Chromatography Forum

Hey does anyone have any ideas how to boost recovery?
I'm assaying Thymol on a kinetex c18, using h20:acn as mobile phase and MeOH (solubility+column pressure forced this) as diluent. I have been trying different things all day and have so far ruled out:
filters
sample/standard (bad sample, etc)
column (tried brand new one)
injection volume/carryover/retention in needle/needle wash compatibility
vials


Currently trying to sonicate sample prep and see if that helps.

There is a considerable lack of detail here.

If your problem is recovery in extraction, then column, injection volume, etc. are not going to be major factors. Matrix and extraction solvent will be. And how have you determined that recovery is low? Spiking matrix and extracting or by some other technique?

I am making a guess given the line "sample/standard (bad sample, etc)" and am assuming that you are getting good results from standards and poor from samples?

I would note that thymol is a relatively non-polar molecule. If you are extracting a matrix which has a non-polar component (like a waxy leaf) with H2O:AcN, you may have a problem with thymol tending to partition into the matrix rather than the extraction solvent.

For more detailed help, we need to know more details about what you are doing.

there is no extraction, the thymol is in a disinfectant

Yes the standards are good and the recovery (from a spiked matrix) is 95%, but I need to get 98-102% here. What other info do you need?

Wow, sorry this is all so unclear!
"sample/standard (bad sample, etc)" I meant: I have ruled out fresh vs. old sample and a single bad sample prep.

May i know how do you calculate recovery of your sample? using standard curve or by comparing standard alone?
Most possible cause is sample preparation. Can you add one more step in your sample preparation: purification step?
If you still experience this kind of problem, i would like to suggest you to use internal standard in your method. However, pipetting and weighing skills are important in using this method.
Hope this information would help you.

I spike a known amount of analyte with a known purity in the matrix.

Some details needed:

A couple of possibilities come to mind. Either the thymol is not making it to the detector or somethign is going on with detection.

How are you prepairing standards - in particular, what solvents.
How are you prepairing the samples - dilutions, solvents etc.
What is the mixture you are analyizing made up of? Alcohol solution of thymol? Water and detergent? What other things are present?

How do you prepare the spiked samles (volumes, concentrations, etc.)

What type of detector are you using? UV, RI, Other? Any indication of coelution with something that might interfere with the detection?

I'm preparing my standards with methanol as a diluent, same with the samples. The mobile phase was 60:40 DI:ACN.

Sample is 2 g in 100 ml MeOH.

The 2 g contains thymol encased in surfactant micelles (just found out), and water.

I spiked samples by creating my own 'concentrate' in the matrix and from that prepare 3 recovery solutions by w/v at 80, 100 and 120%.

I am using a PDA. I guess it is important to note that I had completed all steps of the validation, which all passed, while waiting for a matrix sample from the supplier (which is proprietary, so I don't think they will divulge what is in it). The peak purities were good compared to the USP standard. We recieve their concentrate and just dilute it and that is our product.

Currently we think the micelles are preventing good recovery, so I am trying different ways in the sample prep to 'break' them.

If you have foam in your samples, the concentration of analyte is likely to be higher in the foam. Even if you don't have any foam, the concentration of surfactant is higher in the surface of the solution than in the "bulk"

I would test to add EtOH to the samples (30-50%), that usually break up the micelles.

I guess it is important to note that I had completed all steps of the validation, which all passed, while waiting for a matrix sample from the supplier (which is proprietary, so I don't think they will divulge what is in it).

Just a side note which might not help you with your recovery problem: You performed the validation WITHOUT matrix available? Selectivity, linearity, everything without matrix??
Uhm....

Well technically OUR matrix is just DI water.

Well technically OUR matrix is just DI water.

I thought to understand it's water plus some surfactant?

how did you check versus the effect of the filters?
and which filter membrane are you using right now?