analysis time is important for routine analysis of environmental samples.

Figure 13.5 Schematic presentation of the procedure involved in coupled-column RPLC: AS, autosampler; C-1 and C-2, first and second separation columns, respectively; M-1 and M-2, mobile phases; S-1 and S2, interferences; A, target analytes; HV, high-pressure valve; D, detector. Reprinted from Journal of Chromatography, A 703, E. A. Hogendoorn and P. van Zoonen, ‘Coupled-column reversed-phase liquid chromatography in environmental analysis’, pp. 149 – 166, copyright 1995, with permission from Elsevier Science.

When a first column of a very short length (and therefore a low selectivity) is used (this is especially suitable for multiresidue methods), we talk about an on-line precolumn (PC) switching technique coupled to LC (PC–LC or solid-phase extraction (SPE)-LC). This is particulary useful for the enrichment of analytes, and enables a higher sample volume to be injected into the analytical column and a higher sensitivity to be reached. The sample is passed through the precolumn and analytes are retained, while water is eliminated; then, by switching the valve, the analytes retained in the precolumn are transferred to the analytical column by the mobile phase, and with not just a fraction, as in the previous cases.

Depending on the kind of sorbent in the precolumn connected on-line to the analytical column, the retention of analytes in the precolumn may be more or less selective.

The sorbents that are most frequently used in environmental analysis are C18-silica based sorbents, polymeric sorbents (usually styrenedivinilbenzene) and graphitized carbon. In order to increase the selectivity of these sorbents, immunosorbents (35, 36) have been developed and used with good results, while recently, molecularly imprinted polymers have started be to used (35, 36).

Polar compounds present the most problems because of their low breakthrough volumes with common sorbents. In the last few years, highly crosslinked polymers have become commercially available which involve higher retention capacities for the more polar analytes (37, 38). Polymers have also been chemically modified with polar groups in order to increase the retention of the compounds previously mentioned (35, 37).

Instead of a sorbent contained in a precolumn, discs can also be used with a special device (38, 39) which enables the number of discs to be changed easily, although this technique is currently limited to the kind of discs that are commercially available.

The characteristics of the sorbent in the precolumn may lead to problems when coupling the two systems. Therefore, when the analytes are more retained in the precolumn than in the analytical column, peak broadening may appear, even when the analytes are eluted in the backflush mode (40). This has been solved with a special design in which the analytes retained in the precolumn are eluted with only the organic solvent of the mobile phase and the corresponding mobile phase is subsequently formed (40, 41).

On-line SPE – LC has been widely used in environmental analysis to solve the problems caused by the low concentrations of the analytes to be detected and also to automate the analysis (42 – 44).

13.3.2 EXAMPLES OF MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY IN ENVIRONMENTAL ANALYSIS

LC – LC is applied to environmental samples with two major aims, i.e. to determine a single analyte and to determine a group of analytes (by the multiresidue methods) at the low levels required by legislation in both cases. Some examples of these are discussed below. In addition, some applications for the particular case of SPE – LC, will also be described.

The single-residue methodology has been used to determine analytes with different characteristics. The main advantage of this technique is the short analysis time. LC – LC methodology has been applied to various polar analytes, such as bentazone (46), and less polar compounds such as isoproturon (46) or pentaclorphenol (47). Most applications refer to water samples (46, 48), although solid samples have also been studied (31, 49, 50). Soil samples contain more interfering compounds so clean-up is even more important when analysing soil extracts. Coupled RPLC is

therefore a suitable technique for efficient on-line clean-up procedures. Some examples of various applications are shown in Table 13.1.

Some of the different methods which demonstrate the suitability of this technique for determining single analytes will now be described in greater detail.

Ethylenethiourea (ETU) is a highly polar metabolite of the ethylenebisdithiocarbamate (EBDC) fungicides. This is a relatively stable degradation product which is also present in EBDC formulations (at concentrations 0.02 – 5%) (48). ETU is mainly determined by GC using a precolumn derivatization, which is time consuming, or by LC using UV or electrochemical detection with a clean-up step. Both techniques are very laborious and are also not sensitive enough. LC – LC is therefore an attractive alternative to these methods and has been used to determine ETU in aqueous samples (48). The experimental conditions are given in Table 13.1. The authors, after studying different packing materials of the alkylbonded columns, decided to work with a 5 m Hypersyl ODS for better separation of the interfering compounds and peak compression. Ammonium acetate (pH 7.5) was also added to the mobile phase since this gave a better peak profile than with pure water at pH 3; the flow rates were 1.0 (M-1) and 1.1 (M-2) ml min 1. Experiments showed that the injection volumes on C-1 should not exceed 200 l and these were eluted with 2.6 ml of mobile phase. C-1 was switched on-line with C-2, and the ETU-containing fraction transferred to C-2 by using 0.44 ml of mobile phase (40 s). The retention time of ETU on C-2 was about 3 min, while the total analysis took less than 10 min.

From Figure 13.6, which compares the direct LC analysis and the LC – LC analysis, we can see that there is a significant increase in selectivity.

With this method, levels of 0.1 g l 1 can be detected in ground water and, if an offline liquid – liquid extraction step is added, levels of 0.1 g l 1 can be detected (48).

Another example is the determination of bentazone in aqueous samples. Bentazone is a common medium-polar pesticide, and is an acidic compound which co-elutes with humic and/or fulvic acids. In this application, two additional boundary conditions are important. First, the pH of the M-1 mobile phase should be as low as possible for processing large sample volumes, with a pH of 2.3 being about the best that one can achieve when working with alkyl-modified silicas. Secondly, modifier gradients should be avoided in order to prevent interferences caused by the continuous release of humic and/or fulvic acids from the column during the gradient (46).

With bentazone, small changes in the composition of the mobile phase have a dramatic effect on the final results (see Figure 13.7).

The methanol gradient from 50 to 60% releases quite a lot of interfering components. Omitting the step gradient does not provide enough selectivity and so the best conditions were obtained with a pH gradient. The experimental conditions are shown in Table 13.1.

This method can quantify levels of 0.1 g l 1 in real samples and reproducibility values are good; the total analysis time was 8 min. Figure 13.8 compares the chromatogram obtained by using this method with one obtained without column switching.