1) Running at 10 C is tricky. That far from ambient you may have a
problem with temperature stability. Can you try running at 20 C?
2) Phosphate has a definite seasoning effect on bare silica capillaries.
If you are using NaOH between runs, don't. Use a phosphoric acid rinse
if you must, otherwise just rinse with buffer. Try using a higher
strength phosphate buffer (100 or 150 mM) to speed equilibration and to
reduce wall interactions. If you can't increase the [buffer] during the
run because of heating at least rinse with the higher strength buffer
before filling with run buffer. Doing a series of blank runs (at least
10) before beginning your sample analyses may also help.
3) What counter ion are you using? Sodium is commonly used, but
triethylamine or triethanolamine may give different results.
4) When preparing your buffer, are you measuring the pH at room
temperature or at the running temperature? In this type of assay small
changes in pH can cause noticeable changes in migration.
Good luck!
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>From: "MARIA THYBOLL" <k93_mtl@k.kth.se>
>Organization: KTH, Kemi
>Old-To: abrf@aecom.yu.edu
>Date: Tue, 10 Feb 1998 13:21:09 +0100
>Subject: CE pep analysis
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>To all ABRF's,
>
>I am trying to analyse dipeptides and tripeptides
>in low concentrations (<<10 mikrogram/ml) with CE.
>I have a problem with the stability and reproducibility.
>
>I am using an underivatized silica capillary and
>precondition it with NaOH, H2O, H3PO4, H2O and
>50 mM Phosphate buffer pH=2,3.
>Run voltage=15 kVand run temperature=10 C.
>Detection 214 nm.
>
>The peaks in the electropherogram seem to have
>different migration time from one analysis to another.
>Does anyone think that a change in voltage or
>temperature would help to stabilize the method?
>Or can any other parameter be changed to improve
>the resulted electropherograms?
>
>Thanks in andvance!
>
>Maria Thyboll
>Royal Institute of Technology
>email: k93_mtl@k.kth.se
>
>
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