Other Sample Injection Methods

An optically gated injection was demonstrated for the CZE separation of four amino acids labeled with 4-chloro-7-nitrobenzofurazan (NBD-F) in a one-channel chip [576] or a four-channel chip [577]. The gating beam was used to continuously photobleach the sample, except for a short time during injection by interrupting the beam (100-600 ms) using an electronic shutter. With only a sample reservoir and a waste reservoir, the sample continuously flowed electrokinetically. Six consecutive separations of the same sample mixture have been accomplished in under 30 s [576,577], 

FIGURE 4.24 Schematic view of the mechanical actuation of the PDMS membrane on the sample reservoir for pressure pulse injection [314], Reprinted with permission from the American Chemical Society. 

FIGURE 4.25 Sequences of nine consecutive pressure pulse injections with 4-s delay using a mixture of 38 iM calcein and 19 iM fluorescein (a). Species B, C, and D are derived from calcein. The durations of pressure pulse are (a) 0.3, (b) 0.35, and (c) 0.4 s [314]. Reprinted with permission from the American Chemical Society. 

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The low concentration levels of the compounds in environmental samples impose specific requirements in terms of sample injection for GC analysis. In addition to the common injection techniques of capillary GC (split, splitless, on-column, and programmed temperature vaporized (PTV) injection), some other sample introduction methods coupled to GC such as solid-phase microextraction (SPME), headspace, etc., have favored the versatility of GC and reduced the time required for sample preparation. These techniques have an advantage over the conventional injection methods, which is that a preconcentration step prior to GC... 

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

Recently a decreased level of CE activity has been noticed with a shift of attention towards other separation techniques such as electrochromatography. CE is apparently not more frequently used partly because of early instrumental problems associated with lower sensitivity, sample injection, and lack of precision and reliability compared with HPLC. CE has slumped in many application areas with relatively few accepted routine methods and few manufacturers in the market place. While the slow acceptance of electrokinetic separations in polymer analysis has been attributed to conservatism [905], it is more likely that as yet no unique information has been generated in this area or eventually only the same information has been gathered in a more efficient manner than by conventional means. The applications of CE have recently been reviewed [949,950] metal ion determination by CE was specifically addressed by Pacakova et al. [951]. 

Of course, to be able to use the direct injection method of sample introduction, the analyte or the polymer system must be soluble in a solvent. Other methods of sample introduction need to be considered in order to eliminate the involatile material from the chromatographic separation. These have become extremely effective in the analysis of matrices such as polymers. 

The dry combustion-direct injection technique provides many advantages over other methods, such as quick response and complete oxidation for determining the carbon content of water. Its primary shortcoming is the need for rapid discrete sample injection into a high-temperature combustion tube. When an aqueous sample is injected into the furnace, it is instantaneously vapourised at 900 °C and a 5000-fold volume increase can be expected. Such a sudden change in volume causes so-called system blank and limits the maximum volume of injectable water sample, which in turn limits the sensitivity [106,107]. 

Over the years, in addition to developments with ELISA reagents such as labels, there have been improvements in automation. This has enabled ELISA to be utilized as a high-throughput tool. Typically, ELISAs can be performed in several hours to days. The most common practice is to precoat the microtiter plate for an overnight incubation period, with the remainder of the steps performed the following day. While ELISAs are fast when compared to other assays such as bioassays, which can take days to weeks, they might be considered slow when compared to methods like HPLC, in which the time from sample injection to chromatogram is a matter of minutes. 

Flow-injection methods are quicker, more precise and use less reagents than other techniques in addition, they are very useful where only a Hmited amount of sample is available. The main advantages of the combination of FIA with DCP/OES are the increase in precision in sample handhng, fewer physical interferences, ahigher throughput of samples and versatihty towards physical and chemical properties of reagents. Some minor disadvantages are the loss of sensitivity compared with continuous nehuHzation, and the fact that only one element can he determined at a time. 

For the red wines (82-84), which were injected directly into the HPLC without sample preparation, a ternary-gradient system using aqueous acetic acid (1% and 5% or 6%), and acidified acetonitrile (acetonitrile-acetic acid-water, 30 5 6) was used for cinnamic acid derivatives, catechins, flavonols, flavonol glycosides, and proanthocyanidins. Due to the large number of peaks, the gradient was extended to 150 min for the resolution of many peaks of important phenolics. This direct injection method was able to separate phenolic acids and esters, catechins, proanthocyanidins, flavonols, flavonol glycosides, and other compounds (such as tyrosol, and rrans-resveratrol) in wine in a single analysis. However, use of acetic acid did not permit the detector (PDA) to be used to record the UV spectra of phenolics below 240 nm (84). 

In addition to the examples discussed above, a number of other xenobiotics are measured by their phase I reaction products. These compounds and their metabolites are listed in Table 20.1. These methods are for metabolites in urine. Normally, the urine sample is acidified to release the phase I metabolites from phase II conjugates that they might have formed, and except where direct sample injection is employed, the analyte is collected as vapor or extracted into an organic solvent. In some cases, the analyte is reacted with a reagent that produces a volatile derivative that is readily separated and detected by gas chromatography. 

Apart from thermodesorption, all other sample preparation techniques are normally directed toward production of a liquid extract that is subsequently injected into a GC/MS system. For the simplest of the samples, neat organic liquids or concentrated solutions, the sample preparation method will consist of diluting the sample with a suitable, clean solvent (most often dichloromethane). 

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