The Operating Manual for UV-Vis Spectrophotometer

UV-Visible Spectrophotometry is an effective method for studying the composition, structure, and interactions between substances based on their absorption spectra. A UV-Vis spectrophotometer allows the selection of different wavelengths of light within the UV-visible range. What issues should be considered when using and calibrating a UV-Vis spectrophotometer?

The absorption spectrum of a substance refers to the result of molecular vibration energy level transitions and electronic energy level transitions after molecules and atoms in the substance absorb specific wavelengths of light energy from the incident light.

Since different substances have distinct molecular and atomic structures, as well as different molecular spatial configurations, their absorption of light energy varies accordingly. Therefore, each substance has its unique and fixed absorption spectrum curve, which can be used to identify or determine the concentration of the substance by analyzing the absorbance at specific characteristic wavelengths in the absorption spectrum.

Applications of UV-Vis Spectrophotometry

1. Substance Identification:

The characteristic absorption features, especially the maximum absorption wavelength (λ-max) and molar absorptivity (ε), are commonly used physical parameters for identifying substances. This has widespread applications in pharmaceutical analysis. The maximum absorption wavelengths and absorption coefficients of many drugs' UV absorption spectra are recorded in pharmacopeias both domestically and internationally, providing a valuable tool for drug analysis.

2. Comparison with Standard Substances and Spectra:

The analysis sample and the standard sample are prepared at the same concentration in the same solvent, and their UV-Vis absorption spectra are measured under identical conditions. If both are the same substance, their spectra should match completely. If no standard sample is available, comparisons can be made with an existing standard spectrum. This method requires highly accurate instruments, high precision, and consistent measurement conditions.

3. Comparison of Maximum Absorption Wavelength and Absorption Coefficient Consistency

4. Purity Testing

5. Inference of Molecular Structure of Compounds

6. Measurement of Hydrogen Bond Strength:

Experiments have shown that the strength of hydrogen bonds varies with different polar solvents. UV spectroscopy can be used to assess the strength of hydrogen bonds in different solvents to determine the most suitable solvent.

7. Determination of the Composition and Stability Constants of Complexes

8. Study of Reaction Kinetics

9. Applications in Organic Analysis:

Organic analysis is a comprehensive science developed on the basis of organic chemistry and analytical chemistry, focusing on the separation, identification, and determination of the composition and structure of organic compounds.

Precautions for Using UV-Vis Spectrophotometers

1. Temperature and Humidity:

Temperature and humidity are important factors affecting instrument performance. They can cause corrosion of mechanical components, reduce the smoothness of metal mirrors, and lead to errors or performance degradation in the mechanical parts of the instrument. Optical components such as gratings, mirrors, and focusing lenses may also suffer from aluminum film corrosion, leading to insufficient light energy, stray light, noise, and even instrument failure, thereby affecting the instrument's lifespan. Regular calibration is necessary during maintenance. The laboratory should be equipped with temperature-controlled equipment, especially in southern regions.

2. Dust and Corrosive Gases:

Environmental dust and corrosive gases can affect the flexibility of the mechanical system, reduce the reliability of limit switches, buttons, and photoelectric couplers, and contribute to the corrosion of aluminum films on key components. Therefore, it is essential to regularly clean the UV-Vis spectrophotometer and maintain a clean environment in the instrument room to prevent dust accumulation.

3. Dust Removal:

After a certain period of use, dust will accumulate inside the UV-Vis spectrophotometer. It is recommended to regularly open the instrument's outer casing for dust removal under the guidance of a maintenance engineer. Additionally, tighten the heat sink of each heating element, clean the sealed windows of the optical box, and calibrate the optical path if necessary. Clean and lubricate the mechanical parts, then restore the instrument to its original condition, perform necessary tests, calibrations, and record the results. Analytical instrument operators should understand the daily maintenance and simple testing methods for major technical indicators to ensure the instrument operates in optimal condition.

Detailed Operating Procedures and Calibration Methods for UV-Vis Spectrophotometer

1. Photometric Measurement with UV-Vis Spectrophotometer

• Select "1. Photometric" option on the mode selection screen to display the parameter configuration screen.

• Set the measurement wavelength using the GOTOWL key.

• Press the F2 key to set the sample introduction control.

• Press the START/STOP key to start the measurement; the measurement screen will be displayed.

• If a blank correction is needed, set the blank sample before measurement, then press the AUTO-ZERO key to set the measurement value to 0 ABS (100%).

2. Calibration of UV-Vis Spectrophotometer

• Preheat the instrument for 10 minutes before use.

• Insert the black block and standard sample (prepare your own if not available) and close the cover.

• Set zero.

• Align the light with the black block and set the transmittance to 0.

• Align the light with the standard sample and adjust the absorbance to 100%.

3. Introduction of Reference Solution

The reference solution, also known as the blank solution, is used for comparison during measurement. It does not contain the analyte but has a matrix as similar as possible to the sample solution. The curve obtained by scanning with the reference solution should typically be a flat straight line. Sometimes, although the matrix does not contain the analyte, it may contain other substances, which must be ensured not to affect the test. A common issue is the presence of the analyte in the reagent blank, which must be purified to remove it; otherwise, the measurement results will be affected.

4. Precautions for Using UV-Vis Spectrophotometer

• Preheat for 15 minutes before turning on the instrument for self-check.

• Maintain humidity around 75% and temperature between 5–30°C.

• Ensure the instrument has a stable power supply and proper grounding. Avoid direct sunlight.

5. Common Faults and Troubleshooting

• Light gate not fully closing: Repair the light gate components to ensure complete closure.

• Transmittance knob "100%" is at its limit: Re-adjust the "100%" knob.

• Severe moisture: Open the phototube dark box, dry with a hairdryer, and replace the desiccant.

Analytical instrument operators should understand the daily maintenance and simple testing methods for major technical indicators to ensure the instrument operates in optimal condition.