1.2.1 UV Spectrophotometry

Suitable for Samples with a Single Component, but Susceptible to Interference
Experimental Procedures
1. Preparation of Absorption Curve
Use a pipette to transfer 2 mL of the standard protein solution (5 mg/mL) into a 10 mL colorimetric tube. Dilute the solution to the marked volume with 0.9% NaCl solution and mix well.
Using a 1 cm quartz cuvette, take the 0.9% NaCl solution as the reference. Measure the absorbance of the prepared solution within the wavelength range of 190 nm to 400 nm.
2. Preparation of Standard Curve
With a pipette, separately draw 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, and 2.5 mL of the standard protein solution (5 mg/mL) into five 10 mL colorimetric tubes respectively.
Dilute each tube to the marked volume with 0.9% NaCl solution and mix thoroughly.
Employ a 1 cm quartz cuvette, with 0.9% NaCl solution as the reference. Determine the absorbance of each standard solution at a wavelength of 278 nm and record the obtained data.
3. Sample Determination
Take the test sample that has been diluted to an appropriate multiple with 0.9% NaCl solution.
Determine the absorbance of the sample at 278 nm following the same method as described above.
Perform the determination in triplicate (parallel measurements) and record the measured values.
4. Data Processing
Plot the standard curve using the data obtained from the determination of the standard protein solutions.
Calculate the concentration of the test sample based on the standard curve.