Hi there! I am trying to quantify total inorganic phosphate from plant samples using a spectroscopy based techniques. I am facing issue with the standards. I have not tested the samples yet. For the first 3-4 standards, the coloration and absorption curves are linear but for standards with higher concentration the color is drastically reduced and curves drops rapidly. I independently repeated it thrice but got the same pattern. Do you have any suggestions to help troubleshoot this issue? Or, what protocol do you use? We are not interested in getting high resolution quantification data for phosphate amount in the samples, so we are looking for protocols which doesn't require sophisticated instrumentation. I have attached the protocol being followed in the attachment below.
Thank you!
I don't know why the standards become lighter as the concentration exceeds a certain value. However, I would say that the range of concentrations you should use for the standards is considerably lower than the concentrations you used. The absorbance is very high even in the first standard. I would choose that concentration as the highest standard.
Adam B Shapiro thank you for your answer. I am gonna try reducing the concentration of standards in the experiment.
You are likely reaching the limits of detection on your measurement instrument with your standards. Many instruments that are specifically intended for OD measurement will give you an error when you exceed a certain OD (typically 2.00), but general purpose spectrophotometers don't.
To overcome this problem when you are running samples with an OD higher than 2.00, you'll need to dilute the samples and then calculate the original OD from the dilution factor.
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