Waters HPLC and Accq-Tag- Serious help!

[ekgibson]

Hello. I am a novice HPLC user and am having serious issues, with no one around to ask for help. Service reps are annoyed with my questions, so I came here.

My system is as follows:
- Accq-Tag column and Accq-Flour Reagent kit
- Waters 2475 Multi wavelength fluorescence detector
- Waters 717+ autosampler
- Waters 1525 Binary Hplc Pump
- Breeze software
- Eluent A directly from Waters
- Eluent B- 60% Acetonitrile

I am trying to run indivdual standards of GABA and GLU (made from chemical stock), plus the Amino Acid Standards from Waters. I want to eventually look for these AAs in the brain, after I validate.

Basically, I have just started getting familiar with the system. I have done some injections, and am getting some peaks, but no where they are supposed to be. Plus I am having issues with the signal beting saturated at a gain of 100, so I changed it to 10.

I used the method of sample prep from the Accq-Tag book- borate, etc.

Is there ANYONE out there who has a SOP for this method and/ or system and would be willing to share?

Please be nice, I am very new to this.

[laserman]

I have Accu-tag experiences of HPLC and UPLC. I did not use exactly the same system as you, and I do not have English SOP for this method. I would like to know if you derivatize properly.

1 Add 1 mL of AccQ-Flouor 2B to 2A
2 Vortex 10 sec and heat 55 Celsius less than 10 minutes
(Let's call this AccQ-fluor reagent)
3 Add 60 uL of AccQ-Flouor 1 to your sample
4 Vortex 10 sec immediately after adding 20 uL of AccQ-fluor reagent
5 Heat 55 Celsius for 10 minutes

Here is some important points in this procedure.
1 Your sample should be more than pH=6
2 You have to vortex immediately after adding 20 uL of AccQ-fluor reagent; otherwise you will fail this derivatization.
3 You'd better use glass vial instead of PP vial. Glass vial can conduct heat better than PP vial.

I think you can get more information from Waters Web.
I hope I can help you.
Best Regers,

[ekgibson]

Yep, derivitizing just like the book says. I use plastic low-volume inserts. Should I try using glass? The vials are amber glass.

I'm not so sure it's the sample that's the problem. I'm wondering if it's how I prep the system.

No english SOP? Do you have one in another language?

Thanks for your response!

[Uwe Neue]

I do not know if this applies here, but in gradients it occurs often that one gets reproducible retention times only in the second and third gradient run.

If your signal is too high, then maybe you should inject less, or dilute your sample.

Please describe the gradient in detail, where you get the gradient from, if it is the same as recommended by Waters etc.

[laserman]

I have an old Japanese AccQ-Tag instruction provided by Nihon Waters. The instruction describes how to set up HPLC and derivatize. I do not know if they translated English instruction into Japanese or just made it themselves.

I think your derivatization is fine. As Mr. Uwe mentioned can you tell us your HPLC method?

Best Regards,

[lmh]

Just a comment, and probably a plain silly one:

If you follow the recipe in even a fairly vague way, it's almost impossible to fail to derivatise something with the AccQ tag system, but does your autosampler have the same (in my view plain daft) system of a needle with a hole 5mm up the side, as found in Waters Alliance systems?

If so, the AccQ tag reaction doesn't produce quite enough volume to reach the hole in a standard tiny glass insert (bottom spring version), or a standard low-volume vial. It will work in a Waters zero-dead-volume vial (the ones with the perfectly formed needle well in the bottom), but anything else, you may not be injecting anything. Under these circumstances, in our hands anyway, you may get one or two minor background bumps and one huge peak for no apparent reason at a variable point half way down the gradient!

[ekgibson]

Laserman,

My gradient is the one dictated in the Waters Accq-Tag Manual for "Using a Multi-Pump Gradient System." "Run time for analysis is 50min, 64min and later is the shut fdown the column" for a total time of 100 min.

If you would like more details, please let me know.

LMH,

Interesting about the inserts. I am using low-volume inserts, however no Waters rep has suggested there may be an issue.

However, what you are explaining is exactly what happens- I get nothing, then a bunch of peaks at once. Curious.

I really like to know how to set up this system fresh, condition the column, the specifics to equilibrate, and proper injecting. Like I said, I am in no way an expert, and essentially flying blind here.

Thank you for your suggestions.

[Uwe Neue]

If you follow the official method, and you are not getting the retention times as you should, there is something wrong with the system. If I understand your problem correctly (although it was not very well described; I am guessing based on your "nothing, then a bunch of peaks at once" and "the peaks are not where they are supposed to be"), I suppose that your B-pump is not working properly. Air bubbles?

Actually, are you using the same B-eluent as is prescribed by Waters? Your first note indicates that this may not be the case...