INTRODUCTION

SPECTROPHOTOMETRY
A spectrophotometer is a combination of two instruments which are spectrometer, an
instrument which can produce light of certain wavelength and photometer, an instrument used
for measuring intensity of light. The basic principle of spectrophotometer is that the light emitted
passes through a sample and the intensity of light reaching the detector is being measured. The
instrument has arrangement that the cuvette containing the sample is placed between the light
source and the detector. The beam of light consist a lot of photons. When the photons that
emitted encounter the molecules of the sample, there is probability for the molecules to absorb
the photons. The absorption of photon by the sample will reduce its number which will detect by
the detector, thus the intensity of beam detected will be decreases.

BIURET TEST
Biuret test is a chemical procedure which is used to detect the present of peptide bonds
between amino acids in the protein molecules by using biuret reagent. This reagent usually is
stable for only few months but its color may be darkened and some precipitate will form. In
order to keep it stable indefinitely, one gram of potassium iodide per liter must be added and it
has to be kept in a dark place. If the peptide bonds are present, Cu2+ will form a violet complex
solution. According to Beers law, absorbance of a compound is equal to its concentration, so
Biuret reaction is suitable for assay the concentration of protein by spectrometric method. This is
because peptide bonds are occurring at the same frequency per amino acid in the peptide chain.

ALBUMIN
Albumin is a most common protein found in the blood. Its supplies the body needs for
protein in growth and repairing tissue cell. It helps in maintaining osmotic blood pressure and
also helps during the dialysis treatment. Albumin acts by absorbing out fluid from swollen
tissues into the blood. Chemically, albumin is a water soluble component which will form
precipitate in acidic medium and coagulated by heating.

METHOD
Part A. Determine the absorbance spectrum and maximum absorbance wavelength,max
1. Pressed the ON/OFF (I/O) button to turn on the spectrometer.
2. To activate the instrument and to let it undergoes self-testing, Val button was
pressed.
3. Transmittance mode was selected.
4. The wavelength was set at 375nm by entering the value directly using the
numerical keys on the instrument. Then, it was confirmed by pressing VAL
button.
5. Before using the cuvette, it was cleaned and rinsed with water. Next, wiped to dry
it.
6. Blank solution then poured into the cuvette and placed into sample holder in
spectrophotometer.
7. ZERO button was pressed.

8. The blank solution was removed and the cuvette was cleaned with water. Wiped it
to dry.
9. Standard solution A was poured into the cuvette and placed into sample holder in
the spectrophotometer.
10. The transmittance (T) value displayed was recorded in Table 1.
11. Wavelength was changed into 425nm by directly entered the value using the
numerical keys and confirmed it by pressing VAL button.
12. The transmittance (T) value displayed was recorded in Table 1.
13. Steps 11 and 12 were repeated by using different wavelengths of 490, 530, 540,
550, 580, 625 and 700 nm.
14. Based on transmittance (T) values obtained, the absorbances (A) values were
calculated using the formula A=2-log T.
15. Graph absorbance (A) values versus wavelength () was plotted in Figure 1. The
curve obtained represents the absorption spectrum of the Standard Solution A in
the visible region.

PART B. Plot a protein standard curve

1. The mode was changed from transmittance mode to absorbance mode.
2. An optimum wavelength, max of 540 nm was selected based on the absorption spectrum
of Standard Solution A in experiment part A.
3. The wavelength was inserted by pressing the numerical keys on the instrument and then
confirmed it by pressing VAL button.

4. The cuvettes were cleaned with water and then wiped to dry.
5. Protein (albumin) was prepared in a serial of concentrations which are 0, 20, 40, 60, 80
and 100 mg/ml.
6. The blank solution which was the standard protein solution of concentration of 0mg/ml
was poured into the cuvette and placed into the sample holder of spectrophotometer.
7. Zero button was pressed.
8. The absorbance value displayed was recorded in the Table 2.
9. The standard protein solution was removed and the cuvette was cleaned with water then
wiped to dry.
10. Steps 7 until 9 were repeated by using different protein (albumin) of concentrations 20,
40, 60, 80 and 100 mg/ml respectively.
11. A graph of absorbance at max versus concentration (mg/ml) was plotted in Figure 3. This
curve represented the protein standard curve.
Part C.
1. Absorbance mode was set at the selected wavelength of 540 nm.
2. A cuvette was cleaned with water and wiped it dry.
3. Standard protein solution with 0 mg/ml concentration, the blank solution was poured into
the cuvette and placed into the sample holder of spectrophotometer.
4. Button zero was pressed to obtain the absorbance value.
5. An unknown solution was poured into a cleaned and dried cuvette then, placed into the
sample holder of the spectrophotometer.
6. The absorbance (A) value obtained was recorded.

7. The concentration of this unknown solution was measured based on the protein standard
curve that had drew on part B.

RESULTS
Part A
Wavelength (nm) %Transmittance (%T) Absorbance (A) Value
375

59.2

0.2277

425

83.0

0.0809

490

69.1

0.1605

530

57.9

0.2373

540

57.7

0.2388

550

58.6

0.2321

580

67.5

0.1707

625

97.9

0.0092

700

142.5

-0.1538

Table 1
The above table shows the percentage of transmittance and absorbance value of the
Standard Solution A at various wavelengths. The absorbance value can be calculated from the
%T by using the formula:
A = 2 log %T

The maximum wavelength, max was selected based on the highest absorbance value. The
maximum wavelength, max is 540nm.

Part B
Standard Protein

Distilled Water

Biuret Reagent

Total Volume

Absorbance,A

(ml)

(ml)

(ml)

(ml)

(at max )

0.1

2.4

2.5

5.0

0.000

20mg/ml

0.1

2.4

2.5

5.0

0.082

40mg/ml

0.1

2.4

2.5

5.0

0.161

60mg/ml

0.1

2.4

2.5

5.0

0.223

80mg/ml

0.1

2.4

2.5

5.0

0.312

100mg/ml

0.1

2.4

2.5

5.0

0.528

0mg/ml (blank
solution)

Table 2
The table above shows the absorbance (A) value obtained at different concentration of
protein prepared which the wavelength selected, max is 540 nm. The absorbance values obtained
in part B are more accurate compared to the value obtained using the method in the experiment
part A.

PART C

The absorbance (A) value obtained is 0.218

DISCUSSION
In part A, the selected wavelength, in which the absorbance (A) value is the highest, is
540nm. As expected, Biuret reagent, the standard solution A used in the experiment, has the
highest absorbance at wavelength about 540nm. At wavelength of 700nm, the absorbance value
obtained is a negative value. This is mainly due to the sample used that may be contaminated.
The contamination is when other small substances are added into the sample unintentionally. The
other molecules will cause the sample to have more trasmittance value than the blank solution
does. So, the sample will have lower absorbance than the blank solution. The absorbance of
blank solution is zero. Thus, the absorbance of the sample will be a negative value.
In part B, the wavelength used is the same, which is 540nm, for all different
concentrations of sample. For the blank solution with the concentration of 0mg/ml, the
absorbance (A) value is zero. As concentration of samples is increasing, the absorbance (A)
values also increases. These observations are obeying the theory, Beers law which states that
absorbance (A) value is directly proportional to the concentration of the sample. Based on the
graph of absorbance at max versus concentration of sample, the gradient is 0.004. There are
maybe some mistakes had done as the best fit line drawn is not stable. The absorbance value
obtained by the sample of 100mg/ml is too high compare to the other sample at different
concentration. This is may occur due to the sample at 100mg/ml that may be contaminated. As it
is contaminated, there will be another molecule inside the sample that may absorb more light

than the sample should. So, the absorbance value at this concentration becomes significantly
higher than the other.
In part C, the concentration of the unknown solution can be obtained from the protein
standard curve that had drawn.
QUESTION & ANSWERS
Part A
1. Based on the graph you drew, which wavelength is the optimum wavelength (max)
at which the compound absorbs light at maximum intensity?
Based on the graph, the optimum wavelength obtained is 540nm.
2. To what colour of light do these wavelengths correspond?
These wavelengths are representing visible light, wavelength of 375 and 425 nm are
corresponding to violet colour, 425 and 490 nm are corresponding to blue colour, 530,
540 and 550 nm are corresponding to green colour, 580nm is corresponding to yellow
colour, 625nm is corresponding to orange colour meanwhile 700nm is corresponding to
red colour.
3. When a solution is blue, does it absorb or transmit blue light?
If the solution is blue, it absorbs all other colours of light but transmit the blue colour
light only.
4. Refer to Table 1. Plot another graph of percent transmittance (%T) versus
wavelength (). Place wavelength on the horizontal axis. Compare the shapes of the
two curves generated using %T and the A = 2 log %T data. Explain why it is

sometimes more useful to use transmittance values and at other times, it might also
useful to use absorbance values?
The graph of percent transmittance versus wavelength was plotted in Figure 2 on the next
page. The curve shape from the Figure 1 is opposite to the curve shape in Figure 2. This
can be proven by the relationship of transmittance and absorbance in which transmittance
is inversely proportional to absorbance, according to Beers law. Sometimes it is more
useful to use the graph of transmittance value versus wavelength because

Part B
1. What is the purpose of using Biuret reagent in this practical?
2. Describe the shape of your standar curve.
3. What is the slope value and Y axis-intercept value?
4. Do you able to get a perfect straight line? If not, what is possible reason?
5. It is possible to use a standard curve without perfect straight line to measure unknown
concentration? Explain.