A spectrophotometer is a machine which operates on the principle of Beer-Lambert's Law; that is: the absorbed light is directly proportional to the concentration of the solution while the transmitted light is inversely proportional; the more colored a solution is, the more concentrated it is.
In the laboratory, absorbance and transmittance are measured and are made use of as the basis in measuring unknown substances in the blood.
Most spectrophotometers nowadays are incorporated into automated machines but it pays to know how to operate manual spectrophotometers so that one would be able to understand the concept behind the procedures.
The following are the steps that should be performed:
FIRST, the spectrophotometer has to be warmed up for 5 - 10 minutes. This is to stabilize the machine and avoid fluctuations in the readings.
SECOND, select the wavelength to be used. This is done by making use of the wavelength selector knob which is one of the operating features of a spectrophotometer.
THIRD, prepare the unknown solution to be read by wiping the cuvete gently with a fine cloth or tissue paper, before transferring the unknown solution . (Never brush your cuvete as scratching on the surface of the glass may cause inaccurate readings.)
FOURTH, place the cuvete in the cuvete well/cell well properly. Be sure to follow any instructions with regards to the positioning of the cuvete.
FIFTH, adjust the mode control knob to 0.0 - if you are utilizing the absorbance measurement and 100 % - if you are using % transmittance. Use a water or reagent blank to adjust the spectrophotometer to these readings.
SIXTH, take note of the absorbance or transmittance readings which will be reflected on the digital read out device or in the display window.
N.B. Take at least 2-3 readings of your unknown and be sure to adjust the spectrophotometer to 0.0 for absorbance and 100 % for transmittance, before each reading. (This is to read out errors caused by the machine or by the color of the reagent itself.)
SEVENTH, take the averages of your two or three readings and compute for the unknown concentration making use of this formula.
Cu = Au /As x Cs
Where: Cu = Concentration of Unknown
Au = Absorbance of Unknown
As = Absorbance of Standard
Cs = Concentration of Standard
These are important pointers to remember in performing this manual procedure:
Cuvetes should never be brushed as any scratches could refract and reflect light and therefore will lead to inaccurate results.
Before reading a solution, the cuvete should be properly wiped with a soft cloth or tissue paper to remove dirt and dust which may lead to inaccurate results.
The cuvete should be at least or 3/4ths full so that the light will strike the solution itself and not the empty cuvete.
Be sure to warm the instrument as insufficient warming, will give fluctuating readings.
If your readings are more than 0.0 away from each other, be sure to take a third reading to validate results.
Using the manual method of Spectrophotometry is accurate and precise, as long as the Technologist observes the proper precautions.