Chromatography Forum

Hi guys,
I'm dealing with muscle milks and protein shakes and quantitate for free amino acid content. However, there is a lot of protein in my samples (dissolved in water) which I really need to clean up. I want to use ACN to precipitate the proteins, but without a hood I cannot evaporate the ACN and collect the supernatant. My question is, what happens to concentration when adding a solvent (ACN) that the analyze is insoluble in to an existing solution of you analyte? Lets say I have a 1 ml of 1 M taurine solution in water. If I add 1 ml ACN, my concentration is halved. But technically the sample is not dissolved in ACN, only water right? Evaporating ACN would not change the concentration of my solution? Thank you


-Mike

Lets say I have a 1 ml of 1 M taurine solution in water. If I add 1 ml ACN, my concentration is halved.

No. 1 mL water + 1 mL ACN does NOT give you 2 mL.

But technically the sample is not dissolved in ACN, only water right?

No. It's dissolved in an ACN/Water mixture.

Evaporating ACN would not change the concentration of my solution?

It WILL change the concentration as you're not able to selectively evaporate only the ACN. Some of the water will go away, too. The only way to get to an accurate concentration is complete evaporation and redissolution in a defined amount of liquid.

Mike

Acetonitrile mixes with water, it does not make a separate layer like hexane or dichloromethane would.

If the water contains a lot of e.g. salt, the acetonitrile will form a separate layer but the water layer will contain a lot of acetonitrile and the acetonitrile layer will contain a lot of water. The taurine will be distributed into both layers depending on its partition between (salty) water + acetonitrile and (wet) acetonitrile.

When you evaporate you lose both acetontrile and water - you cannot "undo" the addition of the acetonitrile.

Peter

Mike,
You can avoid having to remove proteins if you derivatize amino acids in suti followed by suitable chromatography I think.

For example:

HPLC or LC-MS: AA -> AA-PTC -> SPE ->LC

GC or GC-MS AA -> AA-TMS -> GC