Hey Ankit,

Assuming you are correctly performing the protocol; either some compounds have irreversibly bound to the cartridge sorbent (if you are using a second-hand SPE cartridge) or your solvents, including methanol or Tris-EDTA buffer are not pure and contain contaminations, which fills the retaining capacity of the cartridge. Also, check the pH of Tris-EDTA buffer as silica-based packings usually have a stable pH range of 2 to 7.5. At pH levels above and below this range, the bonded phase can be hydrolyzed and cleaved off the silica surface, or the silica itself can dissolve.

Good Luck

Thanks Seyed,

But I am using fresh SPE column every time. The Tris-EDTA buffer pH is 8.5.

(i) Will the column not work at 8.5 pH?

(ii) Is it possible that because of the polar nature of Tris and EDTA, these compounds bind to the cartridge sorbent and then no free charged group left on the sorbent to bind with DNA and also DNA no able to replace these molecules from sorbent? Then in this case, should I replace the Tris-EDTA buffer with some other buffer?

Please note that I'm not familiar with this particular protocol, but I have used ion exchange columns.

Is the DNA charged at pH 8.5? If it isn't charged, no ion exchange can occur.

Is there any other conditioning required? As the column is an

quaternary amine, the amine has a positive charge and must have a counter-ion. What is this counter-ion, and can DNA displace it? I've run ion exchange on some phenolic compounds where I needed to change the counter-ion from chloride (which was the counter-ion on the column as delivered) to acetate. Some ions are easier to displace than others.

Both of my questions relate to running the protocol correctly.

Well, I dont particularly know about DNA SPE methods. However, most DNA SPE methods require high concentrations of chaotropic salts and organic alcohols to drive DNA adsorption to the silica surface, followed by washing, and finally elution with a high pH low ionic strength buffer. If you use new cartridge every time, then pH 8.5 is not that much matter; therefore, column works on that pH too.

The second possibility is also refused as Tris-EDTA buffer is an eluant and wont bind to SPE.

How did you get the method? Is it a valid article?

There is one possible mistake, I think you must condition the cartridge with the solvent used for washing (such as phenol-chloroform) not your eluant solvent which discourage/prevent the binding of your desire analyte i.e. DNA.

Please check these and let me know

Regards

Thanks Seyed,

I got this protocol as the handout attached with the SPE column from Sigma-Aldrich.

I will change the Tris-EDTA buffer with washing solvents and try to reach to the result.

Thanks and Regards