Hydrolysed protein is to be analysed on HPLC so want to get rid of 6N HCL from the sample before loading it onto column.
so it's more amino acid sample than protein sample, right? The best way is probably to neutralize. The problem is, that amino acids and HCl/NaCl have too similar properties (size and charged). You could get rid of at least some on ionex.
@Tomas, Thanks for the reply. I am more interested in peptides rather than amino acids. I was wandering if you could suggest a protocol for ion exchange.
@ Steingrimur Since my HCl is 6N, the freezing point is too low nearly -76 C. Furthermore, Acid vapours could also damage the lyophilizer as we do not have an acid resistant one.
Hi Swati. How big are the peptides you are after? If they are > 1800 you could try desalting.
http://www.piercenet.com/product/polyacrylamide-desalting-columns
Thanks Steingrimur for your reply. Actually these can be even less than 1800 daltons. May I know why desalting is not acceptable if size is less than 1800 Da? Also, If you could briefly describe the procedure of desalting, it would be really appreciated.
Hey Swati. The procedure of desalting is applying your sample onto a column of porous beads. Depending on the diameter of the pores of the beads, small molecules will flow into the beads while larger molecules will flow outside the beads because they dont fit into the pores. This is illustrated in:
http://www.mikeblaber.org/oldwine/bch5425/lect31/lect31.htm
But if you are after peptide that are < 1800 Da, I seem to remember a procedure to isolate very small peptides using hydrophobic chromatography. I will look for it. Please remind me.
If you have phenyl sepharose you can follow this procedure.
http://www.animal-science.org/content/68/3/659.full.pdf
Instead of adding salt to the sample before loading I think you should try loading the sample as is. The 6 M HCl should be ionic enough to make the peptides stick.
I'd neutralize it, at least partly to ensure the column is not damaged. And the salt will work to keep peptides stuck.
@Swati
The gel filtration has low resolution, so you will not differentiate small peptides from salts.
Hydrochloric acid is removed from the samples by drying under vacuum at room temperature, achieved by con- necting the vial to the work station manifold and opening the vacuum control valve. The samples are properly evaporated when the reading on the vacuum gauge reaches about 65 millitorr. In practice, values lower than this are easily achieved with the pump used in our system, namely an E2M5 model two-stage rotary vacuum pump (Edwards High Vacuum, Crawley, UK).
Thank you all for your response. I will try with the methods and will see which works best for me.
Medhat; thank you for sharing the picotag system. I have similar issue drying excess Hcl from my hydrolysate prior to derivitization and HPLC analysis. However, i use the microwave assisted hydrolysis and not the Pico.Tag. In the microwave system, the acid is directly added to the samples in a test tube inserted in an activent where microwave energy is applied to heat the samples with in-situ stirring, after complete hydrolysis, the hydrolysates is automatically cooled and released from the system for further workup. Could you help with some tips to remove the excess HCl in the hydrolysate?
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