Chromatography Forum

Hi

I've been doing fatty acid profiling of human plasma samples, and up to now several problems happened. After solving many of them, one remains. It seems that Cholesterol is being retained in the column, and this interferes with the analysis of the following samples. I tried to run several blanks and ageing the column, but this only worked a few times. Does anyone know how to remove the Cholesterol from the column? Is there any derivatization that allows me to extract specifically free fatty acids and esterified fatty acids, and not extract Cholesterol?

Some intrument details: 7890A GC with 5975C MSD and a DB23 column (high polarity, (50%-Cyanopropyl)-methylpolysiloxane).

First of all, what is your inlet temperature? I'm thinking that a lower inlet temperature may trap the cholesterol on some glass wool in a split liner.

Oooooh - let's make this a soap question, as I've assayed tons of fatty acid and soap samples, and also cholesterol. What about making the plasma solution basic (either as is or in sample-methanol), and extracting out the cholesterol. Then add sulfuric acid-methanol or BF3-methanol to acidify and esterify, extracting the methyl esters into organic solvent such as hexane and injecting that.

I will also state that when we've saponified then esterified tallow triglycerides, we haven't had issues with cholesterol build-up, but we've never had a ton of samples in a short time; typically such samples for us would be tallow fatty acids or sodium soaps.

First of all, what is your inlet temperature? I'm thinking that a lower inlet temperature may trap the cholesterol on some glass wool in a split liner.

Oooooh - let's make this a soap question, as I've assayed tons of fatty acid and soap samples, and also cholesterol. What about making the plasma solution basic (either as is or in sample-methanol), and extracting out the cholesterol. Then add sulfuric acid-methanol or BF3-methanol to acidify and esterify, extracting the methyl esters into organic solvent such as hexane and injecting that.

I will also state that when we've saponified then esterified tallow triglycerides, we haven't had issues with cholesterol build-up, but we've never had a ton of samples in a short time; typically such samples for us would be tallow fatty acids or sodium soaps.

About the inlet, we have a temperature of 250ºC. We changed the liner wool several times and it doesn't seem to be the problem. Actually we are making a profiling of esterified fatty acids after extraction with KOH-MeOH and free fatty acids after extraction with H2SO4-MeOH, and as you say, the problem is in the former one, when the cholesterol is extracted together with the EFAs. In both cases we are extracting using hexane and injecting in this solvent. As you probably noticed, we are doing highthroughput analysis, having run more than 100 samples, and still have to run more than 150. Per day I think we spend more than 20h of injections accounting with the QCs and the blanks. But when we have such a problem with cholesterol, we have to stop the process to try to clean the column by running some blanks and ageing. But it seems to be hard. We already quited in continuing the analysis of EFAs and we sill stay with the FFAs, however we still have to clean the column from the Cholesterol. Is there an easy way to do it apart from buying a new column?

Regards

What about reverse-phase SPE to hold up cholesterol and let fatty acids (or maybe better yet, made basic to be sodium soaps) pass through?

What about reverse-phase SPE to hold up cholesterol and let fatty acids (or maybe better yet, made basic to be sodium soaps) pass through?

Thank you very much for the advice. It may work. As soon as I can clean this column from the previous Cholesterol contamination, I'll try it.

Regards

The persistent contamination that you see could be coming form the inlet as well as (or instead of) from the column. Use whatever drastic inlet cleaning protocol is appropriate for your machine (on Agilents it is mechanical cleaning, Brukers have a high temp bakeout for example). If the contamination is still there put the column in in reverse i.e. connect the previous detector end to the inlet and do a column bakeout. Baking with the column in its orignal direction is just pushing heavy crud deeper and deeper into it.

Peter

The persistent contamination that you see could be coming form the inlet as well as (or instead of) from the column. Use whatever drastic inlet cleaning protocol is appropriate for your machine (on Agilents it is mechanical cleaning, Brukers have a high temp bakeout for example). If the contamination is still there put the column in in reverse i.e. connect the previous detector end to the inlet and do a column bakeout. Baking with the column in its orignal direction is just pushing heavy crud deeper and deeper into it.

Peter

Thanks Peter. The inlet doesn't seem to be the reason, I'm using Agilent's and after cleaning I still see the peaks. Now I'm having very low levels and probably these small peaks will not cause interference in my analysis, but turning the column backwards seems to be a good way to deal with it in future.

Regards

Hi

I've been doing fatty acid profiling of human plasma samples, and up to now several problems happened. After solving many of them, one remains. It seems that Cholesterol is being retained in the column, and this interferes with the analysis of the following samples. I tried to run several blanks and ageing the column, but this only worked a few times. Does anyone know how to remove the Cholesterol from the column? Is there any derivatization that allows me to extract specifically free fatty acids and esterified fatty acids, and not extract Cholesterol?

Some intrument details: 7890A GC with 5975C MSD and a DB23 column (high polarity, (50%-Cyanopropyl)-methylpolysiloxane).

Just curious, what do cholesterol peaks/spectra look like? I recently analyzed some egg yolk samples and wonder if I'm going to have the same problems.