Chromatography Forum

HELP! New to this & the guy training me has just left! Performing a method to quantify Glycine & Lysine (on proteinaceous samples), using Arginine as an internal standard using a Waters Spherisorb ODS2 column on a Gilson RP-HPLC system. Prepare my own buffers (2) with acetonitrile, methanol, DMSO & purified water which runs on a gradient over 35 mins per injection (flow rate 2.5ml/min). Iodoacetic acid & OPA are used to when the sequence is running.
What I'd like to know is -

1. Is there a better, quicker way of doing this? I have a HP1100 at my disposal.

2. The plate count on the column seems to decrease very rapidly. Being in a GMP facility, we cannot let this fall below 2000. Anyway of increasing the life of the column?

3. I don't like Unipoint software! Anything better out there?

If I'm gonna do this, I want to do it right. Hope someone can help. Thank You.

I don't think the use of a different HPLC machine is going to help you with your method.
I would not be surprised, if you could improve the method, but it will be a lot of work, requiring a lot of detailed knowledge + validation work. You have to decide if you are up to this.

Neil,

If you are in a hurry and can afford to buy some reagents, standards and columns, I recommend you try to implement the AminoQuant method in your Agilent 1100. This method was developed and validated by Agilent and they should be able to provide you with all the conditions, columns, reagents, etc.

The Aminoquant method does the OPA derivatization online and uses two internal standards for quantitation. Depending on the levels you have, UV or fluorescence detection can be used. If you have an Aminoquant manual around just take a look at it and see if you like it.

I used Aminoquant for a number of years and works well, the column is not as stable as I would like it, but it lasts for about 100 determinations. The mobile phases are somewhat complicated, and the analysis is done quickly (20 min, but in your case can be be shorten) and the calculations are simple.

Out of curiosity, how can you use Arg as an IS?, proteins have it very often. Do you hydrolyze the protein?

Good Luck,

josebenjamin