Introduction to spectrophotometry lab
The spectrophotometer is an essential tool for biologists and chemists in analyzing chemical and biological samples. Gaining familiarity with its operating protocols and understanding what its outputs mean are very important in the development of lab technique for students of cell biology. This experiment will help laboratory students gain experience in using the spectrophotometer.
Basic Laws of Light Absorption - For a uniform absorbing medium (solution: solvent and solute molecules that absorb light) the proportion of light radiation passing through it is called the transmittance, T, and the proportion of light absorbed by molecules in the medium is absorbance, Abs.
Transmittance is defined as:
T = I/Io where: Io= intensity of the incident radiation entering the medium.
I = intensity of the transmitted radiation leaving the medium. T is usually expressed as percent transmittance, %T: %T = I/Io x 100 The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as:
A = a() * b * c where A is the measured absorbance, a() is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as:
A = * b * c where is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1.
Results
Table 1: Observations for the maximum absorbance of Fast Green
Wavelength
Measured Absorbance
460 nm
0.043
480 nm
0.017
500 nm
0.016
520 nm
0.033
540 nm
0.074
560 nm
0.165
580 nm
0.321
600 nm
0.528
620 nm
0.978
640 nm
0.601
660 nm
0.140
680 nm
0.023
700 nm
0.006
Table 2: Observations of maximum absorbance in Chlorophyll a
Wavelength
Measured Absorbance
400 nm
0.367
420 nm
0.478
440 nm
0.328
460 nm
0.116
480 nm
0.076
500 nm
0.021
520 nm
0.018
540 nm
Basic Laws of Light Absorption - For a uniform absorbing medium (solution: solvent and solute molecules that absorb light) the proportion of light radiation passing through it is called the transmittance, T, and the proportion of light absorbed by molecules in the medium is absorbance, Abs.
Transmittance is defined as:
T = I/Io where: Io= intensity of the incident radiation entering the medium.
I = intensity of the transmitted radiation leaving the medium. T is usually expressed as percent transmittance, %T: %T = I/Io x 100 The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as:
A = a() * b * c where A is the measured absorbance, a() is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as:
A = * b * c where is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1.
Results
Table 1: Observations for the maximum absorbance of Fast Green
Wavelength
Measured Absorbance
460 nm
0.043
480 nm
0.017
500 nm
0.016
520 nm
0.033
540 nm
0.074
560 nm
0.165
580 nm
0.321
600 nm
0.528
620 nm
0.978
640 nm
0.601
660 nm
0.140
680 nm
0.023
700 nm
0.006
Table 2: Observations of maximum absorbance in Chlorophyll a
Wavelength
Measured Absorbance
400 nm
0.367
420 nm
0.478
440 nm
0.328
460 nm
0.116
480 nm
0.076
500 nm
0.021
520 nm
0.018
540 nm