More method validation

I have a question related to my previous question of the last three weeks. To review, I am working on method development for analysis of solvents used in vapor degreasing. My starting solutions are all 99.9% purity. To me, this translates out to 99.9%=999000 ug/g. Before, I used 100g +/- 0.001g of each compound and combined them into one mixture. Combining them in this way caused my previous calibration method to quarter every sample, thereby not allowing a higher quantitation than 25.0%. I tried to create a calibration curve by creating each calibration level with one component per level instead of four components per level, even by entering in each area, level, and response factor in my calibration table, it would not link the like compounds into a viable curve. So, if I take instead 100g of each compound and combine them together ( resulting in a 25.0% limit), could I take less, like 100000 ug, effectively 10.0% in terms of purity, and call that 100% and create my curve based on this. Another idea I had is to weigh out exactly the amount for each component that reflects the upper limit of quantitation, e.g. weigh out 999000ug/g of component 1 to DCM 1000 ug, 999900ug/g of component 2 to DCM 100g, etc to refect the exact weight/percentage. This is a problem, however, since each sample vial is approximately 2000000ug. Perhaps if I made one reference standard for each 2 compounds, that might work.....but then I would need one curve for each standard in the mixture. Or create a four component mixture, dilute that down to 100000 ug, call that 100% and then dilute every  testing sample 1:10. Any feedback is appreciated!!

  • megaflorch -

    this was fairly old topic and I wasn't sure if you completed your standards/sample prep.


    I use a similar technique to get my analytes within a better working linear range with respect to my GC-FID.  Your logic is good.... here is my standard/sample prep breakdown:


    Analyte 1 and Analyte 2.  I like my linear range to be from 0.25% to 2.5% true value (or percent).


    I prepare primary standards to the true value.  Equal portions of Analyte 1 and Analyte 2, thus 50% each.


    I then perform a 20 fold dilution to get the 50% down to the top of my linear range at 2.5% which is my secondary standard.  This standard calibration Level is still represents my declared 50%.  This 20 fold dilution is applied to all samples (and all standards) with the same solvent.  Based on my equipment, I pipet 500uL diluted to 10mL.  My declared sample volume is then 500uL diluted to 10mL for the 20-fold (defined dilution factor of 1).


    So if I'm too far outside my linear range, lets say 4 times.... my sample amount would be 125uL diluted to 10mL and the final result multiplied by 4.  If my signal is too low, I may use 750uL with a dilution factor of 3/2 and the final result multiplied by 2/3.


    I tailored this method around incoming samples approximately 10-40%.  If you are trying to determine high purity then you can apply this same logic as you have previously stated.  This starts getting interesting when you use buffered liquid mobile phases where one would want to perform a "solvent match" of an unknown sample that will buffer similarly to their standard.

  • To answer your other question... if I was running 25% (you are stuck with 4 analytes in one standard) I wouldn't report any results over 50% (this is a good rule of thumb, if your sample is greater than 2 times the amount of your highest standard, dilute closer to your linear range).  If this is common I would recommend you develop a new scheme tailored to higher %.

  • 8/2/19


    Hi holtsy907,


    Thanks for your replies. The explanation of your method did give me insight into mine, as I am considering adding some new analytes to my calibration mixture. Since I was able to use dilution for my four analytes, that has translated to a robust method that seem to be operating as expected. My linear range is 1.0%-99.0%(w/w), with my 99.0% standard at 499950ug/g. So far this has worked well, aside from some adjustment to retention time windows and the fact that my amounts are very small. I am sure that I will have more questions in the future, and I hope that we can continue corresponding.

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