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UV Spectrophotometry Method for Preparing a Phenol Standard Curve
Keywords: Ultraviolet spectrophotometry; standard curve preparation; analytical instrument; UV-1300; UV-1500
I. Experimental Objective
1. Understand the basic principles of UV spectroscopy for material characterization and quantitative analysis.
2. Learn how to operate a UV-visible spectrophotometer effectively.
II. Experimental Principle
Organic compounds containing benzene rings and conjugated double bonds exhibit characteristic absorption in the ultraviolet region. Different molecular structures selectively absorb UV and visible light. The maximum absorption wavelength (λmax), molar absorptivity (ε), and the shape of the absorption curve are crucial for qualitative identification. Since absorbance (A) is highest at λmax, changes in A with concentration are most significant at this wavelength, making it ideal for sensitive detection. By analyzing the absorption spectrum of a phenol aqueous solution using a UV spectrophotometer, we can determine λmax and quantify the substance accordingly.
When using a UV spectrophotometer for quantitative analysis, if the analyte concentration is too low or high, the transmittance reading can be adjusted by a factor of 10, which helps improve accuracy for both low and high concentrations. This method is based on the Lambert-Beer Law: A = εbc.
III. Main Instruments and Reagents
Instruments: UV-visible spectrophotometer (UV-1300, UV-1500); 1 cm quartz cuvette (2 pieces); 50 mL colorimetric tubes (5 pieces); 5 mL pipette (1 piece).
Reagents: Phenol standard solution (100 mg/L).
IV. Experimental Procedure
(1) Preparation of Analytical Solutions:
Take 5 50 mL colorimetric tubes and accurately add 0.5 mL, 1 mL, 1.5 mL, 2.0 mL, and 2.5 mL of the 100 mg/L phenol standard solution using a pipette. Dilute each to the 25 mL mark with deionized water and shake well. Record the experimental data in a standard table.
(2) Determination of Maximum Absorption Wavelength:
Prepare a phenol solution with a concentration of 8 mg/L by dilution. Use a 1 cm quartz cuvette and deionized water as the reference. Scan the absorption spectrum between 200–330 nm on the UV-Vis spectrophotometer. Identify λmax1 and λmax2 from the curve.
(3) Preparation of the Standard Curve:
Measure the absorbance of each phenol solution using a 1 cm quartz cuvette and deionized water as the reference. Measure the absorbance in order of increasing concentration at the selected λmax. Plot the absorbance versus concentration to create a standard working curve.
V. Data Recording and Result Analysis
Record all absorbance values and concentrations in a table. Use the standard curve to determine unknown concentrations. Ensure accuracy by repeating measurements and checking for consistency.
Keywords: ultraviolet spectrophotometry; standard curve drawing; analytical instrument; UV-1300; UV-1500
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