Tips on sterol Analysis from plasma/serum

[skibrett15]

Hi, I was wondering if anyone had tips for maintaining column life with this type of application. I have done a search here and found some posts on sterols, but my chief concern is that we are burning through columns (likely because of nonvolatile impurities from the plasma are interfering).

Should I be using a guard? Backflushing? Baking out at a high temp to try to get these to elute?

[Don_Hilton]

What kind of sample prep are you doing? That can make a big difference.

[skibrett15]

We are doing a methanol saponification and extraction into petroleum ether followed by dry down and dissolve into chloroform.

[Peter Apps]

It may not be the samples that are killing the columns; do you have regularly maintained scrubbers in the carier gas lines ?

Peter

[skibrett15]

Scrubbers as in dessicators/filters etc? Yes, we have that.

Really this has been an ongoing problem, I was curious if others are using a similar extraction method and getting more than 150 injections from their columns. I am using a 5% fairly inert column (0.25 ID 30m Agilent DB-5) but running at around 270-290C. This was done to get better throughput. I fear the high temps and relatively large injection are causing some heavy molecules to just stick in the column and resolution fades very quickly after 70+ runs. using a 60m column was a little better, but not in terms of a per meter or price basis.

Additional details:
22 psi He carrier gas (about 1 ml/min)
2.5uL injection with a 5:1 split (I know this is high, but to detect the sterols using this extraction it is necessary to put a lot on the column)

run time is about 20 minutes.

Is anyone else doing a similar method out there? Or analyzing sterols at all using GC? Any help is much appreciated.

[Don_Hilton]

A direction to try (from a differenty type of analysis): If your analytes will make it thorught the liner, use a liner that has a large piece of glass wool in it. Run the liner below the max temperature you use on the column and change it often. If your liner is hot enough to pass the analytes but cool enough that it catches the heavy compunds, you eat liners rather than columns.

A second thought. If the saponification does not go to completion, you have mono and di glycerides present - these take heat, at least, to get them to move down the column. A rapid ramp to the programed tempemperture maximum for the column and holding the temperature there for a few minutes may help - and if it is glycerides, backflushing the column may be a good thing to try. It seems to me that in days gone by, I've destroyed a DB-5 type column trying to get glycerides through it.

[skibrett15]

Ok, thank you for the advice.

I will try this, but i need to order some extra glass wool as we are not packing our own liners at this time. How much is a large piece... 30% of the liner, 50%?

[Don_Hilton]

I would suggest purchsing packed liners and see if you can get the vendor to assure you that the wool is deactivated after packing into the liner. Wool is nice because it collects garbage, but bad because it is full of active sites. I know that Agilent and Termo both make these liners - I had the drawer open and spotted some earlier today, so I know these are two sources, and I am sure there are more.

If you pack your own, I would look for a packed section about 1-2 cm long. You also need to get the same quantity of wool in each liner as the density of the liner makes a difference. You want these to be consistant. It will help you to figure out if this liner is actually helping.

[skibrett15]

Ok, we have been using packed liners with glass wool from restek, and I do notice that after 70 injections there is a brownish residue at the top of the wool. I thought you were suggesting adding more wool to the already packed liners because in all likelihood if I can see the residue, some of it has moved into the column, no?

[Don_Hilton]

If you can see the brown residue, material may (or may not) have moved onto the column. You have two concerns: 1) materials moving onto the column and 2) the brown stuff will show activity and may decompose or retain analytes of interest. After 70 (or fewer) injections discard the liner that you have been using and install a new one. Always install a new liner at the beginning of the day - because the stuff collecting in the inlet has had the opportunity to cook over night. Don't try to add wool to a liner that already has wool in it. Just be sure that you purchase a liner with enough wool in it to do the job. (I've see liners with a wisp of wool at the bottom - which is good for catching septum crumbs - and plugs of glass wool which will catch non-volatile analytes.)

You need to watch peak shapes and abilty to see low level samples to determine if you can run 70 injections per liner or not.