Arg and Thr seperation with OPA precolumn derivatization

[fantasylin]

Hi:
Due to the death of previous NovaPak column , I bought a new NovaPak RP C18 since the old one give a good seperation on amino acids mixture. However, now I have problem seperating Arginine and Threonine, sometimes G-aminobutyric acid and Tyrosine. The gradient mobile phases I am using are acetate buffer (pH=40mM) and methanol.

Hope there is someone here who could help me with this:)

Thanks

Regards

Lin

[Rafael Chust]

Sometimes there are tiny differences from columns batch-to-batch, but if the difference is "severe", then I would recommend that you copy examples of your previous chromatogram, the actual one and file a written complain with your local supplier. Usually the manufacturer analyse it and replace the column free of charge.

Of course I am assuming the PN is the same and the conditions remains unchanged!

[skunked_once]

I used this method several years ago for amino acid analysis and was told that the Nova-Pak columns were screened to pick the best ones. These were sold specifically for amino acid analysis (at a higher price of course). Rafael is right. You should contact your supplier. Good luck!

[josebenjamin]

Dear Fantasylin,

You may want to consult with Agilent and consider switching to the AminoQuant column. This is employed for AA separations (as OPA derivatives) using an acetate pH 7 buffer. These columns are also batch-screened to make sure some AA pairs are adequately resolved, and I seem to remember that the pair you are interested on is one of them.

Good Luck,

josebenjamin

[Uwe Neue]

I do not think that a complaint has merit. Any packing has a certain breadth of retention characteristics. The older packings more so than newer one. The Nova-Pak column will have passed the specifications for a Nova-Pak column...

There are batches of Nova-Pak material that are set aside for the amino acid analysis. If you want a guaranteed amino acid analysis, you shall buy this column. In the unlikely event that this column does not work for an amino acid analysis, it will be replaced free of charge.

Of course you pay more for this guarantee and this QC. If you are not willing to pay for this additional guarantee and this additional service, you are on your own with the cheaper standard Nova-Pak columns. Occasionally, one of these will not work for you.

[Rafael Chust]

Dear Uwe:

Just to clarify: eventually complaint is an excessive term (excuse my bad english). I mean communicate with the supplier, that intelligently will analyse the reasons for this communication.

A responsive and responsible manufacturer will certainly take every point into account and replace the column or argument why not.

I personally always avoid to "complaint" but I do communicate a lot!

[josebenjamin]

Dear Rafael,

I totally agree with you, your comments were not really phrased as a complaint. The problems here described tell us how much column manufacturers do not know about what controls the characteristics of their products. In theory, if a given coloumn works for AA analyses, all columns of the same description should work as well.

I have personally lived through several such column reproducibility problems. Part of the annoying character of this, is that manufacturers never quite explain or admit that something is not known or controlled. Some other more concious ones, argue that in spite of all their efforts, not everything turns out the same all the time.

I guess we will have to live through these problems, until the perfect column and process comes along.

Good luck to you all,

josebenjamin

[Kostas Petritis]

So, just to clarify...

Are there two different Nova-Pak columns, one of which is sold with a gurantee that it will wok for OPA derivatized amino acid analysis? Did Lin (Fantasylin) bought the exact same column she had or another one?

If she did buy the same column I can see the point, if she bought a "cheaper" alternative of the same column family then I will agree with Uwe...

[Uwe Neue]

Let me clarify the situation again. There is a general Nova-Pak column that is sold for all general purposes, and there is a special Nova-Pak that is sold for amino acid analysis under the name of Pico-Tag column. The Pico-Tag column is more expensive than the standard Nova-Pak column, but it is guaranteed to work for the amino acid analysis. It is QCed for this purpose.

Since it is not a secret that the amino acid column is a Nova-Pak column, people have used the cheaper Nova-Pak column for the amino acid analysis. Sometimes it works, sometimes it doesn't. It is like gambling. Sometimes you win and save money, sometimes you loose and you have to buy another column.

As to Rafael's comments: the standard Nova-Pak column must pass a stringent QC test. While the specifications are wider than those of say for example a Symmetry material, they are still quite stringent. Furthermore, to replace a Nova-Pak with another Nova-Pak column is not likely to solve fantasylin's problem.