amino acids analysis with Angilent Zorbax Eclipse-AAA column

[liangli]

Hi, Dear members,

I am a new member. It is so great that there is a place where we could talk and share the experiences.
I don't think I could go backward all the way to see if someone has posted above topic or similar one. What I would like to ask is if anyone has the experience in amino acids analysis with Angilent Zorbax Eclipse-AAA column with precolumn derivatization with OPA. If yes, do you think it is a robust method. What I experienced is the col. pressure was easily built up and peak shape and separation were getting poor. I use 0.02M phosphate buffer as A and acetonitrile/methanol/H2O as B to run the gradient elution. Do you think the phosphate buffer is harmful to the silica-based packing material at the intermediate or high PH(my mobil phase pH is 7. Is there a better buffered mobil phase instead of using phosphate buffer? Another point should be mentioned. My samples are the cereal product extract. The extract solution was cleaned up with Sep-pak C18 cartridge before put into running. I am not sure about the cleanup efficiency yet. I only know the spike recovery after cleanup was fine.

Thanks in advance

Li

[josebenjamin]

Dear Ingli,

I briefly tried that same column and mobile phases and I experimented the same problems you describe. I believe you are trying to use the Agilent recommendation of changing the AminoQuant column.

I did not pursued the procedure any further, but I suspect it is bacterial growth in the phosphate buffer (pH 7) that causes the pressure. look at your reservoir, any haze indicates bacterial presence.

You may want to try adding some methanol or ACN (perhaps 2%) to mobile phase A to prevent bacterial growth. But this would require changing the gradient conditions to maintain the separation.

The idea of using the Zorbax column is to improve the resolution of the early eluting amino acids (Glutamic acid, aspartic acid). This páir is poorly retained in the AminoQuant Column

Good Luck,

josebenjamin

[liangli]

Dear Josebenjamin,

Thanks for the suggestions. Yes, you are right. What I am tring is the Agilent method which is HPLC analysis of hydrolysed protein amino acids with OPA prcolumn derivatisation. The method is based on using Zorbax column and HP 1100 system. I am tring to apply it on analysis of free amino acids in food sample. The hplc system i am using is TSP HPLC.
I wouldn't think there is bacterial growth in the mobile phase because the mobile phase was freshly prepared everytime.

Regards

Li

[josebenjamin]

Dear Friend,

I still feel bacterial growth is related to your problem. The pH 7 buffer you prepare is an ideal media for bacteria. Even if you prepare that fresh everyday, it is likely that in a few hours bacteria will be present.

Do the following experiment. Prepare the buffer, and leave it unopened for a day or overnight exposed to the atmosphere. Quite likely after 12 hours the buffer will bge visibly cloudy with bacteria. Obviously to have high pressure problems you need only a small growth that may not be visible to the eye.

Good Luck,

josebenjamin

[HW Mueller]

On this propensity for phosphate buffers to support microbe colonies: I have had such buffers stand in the lab, closed of course, for years without any sign of any microbial growth. If you get some carbon, nitrogen, etc. into it via dust then a fast deterioration of the mentioned type can occur. So it appears that highly pure reagents are not at all conducive to sustaining bacteria and friends. Microbes are not wizzards that create matter.

[liangli]

Your opinions are appreciated very much.

I agree that the becteria growth may be one of the sources for high col. pressure. There may be another one I would like to consult you about. The gredient program which is refered to Agilent application note runs the organic mobile phase B(ACN/MeOH/H2O) to 100% at around 20 mins. and holds for a few mins. I doult if the phosphate salt(0.02M) may precipitate at high ratio of organic mobile phase. I did flush the col with H2O/ACN(85/15) for 50 mins at the end of every batch. However if precipitation does happen, can the flush dissolve the precipitated salt?

Regards

Li

[Uwe Neue]

If you follow precisely the procedure developed by Agilent, it is very unlikely that you will run into a buffer precipitation problem.

After all, your flush is with a mixture of acetonitrile, methanol and water. Thus the flush does contain water, which does dissolve the buffer.