Kindly let me know about the use of standard for quantitative analysis of plant sample metabolites using UV VIS spectrophotometry calibration method. Is the presence of that particular standard compound necessary in the sample to be analyzed.
You need a solutions of known concentration of substance (standard) that you will determine. Read about external standard calibration.
Per ICH Q3 the method must be able to quantify 0.05% wt/wt of a related substance relative to the active. As far as I know this is the only requirement/regulation. But many chemicals, particularly in the same class will absorb at the same UV wavelength. Thus, in order to meet specificity requirements, chemists use reagents to derivatize a compound and shift the wavelength of absorption.
Dear Musadiq,
In this case, I work on the following assumptions:-
- You have a quantity of the pure standard.
- The standard has a UV/Visible chromophore, or can be derivatised to add a UV/Visible chromophore.
- You are able to adequately isolate that chromophore from the sample matrix of your samples.
You need to find out the Lambda max of your standard. This is the specific wavelength which gives rise to the highest clearly resolved Absorbance value. You may use a published value and/or determine it for yourself.
The Lambda max should help you to determine the specific wavelength, at which you perform measurements for that particular standard dissolved in an appropriate liquid.
The Lambda max is obtained by performing an automatic or manual wavelength scan. A wavelength scan is a plot of Absorbance on the y axis, and wavelength on the x axis, for your standard, dissolved in an appropriate liquid.
Once a specific wavelength has been chosen, a calibration or standard curve of Absorbance versus concentration needs to be plotted. Here various levels of the known concentrations of your standard and the resultant Absorbance readings are plotted. A factor is then derived from the calibration curve.
The factor is generally the gradient of a calibration curve and its intercept on the y axis.
The amount of the unknown concentration/s of the standard analyte in your sample/s is read off against the curve, using the factor.
In general, Concentration = Factor x Absorbance.
Kind regards,
Ade Kujore
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