d hptlc 36 milton aminoacids profile

7/26/2019 D HPTLC 36 Milton Aminoacids Profile

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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 (BOROSI!" #ml of $% &ydrochloric acid solution

was added with sample in specified test tubes" These test tubes were sealed at the top under

'acuum by high temperature gas flame conducted triplicates of samples" )ll the sealed tubes

were *ept in a hot+air o'en at ##0

o

, for -.hrs continuously"

Test solution pepaation

)fter completion of digestion bro*en the tubes at the top and transferred the digest into glass

bea*er (BOROSI! rinsed the tubes 5 times with distilled water" The acid in the digest was

e'aporated to core dry under 'acuum using Roto+'ac e'aporator" The residual content was

dissol'ed with distilled water and made+up to $ml in a centrifuge tubes" This solution

contains -#"$/g dried raw sample in #/l distilled water and used as test solution for amino+

acid profile analysis by &T, techni1ue"


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HPTLC ANALYSIS

!oup I !oup II !oup III !oup I"

)sparagine )spartic acid ysine &istidine

lutamine lutamic acid lycine )rginine

Serine )lanine Threonine ,ystine

roline 3aline Tyrosine Tryptophan

%ethionine henyl alanine Isoleucine eucine

Sample and Standad amino a#id loading

#"2/l of each test solutions were loaded as $mm band in pre+coated Silica gel $04 $%& T,

plate (#0cm #0cm! using #00/l &amilton syringe and ,)%)+I6O%)T 5 instrument"

2/l of each roup I II III 7 I3 standards were loaded in the plate for analysis as separate

trac*s"

Spot de'elopment

The samples loaded plate was *ept in T, twin trough de'eloping chamber with respecti'e

mobile phase ()mino acids! 20min for ,hamber saturation" )fter chamber saturation the

plate was de'eloped in respecti'e mobile phase up to 80mm (9ouble run with the same

mobile phase!"

P(oto)do#umentation

The de'eloped plate was dried by hot air to e'aporate sol'ents from the plate" The plate was

documented using hoto+documentation chamber (,)%)+R:ROST)R ;! at 9aylight


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Dei'ati*ation

The plate was sprayed with respecti'e spray reagent ()mino+acids! and dried at ##0o, in &ot

air o'en" )fter 9eri'ati=ation the plate was documented at 9aylight using ,)%)+

R:ROST)R ;"

S#anning

4inally the plate was fied in scanner stage and scanned at 500nm using ,)%)+T,

S,)66:R ;" The Rf 'alue and ea* area of each trac* were obser'ed for 1uantification

study"

Cal#ulations

Sample #on#entation +250mg dried raw material in $ml distilled water

Loaded 'olume o, test solution +#"2/l (50g of dried raw material!

Indi'idual Amino a#id #ontent in -+ ,onc" of amino acid in /g > 50/g #00


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AMINO)ACIDS PROFILE

C(omatogam

Before deri'ati=ation

9ay light


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6/28


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A,te dei'ati*ation

Da. lig(t

U" $%&nm


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Sample #ode

!I ) Standard amino+acids roup #

!II ) Standard amino+acids roup 2

!III )Standard amino+acids roup ;

!I" )Standard amino+acids roup -

/ ) Sample coded as ,ontrol

$ )Sample coded as 9#

0 )Sample coded as 92

& ) Sample coded as 9;

Mo1ile p(ase

2+Butanol+)mmonia+yridine+?ater (;"8 @ # @ ;"$ @ 2"-!

Spa. eagent

6inhydrin reagent prepared freshly" )fter spray the plate was dried in hot+air o'er at ##0o,

for ;min"

Dete#tion

3iolet+pin* colored =ones appeared in Standard amino+acids groups and Samples confirms

the presence of )mino+acids" )mino+acids which are present in the samples were 1uantified

with respect to their pea* area with single le'el standard amino+acids pea* area (*nown

concentration! of all 20 amino acids"


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Pea2 ta1le

Ta#2 Pea2 Assigned

su1stan#eR,

Aea

3in AU4

Con#entation

3in 5g4

Content 3in

-4

I # )sparagine 0"2. 7505.9 0"- +

I 2 lutamine 0";A 14495.4 0"- +

I ; Serine 0"-2 12253.2 0"- +

I - roline 0"5# 3203.5 0"- +

I 5 %ethionine 0"$# 7302.7 0"- +

II # )spartic acid 0"2# 4707.6 0"- +

II 2

)lanine

lutamic acid 0";$ 31050.6

0"- 0"- +

II ; 3aline 0"5- 13880.6 0"- +

II - henyl alanine 0"$- 7102.2 0"- +

III # ysine 0"0; 4877.7 0"- +

III 2 lycine 0";0 14535.7 0"- +

III ; Threonine 0"-$ 10659.0 0"- +

III - Isoleucine 0"5. 8133.3 0"- +

III 5 Tyrosine 0"$2 4221.5 0"- +

I3 # )rginine 0"0$ 1773.1 0"- +

I3 2 &istidine 0"#8 4507.4 0"- +


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I3 ; ,ystine 0";$ 15007.7 0"- +

I3 - eucine 0"$0 11288.4 0"- +

I3 5 Tryptophan 0"$5 5192.0 0"- +

# # ysine 0"0; 7008.2 0"5. #"#$

# 2 )rginine 0"0$ 5$28"5 #"2$ 2"52

# ; &istidine 0"#8 A##-". 0"$- #"2.

# - lycine 0";0 200##"8 0"5$ #"#2

# 5 )laluacid,ys 0";A -0$;0"0 0"520"52#"0. #"0-#"0-2"#$

# $ Threonine 0"-$ 14699.6 0"5$ #"#2

# A 3aline 0"5- 15947.1 0"-$ 0"82

# . Isoleucine 0"5. 31714.7 #"5$ ;"#2

2 # ysine 0"0; 8866.5 0"A2 #"--

2 2 )rginine 0"0$ 6401.7 #"-- 2"..

2 ; &istidine 0"#8 7206.4 0".. #"A$

2 - lycine 0";0 19638.1

0"5- #"0.

2 5 )laluacid,ys 0";$ 39995.9 0"520"52#"0$ #"0-#"0-2"#2

2 $ Threonine 0"-$ 15360.6 0"5. #"#$

2 A 3aline 0"5- 15560.6 0"-- 0"..

2 . Isoleucine 0"5. 12385.3 0"$ #"2

2 8 eu%et 0"$0 20430.9 0"A2#"#2 #"--2"2-

; # ysine 0"0; 9228.8 0"A$ #"52

; 2 )rginine 0"0$ 6485.8 #"-$ 2"82

; ; &istidine 0"#8 7002.3 0"$2 #"2-

; - lycine 0";0 #8$;2"8 0"5- #"0.


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; 5 )laluacid,ys 0";A ;8A22". 0"520"52#"0$ #"0-#"0-2"#2

; $ Threonine 0"-$ 14732.0 0"5$ #"#2

; A 3aline 0"5- 15524.7 0"-- 0"..

; . Isoleucine 0"5. 12516.4 0"$2 #"2-

- # ysine 0"0; 8801.4 0"A2 #"--

- 2 )rginine 0"0$ 6441.9 #"-$ 2"82

- ; &istidine 0"#8 5577.3 0"5 #

- - lycine 0";0 17722.7 0"-$ 0"82

- 5 )laluacid,ys 0";A 38763.7 0"50"5#"0- ##2"0.

- $ Threonine 0"-$ 13518.2 0"5 #

- A 3aline 0"5- 13946.3 0"- 0".

- . Isoleucine 0"5. 11230.5 0"5$ #"#2

- 8 eu%et 0"$0 18224.8 0"$-# #"2.2


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Ta#2 !I ) !oup I Standad amino a#ids ) 6aseline displa. 3%77nm4


13/28

Ta#2 !I ) !oup I Standad amino a#ids 8 Pea2 densitogam displa. 3%77nm4


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Ta#2 !II ) !oup II Standad amino a#ids 86aseline displa. 3%77nm4


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Ta#2 !II ) !oup II Standad amino a#ids 8 Pea2 densitogam displa. 3%77nm4


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Ta#2 !III ) !oup III Standad amino a#ids )6aseline displa. 3%77nm4


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Ta#2 !III ) !oup III Standad amino a#ids 8Pea2 densitogam displa. 3%77nm4


18/28

Ta#2 !I" ) !oup I" Standad amino a#ids )6aseline displa. 3%77nm4


19/28

Ta#2 !I" ) !oup I" Standad amino a#ids 8Pea2 densitogam displa. 3%77nm4


20/28

Ta#2 / 8 Sample no9 / Contol 6aseline displa. 3s#anned :%77nm4


21/28

Ta#2 / 8 Sample no9 / Contol Pea2 densitogam displa. 3s#anned :%77nm4


22/28

Ta#2 $ 8 Sample no9 $ D/ 6aseline displa. 3s#anned :%77nm4


23/28

Ta#2 $ 8 Sample no9 $ D/ Pea2 densitogam displa. 3s#anned :%77nm4


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Ta#2 0 8 Sample no9 0 D$ 6aseline displa. 3s#anned :%77nm4


25/28

Ta#2 0 8 Sample no9 0 D$ Pea2 densitogam displa. 3s#anned :%77nm4


26/28

Ta#2 & 8 Sample no9 & D0 6aseline displa. 3s#anned :%77nm4


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Ta#2 & 8 Sample no9 & D0 Pea2 densitogam displa. 3s#anned :%77nm4


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0D Displa. o, all Ta#2s

d hptlc 36 milton aminoacids profile

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