Analysis of UV Visible Spectrophotometer

Every compound, that is present in the nature, has a property to absorb, transmit, or reflect light (electromagnetic radiation) at a certain wavelength.

This property of the compounds, helps to measure quantitatively by using spectrophoto-metric techniques.

Spectrophotometry is a technique which deals with the measurement of the interaction of light with materials

Spectrophotometry is regarded as an instrumental technique, based on the measurement of the absorption of electromagnetic radiation in the ultraviolet (UV, 200–380 nm), visible (VIS, 380–780 nm), and near infrared region

Several types of spectroscopic and spectrophotometric methods are applied to analyze the samples. Among them, there are two primary methods which are highly utilized;

Absorption spectrophotometry, which is based on the absorption of radiation at specific wavelength of light to get absorption spectrum

UV-visible spectrophotometry, which is apprehensive with the reflectance of specific spectra of a given material within UV and visible range of electromagnetic radiation spectrum

Principle

Law of absorption is the basic principle of UV-visible spectrophotometry. This law discusses the relation between thickness of the absorbing material and the concentration of the sample solution, which is popularly known as Beer-Lambert law or simply Beer's law. This law states that the amount of light absorbed is proportional to the concentration of the absorbing substance and to the thickness of the absorbing material.

Standard operating procedure of taking absorbance or transmittance reading on UV- Visible Spectrophotometer:

1. OBJECTIVE

To describe the procedure for analysis on UV-VIS Spectrophotometer

2. SCOPE

This procedure shall be applicable for analysis on UV-VIS Spectrophotometer

3. RESPONSIBILITY

Officer/ Executive - Quality Control Dy. Manager - Quality Control

4. ACCOUNTABILITY

Astt. General Manager - QA/QC

PROCEDURE

Ensure that the area is clean.
Switch on the main power.
Switch on the instrument.
Allow stabilizing for 15 min.
Set the desired wavelength with the knob in increasing order.
Keep the sensitivity switch at the highest position.
Put the mode selector at % T position.
Set the filter wheel as per the wavelength.
Adjust zero percent transmittance with the help of set zero control.
Rinse reference cuvette and sample cuvette with blank solution 2 to 3 times.
Adjust 100% T with the help of % knob.
Remove the cuvette and drain out the blank solution from it. Rinse it with sample 2-3 times and filled with sample.
Clean the outer surface of the reference cuvette with tissue paper.
Place the cuvette into the sample holder and cover the cuvette compartment.
The reading shown in the data position is the absorbance or % T of the sample.
Record the results and make the entry in instrument usage log book.
Remove both the cuvette and wash with purified water or methanol as required.
Calibrate the instrument by running similar way and make an entry in the calibration record. Calibration Frequency: Monthly and after maintenance