EXPERIMENT 3

SPECTROPHOTOMETRIC MEASUREMENT OF
ABSORPTION

1. INTRODUCTION AND AIM

What is the aim of using a spectrophotometer in biological experiments?

The aim of using a spectrophotometer is measuring the relative amounts of

radiant energy as a function of wavelenght, usually it is used to analyze unknown
substance in a solution or concentration of a solution according to the substance's
capacity to absorb radiant energy.

What are the basic components of a spectrophotometer? Describe their functions.

• Light source: Releasing light

• Diffraction grating: splitting the light into its components colors.
• Aperture: it is used to control the passing of the light with the selected
wavelength.
• Photoelectric tube: Creating an electric current proportional to the number of
photon striking it.
• Meter: Displaying the out put of the phototube.

Please define transmittance (T) and absorption (A) in your own words.

Transmittance: The ratio of the radiant power transmitted by an object to the

incident radiant power.
Absorption: The process in which radiant energy is retained by a substance.

Which property of a solution affects its absorbance value? Explain.

Concentration of a solution affects the absorbance value. Because absorbance

value is the radiant energy retained by a substance and concentration is related to the
amount of the substance, concentration affects the absorbance value and they have
directly and linearly a relationship.

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2. MATERIALS AND METHODS

Part 1:
How did you operate the spectrophotometer?

First of all I switched it on and waited for a while to be ready to use. After a few
minutes it is ready. To operate the spectrophotometer I put a cuvette of dH2O in it and
pushed the button “REF” to reference the absorbance value to zero. I used dH2O
because it is the most transparent element we know, choosing it enables us to get the
most accurate calculations. Finally, it is ready to use the wavelength that I decide.

Describe the protocol you have used to determine the wavelength of maximum
absorbance by bromophenol blue.

Firstly I chose minimum and maximum wavelengths (525nm-650nm). From the

525nm to 650nm I increased the wavelength 25nm. At each wavelength I referenced the
spectrophotometer by using dH2O then I measure the absorbance value of 0,02mg/ml
bromophenol blue. After I finished the measuring, I looked at the values; there was a
decrease after 600nm. In conclusion, this decrease meant that 600 nm is the wavelength
of maximum absorbance by bromophenol blue.

Part 2:
Describe the protocol you have used to demonstrate the linear relationship between
absorbance and concentration.

In the beginning, I set the wavelength 600 nm because t is the wavelength of

maximum absorbance by bromophenol blue. I measured the dilutions that I have five
different concentrations of bromophenol blue from 0,0025mb/ml to 0,0200mg/ml. Then I
calculate the absorbance value of all five concentrations. I realized that, when the
concentration is increase absorbance value is also increase.

How did you determine the concentration of bromophenol blue in the unknown
solution?

I measured the absorbance value of different concentration which were 0,0025,

0,0050, 0,0100, 0,0150, 0,0200mg/ml. Then I drew a graph. After I drew the graph of
absorbance value and concentration I wrote equation. After I wrote the equation I
decided the slope of the graph. Finally I found the unknown with mathematic.

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3. RESULTS
Part1:
Record the absorbance values (OD) of the 0.02 mg/ml dye solution for 475-675 nm
interval in the following table.

Wavelength 525 550 575 600 625 650

(nm)
Absorbance 0.547 0.856 1.452 1.583 0.248 0.015
(OD)

Plot these values on a graph (independent variable should be on the X-axis).

What is the wavelength of maximum absorbance?

1,7
1,6 1,583
1,5
1,452
1,4
1,3
1,2
Absorbance (OD)

1,1
1
0,9
0,856
0,8
0,7
0,6
0,547
0,5
0,4
0,3
0,248
0,2
0,1 0,015
0
525 550 575 600 625 650
Wavelength (nm)

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Part 2:
Record your absorbance values to the table below.

Concentratio 0.0025 0.0050 0.0100 0.0150 0.0200 Unknown

n (mg/ml)
Absorbance 0.213 0.401 0.818 1.237 1.583 0,622
(OD)

Plot these values on a graph.

1,7
1,6 1,583
1,5
1,4 y =0,273x
Absorbance (OD)

1,3
1,2 1,237
1,1
1
0,9
0,8 0,818
0,7
0,6 0,622
0,5
0,4 0,401
0,3
0,2 0,213
0,1
0
0,0025 0,0050 Unknown 0,0100 0,0150 0,0200
Concentration (mg/ml)

What is the concentration of the dye in the unknown solution?

UNKNOWN1
Because the graphic is not linear, I choose 2 slope points to obtain the most
correct point.

SP1(0,0050, 0,401)
SP2(0,0100, 0,818)

y2 – y1
Slope= -------------- =83,4
x2-x1

83,4= (0,622-0,401) / (Unknown-0,0050)

Unknown= 0,0076 mg/ml

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 4. DISCUSSION:

Evaluate your results for part I.

In the first part of the experiment, I calculated the absorbance value of 0,02mg/ml
bromophenol blue for each wavelength of 525nm,550nm,575nm,600nm,625nm, and
650nm. Then, I determined the highest value for bromophenol blue by looking the
absorbance values. It was 600nm but because we made it for 5 value, we cannot say it is
600nm exactly. To make the most accurate value we must make the experiment again
between 575nm and 625nm. After finding a new value again we must decrease the
interval of wavelengths that we use. We can reach the real absorbance value of
bromophenol blue.

Evaluate your results for part II.

In the second part of the experiment, I measured absorbance value of five different
concentration of bromophenol blue in 600nm. After the measurement when I draw the
graph, it must be linear because of the Lambert-Beer Law. Because of some problems
probably concentrations we could not get a linear function. To minimize the mistakes we
must be more careful and first keep the interval wide then slowly make the interval small.
However I saw the relationship between concentration and absorbance. Lambert-Law is
correct.

Write a short paragraph describing what you have learned in this lab session.

First of all I learnt what spectrophotometer is and using spectrophotometer, and

what we mean by transmittance and absorbance. After this experiment I saw the
relationship between concentration and absorbance value which is linear.