HPTLC ANALYSIS OF SAMPLES FOR AMINOACIDS

PROCEDURE
Sample digestion
The powdered samples each 250mg were weighed accurately in an electronic balance and
transferred into labeled glass test tubes (BOROSIL). 1ml of 6M Hydrochloric acid solution
was added with sample in specified test tubes. These test tubes were sealed at the top under
vacuum by high temperature gas flame, conducted triplicates of samples. All the sealed tubes
were kept in a hot-air oven at 110oC for 48hrs continuously.

Test solution preparation

After completion of digestion, broken the tubes at the top and transferred the digest into glass
beaker (BOROSIL), rinsed the tubes 5 times with distilled water. The acid in the digest was
evaporated to core dry under vacuum using Roto-vac evaporator. The residual content was
dissolved with distilled water and made-up to 6ml in a centrifuge tubes. This solution
contains 41.6g dried raw sample in 1l distilled water and used as test solution for aminoacid profile analysis by HPTLC technique.

HPTLC ANALYSIS

Group I

Group II

Group III

Group IV

Asparagine

Aspartic acid

Lysine

Histidine

Glutamine

Glutamic acid

Glycine

Arginine

Serine

Alanine

Threonine

Cystine

Proline

Valine

Tyrosine

Tryptophan

Methionine

Phenyl alanine

Isoleucine

Leucine

Sample and Standard amino acid loading

1.2l of each test solutions were loaded as 6mm band in pre-coated Silica gel 60F 254 TLC
plate (10cm x 10cm) using 100l Hamilton syringe and CAMAG-LINOMAT 5 instrument.
2l of each Group I, II, III & IV standards were loaded in the plate for analysis as separate
tracks.
Spot development
The samples loaded plate was kept in TLC twin trough developing chamber with respective
mobile phase (Amino acids), 20min for Chamber saturation. After chamber saturation the
plate was developed in respective mobile phase up to 90mm (Double run with the same
mobile phase).
Photo-documentation
The developed plate was dried by hot air to evaporate solvents from the plate. The plate was
documented using Photo-documentation chamber (CAMAG-REPROSTAR 3) at Daylight,
UV 254nm and UV366nm mode.

Derivatization
The plate was sprayed with respective spray reagent (Amino-acids) and dried at 110 oC in Hot
air oven. After Derivatization, the plate was documented at Daylight using CAMAGREPROSTAR 3.
Scanning
Finally, the plate was fixed in scanner stage and scanned at 500nm using CAMAG-TLC
SCANNER 3. The Rf value and Peak area of each track were observed for quantification
study.

Calculations
Sample concentration

: 250mg dried raw material in 6ml distilled water

Loaded volume of test solution

: 1.2l (50g of dried raw material)

Individual Amino acid content in %: Conc. of amino acid in g / 50g x 100

AMINO-ACIDS PROFILE
Chromatogram
Before derivatization
Day light

UV 366nm

UV 254nm

After derivatization

Day light

UV 254nm

Sample code
GI

- Standard amino-acids Group 1

GII

- Standard amino-acids Group 2

GIII

- Standard amino-acids Group 3

GIV

- Standard amino-acids Group 4

- Sample coded as Control

- Sample coded as D1

- Sample coded as D2

- Sample coded as D3

Mobile phase
2-Butanol-Ammonia-Pyridine-Water (3.9 : 1 : 3.6 : 2.4)
Spray reagent
Ninhydrin reagent prepared freshly. After spray, the plate was dried in hot-air over at 110 oC
for 3min.
Detection
Violet-pink colored zones appeared in Standard amino-acids groups and Samples confirms
the presence of Amino-acids. Amino-acids which are present in the samples were quantified
with respect to their peak area with single level standard amino-acids peak area (known
concentration) of all 20 amino acids.

Peak table

Track

Peak

Assigned
substance

Rf

Area
(in AU)

Concentration
(in g)

Content (in
%)

GI

Asparagine

0.28

7505.9

0.4

GI

Glutamine

0.37

14495.4

0.4

GI

Serine

0.42

12253.2

0.4

GI

Proline

0.51

3203.5

0.4

GI

Methionine

0.61

7302.7

0.4

GII

Aspartic acid

0.21

4707.6

0.4

GII

Alanine +
Glutamic acid

0.36

31050.6

0.4 + 0.4

GII

Valine

0.54

13880.6

0.4

GII

Phenyl alanine

0.64

7102.2

0.4

GIII

Lysine

0.03

4877.7

0.4

GIII

Glycine

0.30

14535.7

0.4

GIII

Threonine

0.46

10659.0

0.4

GIII

Isoleucine

0.58

8133.3

0.4

GIII

Tyrosine

0.62

4221.5

0.4

GIV

Arginine

0.06

1773.1

0.4

GIV

Histidine

0.19

4507.4

0.4

GIV

Cystine

0.36

15007.7

0.4

GIV

Leucine

0.60

11288.4

0.4

GIV

Tryptophan

0.65

5192.0

0.4

Lysine

0.03

7008.2

0.58

1.16

Arginine

0.06

5629.5

1.26

2.52

Histidine

0.19

7114.8

0.64

1.28

Glycine

0.30

20011.9

0.56

1.12

Ala+Gluacid+Cys

0.37

40630.0

Threonine

0.46

14699.6

0.56

1.12

Valine

0.54

15947.1

0.46

0.92

Isoleucine

0.58

31714.7

1.56

3.12

Lysine

0.03

8866.5

0.72

1.44

Arginine

0.06

6401.7

1.44

2.88

Histidine

0.19

7206.4

0.88

1.76

Glycine

0.30

19638.1

0.54

1.08

Ala+Gluacid+Cys

0.36

39995.9

Threonine

0.46

15360.6

0.58

1.16

Valine

0.54

15560.6

0.44

0.88

Isoleucine

0.58

12385.3

0.6

1.2

Leu+Met

0.60

20430.9

0.72+1.12

1.44+2.24

Lysine

0.03

9228.8

0.76

1.52

Arginine

0.06

6485.8

1.46

2.92

Histidine

0.19

7002.3

0.62

1.24

Glycine

0.30

19632.9

0.54

1.08

0.52+0.52+1.08 1.04+1.04+2.16

0.52+0.52+1.06 1.04+1.04+2.12

Ala+Gluacid+Cys

0.37

39722.8

0.52+0.52+1.06 1.04+1.04+2.12

Threonine

0.46

14732.0

0.56

1.12

Valine

0.54

15524.7

0.44

0.88

Isoleucine

0.58

12516.4

0.62

1.24

Lysine

0.03

8801.4

0.72

1.44

Arginine

0.06

6441.9

1.46

2.92

Histidine

0.19

5577.3

0.5

Glycine

0.30

17722.7

0.46

0.92

Ala+Gluacid+Cys

0.37

38763.7

0.5+0.5+1.04

1+1+2.08

Threonine

0.46

13518.2

0.5

Valine

0.54

13946.3

0.4

0.8

Isoleucine

0.58

11230.5

0.56

1.12

Leu+Met

0.60

18224.8

0.64+1

1.28+2

Track GI - Group I Standard amino acids - Baseline display (500nm)

Track GI - Group I Standard amino acids Peak densitogram display (500nm)

Track GII - Group II Standard amino acids Baseline display (500nm)

Track GII - Group II Standard amino acids Peak densitogram display (500nm)

Track GIII - Group III Standard amino acids -Baseline display (500nm)

Track GIII - Group III Standard amino acids Peak densitogram display (500nm)

Track GIV - Group IV Standard amino acids -Baseline display (500nm)

Track GIV - Group IV Standard amino acids Peak densitogram display (500nm)

Track 1 Sample no. 1 Control Baseline display (scanned @500nm)

Track 1 Sample no. 1 Control Peak densitogram display (scanned @500nm)

Track 2 Sample no. 2 D1 Baseline display (scanned @500nm)

Track 2 Sample no. 2 D1 Peak densitogram display (scanned @500nm)

Track 3 Sample no. 3 D2 Baseline display (scanned @500nm)

Track 3 Sample no. 3 D2 Peak densitogram display (scanned @500nm)

Track 4 Sample no. 4 D3 Baseline display (scanned @500nm)

Track 4 Sample no. 4 D3 Peak densitogram display (scanned @500nm)

3D Display of all Tracks