If the targeted compounds are polar, major cause of low recovery would be the extraction efficiency. Relevant factors are strength of elution solvents, amount of sorbent in the column/cartridge, volume of sample, and pH of the sample solution. In your case:I assume your SPE cartridge could be blocked by air bubble or trap components from sample matrix and particles or large molecules may also block the column as well for frequent use. As you said you are getting better result by reversing the cartridge position which indicates your cartridge efficiency as normal position has been lost or decreased. Although, you can use it as reverse way but It is may not the exact result what you are getting as better. For normal phase column and ion exchange column, removing the trapped components is not easy.

When elution is by gravity, air in the sorbent can change the flow path of solvents and reduce the extraction efficiency. As a standard practice in old type column chromatography, the sorbent must be covered with solvent all the time to avoid air bubble. However in the new SPE practice, the sorbent particle is much smaller and the elution is by a pump or pressure. Air bubble in sorbent can be pushed out quickly. So it is not a problem anymore. Actually the column/cartridge is often purged with air for solvent exchange.

Thanks very much Md. Saddam,

I already optimized the sample pH, elution strength, and volume of sample.

Even after i eluted the analytes with certain amount with elution solvent, i load more solvent to check if there is anything left in the cartridge.

I believe the only way to compare between reverse and normal flow is to compare the recovery.

Many thanks,

Alyah

I assume that you did check that no analyte was found in the solvent 1 and 2 during load and wash. It sounds like the analyte is strongly retain on the first mm of the packing. It is easier for analyte to be eluted at the head of the sorbent than travel all the way to the bottom. May be you can check by use less sorbent say if you use 1 g, try 250 mg in the forward flow. Check that no break thru and check the recovery in the forward flow with less sorbent. Why in the world you try the reversed flow the first place?

An easy experiment would be to check the recovery of your analyte for reversed vs. forward flow with increasing elution volumes. If Narong's theory is correct (what I believe) the recovery should align for higher elution volumes.

Another question: Do you see any deficiency in specificity by reverse flow (base line unsteadiness or additional/new peaks in comparison to forward flow)?

@Mueller  I visited Heidelber in May, it was a very nice touristy town.

Thanks very much for you all,

I already checked loading and washing solvents, there was no analytes.

I do not see any addition peaks, even baseline is almost the same to normal flow.

For normal flow, need more elution volume than reverse flow as Narong mentioned the analytes retained at the top of the stationary phase.

you can fix this by using less stationary phase or use stronger solvent. I read your mail again and found that you use cation exchange. There are two types of stationary phase. You may use the strong cation exchange phase and that may give you issue, try to use the weaker one. Look at your SPE supplier, and they should have more selection for you. That way you don't have to use a harsher solvent or higher ionic strength that may cause more issue of what you would do with the sample after the cleanup.Even the mixed phase mode may be a good one so you can wash it with methanol and your analyte will still stay in the SPE. That way you don't have to use a harsher solvent or higher ionic strength that may cause more issue of what you would do with the sample after the cleanup.

Thanks Narong,

Yes, am using strong cation exchange SPE with mixed mode. will looking to fine weak cation exchange.