I am thinking of measuring the degree of substitution of esterified polysaccharides by alkaline hydrolysis, followed by extraction with water-immiscible solvents such as ethyl acetate. This is an approach I have used successfully before. Unfortunately our lab does not have a speed-vac, which would enable me to dry down the sovent extracts before RP-HPLC. Would SPE cartridges be suitable, and if so, which type?
The carboxylic acid substituents are mostly non-polar (i.e. oleic acid, lauric acid, ferulic acid), but there may be some with cationic or anionic substituents (i.e. betaine, succinic acid).
The elution solvent for the HPLC system would probably be acetonitrile : water.
Generally, for non-polar compounds the C18 SPE shows high extraction efficiency.
Dear Sally,
As you are not going to evaporate samples you need small volumes of eluent after SPE and acetonitril is an eluent of choice being a part of your HPLC mobile phase. As I can see most of the analytes are either nonpolar or weak anions. I would try to use acidic buffer (pH 3,0 or so) to load your sample on C18 SPE cartridge and wash down your substances of interest with either mixture of AcCN:H2O (say 90:10) or the same mixture, but with some ammonia or DEA. It is also possible to try mixed-mode SPE sorbents like Agilent (former Varian) Certify II with RP and anion-exchange properties. You can load the sorbent with basic or neutral sample and wash down substances of interest with AcCN or AcCN:H2O mixtures with neutral or acidic pH.
Your life would be much easier if you can find a neighboring lab with a speed vac or lyophilizer.
Since your analytes of interest are mostly non-polar C18 would bind them well, but you would need to elute in ACN. Since you don't have a speed vac, you could dry them with a gentle flow of nitrogen gas in the hood and then resuspend in your HPLC mobile phase. Since you are doing a liquid/liquid extraction you might not need an additional solid-phase extraction depending on the source of your sample. You definitely would need to still dry your aqueous phase someone because ethyl acetate will make up about 10% of your aqueous phase (your nose is a sensitive way to know that you have EtAc leftover or not but be careful not to inhale too much EtAc!!). You might just try using a small percent of the aqueous phase for an injection on your HPLC (make sure you either sub-micron filter the same or centrifuge aggressively to ensure insoluble components are gone) to check if it causes a problem. You can know if this method is OK by monitoring the backpressure of your HPLC system. If you are accumulating problematic compounds on the column, the pressure will slowly increase and you will need to reverse the column and clean with a more aggressive solvent system (IPA, etc.). Hope this helps.
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