By Dina on Thursday, February 24, 2000 - 08:59 am:
Hi,
I need to separate/quantitate Albuterol sulfate in the presence of phospholipids. I dissolve mixture in methanol and use C8 column, ACN-0.05%TFA in water mobile phase. Problem is that I can inject only 10ul on the column, after that peak shape is changing, it has shoulders or doubles.
By jclark on Thursday, February 24, 2000 - 10:36 am:
Your injection solvent is too strong. It should be no stronger than the starting mobile phase composition if you inject more than 10 ul on a 4.6 mm column. I would suggest diluting the sample in mobile phase.
By Uwe Neue on Saturday, March 4, 2000 - 01:00 pm:
even better: just dilute your sample with water, until there is more water in the sample than in the mobile phase, and then inject more. If you do this right, you can inject more, much more...
By dina on Tuesday, March 7, 2000 - 02:23 pm:
Thanks! But the trick is that I cann't add water. Phospholipids will precipitate even at 15% water-methanol mixture. Dina
By Uwe Neue on Tuesday, March 7, 2000 - 05:35 pm:
OK, that is a problem. How about doing the dilution with water, precipitating the phospholipids, centrifuging or filtering the stuff and injecting the supernatant or the filtrate?
Alternatively, you can use a simple SPE method to leave the phospholipids behind. You do not really want to inject them onto your column, do you?
Since all of this is work, may I ask you why you want to inject more. I assume that this is a question of sensitivity. Maybe there are simpler solutions to get a higher sensitivity, such as detecting at a different wavelength.
By dina on Thursday, March 9, 2000 - 08:20 am:
I tried precipitation of phospholipids with water and estimated about 20-25% loss of Albuterol during separation. I need to increase total amount of injected Albuterol to be able to see impurities at 0.1% level. Changing wavelength to 220 nm vs. 275 does not make a big difference. Dina
By Uwe Neue on Friday, March 10, 2000 - 03:29 pm:
Well, this is now pointing to the need of sample prep. The sample is dissolved in MeOH. You could run it through an Oasis SPE thingy, I bet that it would retain your phopholipids, and the albuterol and the impurities will go through without a problem. If this would not work, you can still add a very small amount of H2O, maybe 10% to the MeOH, but I bet that MeOH is just fine. After this, you can now do the dilution game since the phospholipids are gone, or even evaporate the MeOH and then redissolve the sampl in mobile phase or better something slightly more polar than the mobile phase (i.e. more water).
Of course, this is work....
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