Chromatography Forum

I am trying to use the Oasis WCX SPE Cartridge for thiamine clean up for an HPLC analysis. I am still on method development.

I am currently only spiking water with thiamine standard.

Here is the current method:

1. Condition Cartridge: 5 mL water, followed by 5 mL methanol.

2. Sample is adjusted to pH 8 and loaded onto cartridge at 1ml/min. (About a quarter of the thiamine is breaking through).

3. Wash with 5 mL of Methanol (No Thiamine).

4. Elute with 0.1% TFA in Methanol. (No Thiamine coming off).

How do I get all of the thiamine to stick to the cartridge?
How do I get all of the thiamine off the cartridge?

Drop the pH to make sure you ionize that primary amino group. I don't know what the transition pH (pKa) of the Oasis MCX material is, so you might also want to switch to the Oasis MCX (strong cation exchanger);

Unfortunately, I had previously tried the MCX but it retains the thiamine so strongly that I am unable to get the thiamine back off.

With the WXC, if I load the sample in just DI water most the thiamine breaks through the cartridge. After looking up more information on the cartridge, to capture an analyte the pH needs to be greater than 7. That's why I made a buffered solution and adjusted the pH to 8 and it helped but did not completely work.

The WCX has a negative charge at a pH greater than 7 and no charge at a pH less than 3. To release the analyte the pH should be less than 3.00. The 0.1% TFA has a pH of ~2.00

http://www.waters.com/waters/en_GB/SPE- ... cale=en_GB

I don't know if you've had any luck yet, but I would give up trying to ionize this compound. It's highly polar so I would use a HLB. Load with water. Wash with 10% MeOH or ACN, dry, etlute with ACN or MeOH.

Don