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HPLC of Nucleic Acids
Posted: Tue Oct 23, 2007 7:50 pm
by Eric Moore
Does anyone have any useful references on this topic? I understand IEX is very common on NAs. Is reversed phase typical, or normal phase? Does anyone have any experience separating polymers from NAs? Are they compatible with an ELSD?
Don't feel obliged to answer all my questions.
HPLC of Nucleic Acids
Posted: Wed Oct 24, 2007 4:43 pm
by skunked_once
I've analyzed nucleic acids on a reverse-phase column with UV detection. Supelco has some application brochures as do other vendors as well. Contact vendors for details. There are method variations depending on which nucleic acids and sample matrix so you will have to do some searching on your own for you particular application.
Posted: Wed Oct 24, 2007 5:27 pm
by rhaefe
Reversed phase ion-pairing (usually done with triethylammonium acetate/acetonitrile but other IP reagents are used as well) is pretty common.
Detection is usually UV. Depending on the IP reagent desalting might be necessary afterwards.
Hamilton does have a "Separation and Purification of DNA" brochure I can send you. Another resource is Gjerde's book: "DNA Chromatography"(ISBN 3-527-30244-1)
Posted: Fri Oct 26, 2007 4:21 am
by Uwe Neue
What is the MW range? Martin Gilar has done some solid homework on optimizing the separation conditions for oligonucleotides, and I can send you a few references.
Posted: Fri Oct 26, 2007 7:24 pm
by Eric Moore
What is the MW range?
They're about 13.5 to 14 kDa, 21mer siRNAs.
Thanks.
Posted: Sat Oct 27, 2007 1:38 am
by Uwe Neue
Here are some relevant references:
Peak capacity in gradient reversed-phase liquid chromatography of biopolymers: Theoretical and practical implications for the separation of oligonucleotides
Journal of Chromatography A, Volume 1169, Issues 1-2, 26 October 2007, Pages 139-150
Martin Gilar and Uwe D. Neue
Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography
Journal of Chromatography A, Volume 1061, Issue 2, 24 December 2004, Pages 183-192
Martin Gilar, Amy E. Daly, Marianna Kele, Uwe D. Neue and John C. Gebler
Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides:: Retention prediction
Journal of Chromatography A, Volume 958, Issues 1-2, 7 June 2002, Pages 167-182
Martin Gilar, Kenneth J. Fountain, Yeva Budman, Uwe D. Neue, Kurt R. Yardley, Paul D. Rainville, Reb J. Russell II and John C. Gebler
Posted: Sat Oct 27, 2007 4:23 am
by kamlesh Patel
Hi,
Neucleic acids can be better seperated and detected by reverese phase chromatography using ion pairing reagents. As acids create anions you require cations for ion pairing. The best cation IP agent in your case is Tetrabutyl ammonium bromide/cetyl ammonium bromide. Try them in your mobile phase @ 0.005 molar concentration.
Best luck
Posted: Mon Oct 29, 2007 12:58 pm
by Bryan Evans
Below are oligonucleotide applications d(T) 12-18 on
Unison US-C18 (5um):
http://www.imtakt.com/TecInfo/TI116E.pdf
For 13.5-14 kDa, you may need to evaluate widepore reversed phase columns.
Posted: Tue Nov 20, 2007 3:47 pm
by Eric Moore
Thank you, everyone! I appreciate the help, and am happy I stumbled upon this website!
Eric