Direct method

1. This method uses a chiral stationary phase; it may be due to either natural chirality of the material as such, like cellulose, or some sort of synthesis of the phase.

2. Chiral discriminating agents are added to the mobile phase.

3. A suitable chiral reagent is incorporated, such as acid, base, an organic compound or a metal complex with the adbsorbent during plate making, or at a stage before developing the chromatogram.

DL-amino acids Separation of d,l-tryptophan on a crystalline cellulose-coated plate in 1980 seems to be one of the first TLC reports. Applying the principle of ligand exchange, (2S, 4R, 2'RS)-4-hydroxyl-1-(2'-hydroxy dodecyl)-proline was used as the chiral

Table 19 hRF values of PTH amino acids on Fe2 +, Co2 +, Ni2+ and Zn2+ impregnated silica plates

Sl. no.

PTH-amino acid

Alone

Fe2#

Co2*

Ni2#

Zn2+

0.2%

0.3%

0.05% 0.1%

0.1%

0.2%

0.2%

0.3%

1.

Alanine

60

42

41

57

51

38

40

50

43

2.

Aspartic acid

0

0

0

0

0

0

0

0

0

3.

Glycine

39

26

21

44

38

29

30

32

27

4.

Glutamic acid

0

0

0

0

0

0

0

0

0

5.

Isoleucine

90

84

75

72

90

65

71

81

72

6.

Leucine

95

87

71

82

81

70

76

85

76

7.

Lysine

23

8

6

15

17

7

4

10

8

8.

Methionine

70

54

47

81

62

58

51

57

58

9.

Phenylalanine

75

61

49

77

68

52

55

66

58

10.

Proline

97

89

89

84

76

83

90

96

89

11.

Serine

13

5

5

11

9

11

12

8

5

12.

Tyrosine

96

867

69

68

95

85

78

83

78

13.

Tryptophan

95

91

70

91

97

77

82

88

81

14.

Threonine

86

78

57

94

83

60

63

78

70

15.

Valine

85

75

73

96

79

57

58

76

67

Solvent, chloroform-ethyl acetate (29: 3, v/v); developing time 35 min; solvent front, 10 cm.

Table 20 hRF values ofPTH amino acids on untreated plates and plates impregnated with sulfates ofMg, Mn, Fe and Co

PTHaminoacid S1(heptane-butylacetate, 15 + 5) S2(heptane-propionicacid, 20+ 4) S3 (benzene-ethylacetate,

Table 20 hRF values ofPTH amino acids on untreated plates and plates impregnated with sulfates ofMg, Mn, Fe and Co

PS1

M1

M2

M3

M4

PS2

M1

m2

M3

M4

PS3

M4

Methionine

28

30

26

32

31

43

45

30

32

35

62

78

Phenylalanine

30

35

29

37

34

50

52

36

38

40

67

80

Tryptophan

63

61

51

60

57

71

67

55

55

57

82

94

Valine

49

46

40

51

47

66

62

52

50

55

73

85

Isoleucine

62

62

50

62

59

77

72

61

60

62

78

65

Tryosine

66

64

53

64

61

80

74

64

64

65

84

89

Threonine

57

52

45

56

53

63

64

53

53

53

72

83

Alanine

23

25

23

26

25

32

34

27

25

29

50

67

Serine

55

55

42

55

51

48

46

38

49

40

70

44

Leucine

69

65

54

63

56

76

71

62

60

67

80

96

Lysine

06

04

02

03

05

06

10

04

06

07

18

35

Glycine

17

15

13

15

15

17

20

15

15

18

37

55

Glutamic acid

04

06

05

06

06

04

04

06

06

05

0

14

Aspartic acid

05

07

06

07

07

05

08

07

07

06

0

22

Proline

44

33

31

34

32

44

42

35

35

45

79

96

aCompounds moved to solvent front on plates impregnated with sulfates of Mg, Mn and Fe. PS,, PS2, PS3, untreated plates; M,, M2, M3, M4, treated with sulfates of Mg, Mn, Fe, and Co, respectively. RF values are the average of at least five determinations. Developed in 30-40 min at 25°C $ 2°C, and exposed to iodine vapours to locate the spots.

selector to resolve several racemic a-amino acids on reversed-phase 18-TLC plates first immersed (1 min) in a 0.25% copper(II) acetate solution (MeOH-H2O, 1 : 9, v/v), dried, and then immersed in a 0.8% meth-anolic solution of the chiral selector (1 min); the results are shown in Table 26. Ready-to-use Chiral-plates® are now marketed by Macherey-Nagel, Duren, Germany, and Chir® plates are marketed by Merck, Germany. Resolution of dl-methyl Dopa, and DL-Dopa is very successful on Chiralplates using methanol-H2O-acetonitrile (50 : 50 : 30, v/v) as the mobile phase and ninhydrin as the detecting reagent (Figure 1). The RF values for l-Dopa and d-Dopa were reported to be 0.47 and 0.61, respectively, and the system is capable of resolving enantiomers in trace amounts, with the lowest level of detection of the d-enantiomer in l-Dopa samples being 0.25%. The resolution of enantiomers of a-substituted a-amino acids, and racemic mixtures of natural and nonnatural amino acids, N-methylated and

Table 21 Values of PTH amino acids on silica plates impregnated with zinc salts

PTHaminoacid S1(heptane-butylacetate, 15+5) S2(heptane-propionicacid, 20+4) S3(benzene-ethylacetate15+3)

Table 21 Values of PTH amino acids on silica plates impregnated with zinc salts

PTHaminoacid S1(heptane-butylacetate, 15+5) S2(heptane-propionicacid, 20+4) S3(benzene-ethylacetate15+3)

L1

L2

L3

L4

L1

L2

L3

L4

L1

L2

L3

L4

Methionine

17

22

18

25

33

33

29

33

42

57

48

58

Phenylalanine

22

25

25

28

37

38

35

36

47

60

52

60

Tryptophan

36

41

41

51

40

50

40

55

67

74

61

82

Valine

40

35

44

42

52

53

54

52

54

68

64

69

Isoleucine

50

46

55

54

50

62

63

63

62

79

74

74

Tryosine

55

48

59

56

62

64

75

65

64

84

79

78

Threonine

47

40

52

48

53

51

52

56

57

72

72

67

Alanine

23

17

21

22

29

27

23

27

35

44

38

44

Serine

49

45

42

50

38

36

37

39

52

66

60

64

Leucine

55

51

55

59

61

50

67

60

65

81

77

76

Lysine

03

02

03

03

04

05

04

07

6

7

6

8

Glycine

15

10

12

13

16

15

15

15

24

29

25

31

Glutamic acid

0

04

0

04

03

02

02

04

0

0

0

0

Aspartic acid

0

05

0

05

04

03

03

05

0

0

0

0

Proline

38

25

32

30

40

40

40

42

70

77

83

72

L-!, L2, L3, L4 plates impregnated with Cl , SO2 , CH3COO and PO4 of zinc, respectively. Other conditions as in Table 20.

L-!, L2, L3, L4 plates impregnated with Cl , SO2 , CH3COO and PO4 of zinc, respectively. Other conditions as in Table 20.

Table 22 TLC of PTH amino acids on impregnated silica gel layers

Solvent system

Ratio (v/v)

Impregnationa

CHCl3-H2O-EtOAc

28

1:

1

Zn2 + , Cd2 +, Hg2 +

CHCl3-MeOH-Benzene

19

1:

9

CCl4-HOAc

19

1

CHCl3-Benzene-EtOAc

25

5:

3

Fe2 + , Co2 +, Zn2 +

CHCl3-EtOAc

29

3

Fe2 + , Co2 +, Ni2 +

n-Heptane-n-butyl acetate

15

5

Cr, ch3coo~, po3~

n-Heptane-n-proprionic acid

20

4

of zinc, or SO2~ of

Benzene-EtOAc

Mg2 + , Mn2 +, Fe2 + , Co2 +

CHCl3-n-butyl acetate

10

5

CHCl3-EtOAc

25

2

aVarious concentrations of each of the impregnating reagent have been used.

aVarious concentrations of each of the impregnating reagent have been used.

N-formylated amino acids, and various other derivatives of amino acids has also been achieved on Chiral-plates; typical results are presented in Tables 27 and 28. A novel chiral selector from (1R, 3R, 5R)-aza-bicyclo-[3,3,0]-octan carboxylic acid has been synthesized and used as a copper(II) complex for the impregnation of reversed-phase 18 plates for ligand exchange TLC separation of amino acids and the results were comparable to those on Chiralplates®.

Chiral selectors such as (— )-brucine and Cu-l-pro-line complex are used to resolve enantiomers of amino acids (Table 29), and (+ )-tartaric acid and (— )-ascorbic acid for the resolution of enantiomeric PTH amino acids (Table 30). The chiral selectors are mixed with silica gel slurry.

Resolution of trytophans and substituted tryp-tophans on cellulose layers developed with copper sulfate solutions has shown that excess of Cu2 + ions decreases the chiral discrimination of the system, and development with aqueous a-cyclodextrin (1-10%) plus NaCl solutions (0.1, 0.5, 1.0 mol L_1) showed the best results with aqueous 4% a-cyclodextrin-1 mol L_1 NaCl solution; the results are comparable to Chiralplate®. It has been observed that chiral effects are essentially additive (for cellulose and a-cyclodextrin) and there is a strong temperature dependence for the chiral separations.

a- and ^-cyclodextrins, hydroxypropyl-^-cyclodex-trin and bovine serum albumin in the mobile phase have provided enantiomeric separations of amino acids and derivatives. Chiral monohalo-s-triazines have been used for the TLC resolution of dl-amino acids. Racemic dinitropyridyl-, dinitrophenyl- and dinitrobenzoyl amino acids are separated on rever-sed-phase-TLC plates developed with aqueous-org mobile phases containing bovine serum albumin as a chiral agent.

Dansyl-DL-amino acids Reversed-phase TLC plates from Whatman are developed before application of dansyl amino acids in buffer A (0.3 mol L_1 sodium acetate in 40% acetonitrile, and 60% water adjusted to pH 7 by acetic acid). After fan-drying, the plates are immersed in a solution of 8 mmol L_1 N,N-di-«-propyl-l-alanine and 4 mmol L_1 cupric acetate in 97.5% acetonitrile, 2.5% water for 1 h or overnight and left to dry in air. After applying the samples, the plates are developed in buffer A with or without N,N-di-«-propyl-l-alanine (4 mmol L_1) and cupric acetate (1 mmol L_1) is dissolved in it. The enantiomers are detected by irradiating with UV light (360 nm) to yield fluorescent yellow-green spots. Use of 25% acetonitrile is preferred for glutamic and aspartic acids and serine and threonine derivatives. N,N-di-«-propyl alanine can be prepared by the following procedure: l-alanine (17.8 g) is dissolved in ethanol (200 mL) and 10% palladium on activated charcoal catalyst (3 g) and

Table 23 Optimum experimental conditions for the separation of PTH amino acids by continuous multiple development HPTLC

Step Mobile phase

Plate length (cm)

Time (min)

PTH amino acid identified

CH2CI2

CH2CI2-propan-2-oI (99 :1, v/v) CH2CI2-propan-2-oI (99 : 1, v/v)

CH2CI2-propan-2-oI (97 : 3, v/v) C2H5COOCH3-CH3CN-CH3COOH (74.3 : 20 : 0.7, v/v)

5 10 10

10 10

Table 24 hRF Values of amino acid standards on reversed-phase layers

Table 26 Enantiomeric resolution of amino acids by TLC

Amino acid

TLC system

Table 24 hRF Values of amino acid standards on reversed-phase layers

Amino acid

TLC system

1

2

3

4

5

6

Aspartic acid

30

59

72

60

83

73

Arginine

28

4

35

28

86

82

Serine

55

36

69

50

82

73

Glycine

50

38

62

45

69

54

Tyrosine

91

77

88

68

77

67

Alanine

78

59

71

63

71

54

Glutamic acid

82

70

86

67

83

69

Proline

56

69

64

40

65

46

Cystine

11

12

39

33

85

84

Methionine

90

74

75

59

75

61

Lysine

31

84

27

24

74

79

Tryprophan

90

77

85

63

72

63

Valine

90

74

75

59

75

61

Threonine

78

52

68

50

72

57

Histidine

21

3

29

23

77

68

Phenylalanine

90

76

83

65

72

61

Leucine

90

77

81

62

75

63

Isoleucine

91

77

81

62

74

61

Table 25 Values of amino acid standards on normal-phase layers

Amino acid

TLC system

1

2

3

4

Aspartic acid

28

27

26

58

Arginine

18

18

17

2

Serine

26

30

27

40

Glycine

26

32

28

43

Tyrosine

46

58

53

78

Alanine

38

32

32

55

Glutamic acid

69

56

50

64

Proline

45

32

28

50

Cystine

10

11

9

30

Methionine

60

59

51

72

Lysine

15

13

10

4

Tryptophan

55

63

57

82

Valine

60

56

49

68

Threonine

34

32

32

53

Histidine

14

14

11

17

Phenylalanine

68

61

55

80

Leucine

79

61

55

78

Isoleucine

78

59

54

77

Table 26 Enantiomeric resolution of amino acids by TLC

Amino acid

Rf value (configuration)

Mobile phase

R

S

Isoleucine

0.37 (2R, 3R)

0.44 (2S,3S)

A

Phenylalanine

0.38

0.45

A

Tyrosine

0.34

0.26

B

Tryptophan

0.39

0.45

A

Proline

0.40

0.59

B

Glutamine

0.53

0.37

A

Development distance: 14 cm; saturated chamber. A, MeOH-water-MeCN (1:1:4, v/v); B, MeOH-water-MeCN (5:5:3, v/v).

a sintered glass filter and the filtrate is evaporated to dryness. The reaction product (N,N-di-w-propyl-l-alanine) is crystallized from chloroform, and the purity may be confirmed by TLC, and carbon, hydrogen, nitrogen analysis.

Layers: 1, 2, Whatman C-18; 3, 5, Merck RP-18; 4, 6, Merck RP-18W. Mobile phases: 1, 3, 4, n-Butanol-glacial acetic acid-water (3:1 : 1, v/v); 2, 5, 6, n-propanol-water (7 : 3, v/v).

propionaldehyde (43 mL) is added. The mixture is hydrogenated for 48 h at 40-50°C at an initial hydrogen pressure of 50 psi; the catalyst is removed using

Table 25 Values of amino acid standards on normal-phase layers

Figure 1 Chromatogram showing separation of different d- and L-dopa samples on Chiralplate. From left to right: 1, L-dopa; 2, D,L-dopa; 3, D-dopa; 4, 3% L-dopa in D-dopa; 5, 3% D-dopa in L-dopa. Developing solvent, methanol-water-acetonitrile (5:5:3, v/v). Developing time 45-60 min. Detection 0.1% nin-hydrin spray reagent.

Layers: 1, Cellulose; 2, 4, Whatman silica gel; 3, Merck silica gel. Mobile phases: 1, Butan-2-ol-glacial acetic acid-water (3:1 : 1, v/v); 2, 3, n-butanol-glacial acetic-water (3:1 : 1, v/v); 4, n-propanol-water (7 : 3, v/v).

Figure 1 Chromatogram showing separation of different d- and L-dopa samples on Chiralplate. From left to right: 1, L-dopa; 2, D,L-dopa; 3, D-dopa; 4, 3% L-dopa in D-dopa; 5, 3% D-dopa in L-dopa. Developing solvent, methanol-water-acetonitrile (5:5:3, v/v). Developing time 45-60 min. Detection 0.1% nin-hydrin spray reagent.

Table 27 Enantiomeric resolution of a-dialkyl amino acids by TLC

Parent amino acid

R1

R2

Rf value

Configuration

Mobile phase

Asp

CH2CO2H

CH3

0.52 (D)

0.56 (L)

A

Glu

(CH2)2CO2H

CH3

0.58 (L)

0.62 (D)

A

Leu

CH2CH(CH3)2

CH3

0.48

0.59

A

Phe

CH2C6H5

CH3

0.53 (L)

0.66 (D)

A

Ser

CH2OH

CH3

0.56 (L)

0.67 (D)

B

Try

CH2-3-indolyl

CH3

0.54

0.65

A

Tyr

CH2-(4-OH-C6H4)

CH3

0.63 (D)

0.70 (L)

A

Val

CH(CH3)2

CH3

0.51

0.56

A

a-Amino butyric acid

CH2CH3

CH3

0.50

0.60

A

Phe

CH2C6H5

chf2

0.63

0.70

A

Phe

CH2C6H5

CH2—CH=CH2

0.57

0.63

A

Phe

CH2C6H5

CH2CH2SCH3

0.57

0.62

A

Mobile phase: A, methanol-water-acetonitrile (1:1:4, v/v); B, methanol-water-acetonitrile (5:5:3, v/v). Development distance 13 cm; saturated chamber.

In a two-dimensional reversed-phase TLC tech- ated in nonchiral mode using 0.3 mol sodium nique for the resolution of complex mixture of dan- acetate in H2O-acetonitrile (80 : 20, pH 6.3) to syW/-amino acids, the Dns-derivatives are first separ- which 0.3 mol L_1 sodium acetate in H2O-aceto-

Table 28 Enantiomeric resolution of racemates by TLC

Racemate

Rf value

Configuration

Mobile phase

Valine

0.54(d)

0.62(l)

A

Methionine

0.54(d)

0.59(l)

A

Allo-isoleucine

0.51(d)

0.61(l)

A

Norleucine

0.53(d)

0.62(l)

A

2-Aminobutyric acid

0.48

0.52

A

o-Benzylserine

0.54(d)

0.65(l)

A

3-Chloralanine

0.57

0.64

A

S-(2-Chlorobenzyl)-cysteine

0.45

0.58

A

S-(3-Thiabutyl)-cysteine

0.53

0.64

A

S-(2-Thiapropyl)-cysteine

0.53

0.64

A

c/s-4-Hydroxyproline

0.41(l)

0.59(d)

A

Phenylglycine

0.57

0.67

A

3-Cyclopentylalanine

0.46

0.56

A

Homophenylalanine

0.49(d)

0.58(l)

A

4-Methoxyphenylalanine

0.52

0.64

A

4-Aminophenylalanine

0.33

0.47

A

4-Bromophenylalanine

0.44

0.58

A

4-Chlorophenylalanine

0.46

0.59

A

2-Fluorophenylalanine

0.55

0.61

A

4-Iodophenylalanine

0.45(d)

0.61(l)

A

4-Nitrophenylalanine

0.52

0.61

A

o-Benzyltyrosine

0.48(d)

0.64(l)

A

3-Flurotyrosine

0.64

0.71

A

4-Methyltryptophan

0.50

0.58

A

5-Methyltryptophan

0.52

0.63

A

6-Methyltryptophan

0.52

0.64

A

7-Methyltryptophan

0.51

0.64

A

5-Bromotryptophan

0.46

0.58

A

5-Methoxytryptophan

0.55

0.66

A

2-(1-Methylcyclopropyl)-glycine

0.49

0.57

A

N-Methylphenylalanine

0.59(d)

0.61(l)

A

N-Formyl-tert-leucine

0.48( + )

0.61( —)

A

N-Glycylphenylalanine

0.51(l)

0.57(l)

B

A, Methanol-water-acetonitrile (1:1:4, v/v); B, methanol-water-acetonitrile (5:5:3, v/v). Development distance, 13 cm; saturated chamber.

Table 29 Resolution data for enantiomers of amino acids from brucine-impregnated plates

Sl. no.

d-l-amino acid

hRF pure l

d

l

1.

Alanine

53

18

53

2.

y-Aminobutyric acid

3.

Isoleucine

35

16

35

4.

DOPA

5.

Leucine

6.

Methionine

29

18

29

7.

Norleucine

8.

Phenylalanine

40

27

40

9.

Serine

50

12

50

10.

Threonine

29

16

29

11.

Tryptophan

31

17

31

12.

Tyrosine

29

22

29

Silica plates impregnated with (-)-brucine, developed in n-butanol-acetic acid-chloroform (3:1:4, v/v), up to 10 cm.

Silica plates impregnated with (-)-brucine, developed in n-butanol-acetic acid-chloroform (3:1:4, v/v), up to 10 cm.

nitrile (70 : 30) is added to give a final acetonitrile concentration of 38% or 47%. For the second dimension, the mobile phase is 8 mol N,N-di-n-propyl-l-alanine and 4 mmol L_1 copper(II) acetate dissolved in 0.3 mol L_1 sodium acetate in H2O-acetonitrile (70 : 30, pH 7); the plates are developed in the second dimension using a temperature gradient. The method is reported to be applicable to the resolution of amino acids in a protein hydrolysate with quantitation by densitometry.

^-Cyclodextrin (^-CD) plates have been used successfully for the resolution of enantiomers of dansyl amino acids and ^-naphthylamide amino acids. The plates are prepared by mixing 1.5 g of ^-CD bonded silica gel in 15 mL of 50% methanol (aqueous) with 0.002 g of binder and acetate in 50/50 MeOH-1% aqueous triethyl ammonium acetate (pH 4.1). Some of the results are shown in Table 31.

Table 30 hRF of pure and resolved enantiomers of PTH amino acids, for tartaric acid-impregnated plate

d,l Mixture of

hRF of

d (resolved)

l (resolved)

PTH amino acids

pure l

Met

83

18

83

Phe

85

15

85

Try

95

95

Val

80

21

80

Ile

92

15

92

Tyr

95

16

95

Thr

85

30

85

Ala

55

12

55

Ser

84

10

84

Solvent, chloroform-ethyl acetate-water (28 :1 :1, v/v). Development time, 35 min, solvent front, 10 cm, room temperature, 25 + 1 °C. Impregnation with (#)-ascorbic acid resolved d,l mixtures of PTH-Met, Phe, Val, Ala, Ser.

Solvent, chloroform-ethyl acetate-water (28 :1 :1, v/v). Development time, 35 min, solvent front, 10 cm, room temperature, 25 + 1 °C. Impregnation with (#)-ascorbic acid resolved d,l mixtures of PTH-Met, Phe, Val, Ala, Ser.

A macrocyclic antibiotic, vancomycin, has been used as a chiral mobile-phase additive for the separation of 6-aminoquinolyl-N-hydroxy succinimidyl carbamate (AQC) derivatized amino acids and dansyl amino acids on chemically bonded diphenyl-Frever-sed-phase plates. Both the nature of stationary phase and the composition of the mobile phase have a strong influence on the enantiomeric resolution; typical results are given in Table 32. Another macro-cyclic antibiotic, erythromycin, has been used as a chiral impregnating reagent for the resolution of 10 dansyl-dl-amino acids on silica gel plates (Figure 2); hRF values and solvent combinations are shown in Table 33. Resolution of dansyl-dl-amino acids has recently been reported (Table 34) on thin silica gel plates impregnated with (1R, 3R, 5R)-aza-bicyclo[3,3,0]octan-3-carboxylic acid, which is an industrial waste material and a proline analogue non-proteinogenic a-amino acid.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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