Sample pre-concentration

Sample stacking is employed in CE separations. In this method, the differences in two types of physiochemical properties between the sample buffer and mn buffer are exploited. What are these two properties (2 marks) 

Describe two methods to pre-concentrate a liquid sample by removing the small molecules in the sample. (2 marks) 

What neutral molecules have been used as EOF markers [670,748]. 

In the use of cross-linked gel in CGE separation, there is the bubble formation problem. Explain its origin. (2 marks) 

In carrying out CGE separation on chip, why is the channel usually coated (2 marks) 

Where none of the above techniques is capable of providing a sufficiently low limit of detection (LOD), it may be necessary to pre-concentrate the analyte relative to the drug substance as part of the method. Alternatively, a cheaper, less powerful detector may be suitable for the analysis given this enrichment. This section describes various approaches. 

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ICP-SFMS (Thermo Finnigan, Flement) with cold vapour generation was developed with a guard electrode and a gold amalgamation device using an Au-sorbent for sample pre-concentration to improve the sensitivity. Instrumental parameters of ICP-SFMS such as take-up time, heating temperature of Au-sorbent, additional gas flow, and sample gas flow were optimized. Detection limit calculated as 3 times the standard deviation of 10 blanks was 0,05 ng/1, RSD = 7-9 %. 

J. T. B. Strode and L. T. Taylor, Supercritical fluid exti action employing a vai iable restrictor coupled to gas chi omatography via a sample pre-concentration cap , ]. High Resolut. Chromatogr. 19 651-654 (1996). 

Typical instrument set-ups for online sample preparation, for example, solid phase extraction and sample pre-concentration, require the control of a six-port valve with an additional special column and a second pump. Ideally, this system will be fully controlled by the data acquisition software (Figure 3.12 (A)). 

Owing to the short path length, the sensitivity of photometric detection in CE is limited. For this reason, procedures are necessary to enhance the sensitivity. This can be achieved by sample pre-concentration, improvement of the optical design and/or alternative capillary geometries [66]. Sample pre-concentration can be done off- or on-column. The off-column procedures are well described in the literature and have been applied extensively in chromatography [67]. The on-column procedures are more specific to CE and are therefore briefly discussed. 

SFC has been used as a technique to analyse nitroaromatics in aqueous environmental samples [29]. It was necessary to use sample pre-concentration methods (see next section) to obtain suitable detection limits. 

An example illustrating the use of the Immuspeed instrument has been shown with the detection of interleukin IB, a molecule for which the relevant detection level for biological applications lies in the sub-pM domain. In order to reach this limit of detection, sample pre-concentration using up to 60 multi-loadings has been introduced, which allows to increase the sensitivity of the assay, yet without generating problems of non-specific adsorption as exemplified by the results shown in Fig. 50.2A. 

Figure 6.12 Schematic diagram of the interface used for direct SFE-CEST coupling without a sample pre-concentration step 1, micro-LC pump 2, heated restrictor 3, six-port valve 4, direct by-pass to the CE unit 5, three-port valve 6, CE instrument, (from ref. 58).

Several sample pre-concentration strategies have been devised to enrich dilute samples. The microfluidic chip has the advantage to integrate various microstructures to achieve sample pre-concentration by stacking, extraction, or other methods. 

Although stack injection has been employed previously [316,582], the benefit of stacking for sample pre-concentration was only studied in detail later [346]. With the sample buffer (0.5 mM) at a 10-fold lower conductivity than the separation buffer (5 mM), simple EK stacking using the gated injection was observed. This was applied to the separation of dansylated amino acids (dansyl-lysine, didansyl-lysine, dansyl-isoleucine, and didansyl-isoleucine) [346]. 

Sample pre-concentration was also achieved based on solid-phase extraction. This was carried out on ODS-coated silica beads (of diameter 1.5 1 4m) trapped... 

Other than beads, porous polymer monoliths, which were photopolymerized in a COC chip, were used for solid-phase extraction. It is known that priming polymeric surfaces is not as simple as priming silica surfaces, which use a common surface primer agent, TMPM. Therefore, the grafting method as initiated by UV should be used to attach the polymer monoliths [588]. A similar strategy was used for sample pre-concentration of PAHs (e.g., pyrene). Pyrene (900 nM) was first concentrated by 400-fold in 24% ACN before switching to 56% ACN for CEC separation (see Figure 5.5) [148]. 

Sample pre-concentration was also achieved for gaseous samples. A flowthrough Pyrex chip consisting of the silica-based solid absorbent was used for pre-concentration of the BTX gaseous mixture. A thin-film heater was used to desorb the adsorbed gas molecules, which flowed downstream for UV absorbance detection. LOD of 1 ppm (toluene) was achieved as compared to 100 ppm without pre-concentration [131,715]. With an additional air-cooled cold-trap channel... 

FIGURE 5.5 Chromatograms of (a) 1-s injection without sample pre-concentration and (b) 320-s injection with concentration. The sample was 900 nM pyrene [148]. Reprinted with permission from the American Chemical Society. 

Another common sample pre-concentration method is dialysis which serves to remove small molecules. For instance, affinity dialysis and pre-concentration of aflatoxins were achieved in a copolyester chip (see Figure 5.10). After affinity binding to the aflatoxin Bi antibody, various aflatoxins (Bj, B2, Gi> G2, G2J in a sample were retained, while the other small molecules passed through a PVDF dialysis membrane. Thereafter, the sample solution was exposed to a countercurrent flow of dry air, leading to water evaporation and analyte concentration. The concentrated and desalted sample was used in subsequent MS analysis [821], More details for MS analysis are described in Chapter 7, section 7.3. 

Lichtenberg, J., Daridon, A., Verpoorte, E., de Rooij, N.F., Combination of sample pre-concentration and capillary electrophoresis on-chip. Micro Total Analysis Systems, Proceedings of the 4th Y7AS Symposium, Enschede, Netherlands, May 14-18, 2000, 307-310. 

For many samples, pre-concentration is essential, and this is commonly achieved by solvent extraction. Often the nickel tetramethylenedithiocarbamate complex is extracted at pH 2-4 into 4-methylpentan-2-one.1 This system has been applied to soil and sediment extracts39 and to water samples.40-42 Kinrade and Van Loon43 used a mixture of ammonium tetramethylenedithiocarbamate and diethylammonium diethyldithiocarbamate to extract a range of elements, including nickel, into 4-methylpentan-2-one from water samples adjusted to pH 5. New solvent extraction-based procedures are still being published regularly for environmental samples such as plant tissues and water samples.44... 

Figure 2, Flow system for seawater sampling/pre-concentrating on controlled pore glass immobilized 8-hydroxyquinoline (CPG-8HOQ)...

Andersson, L. I., Efficient sample pre-concentration of bupivacaine from human plasma solid-phase extraction on molecularly imprinted polymers, Analyst 2000, 125, 1515-1517... 

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