By pillin on Monday, November 17, 2003 - 06:13 am:
Hi all,
I have to perform an impurity method of Amiodaron with about 40µg/ml Polysorbate 80 (Polyoxyethylene 20 sorbitan monooleate, CAS:9005-65-6) in my sample (mobile phase is 40% phosphate buffer with triethylamine and 60% acetonitrile). My column is Spherisorb S10 ODS1 10µm. After at most 200 injections the resolution between Monoiodo-amiodaron and Amiodaron gets lost. Further testing showed, that Polysorbate gets stucked on the column and lowers the resolution. Washing the column with acetonitrile or methanol is only partially succesfull.
Is there any possibility to increase the column lifetime without washing the column with a gradient after each injection? Has anybody else the same problem? Is any HPLC-column available, which can handle with Polysorbate in sample solutions?
By Uwe Neue on Monday, November 17, 2003 - 04:58 pm:
What is the pH of your triethylamine phosphate buffer?
Assuming that the problem is not in the pH value, you may get reasonable results by running a gradient every 10th or 20th injection. If this does not imporve the situation good, then the issue is that whatever deteriorates the separation is stuck really strongly on the column. In cases like this, a guard column (packed with the exact same packing) and a frequent replacement of the guard column will do wonders.
By rick storoni on Tuesday, November 18, 2003 - 06:07 am:
I would try:
washing with a gradient of water (not buffer)/Acn for awhile,
then switch for a longer wash with hexane/Acn.
Go back to water/Acn
Then finish up with the original gradient.
By pillin on Wednesday, November 19, 2003 - 04:40 am:
Thank you for your response! I will try your suggestions and report later.
to Uwe Neue:
The pH value of buffer is 2.0.
By pillin on Monday, January 12, 2004 - 04:24 am:
Hi all,
another problem related to my HPLC-method for assay and rel. subst. of amiodarone: With newer batches of Waters Spherisorb S10 ODS1 there is a change in the elution order of amiodaron and Impurity D ((2-butylbenzofuran-3-yl)(4-hydroxy 3,5-diiodphenyl)methadone - related subst. in Europ. Pharmacopoeia) if they are compared with batches of Spherisorb S10 ODS1 10µm from 1995 and earlier.
Unfortunately a lot of alternative Materials (Purospher, Symmetry, Hypersil,and so on) give the same elution-order like the newer batches of Spherisorb S10 ODS1.
Why is there such a great difference between different batches of the same material? Does anyone know a material that behaves like the older batches of spherisorb? Are older batches of spherisorb available?
By Anonymous on Tuesday, January 13, 2004 - 03:38 pm:
I have not used Spherisorb in a while, but lots of companies used to make columns.
By Russ on Thursday, January 15, 2004 - 05:24 am:
Glad to see we aren't the only ones who have had a problem with the "newer" Spherisorb, though ours was with the ODS2. We tried Spherisorb columns from different suppliers with no success, and ultimately had to develop a method using a different column.
By pillin on Monday, January 19, 2004 - 01:41 am:
Hi Russ!
Did you find a alternative material that chromatographically behaves like the older batches of spherisorb?
It would be nice to hear some comments from the WATERS-staff!
By Uwe Neue on Monday, January 19, 2004 - 05:19 pm:
Pillin,
Contact me and we will discuss your problem!
Uwe
By Russ on Friday, January 23, 2004 - 05:18 am:
Pilin: Sorry, but we did not find a direct replacement. Of course, even if we had it may not be a direct replacement for your application.