Amino acids chromatograms

Figure 3. Spinco amino acid chromatogram of H2SO4 extract of MarceUus shale run in sodium citrate buffer column 1 to right eluted 2Yz hours at pH 3.25, then 2l/s hours at pH 4.25, [Na], 0.2N column 2 to left eluted with pH...

Figure 25-4 Part of amino-acid chromatogram obtained by the method of automatic amino-acid analysis from a hydrolyzed sample of the enzyme ribonuclease. The component amino acids listed are present in the ratio Asp Thr Ser Glu Pro Gly Ala = 15 10 15 12 4 3 12, as determined by peak intensity. The volume of effluent is a measure of the retention time of the amino acids on the column.

For calculation of the molar ratio of Amadori compounds of peak C in the amino acid chromatogram (Figure 1) the following formula was used ... 

Fig. 4. Composite diagram of an amino acid chromatogram of urine from normal individuals. The specimen was applied at the origin (X) and developed as described in Section 4. The solvent systems employed were the same as those listed in Fig. 2, and the completed chromatograms were visualized with ninhydrin-collidine. Not all the amino acids shown need be present in any one sample. The numbers correspond to the amino acids listed in Table 2.

Fig. 1. Amino acid chromatogram by an automatic amino acid analyzer.

E. Kawerau, Th. Wieland, Conservation of amino acid chromatograms. Nature 168 77-79 (1951)... 

There are some areas of the amino acid chromatogram in which several amino acids are eluted very close to each other and in which obtaining a clear separation can be a problem. Often, an improved separation of amino aads in one region of the chromatogram comes at the expense of poorer separation of... 

Amino acid chromatograms have been preserved by impregnation with collodion to which a definite amount of glycerol had been added [42, 66]. Chromatograms have also been pasted over with self-adhesive plastic film and the layer subsequently peeled off the plate [354]. Spraying with polymer dispersions (polyacrylates, polyvinylidene chloride or polyvinyl propionate) is usually more suitable [402]. Such aerosols are commercially available (Firms 88, 153). Mention may be made also of impregnation of layers by immersion in solutions of polymers although this has a restricted application (Firm 60). 

Sequence analysis can only be conducted on a pure protein. First, the amino acid composition is determined after acidic hydrolysis. The procedure (separation on a single cation-exchange resin column and color development with ninhydrin reagent or fluorescamine) has been standardized and automated (amino acid analyzers). Figure 1.10 shows a typical amino acid chromatogram. 

Fig. 1.10. Amino acid chromatogram. Separation of a mixture of amino acids (10 nmol/amino acid) by an amino acid analyzer. Applied is a single ion exchange column Durrum DC-4A, 295 x 4 mm buffers Pi/P2/P3- 0.2 N Na-citrate pH 3.20/0.2 N Na-citrate pH 4.25/1.2 N Na-citrate and NaCl of pH 6.45. Temperatures T1/T2/T3 48/56/80 °C. Flow rate 25 ml/h absorbance reading after color development with ninhydrin at 570/440 nm ...

Formation of D-amino acids occurs through abstraction of a proton via a C2-carbanion. The reaction with L-isoleucine is particularly interesting. L-Isoleucine is isomerized to D-alloisoleucine which, unlike other D-amino acids, is a diastereoisomer and so has a retention time different from L-isoleucine, mtddng its determination possible directly from an amino acid chromatogram ... 

In practice a few iodine crystals are usually placed on the bottom of a dry, closed trough chamber. After the chamber has become saturated with violet iodine vapor the solvent-free plates are placed in the chamber for 30 s to a few minutes. The iodine vapor condenses on the TLC layers and is enriched in the chromatogram zones. Iodine vapor is a universal detector, there are examples of its application for all types of substances, e.g. amino acids, indoles, alkaloids, steroids, psychoactive substances, lipids (a tabular compilation would be too voluminous to include in this section). 

Fig. 1 Reflectance scan (A) and fluorescence scan (B) of a chromatogram track with 50 ng of each amino acid per chromatogram zone hydroxyproline (1), proline (2).

The blue-violet stain which forms on thin-layer chromatograms when amino acids are stained with ninhydrin is only stable for a short time. It rapidly begins to fade even on cellulose layers. The stability can be appreciably enhanced by complex formation with metal ions [3]. 

The chromatograms stained with ninhydrin are immersed in the reagent solution for 1 s or sprayed evenly with it and then placed in the free half of a twin-trough chamber containing 25% ammonia solution. Apart from proline and hydroxyproline, which yield yellow copper complexes, all the amino acids yield reddish-colored chromatogram zones [3],... 

Before treatment with ammonia valine (hRf 45 — 50), isoleucine (hRf 65 — 70) and leucine (hRf 70 — 75) produced red chromatogram zones and phenylalanine (hRf 55—60) violet chromatogram zones on a pale pink background. After treatment with ammonia all four amino acids appeared as violet chromatogram zones on a flesh-colored background these zones were stable over an extended period. 

Glycine (hRf 20—25), DL-alanine (hRf 30 — 35), DL-valine hRf 45 — 50) and L-leucine (HRf 55—60) yielded red-violet chromatogram zones on a pale yellow background. The detection limits for these amino acids were 5 ng substance per... 

Fig. 1 Scanning curve of a chromatogram track with 100 ng per chromatogram zone of the amino acids glycine (1), alanine (2), valine (3), leucine (4).

The detection limits for amino acids and peptides are between 50 and 200 pmol per chromatogram zone [9], 400 pg for 5-hydroxyindolylacetic acid [11] and 300 pg for dihydroxyergotoxin [19]. 

FIGURE 4.22 HPLC chromatogram of amino acids employing precolumn derivatiza-tion with OPA. Chromatography was carried out on an Ultrasphere ODS column using a complex tetrahydrofuran methanol 0.05 M sodium acetate (pH 5.9) 1 19 80 to methanol 0.05 M sodium acetate (pH 5.9) 4 1 gradient at a flow rate of 1.7 mL/min. 

The percentages of amino acids in silk fibroin which Poison et al. (224) found by direct visual and indirect photometric analysis of ninhydrin paper-partition chromatograms are shown in Table VII. The percentages obtained for alanine, glycine, and serine appear to be reasonably accurate, inasmuch as they agree closely with those found by other methods. It would be of interest to determine alanine by the microbiological method reported recently by Sauberlich and Baumann (238), in view of the widely different values found for this amino acid by the described ninhydrin-chromatographic procedure and the selec-... 

Reddish or bluish chromatogram zones [3, 9] are produced, amino sugars and amino acids also react. Unexpectedly ascorbic acid also reacts. 

---