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# EXPERIMENT 3

SPECTROPHOTOMETRIC MEASUREMENT OF
ABSORPTION

## 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

## • 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.

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

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

## 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.