By Anonymous on Saturday, May 29, 2004 - 01:57 am:

Hi all,

I'm trying to separate polar plant fractions using TSKGel Amide 80 column (2.0 mm ID x 25.0 cm L). But before doing so, I've decided to run some polar standards like sugars first using acetonitrile (A) and ammonium acetate (B) as my mobile phase. My gradient program starts with 10 min isocratic run at 0% B for 10 min, gradient to 15% concluded at 10 min, then to 100% B for 60 min. Flow rate is 0.15 mL/min.

Regardless of whether I'm using blanks or standards, this particular peak comes out at the same point (around 0.7 mL or around 4.5-5 min after injection). I've tried varying concentrations of acetonitrile:H2O for my samples and blanks (50:50, 60:40, 70:30 and 80:20) but the same peak keeps on coming out. Do I have reason to suspect that my column is damaged?

I thought this may be due to contaminants in my ACN buffer so I've tried using isopropanol as my (A) and this peak vanished so I was relieved. And I've tried using both blank and standards again but a peak keeps on appearing again regardless of whether a compound was present or not (this time, at around 40 min).

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By Anonymous on Saturday, May 29, 2004 - 06:59 am:

Oxygen peak.

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By Uwe Neue on Saturday, May 29, 2004 - 10:29 am:

If a peak shows up, there is no reason to think that the column is damaged.
What is your detection system?

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By Anonymous on Friday, June 4, 2004 - 02:28 am:

Thanks for the replies.
Uwe, I'm using UV detector from Amersham Aktas purifier.
By the way, how do I remove this oxygen peak?
I think my analytes aren't being retained in the column as there is no peak in my gradient AT ALL. Only the large peak I was talking about appears regardless of whether a compound was present or not in my sample.
Would appreciate any enlightenment... :-)

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By Chris Pohl on Friday, June 4, 2004 - 11:02 am:

If it's an oxygen peak you have several options. If you are degassing your mobile phase and you are seeing an oxygen peak with every injection then you can eliminate (or at least minimize the peak) by sparging a sample with helium (or nitrogen). Alternatively you can skip the degassing process for your mobile phase assuming that doesn't create new problems with your pump maintaining proper prime. If the above the option isn't viable, a third option is to "resaturate" the mobile phase with oxygen postseparation. The easiest way to do this is to add a length of gas permeable tubing between the column outlet in the detector inlet. Teflon AF tubing works best for this but if you don't have ready access to this tubing material you can use Teflon tubing but you may need to increase the tubing lengths. Some have reported that using a length of Teflon tubing that is knitted works better for this application.

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By Uwe Neue on Friday, June 4, 2004 - 02:26 pm:

Your sample is composed of sugars You will see them only at very low UV. Under these circumstances you will also get much background from ammonium acetate. Your detector would potentially go blind. If you want to see the sugars, you need to work around 210 nm. Then you should not use ammonium acetate in your mobile phase. If you have other samples, you may be able to see them at higher wavelength and ammonium acetate could be a useful additive to the mobile phase.

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By Einar Pontén - SeQuant AB on Friday, June 4, 2004 - 02:56 pm:

Why make it difficult. You are actually running a HILIC gradient.

Apparently the column is not hydrophilic enough. Try other alternatives for HILIC separations.