Measure 1cm from bottom of strip and draw a line across Draw a cross in the centre of this line Attach a pin to the top of the chromatography paper, and put pin in bung Place inside test tube, and measure 5mm from where the bottom of the paper lies, draw a line across the test tube Do the same with both strips On the cross put 10 drops of a known amino acid, wait for each drop to dry in between * On other strip put 10 drops of unknown amino acid Pour paper solvent into both test tubes up to line drawn.
Put bung in both test tubes, let solvent work its way up chromatography paper When near top of paper, take out, and draw line across where solvent has reached. Method (Part 2): Place TLC on paper towel, draw line across at 1cm from bottom Draw 2 crosses each 1/4 way across line On 1st cross put 10 drops of treated orange juice, letting each drop dry in between On second dot put 10 drops of untreated orange juice Pour TLC solvent into bottom of jam jar * Using tweezers place TLC into jam jar Let TLC soak up solvent When nearly at top take out and draw solvent line across Method (Part 3):
Take the results of method part 1 and 2 and spray all with ninhydrin spray Place in incubator until amino acids can be seen * Draw lines across where colours end, and work out Rf values for all lines Results Chromatograms: Results Table: Rf value = distance moved by solvent / distance moved by solvent front My Results Solvent Front Substance Front Rf value Treated Orange 9.
Class Results Calculated Rf value Average Actual Rf Proline 0.76 0. 87 0. 82 0. 48 Leucine 0. 79 0. 59 0. 69 0. 73 Lycine 0. 41 0. 45 0. 43 0. 14 Asparagine 0. 26 0. 54 0. 40 Arginine 0. 53 0. 53 0. 2 Conclusion and Evaluation: From these results I can say that in the unknown substance, were two amino acids, and from what my results tell me, I can predict that they were Arginine and Leucine. The Treated orange juice appeared to have three amino acids in it, my results tell me that these are Asparagine, Arginine and Leucine. The untreated Orange Juice seemed to have four unknown amino acids, my results tell me that these were Asparagine, Lycine, Arginine and Leucine.
I could tell this as I matched up the Rf values with the ones that seemed to be the closest match. I know that these results were not that accurate as I know the Rf values for four of them. For Proline the real Rf value is 0. 48, but our class results got 0. 76 and 0. 87 with an average of 0. 82. This is clearly wrong. Therefore anything that I have predicted to have Proline in, might not have it in. For Leucine the real Rf value is 0. 73, and our class results are 0. 79 and 0. 59 with an average of 0. 69. This is a very close result, so it is quite accurate. So it is likely that if I have predicted it to have Leucine in it, it probably does.
For Lycine the real Rf value is 0. 14, and our class values are 0. 41 and 0. 45, with and average of 0. 43. This is not a very good match. From this I can tell that anything I have said to have Lycine in it, probably does not have it in. For Arginine the real Rf value is 0. 2, and we only have one class result for that and it is 0. 53 which is clearly wrong. This shows us that anything I have predicted to have Arginine in, probably won’t. For Asparagine, we are not sure of the real Rf value, so I can not tell if our results are right, but guessing from the rest not being accurate, it probably is not.
Saying this, not only could our results from the known amino acids be wrong, but also so could our results from the unknown amino acids, and the treated and untreated Orange Juice. This tells me that our results could have been wrong on both accounts, and so could possibly be right! We can not be sure, but we do know that the results for the known amino acids were not as accurate as they should have been. These errors could be down to a number of things. Firstly if we had touched the paper chromatography or TLC at any time we would have left our own amino acids on it, and so our experiment would have been inaccurate.
It was very hard not to touch either of these while cutting, or moving to place in test tubes or in jars. It could have been very easy to accidentally touch it, thus messing up the experiment. Another way these errors could have occurred could have been down to making the amino acid drop too big, due to not waiting until it is dry enough to put another drop on. This would have meant that when put in the solvent, the dot would have been emerged in it, and made our results inaccurate.
Another way an error could have occurred could be not making the dots as concentrated as they should have been, not counting enough drops of the amino acid onto the paper or TLC. This would have changed our results. Another way would be if when put in the solvent the test tube or jar moved, and splashed the paper or TLC, making the solvent front longer then it should have been, so our calculations would be inaccurate. Another way could be putting too much TLC or Paper solvent into the jar or test tube, thus emerging the dot, making more inaccuracies.
If the paper chromatography had been touching the side of the test tube while the solvent was working its way up, then this would have made our results inaccurate as well. All of these errors would make our results inaccurate and less reliable. One way of making our results more accurate would be to get rid of all these errors, which is very difficult as there are so many errors that could be made during this experiment. If I were to do this experiment again, I would start by wearing some rubber gloves, so that if I did touch the paper it would not matter as much.
The second thing I would change would to be more accurate when putting the dots on the paper or TLC, making sure that they are dry properly, and that they do not spread too far. Another improvement I would make would be to make sure the paper was not touching the side of the tube, to not move the tube once the paper is in, and to make sure I draw my lines as accurate as I can. Also when I measure them, I will be more precise so that my results will be a lot more accurate then they were this time round.