Subject: separation of amino acids by paper chromatography

Iam presently doing a biology prac on the separation of amino acids in egg 
white, by means of ascending paper chromatography. On my chromatogram, all 
of the spots were purple, except for one, which was yellow. The Rf value 
table we were to use to identify the amino acid responsible for the spot 
said that the yellow spot should be proline (Rf value:0.43) My Rf value 
for the yellow spot was 0.37. Hence, I have made an error of 0.6. How 
could this have happened??? Did I not leave the chromatogram in the solute 
long enough? Also, albumin is supposed to conatin 15 amino acids, my 
chromatogram only contained 9 why is this so?? How could I have improved 
my experiment to obtain better results??? 
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Hi!!! thank you for reading my question. You asked for a procedure......well
here it is!!!!! Hopefully you'll be able to derive something from this. It
means a lot to me that you are willing to answer my question. Thanks!!
Amanda.
Procedure:    part A.

1.      0.5 of powdered trypsin was weighed on the electronic balance in the
small plastic container and placed into the large test tube.
2.      The test tube was half filled with distilled water, and shaken until
the trypsin had dissolved.
3.      5ml of sodium bicarbonate solution was added.
4.      5ml of egg white was added.
5.       A rice grain of crystal thymol was added.
6.       The mixture was left to incubate at 37 *C for approximately 48
hours.

Part B:

1.      A strip of chromatography paper with dimensions 4 x 40 cm was cut out, 
and a pencil line was drawn across the strip about 4 cm from one end of
the strip of paper.
2.      100ml of the butan-1-ol solvent was poured into the large measuring
cylinder. The open end of the cylinder was then quickly covered with the
paper clip attached lid.
3.      A small drop of the part A solution was placed in the centre of the
pencil line drawn on the paper, using the fine pipette. The spot was dried
using the hair drier and another drop was placed on top of the first and
blown dry again.  This procedure was repeated 6 times with the spot being
kept as small as possible. The paper was left for approximately 2 and
half-hours.
4.      The strip was carefully (without allowing contact with the sides of
the jar) lowered (pencilled end first) into the large solvent-filled
cylinder. The bottom of the strip was allowed to dip about 5mm in the
butan-1-ol solvent, and then the top end of the paper was attached to the
paperclip on the lid of the cylinder.  This was left for approximately 16
hours, and the following procedure was carried out by the laboratory
assistant:

Materials:      nindrin in butan-1-ol (1% solution nindrin in butan-1-ol)
                Crystallizing dish (125mm diameter)
Procedure:
*       A small amount of a dilute solution, ninhydrin in butan-1-ol was
poured into the crystallizing dish to a depth of 10mm, and the
chromatography strip was drawn through the liquid.
*       The strip was rapidly dried by means of close exposure to a source
of heat, until spots of maximum purple density appeared along the length of
the paper.

Response:

Re: separation of amino acids by paper chromatography
Iam presently doing a biology prac on the separation of amino acids in egg 
white, by means of ascending paper chromatography. On my chromatogram, all 
of the spots were purple, except for one, which was yellow. The Rf value 
table we were to use to identify the amino acid responsible for the spot 
said that the yellow spot should be proline (Rf value:0.43) My Rf value 
for the yellow spot was 0.37. Hence, I have made an error of 0.6. How 
could this have happened??? Did I not leave the chromatogram in the solute 
long enough? Also, albumin is supposed to conatin 15 amino acids, my 
chromatogram only contained 9 why is this so?? How could I have improved 
my experiment to obtain better results??? 


_______________________________________________________________
Greetings - Your question was not submitted to the
MAD Scientist Network for the following reason:

Please provide further specifics. As it stands it would be
difficult for us to find an answer to your question. Questions may
be resubmitted via http://www.madsci.org/submit.html

Hi Amanda - It would help to know what procedure you used to isolate the 
proteins, or specific amino acids.
Thanks..

	Lynn Bry, Admin MadSci Network

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Re: separation of amino acids by paper chromatography