https://www.academia.

edu/15363986/spectrophotometry

Food chemistry II: using

spectrophotometry

Ntokozo Vusumuzi Gumede

Nv.gumede58@gmail.com
0612047986

8/4/2015
Contents
Abstract.............................................................................................................................................2
Introduction.......................................................................................................................................2
Aim....................................................................................................................................................3
Methodology.....................................................................................................................................3
Results...............................................................................................................................................5
Discussion.........................................................................................................................................7
Conclusion.........................................................................................................................................8
References.........................................................................................................................................9

1
Abstract

This lab report contains the details of how an assay using spectrophotometry was done.
Spectrophotometry is the use of light energy to detect molecules in a solution. The light
energy is reported back to the user as wavelength in nanometres. This is done using a
spectrophotometer (Nkaissieh,D. 2008). . The method used when conducting this experiment
was firstly the identification of the Amax of the solution which was recording different
absorbance values for the different wavelengths. Then using the Amax wavelength to
measure the absorbance of the bromophenol blue solution that has been double diluted which
therefor have different concentrations. Then the graph was used to identify the possible
concentration of the given bromophenol blue solution with an unknown concentration. The
results are recorded below and a discussion that has our observations and briefly describing
the significance of the results recorded. Then finally the conclusion that will sum up the
report and state whether we have achieved what we aimed to achieve at the beginning of the
experiment.

Introduction

When light is transmitted through a solution, some of it may be absorbed. If the absorption
occurs in the ultraviolet or infrared regions of the electromagnetic spectrum, the solution will
appear colourless. But if the absorption occurs in the visible region of the spectrum, the
solution will appear coloured. Quantitative photometric of the process is measured using a
spectrophotometer. A spectrophotometer is an instrument that measures the amount of light
that can pass through a solution. It is apparent that less light allowed to be permeated through
a highly turbid or coloured solution than through a clear solution. Spectrophotometer is a
device that can quantify the amount of light transmitted through solutions. The
spectrophotometer determines the Amax of the solution, this is the point the solution where of
absorbs more light. In any given liquid solution some wavelengths of light will be absorbed
in greater amounts than others. Spectrophotometry directs a beam of light through the sample
solution being studied. The spectrophotometry absorbance is the amount of light that is
absorbed by the under study (Sanker, 2003).
Cuvettes (sample container) are normally used while determining the absorbance of light,
containing Bromophenol Blue. Distilled water is used as a blank.

2
Lambert worked on transmission of monochromatic light (light composed of only one
wavelength) by homogenous solid substance.
He states that the thickness of solid material played a major role in the amount of light being
transmitted through (Lambert, 2003). The concentration as well as the length of the oath that
the light travels affected the result. He takes into account the concentration and the
wavelength of the solution (Beer, 2007).
Aim
To find the Amax value and wavelength of bromophenol blue and calculate different
concentrations using that Amax value.

Methodology

Plastic pipettes were used to discard 1500 µml distilled water (which is used as a blank for
this experiment) and bromophenol blue solution into their respective cuvettes. The cuvette
containing distilled water was inserted into the spectrophotometer’s sample compartment
door. It was then further set as the blank sample at starting wavelength of 480nm. A cuvette
containing bromophenol blue was then inserted in place of the blank. the absorbance reading
was obtained and recorded. The wavelength was increased by 10nm from 480nm up to
620nm while the blank was zeroed each interval and the absorbance of bromophenol blue
recorded after every blank till 620nm and noted down. This was to get the A max of the
bromophenol blue (peak absorbed light). The then used solutions were disposed of
aseptically.
Four test tubes were taken and numbered from 1-4. The aseptically prepared 1st glass pipette
was used to discard three mills of distilled water into each test tube. The 2 nd glass pipette was
used to discard 3ml of 18.6µM bromophenol blue solution into test tube 1. Test tube 1 was
run by the vortex to diffuse the mixture of bromophenol blue and distilled water, making a
dilution of ½. It was further continued to pipette 3ml from test tube 1 to test tube 2 and ran
through the vortex to make up a complete solution of ¼ dilutions. Again 3ml was pipetted
from 2nd test tube to the 3rd and ran through the vortex to make up a dilution of 1:8. Finally,
another 3ml was pipetted from the 3rd test tube to the 4th and ran through the vortex to make a
dilution of 1:16. To equalise the amounts of the solutions, 3ml was pipetted from the last test
tube and discarded down the sink. The distilled water cuvette was placed into the sample
compartment chamber of the spectrometer and was blanked using the wavelength
corresponding with the Amax. The 4th test tube`s cuvette was inserted into the chambers and

3
recorded the absorbance. We continued checking the absorbance of cuvettes in descending
order, till the first cuvette and recorded the absorbance results.
The cuvettes with the bromophenol blue solutions of known concentrations were removed
from the chambers except the one with distilled water.
Bromophenol blue of unknown concentration was pipetted from the bottle into the last
cuvette and inserted into the chambers. The distilled water in the chambers was then blanked
and the absorbance reading of the bromophenol blue cuvette was recorded.

Dilution ratio
1:2 1:4 1:8 1:16

1 2 3 4

Cuvette with distilled water

7
Spectrophotometer
Figure 1: serial dilution

4
Results
After measuring absorbance of bromophenol blue at different wavelengths it was
further concluded that its Amax of 1.990 was obtained from the wavelength of 590. The
absorbance of other wavelengths were recorded and graphed. The absorbance at different
concentrations were also tabled and represented graphically. Finally the unknown
concentration bromophenol blue`s absorbance was also tabled together with its theoretical
concentration.

Table 1: finding Amax

Wavelength (nm) Absorbance

480 2.388
490 2.000
500 1.643
510 1.430
520 1.364
530 1.458
540 1.645
550 1.639
560 1.755
570 1.866
580 1.927
590 1.990
600 1.890
610 1.811
620 0.971

Table 2 concentration versus absorbance

Test tube Dilution [BPB]µM Absorbance(nm)
1 ½ 9.3 3.010
2 ¼ 4.65 0.531
3 1/8 2.325 0.591
4 1/16 1.1625 0.282

Table 3 finding unknown concentration

Absorbance at Amax Concentration µM
Unknown 0.299 1.625

5
 wavelength (nm) versus absorbance (nm)
2.6
2.4
2.2
2
1.8
absorbance (nm)

1.6
1.4
absorbance (nm)
1.2
1
0.8
0.6
0.4
0.2
0
460 480 500 520 540 560 580 600 620 640

wavelength (nm)

Figure 2: graph of wavelength and absorbance

3.2
3
2.8
2.6
2.4
2.2
absorbance (nm)

2
1.8
1.6
1.4 relationship
1.2 Linear (relationship)
1
0.8
0.6
0.4
0.2
0.299
0.282
0
0 1 2 3 4 5 6 7 8 9 10

concentration (µM)

Figure 3: graph of absorbance at varying concentrations

6
Discussion

A spectrophotometer is a photometer that can measure the intensity of light as a function of

its wavelength. Single beam and double beam are the two major classes of
spectrophotometers. Linear range of absorption and spectral bandwidth measurement are the
important features of spectrophotometers. In Single Beam Spectrophotometers, all the light
passes through the sample. To measure the intensity of the incident light the sample must be
removed so that all the light can pass through. This type of spectrometer is usually less
expensive and less complicated. The single beam instruments are optically simpler and more
compact; this can also have a larger dynamic range. In a Double Beam Spectrophotometer,
before it reaches the sample, the light source is split into two separate beams. One beam
passes through the sample and the second one is used for reference. This gives an advantage
because the reference reading and sample reading can take place at the same time ( Rouessac,
F.A. 2000).

Many spectrophotometers must be calibrated before they start to analyse the sample and the
procedure for calibrating spectrophotometer is known as "zeroing." Calibration is done by
using the reference substance, and the absorbencies of all other substances are measured
relative to the reference substance. Percentage ability to transmit which is the amount of light
transmitted through the substance relative to the initial substance is displayed on the
spectrophotometer (Willard, H.1998).

The results show that at the initial wavelength the absorbance of Bromophenol Blue was
high. As the wavelength of the spectrophotometer increases the absorbance decreases, it then
increases Bromophenol blue (3',3",5',5"-tetrabromophenolsulfonphthalein, BPB, albutest) is
used as a pH indicator, a colour marker, and a dye. It can be prepared by slowly adding
excess bromine to a hot solution of phenolsulfonphthalein in glacial acetic acid. Again until it
reaches its maximum absorbance for Bromophenol Blue which is 590 nm. The amplitude of
the graph is the Amax of Bromophenol Blue.
The relationship between absorbance and concentration is direct proportionality relationship
from the serial dilutions the ones with the highest concentration had the highest absorbance
indicating a direct proportionality.

7
Because of the 1:1 dilution of the unknown with water, the concentration of the undiluted
solution would be twice that of the diluted solution. Although this does fit well with the
standard solutions, it has already been noted that the trend line analysis was not very accurate
(http://webs.anokaramsey.edu/chemistry/chem1061/Labs/SampleReport/Sample%2520Lab
%2520Report).

Conclusion
The aim of the experiment has been achieved. The Amax value is at 590 and the
concentration of the unknown 2 sample was found to be 1.1625 which is the concentration of
the 1:16 dilution of bromophenol blue. The concentration was found using the line of best fit
in the scatter plat graph above. We can also conclude that the experiment was a success
because the results were conclusive.

8
References
1. Nkaissieh, D. (2008). spectrophotometry lectures. Available:
https://wfsolutions.workforce3one.org/.../CB094-11.24_Spectrophotome.. Last
accessed 10th august 2015.
2. Sanker, J (2003). Biochemical calculations. USA: Pearson. p90-91.
3. Lambert, S (2003). Spectrometry. New York: Gill & Johnson. p18-20.
4. Beer, C (2004). Physical sciences for the biosciences. California: Oxford publishers.
p48.
5. Rouessac, F.A (2000). Modern Instrumental Methods and Techniques . England:
John Wiley & Sons. p58-60.
6. Willard, H (1998). Instrumental methods of analysis. Belmont, California:
Wadsworth publishing company. p802.
7. http://webs.anokaramsey.edu/chemistry/chem1061/Labs/SampleReport/Sample
%2520Lab%2520Report- (accessed 07-08-2015)