DNP Amino Acids

Use of DNP amino acids, formed by condensation of 1-fiuoro-2,4-dinitrobenzene (FDNB) with the free amino group of an amino acid, was first described by Sanger in 1945, who identified DNP amino acids by paper chromatography. Since then many modifications in the methods of obtaining derivatives of

Table 12 hRF Values of 10 dansylamino acids on silica gel thin layers

N-DNP-l-amino acid

Solvent system

S1

S2

S3

S4

S5

1.

Phenylalanine

53

48

85

70

55

2.

Isoleucine

68

82

96

97

60

3.

Tyrosine

25

30

60

52

36

4.

Alanine

40

36

68

50

42

5.

Glycine

28

17

35

25

27

6.

Leucine

65

73

93

90

52

7.

Tryptophan

48

33

53

47

34

8.

Methionine

45

40

75

57

42

9.

Valine

62

65

90

85

47

10.

Proline

41

45

74

60

38

11.

Norvaline

61

62

88

83

45

Solvent system

A1

A2

A3

A4

a5

12.

N-DNP-L-serine

51

68

70

70

70

13.

N-DNP-lysine

21

26

11

7

27

14.

N-S-di-DNP-L-cysteine

82

87

77

85

85

15.

N-DNP-L-glutamic acid

67

80

83

92

82

cyclohexyl-amine salt

16.

N-DNP-L-aspartic acid

38

70

75

60

60

17.

N-DNP-L-asparagine

30

64

45

38

55

18.

N-DNP-L-arginine

10

6

5

3

18

19.

N,N-di-DNP-L-cystine

48

70

55

65

82

A-!, Dichlormethane-MeOH-propionic acid (21:3:2, v/v); A2, ethyl acetate-MeOH-propionic acid (22 : 10 : 3, v/v); A3, chloro-form-MeOH-HOAc (28 : 4 : 2, v/v); A4, chloroform-acetone-HOAc (20 : 8 : 4, v/v); A5, chloroform-acetone-propionic acid (24 : 10 : 5, v/v) RF values are the average of five determinations.

Si, n-heptane-n-butanol-acetic acid (20 : 4 : 1, v/v); S2, chloroform-propionic acid (26: 2, v/v); S3, chloroform-acetic acid (21 : 1, v/v); S4, chloroform-ethanol-propionic acid (30 : 2 : 1, v/v); S5, benzene-n-butanol-acetic acid (34:1 :1, v/v); A1, chloroform-methanol-acetic acid (25 : 5 : 1, v/v); A2, chloro-form-propionic acid-methanol (15:10:1, v/v); A3, n-heptane-butanol-acetic acid (16:8:4, v/v); A4, n-butanol-ethyl acetate-acetic acid (20 : 8 : 2, v/v); A5, n-butanol-methanol-propionic acid (18:8:2, v/v). RF values are average of five determinations.

amino acids for sequence analysis and in identification of such derivatives have been reported, and the use of DNP amino acids for sequencing purposes is rapidly going out of practice. Nevertheless, the importance of DNP amino acids has not yet disappeared.

Kirchner presented considerable information on the analysis of DNP amino acids based on the literature available up to 1970. In one of the earlier methods, thin-layer plates (20 x 20 cm x 0.25 mm) were prepared from a mixture of 10 g of cellulose MN-300 and 4 g silica gel H (Merck), homogenized in 80 mL of water, dried overnight at 37°C and developed in the first dimension with two solvents successively: /so-propanol-acetic acid-H2O (75:10:15) for 15 min and w-butanol-0.15 mol L_1 ammonium hydroxide (1 : 1, upper phase). The dried chromatograms were developed in 1.5 mol

L_1 sodium phosphate buffer (pH 6.0) in the second dimension.

In almost all methods reported, the separation has been carried out in groups of water-soluble and ether-soluble DNP amino acids, and for each group mostly two-dimensional TLC has been performed. A few solvent systems for one-dimensional resolution of DNP amino acids on silica gel plates are shown in Table 13.

Detection of DNP amino acids The DNP amino acids have been visualized by UV light (360 nm with dried plates, or 254 nm with wet ones) or by their yellow colour, which deepens upon exposure to ammonia vapours. Thin layers of silica gel usually give an intense purple fluorescence for DNP amino acids under UV light, which masks the presence of very faint spots and decreases the colour contrasts. The cellulose-silica mixed layer gives much lower fluorescence and preserves the colour contrasts between various derivatives. Because of the photosensitivity of these derivatives, it is advisable to carry out their preparation and chromatography in the absence of direct illumination.

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