Methods for Impregnation

Of the various methods used for impregnation, one is mixing of the impregnating reagent with the inert support. A second approach is the immersion of the plates into an appropriate solution of the impregnating reagent carefully and slowly so as not to disturb the thin layer. Alternatively, a solution of the impregnating material is allowed to ascend or descend the plate in the normal manner of development; this method is less likely to damage the thin layer. Exposing the layers to the vapours of the impregnating reagent or spraying the impregnating reagent (or its solution) on to the plate have also been employed; spraying provides a less uniform dispersion than the other methods. Another approach is to have a chemical reaction between the inert support and a suitable reagent: the support is chemically modified before making the plate, the compounds of interest are bonded to the reactive groups of the layer.

The impregnating agent participates in various mechanisms in the resolution process, including ion-pairing, complex formation, ligand exchange, coordination bonds, charge transfer, ion exchange and hydrogen bonding.

Amino acids Resolution of amino acids has been reported to be very rapid and improved by using copper sulfate, halide ions, zinc, cadmium and mercury salts, and alkaline earth metal hydroxides as impregnating materials and some of the results are described in Tables 14-17. The chromatograms developed in these systems provide compact spots, without lateral drifting of the solvent front. C18 layers impregnated with dodecylbenzene sulfonic acid are helpful in confirming the presence of an unknown amino acid in a sample and the migration sequence on these impregnated plates is reversed, probably due to an ion exchange mechanism. Separation of a-amino acids with butan-1-ol-acetic acid-water (3:1:1, v/v), butan-1-ol-acetic acid-chloroform (3:1:1, v/v), and butan-1-ol-acetic acid-ethyl acetate (3:1:1, v/v), on plain and nickel chloride impregnated plates has been reported; the partition and adsorption coefficients for the amino acids under study were determined on both untreated and Ni2 + impregnated silica gel in a batch process and correlations were drawn between TLC separation of amino acids on the impregnated gel with adsorption/partition characteristics. The results indicate a predominant role of partitioning in the separation. Application of antimony (V) phosphate-silica gel plates in different aqueous, nonaqueous and mixed solvent systems has also been reported. Some impregnated TLC systems for resolution of amino acids are summarized in Table 18.

PTH amino acids As mentioned above, certain difficulties in resolving or identifying various PTH amino acid combinations have successfully been removed and multicomponent mixtures separated with metal impregnated silica gel layers, while other reagents such as (+ )-tartaric acid and ( — )-ascorbic acid have been used for the resolution of enantiomeric mixtures. The methods reported provide very effective resolution and compact spots (by exposure to iodine vapours) and can be applied to the identification of unknown PTH amino acid; some of these are given in Tables 19-21. Some of the successful solvent systems for TLC of PTH amino acids on impregnated plates are summarized in Table 22.

High performance TLC (HPTLC)/overpressured

TLC (OPTLC) Improvements in the solid-phase materials for TLC have resulted in an increase in

Table 14 of amino acids in presence of halides

Sl. no.

Amino acid

Control plate

Amino acids pretreated with

Plates impregnated with

cr

Br

r

cr

Br

r

1.

Gly

07

08

09

12

07

08

09

2.

Tyr

30

35

40

47

29

30

31

3.

Pro

12

15

19

22

08

09

10

4.

Thr

15

14

15

19

13

14

16

5.

Cys

22

22

25

27

19

20

22

6.

Leu

32

40

47

50T

50T

55T

60T

7.

Met

23

35

36

37

22

23

24

8.

Ile

30

38

44

44

30

30

31

9.

Ala

15

19

13

16T

16T

16

16

10.

Try

35T

40

50T

53

30

31

34

11.

Phe

36T

41

48

48

365

37

38

12.

Val

19

32

25

29

25

26

26

13.

Asp

08

13

14

15

08

09

10

14.

Ser

09

13T

13

14T

08

08

09

15.

His

01

03

04

05

02

02

02

Time

(min)

50

64

67

67

50

50

50

Solvent system: n-butanol-acetic acid-chloroform (3:1 : 1, v/v); temperature 25 $ 2°C. T = tailing.

Solvent system: n-butanol-acetic acid-chloroform (3:1 : 1, v/v); temperature 25 $ 2°C. T = tailing.

separation efficiency, sample detectability limits and reduced analysis time. HPTLC can be used with advantage for the separation of PTH amino acids but separation of all 20 common PTH amino acids was

Table 15 values for amino acids on copper sulfate and polyamide mixed silica gel plates

Amino acid

A

B

C

L-Leucine (Leu)

65

63

71

D,L-Isoleucine (Ile)

66

72

81

D,L-Tryptophane (Try)

63

68

75

D,L-Methionine (Met)

64

64

72

D,L-Valine (Val)

64

60

77

L-Lysine-HCl (Lys)

16T

12

33

L-Histidine-HCl (His)

22T

20

39

D,L-^-Phenylalanine (Phe)

64

65

82

D,L-Threonine (Thr)

50

51

67

D,L-Alanine (Ala)

46

45

64

D,L-Serine (Ser)

40

43

56

L-Tyrosine (Tyr)

58

61

71

L-Glutamic acid (Glu)

41

48

58

D,L-Aspartic acid (Asp)

28

25

44

L-Arginine HCl (Arg)

24T

19

39

Glycine (Gly)

36

46

49

L-Proline (Pro)

37

36

58

L-Cysteine HCl (Cys)

20T

17

29

D,L-2-Aminobutyric acid (Aba)

51

54

61

L-Ornithine

27T

23

35

The values are average of two or more identical runs, 10 cm in 35 min. A, untreated silica gel plate; B, copper sulfate-impreg-nated silica gel; C, polyamide mixed silica gel layers; T, tailing Solvent, methanol-butyl acetate-acetic acid-pyridine (20 : 20 : 10:5, v/v).

The values are average of two or more identical runs, 10 cm in 35 min. A, untreated silica gel plate; B, copper sulfate-impreg-nated silica gel; C, polyamide mixed silica gel layers; T, tailing Solvent, methanol-butyl acetate-acetic acid-pyridine (20 : 20 : 10:5, v/v).

not achieved initially. A continuous multiple development on silica gel was able to separate 18 samples and standards simultaneously using five development steps with four changes in mobile-phase and scanning densitometry; typical results are given in Table 23. PTH-Leu/Ile/Pro have been identified by HPTLC using multiple wavelength detection. OPLC using chloroform-ethanol-acetic acid (90 : 10 : 2) for polar, and dichloromethane-ethyl acetate (90 : 10) for nonpolar PTH amino acids has been successful in their separation and quantitation; the method is claimed to be superior to HPTLC in having relatively increased migration distance, resulting in the resolution of complex mixtures containing a large number of derivatives. OPTLC and HPTLC on RP-8, RP-18, and home-made ammonium tungstophosphate layers have also been used for the analysis of DNP amino acids.

Separation of 18 amino acids on cellulose, silica gel and chemically bonded C18 HPTLC plates has been achieved. All of these plates contain a preadsorbent zone except the cellulose. Quantification is carried out by scanning standard and sample zones at 610 nm. hRF values of amino acid standards on rever-sed-phase and on normal-phase layers in different solvents are given in Tables 24 and 25, respectively.

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