I am working on a separation of plasmid DNA using a DEAE-NP column from TosoHaas. The DNA are in different sizes (7.2 and 9.0 kb). Their retention times are about 0.5 min if I inject them separately. They form one peak when I inject them together (a co-injection). I am desperate. Can anyone give me some suggestions on column, gradients etc.? Please! Thank you.

Check with Dr. Barbara Vogler at Supelco Technical Service, 800-359-3041.

Would you please give more detail information for your current chromatographic condition? (mobile phase, gradient or isocratic)

Thank you very much for the information.
I will check with Dr. Vogler today.
I have tried so many chromatographic conditions. Basically, the buffer A was 20 mM-tris, pH=9.0 and the buffer B was 2.0 M Nacl in buffer A. Using these buffers I knew that the plasmids could be eluted out at ~0.6 M-NaCl. Now, I am using buffer A=0.5M Nacl and buffer B=0.75M to find a good gradient. It turns out I can not see sharp peak(s) any more. The peak(s) are very broad and the two plasmids are not separated at all.
Again, Thank you very much for your help and time. Have a good weekend.

Hamilton has a column called PRP-infinity which was used by some reseearchers for purification of plasmid and chromosomal DNA. I have one page literature, please let me know your fax number, I will fax information to you for review.

Best regards,

sha
email:hplc@hamiltoncompany.com

Regarding anion exchange for resolution of plasmids, there are eluent- and phase-dependent limits to gradient slopes that result in sharp peaks.

For an alternate column and eluent system that will resolve oligonucleotides, restriction fragments, PCR products, and Plasmids (supercoiled from linear/nicked) see Methods in Enzymology 271: 147-174, 1996 " High resolution Nucleic Acid Separations by HPLC". In that article, a 7.2 Kbp plasmid eluted at ~31 min, and a 4.3Kbp plasmid eluted at ~ 16 min.

We have not examined plasmids, but have worked at two other extremes with polynucleotides. In both cases, we use size exclusion chromatography. In a rather low resolution system using SynChropak columns, we resolved 1.2 MDa and 0.6 MDa rRNA (Anal. Chem. 71:2951-2955, 1999). Much better resolution is obtained with a TSK300SWXL column where we can resolve a mixture of 20-mer, 30-mer, 40-mer, 50-mer and 60-mer synthetic oligos with baseline resolution (not published). You ought to try SEC.