PTH Amino Acids

The quantitation of PTH amino acids is carried out in situ or after elution. For in situ determination, the fluorescence-quenching areas of PTH derivatives are usually measured against the fluorescent background

Table 31 Separation data for enantiomeric compounds on /-CD-bonded-phase plates

Compound, d,l mixture RF Mobile Detection

- phasea method

Dns-leucine

0.49

0.66

40/66

Fluorescence

Dns-methionine

0.28

0.43

25/75

Fluorescence

Dns-alanine

0.25

0.33

25/75

Fluorescence

Dns-valine

0.31

0.42

25/75

Fluorescence

Alanine-ß-naphthylamide

0.16

0.25

30/70

Ninhydrin

Methionine-ß-naphthylamide

0.16

0.24

30/70

Ninhydrin

aVolume ratio of methanol to 1 % triethylammonium acetate (pH 4.1).

aVolume ratio of methanol to 1 % triethylammonium acetate (pH 4.1).

at 254 nm. While using a Turner fluorometer fitted with a door for scanning chromatoplates, the position of the scanner, the standardization of time between scanning and the end of chromatography, the loading volume, the developing distance and the layer thickness are the important influencing factors for repro-ducibility. The quantitation of PTH amino acids is also carried out by measuring their UV absorbance after they have been eluted from the layer. The scraped layer is extracted with methanol overnight, centrifuged for 30 min at 300 rpm, and the spectra of the extracts are recorded in the range from 320 nm to about 230 nm. To obtain reproducible UV absorban-ces the layers must be washed with methanol prior to development, and with chloroform after the separ ation has been carried out. The quantitation of PTH amino acids has also been practised as follows: the developed chromatograms are exposed to iodine vapours and the brownish spots scraped off, eluted with 95% ethanol or ethyl acetate, and the iodine removed by warming the sample tubes in a warm-water bath. The optical densities are read at 269 and 245 nm, appropriate blank determinations are carried out, standard plots are drawn, and the concentration of the unknown sample is calculated.

Table 32 RP-TLC enantiomeric separation using vancomycin as chiral mobile-phase additive

Compound

l

d

Vancomycin concentration (mol L -')

AQC-allo-iso-leucine

14

21

0.025

AQC-methionine

19

23

0.025

AQC-nor-leucine

13

16

0.025

AQC-nor-valine

21

25

0.025

AQC-valine

23

27

0.025

Dansyl-glumatic acid

21

23

0.04

Dansyl-leucine

03

09

0.04

Dansyl-methionine

05

12

0.04

Dansyl-nor-leucine

03

07

0.04

Dansyl-nor-valine

05

12

0.04

Dansyl-phenylalanine

03

05

0.04

Dansyl-serine

15

20

0.04

Dansyl-threonine

13

17

0.05

Dansyl-tryptophan

01

03

0.04

Dansyl-valine

06

10

Mobile phase, acetonitrile-0.6 mol L~1 NaCl (2 : 10). Plates developed at room temperature (22°C) in cylindrical glass chambers. Time, 1 -3 h for 5x20 cm plates. Visualization, UV. AQC, 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate, a fluorescent tagging agent; reaction mixture of AQC and amino acid was used without purifying the derivatives.

Figure 2 Chromatogram showing resolution of Dns-DL-phenylalanine, valine and leucine. From left to right: 1, Dns-DL-phenylalanine; 2, Dns-L-phenylalanine; 3, Dns-DL-valine; 4, Dns-L-valine; 5, Dns-DL-leucine; 6, Dns-L-leucine. Developing solvent, aq. 0.5 mol L!l sodium chloride + acetonitrile (15 + 1). Developing time 20-25 min. Detection 254 nm.

Table 33 Values of enantiomers of dansyl amino acids resolved on plates with erythromycin

Dansyl dl-amino acid Pure l hRF from dl mixture Solvent system

d l NaCl-MeCN-MeOH

Dansyl dl-amino acid Pure l hRF from dl mixture Solvent system

d l NaCl-MeCN-MeOH

Serine

64

68

64

10

4

1

30

36

30

15

:1

1

Glutamic acid

45

56

45

22

:1

0.5

56

65

56

22

:1

0

52

59

52

26

:1

0

Phenylalanine

50

65

50

15

:2

0

20

27

20

15

:1

0

Valine

22

30

22

15

:1

0

Leucine

24

32

24

15

:1

0

Tryptophan

38

47

38

18

:1

0.25

Methionine

56

63

56

25

:2

0.5

Aspartic acid

50

63

50

28

1.5:0.5

a-Amino-n-butyric acid

42

51

42

12

:1

0

Norleucine

63

71

63

16

:1

0 : 0.

Temperature 25 $ 2°C. Solvent front, 10 cm. Time, 20-25 min. Visualization, UV, 254 nm.

Temperature 25 $ 2°C. Solvent front, 10 cm. Time, 20-25 min. Visualization, UV, 254 nm.

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