Amino acid analysis running conditions

Following affinity chromatography of the MIANS-ricin A-chain digest over the anti-MIANS column, the eluted fraction was run over the C,g RP-HPLC column utilizing gradient elution conditions. Individual peptides were collected for amino acid analysis (data not shown) and for sequencing. 

One other general point concerns the question of stability of samples. We always store our acidified samples at -20°C or below before analysis, and under these conditions amino acids are stable for 2-3 wk. The main stability problem is that of Gin. This ammo acid decomposes at temperatures above -20°C One of the problems associated with the instability of Gin is that it may decompose not only to Glu, but to pyroglutamic acid, which is ninhydrin negative. Thus the inaccuracy of Gin determinations may not be readily apparent from increases in Glu values Finally, because many ammo acid analyzers are fully automated, and samples are loaded into the machine for days prior to analysis, samples may be stored in a machine under varying conditions All investigators should verify that Gin is stable under the conditions of analysis used In a particular system, for instance. Gin may decompose at a rate of approximately 2-3% during the time period of each analysis. It may not be possible to report Gin values unless the sample is one of the first three run through a particular machine. 

This gradient system has been adapted for the analysis of coastal tind interstitial waters where compounds derived from amino acid degradation such as P-alaavae, taurine or amino butyric acids may occur in addition to the standard amino acids given in Table 26-1. For less complex samples such as, e.g., hydrolysates the gradient run time may be abbreviated and a linear gradient employed. It should, however, be noted that under these conditions glycine and threonine are usually not separated. 

Figure 7 Analysis of the 1-kb ladder by capillary zone electrophoresis. Conditions coated capillary of 100pm i.d., 47.4cm length, filled with 4.5% polyacrylamide strings in 100 mmol r Tris-borate-EDTA, pH 8.0. Sample a0.25pgmr solution of the 1-kb ladder was injected electrophoretically for 3 s at 4kV run at 4kV and 11.4pA. The numbers on each peak represent the length of each fragment, in number of bases. Tris, 2-amino-2-hydroxymethylpropane-1,3-diol EDTA, ethylenediaminetetraacetic acid.

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