MUJOMBI TALENT CHIPO

R143547B

MBChB BIOCHEMISTRY
PART 1
 EXPERIMENT 2: USE OF
THE
SPECTROPHOTOMETER

Introduction
A spectrophotometer is employed to measure the amount of light that a sample absorbs. The
instrument operates by passing a beam of light through a sample and measuring the intensity of
light reaching a detector. The beam of light consists of a stream of photons. A beam of light
passing through a substance can be absorbed if the energy of the photons happens to coincide
with a particular energy transition available in the absorbing substance. When a photon
encounters an analyte molecule (the analyte is the molecule being studied), there is a chance the
analyte will absorb the photon. This absorption reduces the number of photons in the beam of
light, thereby reducing the intensity of the light beam. Spectrophotometry can be used to analyze
fluid samples from patients in hospitals (Harper, 2003). A wide range of biomolecules
absorb light at characteristic wavelengths. Measurement of light absorption by a
spectrophotometer is also used to detect and identify molecules and to measure
their concentration in solution.(Koolman and Roehem,2005) The fraction of the
incident light absorbed by a solution at a given wavelength is related to the
thickness of the absorbing layer (path length) and the concentration of the
absorbing species. These two relationships are combined into the Lambert-Beer law,

Log Io = Ecl
I
(Nelson and Cox,2005)

where Io is the intensity of the incident light, I is the intensity of the transmitted
light, E is the molar extinction coefficient (in units of liters per mole-centimeter), c
is the concentration of the absorbing species (in moles per liter), and l is the path
length of the light absorbing sample (in centimeters). The Lambert-Beer law
assumes that the incident light is parallel and monochromatic (of a single
wavelength) and that the solvent and solute molecules are randomly oriented. The
expression log (I0 /I) is called the absorbance, designated A. It is important to note
that each successive millimeter of path length of absorbing solution in a 1.0 cm cell
absorbs not a constant amount but a constant fraction of the light that is incident
upon it. However, with an absorbing layer of fixed path length, the absorbance, A, is
directly proportional to the concentration of the absorbing solute. The molar
extinction coefficient varies with the nature of the absorbing compound, the solvent,
and the wavelength, and also with pH if the light-absorbing species is in
equilibrium with an ionization state that has different absorbance properties.
Absorptance is the simple ratio of the radiation absorbed by a surface to that incident upon it.
Total absorptance refers to absorptance measured over all wavelengths. Spectral absorptance
refers to absorptance measured at a specified wavelength. Transmittance refers to the fraction of
light transmitted through a particular medium and is a simple ratio of I/Io. Total transmittance
refers to transmittance measured over all wavelengths. Spectral transmittance refers to
transmittance measured at a specified wavelength (Stenesh,1989). In most applications, one
wishes to relate the amount of light absorbed to the concentration of the absorbing molecule. It
turns out that the absorbance rather than the transmittance is most useful for this purpose. If no
light is absorbed, the absorbance is zero (100% transmittance). Each unit in absorbance
corresponds with an order of magnitude in the fraction of light transmitted. For A = 1, 10% of the
light is transmitted (T = 0.10) and 90% is absorbed by the sample. For A = 2, 1% of the light is
transmitted and 99% is absorbed. For A = 3, 0.1% of the light is transmitted and 99.9% is
absorbed.

When using a spectrophotometer first, the intensity of light (I0) passing through a blank is
measured. The intensity is the number of photons per second. The blank is a solution that is
identical to the sample solution except that the blank does not contain the solute that absorbs
light (Clark, 1993). This measurement is necessary to calibrate the spectrophotometer for that
wavelength since the cell cuvette itself scatters some of the light. If light of many wavelengths is
passed through a substance, the adsorption of that light will rise to a peak value and then fall
again as the wavelength coinciding with energy transition is reached and passed. For some
substances, several such peaks may be found and may superimpose to form a broad peak. If the
amount of absorption is measured and plotted against wavelength the resulting curve is called an
absorption spectrum and is characteristic of the absorbing substance. The absorption wavelength
where absorption is greatest is termed absorption peak. (Biochemistry practical schedule, 2014).
Therefore the objective of this practical is to show that absorption values depend on the
concentration of the absorbing matter and the efficiency at which matter absorbs light.
 Materials and Methods
“As per biochemistry practical schedule MBChB/BDS 2014-2015”

Results

Table 1: Absorbance of 0.000625 mM fast Green and o.1 M CoCl2 at different wavelengths
Wavelength (nm) Absorbance
0.000625 mM Fast green 0.1 M CoCl2

400 0.075 0.099

425 0.091 0.161
450 0.046 0.273
475 0.029 0.387
500 0.023 0.511
525 0.042 0.466
550 0.087 0.273
575 0.204 0.125
600 0.395 0.088
625 0.682 0.084
 650 0.192 0.075

Absorption peak 625 nm 505nm

Table 2: % Transmission of CoCl2 at different volumes (at 505nm)

CoCl2 (ml) HCl (ml) % Transmission

0.50 4.50 73.2

1.00 4.00 58.8

2.50 2.50 29.7

3.50 1.50 19.3

5.00 0.00 11.9

Pure CoCl2 transmittance is 11.9%

Calculation
1) Molarity of CoCl2 after dilution

Tube 1: C1V1 = C2V2

0.2 x 0.5 = C2 x 5
C2 = 0.02

Tube 2 : C1V1 = C2V2

0.2 x 1 = C2 x 5
C2 = 0.04

Tube 3 : C1V1 = C2V2

0.2 x 2.5 = C2 x 5
C2 = 0.1
Tube 4 : C1V1 = C2V2
0.2 x 3.5 = C2 x 5
C2 = 0.14

2) Conversion of % transmission to absorbance

Absorbance = -log %T ,where %T is the % transmission
100

Tube 1: -log 73.2

100
= 0.135

Tube 2: -log 58.8

100
= 0.231

Tube 3: -log 29.7

100
=0.527

Tube 4: -log 19.3

100
= 0.714

Discussion
Absorption maximum for Fast Green is 0.682 at a wavelength of 625nm(Fig 1), whereas
that for CoCl2 is 0.537 at 505nm( Fig 2). Fast Green has two peaks, the smaller peak is a
possible result of the absorption of energy by impurities in the solution. As has been
stated before; the peak for fast Green is higher than that for CoCl2,therefore absorption
coefficient, that is, efficiency of 0.1M CoCl2 in absorbing energy, is lower than for
0.00625mM fast Green at their different wavelengths. This coincides with Michael
Gore’s results in which CuSO4 solution had a higher absorbance peak than CoCl2 at 0.1M
concentration of both solutions. Thus amount required to raise the molecule of CoCl2 to a
higher energy state is relatively lower than that for fast green. ( Gore,2000). Absorption
or emission of visible light by a molecule depends on electron transitions between
molecular orbital energy levels. When an electron goes from a higher to a lower energy
state, a photon of definite wavelength and frequency is emitted.

Fig 4 shows a linear relationship between molarity and absorbance of CoCl2 at 505nm.
Spectrophotometric absorbance measurements are ordinarily made at a wavelength
corresponding to an absorption peak, because the change in absorbance per unit
concentration is greatest at that point. Maximum sensitivity is thus realized. The
derivative of Lambert-Beer’s law: Ecl implies that the graph of absorbance against
molarity(concentration) should be a straight line passing through the origin, with gradient
El. The straight line obtained also tallies with the graph obtained by M.Gore on his
experiment on absorbance of CoCl2 at different concentrations (molarity). This linear
relationship proves that CoCl2 follows the Lambert-Beer law.(Gore,2000)

Fig 3 shows an exponential/inverse relationship between % transmission and molarity of

CoCl2 at 505nm.This phenomena is according to Beer’s law which states that ‘the
intensity ray of monochromatic light passing through an absorbing medium decreases
exponentially as the concentration of absorbing medium increases’. (Holmes, 1997)

Answers to questions
1)
 2) K2Cr2O2 – 625nm
CoCl2 – 505nm

3) 626nm – Pink
505 – Blue

4)Color absorbed from the visible region of the spectrum is complementary to color
reflected.

5) % transmission against molarity has exponential curve.

Absorbance against molarity is a linear graph. It is absorbance against morality that is
more useful in checking for errors and for references.

6) It does obey the Beer-Lambert law

References
Clark, B.J. (1993). Spectroscopy Techniques-Instrumental Data Handling. 2nd Edition (Chapman
and Hall: London UK), pg 20

Holmes, D.J, Peck, H. (1997). Analytical Biochemistry. 3rd Edition (Pearson Education Limited:
Essex, England)
Koolman, J., Roehm, K.H. (2005). Color Atlas of Biochemistry. 2nd Edition (Thieme Stuttgart:
New York), pg 102

Murray, R. K., Granner, D. K., Mayes, P. A., Rodwell, V.W. (2003). Harper’s illustrated
biochemistry. 26th edition. (McGraw Hill Companies: Toronto)

Nelson, D.L., Cox, M.M. (2005). Lehninger, Principles of Biochemistry. 4th Edition (W.H
Freeman: New York), pg 82

Stenesh, J. (1989). Dictionary of Biochemistry and Molecular Biology. 2nd Edition (Jon Wiley
and Sons: Canada)