Back pressure and cleaning

[sluggo]

Hello,

I am conducting studies on drug elution from various formulation matrices. All my samples pass through a C18 SPE procedure prior to injection in HPLC. Over time the back pressure in the HPLC system increases. I'll take a guess that I see a rise from 1400psi to 2000psi in 200 injections. Replacing the guard column lowers the pressure, but the pressure builds back up quickly. The retention time does not seem to move.

The only thing that I can think that is coming from my samples that may cause problems are oils or lipids. The lipid must be molecularized in order to pass through the SPE I guess. I have seen the lipids solidify in the HPLC sample vial when cooled though.

HPLC method 1: MeCN:water 60:40, flow 1ml/min, C18 column

My questions are:
1. could this pressure rise be seen as "normal"?
2. is it likely that an oil such as castor oil, or a lipid cause backpressure increase?
3. is it common for labs to implement a regular column cleaning procedure? for example, run 95% acetone, IPA, etc for 30min after each shift/sequence?

thanks

[Uwe Neue]

When a guard column cloggs, it is doing its job. Therefore the problem must be the previous step, in your case the SPE procedure. Please describe in detail your SPE procedure, and what it is supposed to remove, and maybe we will find a solution.

[sluggo]

SPE is primarily used to remove the drug from a PBS solution and to concentrate the sample. I'm not intentionally removing any excipients, as I believe that everything in the sample is dissolved and will remain soluable in the mobile phase. This is why I find my pressure problem to be so strange.

SPE procedure
conditioning: 1ml methanol, 1ml water
sample addition: 3ml of sample or more
water wash: 3ml of water
dry
elution: 1ml MeOH

I've verified this procedure in terms of recovery, washout, etc. and it seems to work well for my purposes.

[Uwe Neue]

Your SPE procedure is rather simple. It may not remove what you want to remove.

I would not dry the cartridge before elution. I do not see the need for this step.

If you can quantify at what methanol concentration the contaminants are eluting, you could potentially improve the method, for example by elution with 90% methanol and 10% water etc.

[sluggo]

I will look into that, thanks for the suggestion. One troublesome issue is that I do not have any easy way of identifying the contaminants (other than the gradual pressure increase I guess).

What about the issue of doing a wash at the end of the day with a strong solvent? Is this advisable?

[syx]

I would not dry the cartridge before elution. I do not see the need for this step.

I'm sorry, Mr. Neue, I'm a beginner in SPE. I always dry the cartridge using vacuum for several seconds before elution because I do not know when to start eluted-solution collection. I feel safe if I dry it first and then collect entire eluted-solution.
Could you please give me advice about the right time to start collection?

[Uwe Neue]

I have spent some time of my life guiding a group in the development and optimization of SPE techniques. I can send you an article describing the techniques. It is not brandnew any more, and in the meantime, even better methods have been developed, but it gives you a general idea how to deal with SPE problems. I have also given many, many seminars on SPE techniques. My focus was on plasma or urine samples, but we have also dealt with other issues like enrichment from waters or sample preparation of animal products and plants.

To sluggo:

A wash at the end of the day with a high concentration of organic, mayby 95% MeCN with 5% water, is a good idea. However, I would do this only for the precolumn, and disconnect the analytical column. You do not want to have the stuff that elutes from your guard column on your expensive analytical column, do you?

To syx:

If you are working from a 96-well plate or even from individual cartridges with a vacuum manifold, there is no detriment to let air pass through your SPE device, but there is also no reason to attempt to "dry" the device. If you are working with an old-fashioned technique with a single SPE device and a syringe, there is no reason to do anything specific to "dry" the device. Just collect everything from the moment you add the elution solvent.

[james little]

Lipids not eluted very well from column with acetonitrile.

See page seven of poster 2 at

http://littledomain.com/james/files/matrix_effects.htm

however, i have let the lipids build up on the column and not noted a lot of pressure increase.

Probaby something else..

[Peter Apps]

Hi Sluggo

Do you really need to do SPE at all ?. With 3 ml of sample and elution with 1 ml methanol you have increased your peak areas by only a factor of 3, which might not be worth the trouble. How about injecting the PBS solution directly ?

The pressure increase is not huge considering that it takes 200 injections to develop, and could be due to suspended particles in the mobile phase or the samples - SPE cartridges can shed fines. Are you filtering your samples before injection ?

Peter

[Uwe Neue]

Peter, this is apparently not a question of concentrating the sample, but one of sample cleanup, which appears to be only partial.

A well designed SPE device should not shed any fines...

[Peter Apps]

Hi Uwe

Check Sluggo's second post - he is using SPE to concentrate the sample, and not to clean it up. If he has lipids (vegetable oils) in the sample they will be suspended rather than dissolved in the aqueous PBS. I am worried that the lipids deposit mechanically (rather than adsorbing) onto the C18 SPE packing, and then dissolve in the eluting methanol. Then when the methanol is injected the lipids come out of solution and clog the guard column.

If he can get a PBS solution with no suspended lipids (centrifuge perhaps) it should be OK to inject as such, as long as the buffer salts are dilute enough.

Injecting samples in pure methanol is also a risk for peak distortion.

"Drying" is a minefield in SPE, and can mean anything from a quick pulse of gas flow to blow some drops out of the tip of the cartridge, gas flow until visible water has disappeared, or rigorous dehydration (which then "collapses" the C18). One problem that we encountered was that unless the cartridge is thoroughly "dry" the analytes would end up dissolved in a drop of water when the eluting solvent was blown down with nitrogen to concentrate it or exchange solvents.

Sluggo,

In what state are the lipids in the samples - suspended or floating as a layer on top ? Are they triglycerides (oils) or fatty acids ?. If they are triglycerides you might be able to de-fat with hexane and then inject the aqueous phase. What is the concentration of the PBS buffers ?

Regards Peter