EXPERIMENT FIX komang.doc

8/10/2019 EXPERIMENT FIX komang.doc

1/22

Experiment Report of BioChemistry 1

I. TITLE OF EXPERIMENT : Determine Amino Acids

Level in

Sample

II. DATE OF EXPERIMENT : October 24 th 2014

III. END OF EXPERIMENT : October 24 th 2014

IV. PURPOSE OF EXPERIMENT :

Determine Amino Acid Contained in The Sample with Paper

Chromatograph

V. BASIC THEORY

A. AMINO ACIDAmino acids are organic compo!nds that have a carbo" l

#!nctional gro!p $%COO&' and amine $!s!all % (& 2') *n biochemistr + it

is o#ten narrowed sense, both are attached to the carbon $C' atoms are the

same and so%called C%alpha) Carbo" l gro!ps giving properties o# acids

and amine gro!ps provide al-alinit ) The general str!ct!re o# an amino

acid ,

.here / is a #!nctional gro!p that determines the t pe o# amino

acid) All amino acids #o!nd in proteins have the same characteristics+

namel the presence o# carbo" l and amine bonded to the same carbon

atom) The str!ct!re o# the amino acids in general is one C atom that binds

#o!r gro!ps, the amine gro!p $(& 2'+ carbo" l gro!p $COO&'+ a h drogen

atom $&'+ and the leaving gro!p $/+ o# the resid!e' or also called gro!ps or

side chains disting!ishes one amino acid with another amino acid)

The central C atom named C atoms $ C %al#a ' in accordance

with the naming o# a compo!nd having a carbo" l gro!p+ the C atoms

directl bonded to the carbo" l gro!p) There#ore+ the cl!ster amines are

also bo!nd to the C atom+ the compo!nd is an acid amino)


2/22


B. PAPER CHROMATOGRAPHY

Chromatograph is !sed to separated the mi"ed #rom the

s!bstansion become the components) All chromatograph t pe have a

same principle wor-s) All chromatograph have a stationar phase $li-e a

solid or li3!id that s!pport in a solid' and mobile phase $a li3!id or gas')

obile phase #low together #rom stationar phase and bring some

components #rom a mi"ed) Di##erent components will move on a di##erent

rate too) *n paper chromatograph + stationar phase is man heart paper)

A mobile phase is s!itable solvent or solvent mi"ed)

Paper Chromatograph !sed thin plat that have been strat!m with

absorbent li-e silica gel+ al!mini!m o"ide $al!mine' or cell!lose) The

absorbent as a stationar phase) The silica gel a!"icle contained hidro" l

gro!p on the top that #ormed a h drogen bond with polar water molec!le)

Some o# compo!nd in mi"ed move #ar with the distance o# solvent+

and the others still near with based line) The relative attac- distance on a

solvent is constant #or certain compo!nd i# o! still -eep all is same+

ma be the t pe p# paper and the e"actl composition solvent)

C. RETENTION FACTOR (Rf)

The distance between the wa o# solvent is relativel ) So+ its

needed a certain calc!lated to ma-e s!re the stained that #ormet have same

distance altho!ght the si5e distance o# plat is di##erent) The val!e o# the

calc!lated is /#+ this val!e is !sed as relative compared val!e between

sample)

/# val!e is e"plained the retension degree o# a component instationar phase+ so the /# val!e is rope called retension #actor) /etension

6actor $/#' is attac- distance o# component divided with the attac-

distance o# el!ent) /# is also e"plain a retension degree o# a component in

stationar phase and its beca!se wh /# also called re#erence #actor+ with

the start dot is the middle spot)


3/22


The ge#e!al $%!&'la %$ Re"e#si%# $ac"%! (R$) :

Ill's"!a"e* ic"'!e %$ TLC la" :

*n the pict!re7 TLC plate $a' plat be#ore improve+ $b' plat a#ter improved)

$6irda!s) 2011')The /# val!e is ver charateristics #or certain compo!nd in certain ele!ent

too) *t can be !sed to ident# there8s a di##erent on sample) A compo!nd that have

bigger /# it means have lover polar+ and viece ver5a) *ts beca!se the stationar

phase is polar) A more polar compo!nd will strength resist in stationar phase+ so

its prod!ce the lower /# val!e)

The s"a!" s %"The A+B+C c%& %#e#"s is se a!a"e*

The li&i" s%l,e#"

The i#i"ial *%"


4/22


Ta-le . P!% e!"ies R$ ,al'e %$ s%&e a&i#% aci*s.


5/22


D. Paper Chromatography I Am! o A"!# M!$t%re

A a&y'!'

Chromatograph paper is applied to the anal sis o# a mi"t!re o#

amino+ beca!se amino acids have a water%sol!ble and non%volatile

properties+ so that it can be separated thro!gh the displacement o# the

mobile phase $el!ent' on the stationar phase $adsorbent' amino) Amino

acids will be Carried b the mobile phase will precipitate or attached to the

stationar phase $adsorbent' a#ter a on certain distance)

Adsorbent in the chromatograph paper is #ilter paper+ namel

cell!lose) Samples to be anal 5ed is spotted to the edge o# the paper which

is then h!ng in the container) Then the basic #ilter paper dipped in a

solvent that #ills the bottom o# the container) 9ach amino acid moves #rom

the starting point along a certain distance) 9ach t pe o# amino acid is

alwa s a distance that is t pical o# each amino acid as long as the paper

t pe+ el!ent+ and the same solvent) Then compared the mileage el!ent #rom

each amino acid)

Amino acids that react with ninh drin #ormed prod!ct that called

P!rple /!hmann) This raection !s!all !se as spot test to identi# there8san amino acid in paper chromatograph )

/i"h "he !eac"i%# :

(inhidrin : & ; (:%C&/%COO% < anion p!rple : /C&O : CO 2 : ;& 2O : & :


6/22


VI. TOOLS AND MATERIALS

T%%ls% Chromatograph paper $1 item'% Capiler pipe $1 item'% =ea-er glass large si5e $1 item'% Spre bottle $1 item'% Oven $1 item'% Pencil $1 item'% /!ler $1 item'% >arn $1 item'% Sewing needle $1 item'

Ma"e!ials

% Acetic acids glacial% n%b!thanol% A3!adest% Amino acid standard


7/22


VII. PROCEDURE

. De"e!&i#i#g C%& %#e#" O$ A&i#% Aci*

. Ma0i#g E&'lsi%# S%l'"i%#

2?ml grams n%b!thanol:@ml acetic

acid glacial : 2?ml a3!adest

% i"ed

% Sha-ed

% P!t the sol!tion into

chromatograph c!pboard

% Sat!rated with it8s vapo!r

9l!ent $mobile phase'

Paper Chromatograph

$si5e 4"10cm'

% Drop 2 sol!tions $A and =' side b side

with the distance 1cm

% Pic- sol!tion 0+?cm #rom border paper

% 9ach drop is dried be#ore the ne"t drop

placed on it

% Large stains is not more than 0+4cm

Paper Chromatograph stained

% Paper chromatograph hanging in

chromatograph c!pboard #or sat!ration

% Once sat!ration is reached recentl !sed

el!sion) .here chromatograph can be

!sed to decrease or increase

chromatograph


8/22


Paper Chromatograph a#ter el!ated

% A#ter el!tion r!nning well+ the paper

removed)

% A sol!tion o# the =o!ndar mar-ed with

a pencil paper spra ed with ninh drine

% Dried at 10?%110oC #or ? min!tes the

stain will loo- amino acids

% Calc!late the /# val!es determined b

comparing the mino acid component o#

the val!e o# the standard amino acids /#

Color+ /# val!e+ name o#

amino acid


9/22


IX. DATA ANALYSIS

1) P!e a!a"i%# %$ e&'lsi$ie! s%l'"i%# (&%-ile hase)

The principle o# chromatograph is the di##erence in distrib!tion

between the mobile phase and stationar ) 9l!ent was !sed as a phase o#

motion) So the #irst step is aimed at the man!#act!re o# the em!lsi#ier sol!tion

or el!ent !sed as mobile phase)

6or the #irst step o# Separated amino acid in sample+ we made

em!lsi#ier sol!tion $mobile phase' with 2?ml n%=!thanol colorless adding

with @ml acetic acid glacial colorless+ than adding 2?ml a3!adest colorless+ its

can prod!ce a t!rbid sol!tion and there8s a b!bble and its ma- esteri#ication

reaction)

/i"h "he es"e!i$ica"i%# !eac"i%# :

1 &2O

The third sol!tion is called the mi"ed el!ent that will serve as the

mobile phase in chromatograph + the el!ent was mi"ed and into two la ers+

namel the !pper and lower la ers) Then po!r the sol!tion in the closet

chromatograph $chamber' in the #orm o# a rectang!lar glass and #ilter paper

was covered in it !ntil covered all over the glass wall on the inside) The#

i&&e*ia"el2 cl%se "he cha&-e! "ha" ha* c%#"ai#e* "he s%l'"i%# 3i"h a

glass cl%se*+ -eca'se el!ent is easier to dissolve+ so the atmosphere in the

chamber becomes sat!rated with vapor #rom the sol!tion and to ma-e s!re i#

the condition in glass is satiated b the steam #rom solvent) Signs that the

atmosphere in the chamber sat!rated is #ilter paper that was in the chamber as

a whole has become wet)

The '! %se %$ "he sa"'!a"i%# cha&-e! is to ma-e the el!ent #!l#ill

the chamber and its #!nction as a mobile phase in a chromatographic r!n well)

*# the el!ent doesn8t #!ll#ill the chamber+ then the distrib!tion rather than the

stationar phase will not be able to wal- so chromatograph #ailed and the

&: -alor


10/22


res!lts obtained are not care#!l) .hereas in chromatograph aims to determine

what compo!nds contained in the sample indicated the presence o#

spots stains that indicate a compo!nd) 9ach compo!nd has a di##erent

distrib!tion in the mobile phase)

Selec"i%# %$ el'e#" is ,e!2 i& %!"a#" -eca'se i# the el!ent !sed has a

concentration that is not in accordance with the sample to be separated+ the

chromatograph can not r!n) *# the el!ent is too polar will ca!se the entire

stain is spotted on the paper rose to the !pper limit witho!t separation+ i# a less

polar el!ent+ then spotted a stain that will not move at all)

2) De"e!&i#e The A&i#% Aci* C%& %#e#"s

6or the #irst step is meas!ring chromatograph paper 4 cm " 10 cm

made the o!tline o# the top and the bottom edge) Line o# the top edge o# 0)?

cm and the bottom edge lines o# 1)? cm) 4i,e* a sig# li#e 3i"h e#cil in

paper chromatograph as show the start position #rom the #irst drop) *# we do

with pen+ the in- color will move li-e chromatogram shaped $/o B)1 1')

Re&e&-e! 3e ha,e 'se* "he g%%* 5'ali"2 %$ la" ch!%&a"%g!a h2 + beca!se i# we !sed bro-e or bad 3!alit o# plat chromatograph + when we

inp!t the plat on chamber+ then r!n process o# el!ent on the plat

chromatograph is r!n not reall good+ can8t to straght and obli3!e inclined)

A#ter it gave a point #or sample A and = at a distance o# 1cm #rom the side

with capiller pipe+ we !se capiler pipe when we spotes in plat chromatograph

beca!se to order the dropped o# amino acid is not #all too m!ch+ so in one plat

chromatograph can patch ; even 4 o# amino acids) Then Appl the sample to

the chromatographic plat in accordance with the point that has been given)

then give the top rope witho!t on%line chromatograph plate)

A#ter chromatograph plate #astened properl + enter the

chromatograph plate into the chamber+ do it ver care#!ll + lest when

inserting chromatograph plate+ bottom plate bo!ndaries e"posed

chromatographic el!ent) The se a!a"e* a&i#% aci* 'si#g ch!%&a"%g!a h2

a e! is ca'se* there is di##erent partision+ that show the di##erent o# creep


11/22


rate on the top o# paper and the organic solvent that creep slowl ) The phase o#

water will strength resist in paper pore beca!se there are strength interaction

between water in h dro" l gro!p #rom cell!lose #ilter paper)

Then wait !ntil the el!ent was wal-ing towards the !pper limit

almost chromatograph plate+ then li#t passage and remove+ and meas!re the

distance o# the bo!ndar el!ent r!nning+ then dr in the oven #or 1? min!tes+

then spra plate chromatograph with ninh drin sol!tion !ntil the color

p!rple) *n this e"periment+ 3e 'se* #i#h2*!i#e to ma-e showed the stained

p!rple color $/!hemann8s p!rple'+ become stained p!rple color beca!se there

are reaction between h drate and three-etones si-li- $ninh drine' with amino

acid)

/i"h "he !eac"i%# :

(inhidrin : & ; (:%C&/%COO% < anion p!rple : /C&O : CO 2 : ;& 2O : & :

*n o!r e"epriment+ a#ter being spra ed with ninh drin color is not an

indication o# an amino acid+ beca!se it is li-el d!e to the chromatographic

plate is still water content is derived #rom several #actors+ s!ch as sweat o!r


12/22


hands+ then we dr the plate chromatograph or reheat in the oven #or 1?

min!tes+ and then dots appear p!rplish color+ it shows that there are amino

acids in the sample+ then comp!te /# val!e )

The ge#e!al $%!&'la %$ Re"e#si%# $ac"%! (R$) :

This meas!red based the attac- distance o# the solvent and the attac- distance

o# each spot)

6rom the data we get the almost right /# is #rom /# = 0+2E its almost

right with the /# o# glic ne 0+2@) And #or /# A 0+12 we get the almost right

/# with /# o# Fl!tamin is 0+1;) .e can8t get the e"actl /# with the theor

beca!se man mista-es in o!r e"periment+ #rom the materials+ tools+ or

h!man error) So its a##eceted the di##erent res!lt is not s!itable with theor )

Than determine the /# o# each sample sol!tion $AG =') .ith the #orm!la+ this is

the calc!lated , 9l!ent distance @+;cm+ Stain A 0+Ecm and Stain = 1+Ecm

/# A

/# =

So+ =ased the data res!lt o# o!r e"periment we get /# A 0+12 and /# = 0+2@


13/22


X. DISCUSSION

*n a second e"periment+ there were some problems+ especiall #rom

the mista-es o# h!man error) A#ter spra ing ninh drine color p!rple

does not immediatel appear in the chromatograph plate+ and this is

beca!se7 The $i!s" !%-le& , A#ter we spots the sample sol!tion $AG='

we dont oven the plat chromatograph !ntil 10 min!tes+ wh we m!st

ovened itH =eca!se on plat chromatograph contain silica gel+ and in

silica gel that contained with water+ so we m!st ovened it be#ore we

inp!t the plat chromatograph in chamber) The sec%#* !%-le& , a

be an error #rom o!r gro!p when we spots the sample on plat

chromatograph its less preass!red+ so that ca!sed o# the el!ent is not

so per#ect) The "h!i* !%-le& , beca!se the ninh drine place is in

bottles o# per#!me+ so the ninh drine sol!tin is containated with the

little o# per#!me) A#* "he las" !%-le& , =eca!se the diameter o# the

ninh drine bottles is too big+ so we can spra completl i# the diameter

is too big)

XI. CONCLUSION

1) Paper chromatograph it can be !sed to ident# or separated

amino acid on a mi"ed sol!tion)

2) The name o# amino acid o# sample A is Fl!tamine 0+1; with

/# A val!e that we get is almost right 0+12

;) The second name o# amino acid o# sample = is Flic ne 0+2@with /# = val!e that we get is almost right 0+2E


14/22


ANS/ER OF 6UESTION

1. The a*,a#"ages a#* *isa*,a#"ages %$ "he &e"h%* %$ se a!a"i%# -2

a e! ch!%&a"%g!a h2 : Answered:

A*,a#"ages %$ TLCTLC is ver simple to !se and ine"pensive) Indergrad!ates can be

ta!ght this techni3!e and appl its similar principles to other

chromatographic techni3!es) There are little materials needed #or TLC

$chamber+watch glass+ capillar + plate+ solvent+ pencil+ and IJ%light')

There#ore+ once the best solvent is #o!nd+ it can be applied to other techni3!es s!ch as &igh per#ormance li3!id chromatograph ) ore than 1

compo!nd can be separated on a TLC plate as long as the mobile phase is

pre#erred #or each compo!nd) The solvents #or the TLC plate can be

changed easil and it is possible to !se several di##erent solvents

depending on o!r desired res!lts) As stated earlier+ TLC can be !sed to

ens!re p!rit o# a compo!nd) *t is ver eas to chec- the p!rit !sing a

IJ%light) *denti#ication o# most compo!nds can be done simpl bchec-ing /# literat!re val!es) >o! can modi# the chromatograph

conditions easil to increase the optimi5ation #or resol!tion o# a speci#ic

component)

Disa*,a#"ages %$ TLCTLC plates do not have long stationar phases) There#ore+ the

length o# separation is limited compared to other chromatographic

techni3!es) Also+ the detection limit is a lot higher) *# o! wo!ld need alower detection limit+ one wo!ld have to !se other chromatographic

techni3!es) TLC operates as an open s stem+ so #actors s!ch as h!midit

and temperat!re can be conse3!ences to the res!lts o# o!r chromatogram)

2. /ha" &e"h%* %$ a e! ch!%&a"%g!a h2 ca# -e 'se* $%! 5'a#"i"a"i,e

a#al2sis7 Answered :


15/22


K!antitative anal sis is per#ormed b comparing the /# o# the

sample s!bstance with /# price o# a standard s!bstance) *n order #or

3!antitative anal sis can wor- well to note it % the #ollowing,

a. The e"perimental conditions sho!ld be the same+ beca!se the pricedepends on the condition /#

b. There is a stain on the chromatogram not impl the e"istence o# a

single s!bstance in the samplec. !st be #itted with a variet o# solvents

K!alitative anal sis is done b identi# ing the amino acid

components o# the sample to a sol!tion o# amino acids which are -nown in

advance based on the val!e o# /#+ *n this e"periment mar-ed b a p!rple

color as well as the price /# investigated sample is then compared withstandard /# price)

3. Fac"%!s "ha" a$$ec" "he ,al'e %$ R$7 Answered :

a. Solvents+ ca!sed b importance o# the partition coe##icient+ the

changes are ver small in solvent composition can lead to changes

in the price o# /#)b. The presence o# other ions+ s!ch as the presence o# chloride in the

separation is done with sol!tions o# nitratec. The nat!re o# the mi"t!re+ the vario!s compo!nds !ndergo

partitioning between e3!al vol!mes o# #i"ed and mobile phase) The

nat!re o# the mi"t!re is almost alwa s a##ects the sol!bilit

characteristics o# one against the other to also a##ect the price o# /# d. Paper+ paper ma or in#l!ence on the price o# /# arising #rom

changes in ion and absorption+ which is di##erent #or the di##erent

t pes o# paper) Paper a##ect the #low rate and a##ects thee3!ilibri!m partitioning)

e. temperat!re+ change in temperat!re alter the partition coe##icient

and #low velocit )f. The si5e o# the vessel+ the vol!me o# the vessel a##ects the

homogeneit o# the atmosphere that a##ect the rate o# evaporation

o# the solvent components o# the paper) *# the el!ent large vessel is

!sed+ there is a tendenc #or longer propagation+ s!ch as solvent

composition changes thro!gho!t the paper+ then the partition


16/22


coe##icient will change as well) Two #actors that a##ect the price o#

evaporation and composition o# /#)g. K!alit o# adsorbenth. The thic-ness o# the la er+ the thic-ness o# its la er getting smaller

/# i. Sat!ration space chromatograph


17/22


REFFERENCES

Ag!stini+ /!diana d--) 1 ;) Petunjuk Praktikum Biokimia ) S!raba a,

Inipress7 *M*P

Lehninger+ Albert L) 1 0) Dasar-dasar Biokimia Jilid 1 ) Ter emahan)

Ba-arta7,9rlangga

(elson+ D)L) Lehninger) 1 0) Principle of Biochemistry 4 th) (ew >or-,

.)&)6reeman

Tim Pen !s!n) 2012) Petunjuk Praktikum Biokimia ) S!raba a, Inesa Press


18/22


ATTACHMENT

N% Pic"'!e E8 la#a"i%#

9l!ent that !se #or paper

chromatograph $2? ml n%b!thanol : @

ml acetic acid glacial : 2? ml a3!adest

all sol!tion is colorless '

9Paper chromatograph si5ed 4 cm " 10

cm made the o!tline !sing pencil o#

the top and the bottom edge)

Line o# the top edge o# 0)? cm The bottom edge lines o# 1)?

cm)

A#ter it give a point and lable #or

sample A and = at a distance o# 1cm

#rom the side with pencil)

Five the rope on the top line o# paper

chromatograph


19/22


;Sample A and = standards that !se #or

paper chromatograph

< Spots the sample A and = in each point

!sing Capiller pipe

= Chamber that contain the el!ent


20/22


> The paper chromatograph is p!t into

chamber in verticall position

? 9l!ent is removed #rom the chamber)

The distance o# el!ent moving is @);

cm

@ Paper chromatograph is dried in the

oven #or 1? min!tes


21/22


Paper chromatograph is spra ed with

ninh drine sol!tion !ntil the p!rple

color

Paper chromatograph is dried in the

oven #or 1? min!tes a#ter spra ing with

ninh drine

9The stain res!lt #rom paper

chromatograph


22/22


Documents

EXPERIMENT FIX komang.doc