UV-VIS Ascorbic Acid Content Measurement

UV-VIS Ascorbic Acid Content Measurement

Materials and Reagents:

Ascorbic acid standard solution or sample solution
Deionized water
Suitable buffer solution (e.g., 0.1 M phosphate buffer, pH 7)
Ascorbic acid dehydrogenase enzyme (optional, for specific assays)
Co-factor solutions (e.g., dithiothreitol, DTT)

Procedure:

Prepare a Blank Solution:

Use a UV-grade quartz cuvette and fill it with the buffer solution (without any ascorbic acid). This solution will serve as blank reference for baseline correction.
Calibrate the Spectrophotometer:

Turn on the UV-Vis spectrophotometer and allow it to warm up.
Set the wavelength to the appropriate value for ascorbic acid, which is typically around 245 nm.
Adjust the spectrophotometer's baseline using the blank solution, so that it reads zero absorbance at the chosen wavelength.
Prepare the Sample or Standard Solutions:

If you are using a standard solution, prepare a series of ascorbic acid standard solutions with known concentrations. These solutions will be used to create a calibration curve.
If you are analyzing a sample, prepare the sample solution by dissolving or extracting the ascorbic acid from sample matrix. Ensure that the sample solution is appropriately diluted to fall within the linear range of calibration curve.
Measure the Absorbance:

Pipette a small volume (usually 1 mL) of each standard solution or sample solution into separate cuvettes.
Wipe the cuvettes with a lint-free tissue to remove any fingerprints or smudges.
Place the cuvette containing the blank solution in the spectrophotometer and set the absorbance to zero.
Then, one by one, place each standard or sample cuvette in the spectrophotometer and record the absorbance at the selected wavelength.
Create a Calibration Curve:

Plot a calibration curve using the known concentrations of the standard solutions and their corresponding absorbance values. Use a linear regression analysis to determine the equation of the line.
Determine the Ascorbic Acid Concentration in Sample:

Measure the absorbance of sample solution at the same wavelength used for the standards.
Use the calibration curve equation to calculate the concentration of ascorbic acid in sample.
Optional: Enzymatic Reaction for Specific Assays (e.g., Total Vitamin C):

For specific assays measuring total vitamin C, an enzymatic reaction involving ascorbic acid dehydrogenase and co-factors (e.g., DTT) may be used. This reaction converts ascorbic acid to its oxidized form, which can be measured at a different wavelength.
Cleanup and Data Recording:

Properly dispose of waste materials and record data, including the absorbance values and calculated concentrations.