Spectrophotometer: Working principle, Calibration and Procedure

 

Spectrophotometer

A spectrophotometer is an instrument that measures the number of photons emitted to estimate the intensity of light spectra absorbed and transmitted by a sample. This provides information on the concentration of a compound in the sample.

Scientist Arnold J. Beckman and his colleagues at the National Technologies Laboratory (NTL) invented the Beckman DU spectrophotometer in 1940. In 1981 Cecil Instruments produced a spectrophotometer that was microprocessor controlled. This automated the device and improved the speed. From 1984 to 1985, development was made in double beam versions of the instrument which developed into the Series 4000 model. With the 1990s came the addition of external software that provided PC control and onscreen displays of the spectra. Today, the development of the spectrophotometer continues and its applications range from science and medicine to crime scene investigation and law enforcement.

Working Principle

Spectrophotometer is based on the photometric technique which states that When a beam of incident light of intensity I₀ passes through a solution, a part of the incident light is reflected (I_r), a part is absorbed (I_a) and rest of the light is transmitted (I_t)

Thus,

I₀ = I_r + I_a + I_t

The mathematical relationship between the amount of light absorbed and the concentration of the substance can be shown by the two fundamental laws of photometry on which the Spectrophotometer is based.

Beer’s Law

This law states that the amount of light absorbed is directly proportional to the concentration of the solute in the solution.

Log₁₀ I₀/I_t = a_sc

where,

          a_s = Absorbency index

            c = Concentration of Solution

Lambert’s Law

The Lambert’s law states that the amount of light absorbed is directly proportional to the length and thickness of the solution under analysis.

A = log₁₀ I₀/I_t = a_sb

Where,

            A = Absorbance of test

           a_s = Absorbance of standard

            b = length / thickness of the solution

The mathematical representation of the combined form of Beer-Lambert’s law is as follows:

Log₁₀ I₀ / I_t = a_sbc

If b is kept constant by applying Cuvette or standard cell then,

Log₁₀ I₀/I_t = a_sc

The absorbency index a_s is defined as

a_s = A/cl

Where,

           c = concentration of the absorbing material (in gm/liter).

             l = distance traveled by the light in solution (in cm).

To sum up, the working of spectrophotometer is based on Beer-Lambert’s law which states that the amount of light absorbed by a color solution is directly proportional to the concentration of the solution and the length of a light path through the solution.

A ∝ cl

Where,

            A = Absorbance / Optical density of solution

             c = Concentration of solution

              l = Path length

or,          A = ∈cl

             ∈ = Absorption coefficient

 

Components of spectrophotometer

• Light source that gives monochromatic/white light.
• Collimator to converge the light into a parallel beam
• Monochromator to split monochromatic light into the component wavelengths These can be prisms that split white light into the component visual colors. The monochromator can also be a grating to get UV, visual, and IR radiation bands.
• Wavelength selector, which is a slit that is used to select the desired wavelength/light band. 
• Cuvettes, usually made of glass or quartz to hold sample solution.
• Photometer, which is a photosensitive detector to measure the amount/intensity of light absorbed and transmitted through the sample.
• Result display section, which can be a meter or a digital screen.

How to use spectrophotometer

Procedure

1. Turn on the spectrophotometer

2. Clean the cuvettes

Take care of cuvettes as they can be quite expensive, particularly if they are made from glass or quartz. Quartz cuvettes are designed for use in UV-visible spectrophotometry. When handling the cuvette, avoid touching the sides the light will pass through (generally, the clear sides of the container). Wipe the cuvette down with a kim-wipe. 

3. Load the proper volume of the sample into the cuvette

4. Prepare a control solution. 

5. Wipe the outside of the cuvette

Before placing the cuvette into the spectrophotometer make sure it is as clean as possible to avoid interference from dirt or dust particles. Using a lint free cloth, remove any water droplets or dust that may be on the outside of the cuvette.

6. Choose and set the wavelength of light to analyze the sample with

7. Calibrate the spectrophotometer with the blank. 

It is a process by which we use a calibration standard to find out the light source’s accuracy. It is vital to make sure that the device functions properly and the correct measurement is obtained.

Calibration Procedure

• Turn on the spectrometer and let it warm up for at least 10 minutes.
• Change the chamber light to the desired wavelength on the spectrometer.
• Prepare a "blank”.

Calibration blank is a standard that does not contain the analyte(s) of interest at a detectable level. For example, if we had salt dissolved in water, our blank would be just water. The blank is the same volume as the solution to be analyzed and kept in the same kind of container.

• Fill the cuvette halfway with the reaction solution that does not contain the unknown sample.
• Wipe off the sides of the cuvette with a Kim-wipe. This removes any oil left from our hands and fingerprints from the side of the cuvette.
• Load the "blank" into the spectrometer chamber.
• Close the lid of the chamber and wait for the measurement to stop.
• Press the "zero" button to calibrate the spectrometer.

8. Remove the blank and test the calibration. With the blank removed the needle should stay at 0 (zero) or the digital readout should continue to read 0. Place the blank back into the machine and ensure the needle or readout doesn't change. If the machine is properly calibrated with your blank, everything should stay at 0. If the needle or readout is not 0, repeat the calibration steps with the blank.

9. Measure the absorbance of experimental sample. Remove the blank and place the experimental sample into the spectrophotometer. Slide the cuvette into the designated groove and ensure it stands upright. Wait about 10 seconds until the needle is steady or until the digital numbers stop changing. Record the values of % transmittance and/or absorbance. The absorbance is also known as the optical density (OD).

10. Repeat the test with successive wavelengths of light. The sample may have multiple unknown compounds that will vary in their absorbance depending on wavelength. To eliminate uncertainty, repeat your readings at 25 nm intervals across the spectrum. This will allow to detect other chemicals suspected to be in the solute.

11. Calculate the transmittance and absorbance of the sample. Transmittance is how much of the light that passed through the sample reached the spectrophotometer. Absorbance is how much of the light has been absorbed by one of the chemicals in the solute. The transmittance (T) is found by dividing the intensity of the light that passed through the sample solution with the amount that passed through the blank. It is normally expressed as a decimal or percentage. T = I/I₀ where I is the intensity of the sample and I₀ is the intensity of the blank. The absorbance (A) is expressed as the negative of the base-10 logarithm (exponent) of the transmittance value: A = -log₁₀T. For a T value of 0.1, the value of A is 1 (0.1 is 10 to the -1 power), meaning 10% of the light is transmitted and 90% is absorbed. For a T value of 0.01, the value of A is 2 (0.01 is 10 to the -2 power), meaning 1% of the light is transmitted.