I am developing a work on HPLC with imidacloprid in waters where I also do the validation of the method. In the concentration of the lowest standard (0.50 ug/mL) the values in the evaluation of accuracy and precision are constantly changing, from one analysis to another, the first one usually being within the expected values and in the following already gives different, but consistent with each other. What could be happening?
You are probably around the LOQ (the area you should not go). Try increasing your injection volume or using the maximal wavelength.
Are you close to the void volume of the HPLC column? Chromatography (baseline separation) occurs when the capacity factor (K) >2 and
Does your retention time vary? What is your sample matrix and analytical method?
There can be a variation in accuracy due to a difference in peak shape. You may find at low concentrations the peak is small and squat and sharp at high concentrations, thus the integration is different. The solution is to use a smaller particle size in the column or a stronger mobile phase.
Daniel J F Santos The concentration of your component is very low. I have faced the same problem in one of my method. Try increasing the concentration of solution. Due to low concentration the peak shape is bit flat and not consistence which leads to failure. You can go up to 40% concentration of your specification level for LOQ.
Dear Daniel J F Santos
many thanks for your question. In order to adequately access your question, it would help, if you would provide us with some details of the method you are applying. Troubleshooting - the way I was taught eons ago - begins with information. The typical culprits: What, when, how, how much, how often, who...
(and to participate in the guess work: How old is your UV-lamp?)
Good luck,
Detlef.
What is de S/N ratio of your peak? Are you using stable isotopes as internal standard in your samples? What is the ratio of your analyte:IS?
With the limited amount of information and assuming that the problem is low signals: try to concentrate your samples if possible. You will probably also use different matrices? Try SPE or evaporation until dryness and reconstituting in a lower volume in the sample preparation.
Maybe the purity of the IS is causing interference with your analyte signal. Inject only IS in the same matix and look for signals at your analyte retention time.
If that doesnt help, more information on the method is needed to find out the problem.
Your concentration is too low and the peak shape probably at this concentration is not good and changing from run to run making the integration more difficult.
Try to increase the concentration at your low end and improve the peak shape
Sorry.excuse my confusion. How do you not know diameter of column?
What is column dimensions.
Ex. 250 x 4.6 x 5um.
Can you show us a picture of how the peak looks like? And to concentrate a sample, you dont spike a higher concentration. You reconstitute it in a lower volume!
Have you ever tried solid phase extraction in order to concentrate the sample
If possible you can concentrate the solution by evaporation and heating on water bath under inert atmosphere, only concern is that your sample should not get degraded on heating.
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