By mda1224 on Wednesday, October 9, 2002 - 07:41 am:
Hello,
I'm curious to know if anyone has experience purifying a 35-mer made via the phosphoramidite method (DMT-ON)on HPLC. Literature methods seem vary but I decided to get a C-8 column, 10µm particle size, 300Å pore size, 250x4.6mm ID.
I'm not exactly sure of the correct mobile phase composition. I'd like to get some more suggestions before I run my sample. Thanks.
Mike
By Anonymous on Wednesday, October 9, 2002 - 03:00 pm:
10 micron particles? The resolution will be rather poor unless you run it at very slow flow.
By Tom Mizukami on Wednesday, October 9, 2002 - 04:09 pm:
I haven't done any work with oligos for a while but ... I think one of the newer polystyrene copolymer columns will give you better recovery (>75%). There are also cartrige purification kits using the same types of stationary phases.
Do you just want to desalt, or do you need to remove all failure sequences also? PAGE and anion exchange are also possibilities but probably less desirable.
By mda1224 on Wednesday, October 9, 2002 - 07:54 pm:
Tom, actually, I have already run the oligo through a PolyPak II cartridge whose purpose is to remove failure sequences, however, I was told HPLC is needed for further purification and PAGE should be performed to determine if indeed I have a 35-mer. Just curious as to the correct LC parameters I should be using. Thanks.
Mike
By Gerhard Kratz on Wednesday, October 9, 2002 - 11:57 pm:
Mike, newer polysyrene copolymer columns with RP function will give you better recovery. Ionexchanger columns are also used. We have some applications available, which might give you an idea for the LC parameters you should use for the start conditions. Please have a look on www.tosohbiosep.com and click on the HPLC database. Good luck. Gerhard
By Anonymous on Friday, October 11, 2002 - 11:16 am:
Take a look here:
http://www.waters.com/watersdivision/waters_website/chemistry/xterra/oligos/xterra_oligo_top.asp