Amino acids post-column derivatization

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

The use of reverse-phase columns with pre-column derivatization of the amino acids offers an acceptable alternative to the dedicated instrumentation of an amino acid analyser or separation by HPLC followed by post-column derivatization. 

As already mentioned the EP wants to replace old TEC tests with separation methods of higher efficiency for example, the purity of amino acids is currently evaluated by a TEC test for ninhydrin-positive substances that is only able to find and limit amino acids to 0.5%. However, this test is only valid in the case the amino acids are produced by the cleavage of peptides/proteins and purification. The ninhydrin method is also used in the amino acid analysis of peptides, utilizing a cation-exchange chromatography with a post-column derivatization and a subsequent UVA is detection. This method is often used in industries for purity evaluation of amino acids. 

The post-column derivatization of amino acids by the ninhydrin technique is a well known method for routine analysis of amino acids [7-9]. The amino acids are usually separated by ion-exchange chromatography and then converted into UV-absorbing derivatives for quantitation. The ninhydrin reaction is often used for TLC detection of amino acids and proteins. 

LIF detection (X,cx = 351.1 nm, Xm = 440 nm) of two amino acids (0.58 mM glycine, 0.48 mM arginine) was achieved by pre-column derivatization with 5.1 mM o-phthaldialdehyde (OPA) [106]. A reaction chamber was constructed before the cross-injection (see Figure 6.33). The chamber is wider than the separation channel to allow for a lower electric field and hence longer residence time for the derivatization reaction [106]. This method may be more advantageous than post-column derivatization [317], when the analysis time is faster than the product reaction time (tm for OPA 4 s [106]). 

Post-column derivatization was employed to detect the separated proteins [657] or amino acids [317,658]. For instance, 2-toluidinonaphthalene-6-sulfonate (TNS) was used to derivatize serum proteins in the post-column format (see Figure 6.34) [657]. With the use of fused silica with substantially lower fluorescence background = 325 nm), a two-fold improvement in S/N for phenylalanine detection was achieved [658],... 

Fluorescence in combination with post-column derivatization Excitation and emission Ammonium, amino acids, and polyamines after reaction with o-phthaldialdehyde... 

Filtrate (total amino acids) or supernatant (free amino acids) from above was diluted (20 1) with 0.2 N lithium citrate buffer, pH 2.8, prior to analysis using a Beckman 6380 amino acid analyzer (Beckman Coulter, Inc.) equipped with a 10 cm ion exchange column and lithium citrate buffer supplied by Beckman. Detection was via post column derivatization using ninhydrin. Calibration was via mixed external free amino acid standards. 

Amino acid analysis is useful for assessment of the nutritional value of food and feed.15 The essential amino acids are methionine, cysteine, lysine, threonine, valine, isoleucine, leucine, phenylalanine, tyrosine, and tryptophan. Glutamic acid is a commercial flavor enhancer. Since amino acids are nonchro-mophoric (possess low UV absorbance), either pre-column or post-column derivatization is typically needed ... 

Post-column derivatization—IEC is used to separate free amino acids followed by ninhydrin or o-phthaldehyde (OPA) post-column derivatiza-... 

In post-column derivatization systems, the derivatlzation reagent is continuously mixed with the column effluent consequently, the presence of trace amounts of amino acids or interfering components such as ammonia in the column eluent limits the ultimate sensitivity attainable. Figure 2 shows the effects of buffer contaminants on the baseline of a typical high sensitivity analysis (12). The basic contaminants are held up on the column until elution by the final buffer. Detection of the basic amino acids below lO-picomole levels is difficult using this approach. The use of a shorter second column with isocratic elution for the analysis of just the basic amino acids is a good solution to the problem but does require the use of a second aliquot of sample. 

The most recent advances[21] in LC-EC include pre- and post-column derivatization (mostly with the aim of increasing the number of electrons transferred and hence also the sensitivity). In the LC-EC determination of amino acids it may proceed via oxidative detection of o-pthaldehyde/ mercaptoethanol. In this case the sensitivity has been increased so that 5.10 mol may be determined. A special method is the photolytic deriv-... 

Table 3-33. Comparison of integrated amperomet and post-column derivatization with ninhydrin as detection methods for determining amino acids in a collagen hydrolysate.

The method was further extended for analysis of d- and L-amino acids in mouse kidney in a similar manner. HPLC methods, for the resolution of amino acid enantiomers that utilized a chiral stationary phase or a chiral mobile phase additive were considered expensive and less satisfactory due to the high retention results in broad peaks. The separation of FDAA derivatives followed by HPLC, as described above, was found to be much more successful as there was neither needed a post column derivatization nor fluorimetric analysis and moreover a subnanomolar sensitivity was attained [33], d- and L-enantiomers of glutamate, aspartate, asparagine, serine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, and histidine were derivatized with FDAA and the diastereomers were separated by 2D TLC. Each was separated except for the two spots comprising Tyr and Val, and He, Leu, and Phe. Only histidine was separated further into D- and L-diastereomers by TLC. The excess hydrolyzed FDAA moved to the front... 

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