SB-806M

Record: 844

Category: Column Problem
Product: SB-806 troubleshooting
Sample:

Question
Could you follow up the following issue?

Best regards
Sammy

We are still experiencing a peak of unknown origin on the SB 806M-HQ column. The mobile phase is 25 mM KH2PO4 at pH 7.0 (PH adjusted with 0.05M NaOH) with 10 ppm sodium azide. The flow rate is 1.0 ml/min.

We are attempting to convert to the use of liquid sodium azide commercially prepared (5% w/v) for safety purposes. We are using 200 microliters of the 5% sodium azide per liter of mobile phase.

Previously, we were using 10 mg of powdered sodium azide per liter.

We are seeing a peak, when using the commercially prepared liquid sodium azide, at about RT= 10.3 min, which coincides with the RT of a peak we have seen in the past when dextran standards deteriorate over time, or when people handle vials without gloves. We have labelled this peak in the past as a contamination peak, but in truth, that is only theory.

Question for you: Is it possible that sodium azide could be retained on this column and show up as a peak at RT=10.3 min?

Some other hypotheses:

1-Liquid sodium azide is interacting with plastics in the automatic pipettor or plastic pipettor tip (not used for powder form).

2-Liquid sodium azide is interacting with plastics in the mobile phase preparation carboy (however, the peak does not appear when using the powder form, using the same type of carboy).

3-Liquid sodium azide is not retaining its potency after opening.

If you have any ideas to help us solve this problem, we would be most appreciative!

Answer
Thank you for your inquiry. Please see the response below:

I do not think it is possible that the unknown peak will correspond to sodium azide. The exclusion limit of the column is approximately 100MW and the MW of sodium azide is 65 g/mol. If you inject something of comparable MW does it also appear at 10.3 min? Is 10.3 min the exclusion limit of the column?

Perhaps, you could make a series of dilutions (0.5, 1.0, 2.0% w/v) of the liquid NaN3 solution and inject them onto the column-preferably without the use of plastic pipette tips. If the peak continues to appear then you know the contamination comes from within the bottle. If you have used plastic pipette tips then perhaps you can repeat the procedure using the powder form in the same procedure making your dilutions with the pipette tips. Then you can deduce whether the pipette tips are causing contamination.

I do not suggest injecting the 5% w/v NaN3 onto the column directly because it may precipitate in the buffer conditions you have been using.

Unlike the decomposition of dextran, the deterioration products of sodium azide would lead to volatile gasses and aqueous soluble ions that would most likely not be detected.

Please let me know if I can provide additional assistance. If your customer would like to send additional data or chromatograms I would be happy to look at them.

 
 
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