Amino acid chromatography - any tips?

Discussion in 'Supporting Biology' started by Miffy, Sep 25, 2018.

  1. My A level lot just did this prac, but didn't get good results. I made up the aa's as directed with IPA and water, and the solvent was ethanol/ammonia/water as per instructions. The prac was done last lesson yesterday so the chromatograms were left to dry overnight, then I sprayed with ninhydrin this morning. Should it all be done in one day? Also, I'm wondering if the chromatograms should have been left in the solvent for longer (they were in for about 90 mins and only half way up the paper)? I have it again today so just wanted any pointers before we get another duff set of results. :/
     
  2. Sadly Miffy the solvent used for the Mobile Phase was not a good one. To answer your questions the Pracc can be done in about 1.5 hours if you use the Solvent System that I've tweaked in earlier posts and the choice of Amino acids I've suggested to give a wide range of Retention Factors. Ask if you need further info. I'll gladly help.:);)
     
  3. Thanks, we are using paper chromatography, let me know what solvent combination you use and I will run it past my head tech.
     
  4. Paper Chromatography is rather messy Miffy as a small amount of solvent spotted gives large diffuse zones. However it is achievable. I suggest that you use these Amino acids:
    http://community.preproom.org/index.php?threads/thin-layer-chromatography-of-amino-acids.1849/
    The solvent system is Butan-1-ol:Glacial Acetic Acid: Water (65:25:15):);)
     
  5. I always have trouble with those 105% mobile phases.
     
    ClaireS and Jade like this.
  6. does anyone know what is the maximum time between running/drying the chromatogram and spraying with ninhydrin? one day, a weekend?
     
  7. As long as it's dry, I don't think there is a time limit. I would keep them in a dessicator and dark.
     
  8. About 90 minutes.;)
     
  9. They both will as they both have 254 nm settings, which is the normal wavelength for the plates (as it says on the label).
     
  10. Thank you.

    I thought that was the case but wanted to check before purchasing the lamp.
     
  11. Miffy - do you subscribe to CLEAPSS? If so, there's a great practical on there (document PP062 - Paper Chromatography of amino acids). Our BTEC lot did it today, and got some really nice chromatograms. Of course, it's much better on a TLC plate, but that's kinda the point of doing the practical, and seeing how some methods are better than others! Ours also do plant pigments on both paper and TLC, and it's a bit "wow" to see what a difference there is :D
     
  12. ;)
    To answer your question Miffy I usually spray on the Ninhydrin within minutes of drying the plate ( warmed on a hotpate at about 30C) but it is important that it is dry. I've been reading many post on this Forum about the use of different Mobile Phases. It is important to use a Mobile Phase which has been completly characterised for the 20 diffent Amino Acids so that you can compare your retention times with the published values, as is the case of the Mobile Phase I use. It is also important to optimise the Run Time so that optimal seperation is achieved. The two plates below were run for 66 and 118 minutes respectively. Hope that this is of use.:);) M is a mixture of all three AA's (Lys, Leu and Ala).

    AA TLC.png
     
    Last edited: Jun 10, 2019
  13. AA-annotated.png
    Thought I'd post my Chromatograms of the Amino acids using my solvent mixture run time was 90 minutes at 20C.
     
  14. My BTecs did this on paper and TLC. Paper gave diffuse spots but the TLC was good on butan-1-ol, acetic acid, water solvent system and the AAs made up in propanol/water mix.The kids ran in for about 1hour, then dried in a warm oven. I forgot them and they stayed there for a couple of days. I made up a ninhydrin spray which was visualising just after spraying and before heating and gave good results when heated. I thought that because I'd forgotten them they would be disastrous, but no.