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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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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
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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
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Ta#2 !I" ) !oup I" Standad amino a#ids )6aseline displa. 3%77nm4
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Ta#2 !I" ) !oup I" Standad amino a#ids 8Pea2 densitogam displa. 3%77nm4
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Ta#2 / 8 Sample no9 / Contol 6aseline displa. 3s#anned :%77nm4
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Ta#2 / 8 Sample no9 / Contol Pea2 densitogam displa. 3s#anned :%77nm4
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Ta#2 $ 8 Sample no9 $ D/ 6aseline displa. 3s#anned :%77nm4
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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
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Ta#2 0 8 Sample no9 0 D$ Pea2 densitogam displa. 3s#anned :%77nm4
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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
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