I have created a standard curve(5 concentrations) for antibiotics using a spectrophotometer. When I measure the concentration of the unknown sample, I get unrealistic results for example, if the sample contains 30 mg of Antibiotics I get 44. Also, when I repeat the process sometimes I get a different result.
In such cases I suggest starting by asking, is there a problem with the calibration curve? This question includes looking at the residuals and at whereabouts the sample falls on the curve. If yes, you fix that. If no, then it's a problem with the uniformity of he sample.
First, make sure that your working sample solution is within the calibration curve and that you are working at the linear range. If you see no problem with your calibration curve, you should also consider tyndall effect or other absorbing compounds.
Tyndall effect is the scattering of light in your sample. If strong enough, it can affect absorbance reading.
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