How to generate standard curve for antibiotic sample in spectrophotometer?
Prepare solutions of the antibiotic at various concentrations in the desired solvent. Use a spectrophotometer to measure the absorbance spectrum of the solutions over the desired range of wavelengths. Make sure to use a clean cuvette, which should be quartz if you plan to make measurements in the UV range. Make sure the samples fill the cuvette.
Discard the concentrations that go off-scale for the instrument. A maximum absorbance of about 1 is desirable. Subtract any background from the solvent.
Choose the wavelength of interest. This is usually the wavelength with the highest absorbance, but you may choose a longer wavelength if the maximal absorbance occurs very far down in the UV.
Plot a graph of absorbance versus antibiotic concentration at the chosen wavelength. Perform a linear regression through the data points. This is your standard curve. The slope of the standard curve is the extinction coefficient for the precise conditions used to make the measurements.
You should prepare solutions of various known concentration of the chemical you want to study in your case it's (an antibiotic) then measure the absorbance of each solution.
Usually 6 solutions are sufficient to draw a curve, how ever the concentrations distribution should be homogenous to obtain a representative curve, make sure then that the samples you will analyse fit kn the interval of the standard curve demited by the minimal and maximal concentration values of the 6 solutions used to make the curve.
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