Spectrophotometer, types, Setting up, uses.

Spectrophotometer

Spectrophotometer

This is an instrument used to measure absorbance at various wavelengths. It is similar to the absorption meter except that it uses diffraction gratings or glass prism to produce monochromatic light.

There are two types of spectrophotometers.

(1) Single beam colorimeter/ spectrophoto meter.

It operates between 325 - 1000 nm wavelength; using a single source of light, e.g., tungsten filament lamp. It has two photocells. The light travels along only one pathway. The test solutions and blank are read in the same position.

(2) Double beam colorimeter/spectrophotometer.

 It operates between wavelength range 185-1000 nm. It has two light sources and two photocells. This instrument splits the light from the monochrometer into two beams. One beam is used for reference and the other for sample reading. It eliminates errors due to fluctuations in the light output, and sensitivity of the detector. 

This is because the final reading is derived from the difference between the intensities of test and reference beams. Figure 1.5 (a) and (b) show the light path in single and double beam spectrophotometers respectively.

Setting up the colorimeter/spectrophotometer.

 An important step in colorimetric procedure is the selection of the correct wavelength or filter to use. The next step is to produce an absorption curve of the color to be measured by plotting the absorbance against the wavelength using the same solution. In this way the wavelength that gives the maximum absorption is determined (Fig. 1.6). For this purpose, serial dilutions should be prepared from a colored solution such as copper sulphate.

the light path in a single cell photo cell photoelectric colorimeter using red filter

 

light path in (a) single beam and (b) Double beam spectrophotometer

 

 

 

 

 

 a wavelength showing maximum absorbance

After selecting the correct filter or wavelength to give maximum absorption, it is still essential to ensure that the following criteria are observed: 

1. Beer's law is obeyed over the range of expected values.

Note 1. The linear relationship exists only for a certain range of concentrations. Beyond this range it is no longer valid.

2. The degree of sensitivity is satisfactory, i.e. increase in concentration corresponds to suitable increase in absorbance.

Checking the wavelength

The calibration curve must be carried out with extreme care. Freshly prepared reagents and standards and scrupulously clean glassware must be used. The accuracy of the wavelength calibration of a spectrophotometer should be checked from time to time, especially after the instrument has been shifted. This is to ensure that a linear relationship is maintained. For this purpose, it is ideal to follow the manufacturer's instruction using the special filters meant for the purpose.

The following two methods can be used to verify Beer's law and to check the degree of sensitivity.

VERIFICATION OF BEER'S LAW

Method 1

Reagent

Copper sulphate (CuSO4.5H2O): Prepare a 0.5 M solution by dissolving 12.5 g in 100 ml of distilled water. 

Technique

Prepare the following dilutions from the above solution

Read the absorbance of Tube no. 6 using different wavelengths. Select the wavelength which shows maximum absorbance. Read the absorbance for each tube. Plot a graph of concentration against absorbance. Results A straight line relationship shows that the Beer's law is obeyed.

Method 2

Reagent 

Dilute Iml of heparin or oxalated blood in 100 ml volumetric flask with 0.4% ammonium hydroxide (NH,OH). The concentration of diluted blood is 1%. 

Technique

From the diluted blood (1%) prepare the dilutions as shown below.

Follow the same procedure as in Method 1, using Tube no. 1 as blank, to set the zero absorbance. Plot the graph of concentration against absorbance. 

Results

 A linear graph shows that the Beer's law is obeyed.

Note

1. Errors arise due to (a) failure to mix solutions properly and (b) inaccurate pipetting.

2. Failure to obey Beer-Lambert laws leads to error if the test concentrations are outside the linear range of the standard.

3. Optically or mechanically faulty instruments as a result of too much stray light or ambient light entering the instruments or inadequate warm-up time can give wrong reading.

4. Dirty cuvettes, impure solution and air-bubbles lead to errors in absorbance values.

5. Avoid precipitation reaction occurring in the cuvettes.

6. Read the results at the recommended time limit to avoid incomplete colour development or colour fading.

For a more accurate and reliable analysis, it is very essential to be conversant with the manufacturers instruction on the setting up, use and maintenance of a particular instrument.