Chromatography Forum

I am analyzing amino acids in sports nutrition products (contain a lot of whey protein). After derivatization, detection is at 360 nm so the proteins do not interfere with the chromatogram. But I have been getting super high pressures lately and have ruined about 4 columns in the past 4 months. Now I am using a C18 3x50 mm 2.7 um pore on the perking elmer fx-15 UHPLC. The size of the proteins are around 14-19 kg/mol. With a mobile phase of 84:16 10 mM phos. buffer pH 6 : acetonitrile at 40 deg C, the pressure is around 10,000 psi. What can I flush with. I've tried all conventional flushes and inject DMSO almost every morning. Can the proteins clog up the column? and is it bad for the needle and inj. valve? Finally, should I be doing SPE to remove proteins from aqueous solution? Thanks a lot guys

-Mike

SPE and guard columns are always a good idea. Maybe some of your Proteins are hydrophobic they can stick on the packing material.

Lower the pH to say 2.4 and you'll be rewarded It goes for flushing as you asked, but the chromatography it self could get better as well.

Best Regards

if you have switching valve you can use our approach for analysis of complex mixtures. With this set up we were able to do milk analysis without SPE and tooth paste analysis without SPE:
http://www.sielc.com/upload/file/pdf/SI ... r_2004.pdf

I have two reports on both experiments including linearity study and can send it to you.

Nice thanks so much. Now auto injector valve is clogged. I'm doing large DMSO injections to try to clear whatever could be there. Probably clogged in almost every port . If I disconnect tubing from injector and let it leak mobile phase will it clean it? Thanks

Honestly the perkin elmer flexar UHPLC is a terrible system. The service is horrible. The engineer called me F***ing stupid. I made a complaint. I was getting electric shocks when touching the pump. The Totalchrom software is garbage . For example after shutting down the system it would say I have a pressure of 8000 psi and would stay like that indefinitely until I restart the software. Pressure is always about 250 psi with 0.00 flow rate. The pump area is so small and designed for baby hands. I always cut myself when trying to do something with the pump. If anyone ever thinks about getting the Perkin Elmer UHPLC you should think twice. It is dangerous. I wouldn't be surprised if the pump blows up. It won't stop pumping at pressures above max.

If I disconnect tubing from injector and let it leak mobile phase will it clean it?

If you use the same mobile phase as you describe it in your first post, then the answer is "No"
It was the same mbile phase that cased the problem in the first plase.

DMSO won't do any good either.

Best Regards

You can sometimes regenerate a protein-fouled RP column by injecting slugs (0.1-1 mL) of HFIP or TFE.

You will be better off if you can get rid of the protein before you inject your sample-- Several strategies here: lower the pH and pass your sample through a SCX SPE/Guard with ionic strength just high enough to prevent retention of your AAs. Also adding a couple volumes MeCN to your sample will precipitate most proteins (Andy Alpert).