I'm trying to modify oligonucleotides (20-30 base) using sugars and other different molecules. But I have difficulty in separation and purification of reaction products due to low amount (low concentration) of sample. Could you suggest me a way to purify these compounds except HPLC and LC-MS?. Our LC-MS detection limit is up to 2000 and my product's molecular weight is approximately 4000.
Thanks in advance for your interest
You could try G50, or preferrably G25 spin columns equilibrated in TE pH 8.0. This may provide a sufficient purification in a single step, and filtrates from multiple columns can be combined and concentrated if necessary.
Good luck!
I suggest PAGE or HPLC for your problem using unmodified oligo as standards.
If you have decided on LC-MS it works as analytical platform for your problem and you can not recover the product. Separation of your oligonucleotide occurs perfectly and the 4000+ amu could be simply and accurately resolve by deconvultion of the doublet and triplet ions. Accuracy of mol. mass analysis and sequencing by LC-MS/MS is obviously a gold standard.
How much exactly is low amount? Acrylamide is the obvious easy option as mentioned. However if you are dealing with very small amounts it may not be practical.
I am assuming your goal is to separate the differently modified oligos and not the oligos from the modifying agents. If it is the latter, the spin column suggestion above would work well.
Do you have a low amount or low concentration? What buffer are they in? You can try lyophilizing first to increase concentration, if you have sufficient quantity. Then, a C8 or C18 column linked to an LC would give you pretty good separation. This is assuming you have a large volume of low concentration sample. If you have patience, you can try loading to the column the entire volume to aid with the concentration as wel - that way you can skip lyophilization.
Good luck!
You may have to first do a sizing spin column, collect the oligo-bio molecule conjugate along with the biomiolecule ( like sugars) or Peg, and then run an ion exchange spin column, to collect the logo-conjugate. This would work for non charged or positively charge bio molecules.
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