Detection techniques chemical separations

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

Solid phase spectrophotometry proved to be an appropriate technique for the determination of colorants in foods dne to its simplicity, selectivity, reasonable cost, low detection limits, and use of conventional instrnmentation. This simple, sensitive, and inexpensive method allowed simnltaneons determinations of Snnset Yellow FCF (SY), Quinoline Yellow, and their nnsnlfonated derivatives [Sndan I (SUD) and Quinoline Yellow Spirit Soluble (QYSS)] in mixtnres. Mixtnres of food colorants containing Tartrazine, Sunset Yellow, Ponceau 4R, Amaranth, and Brilliant Blue were simultaneously analyzed with Vis spectrophotometry without previous chemical separation. ... 

Once the analyte has been separated from the matrix in LC, the best approach to the detection of the molecule must be determined. This section will discuss the detection techniques of ultraviolet/visible (UVA IS), fluorescence (FL), and electrochemical (EC) detection, with MS being addressed separately in Section 4.2. When deciding on the most appropriate detector for an LC separation, the appropriate chemical data on the analyte should be collected by using a spectrophotometer, fluorimeter, and potentiometer. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

Figure 14 shows the Cl data [13] where the events are plotted on a two-parameter display of range vs. energy. When improved chemical separation preparation techniques were used in later experiments on antarctic meteorites [14], the lower limit of Cl detection that was reached was 2 x 10 16 for 36C1/C1, with a quoted accuracy between 5 percent and 10 percent. 

Capillary electrophoresis, a relatively new technique, uses an electric current to separate compounds based on their size, charge, and mobile phase solubility. This technique requires small amounts of sample. An analytical technique that provides enhanced specificity and sensitivity for detection of chemicals is LC/MS/MS. This technique separates compounds by HPLC and then uses the MS to fragment the separated compounds. Unlike... 

Conventional radiochemical methods for the determination of long-lived radionuclides at low concentration levels require a careful chemical separation of the analyte, e.g., by liquid-liquid, solid phase extraction or ion chromatography. The chemical separation of the interferents from the long-lived radionuclide at the ultratrace level and its enrichment in order to achieve low detection limits is often very time consuming. Inorganic mass spectrometry is especially advantageous in comparison to radioanalytical techniques for the characterization of radionuclides with long half-lives (> 104 a) at the ultratrace level and very low radioactive environmental or waste samples. 

HPLC units have been interfaced with a wide range of detection techniques (e.g. spectrophotometry, fluorimetry, refractive index measurement, voltammetry and conductance) but most of them only provide elution rate information. As with other forms of chromatography, for component identification, the retention parameters have to be compared with the behaviour of known chemical species. For organo-metallic species element-specific detectors (such as spectrometers which measure atomic absorption, atomic emission and atomic fluorescence) have proved quite useful. The state-of-the-art HPLC detection system is an inductively coupled plasma/MS unit. HPLC applications (in speciation studies) include determination of metal alkyls and aryls in oils, separation of soluble species of higher molecular weight, and separation of As111, Asv, mono-, di- and trimethyl arsonic acids. There are also procedures for separating mixtures of oxyanions of N, S or P. 

Many different separation and detection systems have been used for speciation. For example, size fractionation and ultra-filtration have been used for separation with the separated species then being determined by neutron activation (Tanizaki et al., 1992). These physico-chemical separation processes are, however, time consuming and the species have to be collected and then determined separately. Although the techniques are invaluable for certain types of speciation where the interaction of the species with colloids and sediments is important, hybrid or coupled techniques are usually preferred. 

The first main part of this chapter opens with general aspects of chemical TA structures and the resulting physico-chemical properties, which determine the adequate choice of sample preparation, separation and detection techniques. Thereafter, pharmacological and toxicological basics of TA are outlined in general, followed by individual introduction of those TA, that are referred to in detail in this chapter. 

Application of LC-MS/MS techniques to the analysis of phthalate ester metabolites in urine have also been developed. For example, Blount et al. (2000b) have developed an assay to quantify the monoester metabolites (including MEHP) of eight phthalate diesters in urine, utilizing HPLC coupled with atmospheric pressure chemical ionization and tandem mass spectrometric (APCI-MS/MS) detection techniques. Urine samples were treated with -glucuronidase to release the free phthalate monoesters followed by a two-step solid phase extraction procedure. After evaporative concentration of the eluant, the analytes in the purified samples are further separated on a phenyl reverse phase HPLC column and quantified by APCI-MS/MS, following careful optizimation of the APCI-MS/MS instrument. The limits of detection for MEHP were determined to be 1.2 ng/ml urine with recovery efficiencies of between 78 and 91%. 

Hydride and cold-vapour techniques represent a special combination of chemical separation and pre-enrichment with AAS determination, resulting in higher powers of detection for elements with volatile hydrides, eg, As, Bi, Se, Sb, Hg. Recent literature on vapour generation has been reviewed by Hill et al. (1991). Some examples of the use of hydride generation for the analysis of plant material are given by Muse et al. (1989), Leuka et al. (1990) and Ainsworth and Cooke (1990). Hydride generation can also be used with ICP-EAS (see below) and applications have been reviewed (Nakahara, 1991). 

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