Amino acids separation indirect

Figure 11 Electropherogram of a mixture of five amino acids using indirect CL detection. Conditions 21-kV separation voltage, and 2 s at 21 kV for sample injection sample concentration 0.5 mM of each amino acid. Peak identities (1) arginine (2) leucine (3) serine (4) cysteine (5) aspartic acid. (From Ref. 86, with permission.)...

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. 

Direct and indirect chromatographic methods were developed and compared in systematic examinations for the enantioseparation of P-amino acids direct separation of underivatized analytes involved the use of commercially available Crownpak CR(-I-), teicoplanin, and ristocetin A CSPs [148], while indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-G-acetyl-f)-D-glucopyranosyl isothiocyanate (GITC) or A - a-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (EDAA, Marfey s reagent), with subsequent separation on a nonenantioselective column. 

Peter, A. et al., A comparison of the direct and indirect LC methods for separating enantiomers of unusual glycine and alanine amino acid analogues, Chromatographia, 56, S79, 2002. 

Pool-Loading Experiments and Possibility that Reduction in Incorporation Reduces Influx into Soluble Pool. In order to empirically eliminate the possibility that the observed reduction in uptake of labelled amino acids into the soluble pool was an indirect result of an inhibition of incorporation, it was necessary to experimentally separate the influx and incorporation systems in vivo. The operational compromise required to accomplish this has been given by Sacher (55), and involves in the present case measuring the rates of label incorporation in control and treated tissues which have had their soluble pools fully loaded with unlabelled amino acid. Thus, the label-specific... 

In order to generally categorize the reaction schemes mentioned previously and the following ones in the course of indirect enantioseparation techniques, it has to be emphasized again, that the reciprocity principle should always be applicable. This means that if a chiral acid as the CDA can be used successfully to resolve the enantiomers of a chiral amine, then this optically pure amine as the CDA will equally well separate the enantiomers of the acid by the indirect method. The OPA reaction (see Figure 4) is therefore equally well suited for analyzing the optical purity of thiols, amines or amino acids. 

OW Lau, CS Mok. Indirect conductometric detection of amino acids after liquid chromatographic separation. Part II. Determination of monosodium glutamate in foods. Anal Chim Acta 302 45 -52, 1995. 

Besides the commonly used direct LIF detection, indirect LIF detection on the microchip has also been reported. This method has been employed to detect explosives in spiked soil samples (see Figure 7.9) [620]. In contrast to a capillary-based system, an increase in E from 185-370 V/cm for MEKC separation did not result in an unstable background fluorescence due to excessive loule heating. This was probably because of the effective heat dissipation in the glass chip. However, upon multiple injection, it was found that the detection sensitivity decreased, which might be caused by the degradation of the visualizing dye (Cy7) [620]. Indirect LIF also allows the detection of unlabeled amino acids [683]. 

Figure 4 Separation of 100 pM amino acid mixture consisting of (1) alanine, (2) valine, (3) glutamine, and (4) tryptophan in an unmodified PMMA microchip using indirect, contact conductivity detection. Electrophoretic conditions 3-s electroki-netic injection time =150 V/cm for the electrophoresis. (Reprinted with permission from Ref. 20.)...

Often it is required to detect compounds with no or only very weak chromophores such as sugars and amino acids. Refractive index detectors and mass sensitive detectors can be used but they are relatively insensitive in the context of biological sample concentrations. Indirect detection using a UV or fluorescent eluent can also be employed. However, the most common approach is the use of derivatisation. Derivatisation of some chemically reactive moiety on the analyte can be performed in two modes. In post-column derivatisation the sample is separated first and then reacted with a flowing stream of derivatising reagent being pumped into... 

Enantiomeric separations of amino acids and short peptides are performed using either a direct or the indirect approach [10]. The indirect approach employs chiral reagents for diasteromer formation and their subsequent separation by various modes of CE. The direct approach uses a variety of chiral selectors that are incorporated into the electrolyte solution. Chiral selectors are optically pure compounds bearing at least one functional group with a chiral center (usually represented by an asymmetric carbon atom) which allows sterically selective interactions with the two enantiomers. Among others, cyclodextrins (CDs) are the... 

FIGURE 4 Indirect laser-induced fluorescence detection of 19 amino acids by microchip electrophoresis. Separation buffer 1.0 mM sodium carbonate, 0.5 mM fluorescein, and 0.2 mM CTAOH at pH 10.3. Separation condition /eff 5.5 cm, 15 s injection at 417 V/cm (reversed polarity), 183 V/cm separation voltage, sample amino acid concentrations of 0.4 mM in 1.0 mM sodium carbonate and 0.2 mM CTAOH. Reprinted with permission from [66]. Copyright 2000, The American Chemical Society. 

If these results can be taken at face value, the middle of domain 4 [726-767 (669-710 in chicken)] is involved in the TM enhancement of CD inhibition and binding to actin. Since this sequence is well separated from the putative TM binding sequence in domain 4, the effects of TM are probably indirect. The C-terminal end of this sequence is particularly rich in proline, a motif known to be present in many protein-protein binding sites (Williamson, 1994). Amino acids 752-771 (695-714),... 

Munro et al. showed separation and detection of amino acids on microchips using an indirect fluorescence detection method. Figure 36.10 shows application of this method to urine samples with no pretreatment other than dilution in the appropriate separation buffer. Abnormal amounts of amino acids can easily be detected in the two patient samples compared to the healthy control sample. An absorbance detection based approach was utilized for the clinical analysis of calcium ion in serum, which is important in the regulation of a number of physiological processes. Beads with an immobilized calcium reactive dye were placed into the detection region, and the samples mobilized past the beads using electrophoretic flow. While a true separation was not intended, the interference... 

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