TABLE OF CONTENTS

OBJECTIVES 3
INTRODUCTION 4
EXPERIMENTAL PROCEDURES 5-7
RESULTS 8-12
DISCUSSION 13
CONCUSION 14
REFERENCES 15

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INTRODUCTION

A spectrophotometer is a combination of words spectro which is the spectrum, photo

which means light, and meter which is defined as an apparatus to measure. Thus, a
spectrophotometer is an instrument that is combined through two major components which are
a spectrometer and also a photometer. The spectrometer is used to produce light by using any
selected wavelength whereas the photometer is to measures the light intensity after it interacted
with any matter. Spectrophotometer has several uses such as in beverages, pharmaceuticals,
food, and so on. The amount of light absorbed by the solution in a cuvette is measured using
spectrophotometer techniques to determine the concentration of solutes in the solution. The
spectrophotometer measures the intensity of light absorbed or transmitted by a substance to
determine its properties. The liquid or sample is placed between the spectrometer and the
photometer in the spectrophotometer. The photometer determines how much light travels
through the sample and sends a voltage signal to the display. When the amount of light
absorbed changes, the voltage signal changes as well. A light source, a digital display, a
monochromator, a wavelength selector, a collimator for straight light beam transmission, a
photoelectric detector, and a cuvette to deposit a sample make up the basic spectrophotometer
equipment. If the initial light intensity which is pointed towards the matter is absorbed 100% or
fully, it will not be transmitted or transmit 0%. Thus, it is called fully absorbed. Meanwhile, if the
initial light intensity is not absorbed or absorbed 0%, it will be transmitted 100% as it is called full
transmission. Thus, the connection between both absorbance and transmittance is inverse.
Absorbance is defined as below:

% 𝑜𝑓 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 = − log10 𝑇

The working principle of the spectrophotometer is based on Beer-Lambert’s law which states
that the amount of light absorbed by the absorbing material or the absorbance, A is directly
proportional to its concentration in the solution, c and the length of a light path through the
solution,b. The equation for the law mentioned above is:

𝐴 = 𝜀𝑏𝑐
which consists of 𝜀, extinction coefficient or molar absorptivity with units of L mol-1 cm-1 which is
a constant value.

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PROCEDURE

A. The absorbance spectrum and maximum absorbance wavelength (λ max)

1. The spectrophotometer was turned on by pressing ON/OFF (I/O) button and was
warmed up for 15 minutes.
2. Val button was pressed for self-test which is for the activation of the instrument.
3. The transmittance mode was selected by pressing button A.

4. A wavelength at 375nm was set and the wavelength was been set by entering the value
directly using numerical keys. It was confirmed by pressing “VAL”.
5. A cuvette was cleaned with distilled water and was wiped until it dried.
6. Blank solution was poured into the cuvette and placed into sample holder of
spectrophotometer and was made sure side with arrow facing light path.
7. Zero button was pressed.
8. The cuvette with blank solution was removed and was put aside.
9. Another cuvette was cleaned with distilled water and was wiped to dry.
10. Standard Solution A was poured into the cuvette and placed into sample holder of
spectrophotometer. It was also made sure the side with arrow facing light path.
11. The transmittance (T) value was read and recorded in Table 1.
12. The cuvette with Standard Solution A was removed and was put aside.
13. Wavelength to 425nm was changed and the wavelength was set by entering the value
directly using numerical keys and was confirmed by pressing “VAL”.
14. The cuvette with blank solution was placed into sample holder of spectrophotometer and
was made sure the side with arrow facing light path.
15. Zero button was pressed.
16. The cuvette with blank solution was removed.
17. The cuvette with Standard Solution A placed into sample holder of spectrophotometer
and was made sure the side with arrow facing light path.
18. The transmittance (T) value was read and recorded in Table 1.
19. The cuvette with Standard Solution A was removed.
20. Step 13 to step 19 were repeated for different wavelengths: 490, 530, 540, 550, 580,
625 and 700nm.

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 21. Based on the T value obtained in Table 1, the absorbance (A) value was calculated.
22. On a piece of graph paper, absorbance (A) value versus wavelength (λ) was plotted.
The wavelength was placed on the horizontal axis and the curve obtained represents the
absorption spectrum of the Standard Solution A in the visible region.

B. A protein standard curve was plotted

1. The absorbance mode and not the transmittance mode was selected by
pressing button A.

2. Based on the absorbance spectrum that was drawn in section A, the optimum
wavelength (λ max) was selected at which the compound absorbs light at maximum
intensity.
3. The selected wavelength was set by entering the value directly using numerical keys
and confirmed by pressing “VAL”.
4. A cuvette was cleaned with distilled water and wiped to dry.
5. Protein (albumin) in serial concentrations (0, 20, 40, 60, 80, and 100 mg/ml) were
prepared.
6. Standard protein solution with 0 mg/ml concentration and also known as blank solution
was poured into the cuvette and placed into sample holder of spectrophotometer and
made sure the side with arrow facing light path.
7. Zero button was pressed.
8. The Absorbance (A) value was read and recorded in Table 2.
9. The standard protein solution (0 mg/ml) was removed and the cuvette cleaned with
distilled water and then was wiped to dry.
10. The standard protein solution with 20 mg/ml concentration was poured into the cuvette
and placed into sample holder of spectrophotometer. Then, it was made sure side with
arrow facing light path and zero was not needed to be pressed this time.
11. The Absorbance (A) value was read and recorded in Table 2.
12. the standard protein solution (20 mg/ml) was removed and the cuvette was cleaned with
distilled water. It was wiped to dry.
13. Step 10 to step 12 was repeated to measure A value of the standard protein solutions at
40 mg/ml, 60 mg/ml, 80 mg/ml and 100 mg/ml concentrations, respectively.

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 14. On a piece of graph paper, absorbance (at λ max) versus concentration (mg/ml) was
plotted in which concentration was placed on the horizontal axis. The curve you obtained
represented the protein standard curve.

C. Determine the concentration of an unknown solution using the protein standard curve

1. Absorbance was set at the selected wavelength.

2. A cuvette was cleaned with water and was wiped to dry.
3. The standard protein solution with 0 mg/ml concentration also known as blank solution
was poured into the cuvette and placed into sample holder of spectrophotometer. It was
made sure the side with arrow facing light path.
4. Zero was pressed.
5. The unknown solution was poured into the cuvette and placed into sample holder of
spectrophotometer. It was made sure the side with arrow facing light path.
6. The Absorbance (A) value was read.
7. The concentration of this unknown solution was measured based on the protein
standard curve drawn in Section B.

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RESULTS

PART A: Percentage of Transmittance, Absorbance of the Standard Solution A at various

wavelengths

Wavelength (nm) % Transmittance (% T) Absorbance Value

A = 2 – log %T
375 66.5 0.177
425 66.4 0.178
490 50.5 0.297
530 35.3 0.452
540 34.1 0.467
550 34.0 0.469
580 37.6 0.425
625 57.4 0.241
700 100.2 -0.001
TABLE 1

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 Optimum wavelength (λmax)
at which the compound
absorbs light at maximum
intensity = 545nm

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The curve for the graph of absorbance versus wavelength is a concave downwards curve. This
is because curve lie below this tangent. Meanwhile, the curve for the graph of percent
transmittance (%) versus wavelength is a concave upwards curve. This is because the curve
lies above this tangent. The transmittance and absorbance have a logarithmic relationship, with
an absorbance of 0 equal to 100% transmittance and an absorbance of 1 equivalent to 10%
transmittance. Transmittance and absorbance are diametrically opposed.

.
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PART B: Protein (albumin) Standard Curve

Absorbance (at λ max)

0 mg/ml (Blank solution) 0
20 mg/ml 0.095
40 mg/ml 0.185
60 mg/ml 0.276
80 mg/ml 0.347
100 mg/ml 0.425

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 The biuret reagent, which is employed in protein reactions, is suitable for
spectrophotometric measurement of total protein. The line, or standard curve, that fits to
points on a graph of absorbance vs concentration for a sequence of known solutions can
be used to figure out the concentrations of an unknown solution. The dependent
variable, absorbance, is plotted on the y-axis (the vertical axis). The x-axis graphs
concentration, the independent variable. y-value on the standard curve is found when
measuring the absorbance of an unknown sample. To see which concentration is closest
to it, the subject must look down on it. As the concentration rises, so does the
absorption. While looking at a graph to estimate concentration of an unknown, a more
accurate technique to evaluate concentration is to utilize the equation of the line which is
the y=mx+c that matches the data points.

𝟎.𝟒𝟏𝟎−𝟎.𝟎𝟎𝟓
2. Slope, m =
𝟕𝟖−𝟐𝟎

m = 𝟎. 𝟎𝟎𝟔𝟗𝟖𝟑

y-intercept = -0.138

In most cases, a series of standard samples are prepared at various

concentrations in a range that includes the unknown of interest, and the instrumental
response at each concentration is recorded. The answer at each concentration can be
repeated to obtain an error bar for more precision and to better comprehend the
inaccuracy. The data is then fitted using a function to predict unknown concentrations.
Typically, the response is linear; however, as long as the function is known, a curve can
be created with different functions.

PART C: The concentration of an unknown solution using the protein standard curve

𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 − 𝟎. 𝟎𝟎𝟖
𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 =
𝟏. 𝟔𝟓

𝟎. 𝟐𝟒𝟗 − ( −𝟎. 𝟏𝟑𝟖)

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 =
𝟎. 𝟎𝟎𝟔𝟗𝟖𝟑

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 = 𝟓𝟓. 𝟒𝟐 𝒎𝒈/𝒎𝒍

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CONCLUSION

In this report, all the objectives of the report have been conducted. The basic
operation of the spectrophotometer Secomam PRIM has been studied. The basic principles
of spectrophotometry including the relationship and the calculation of transmittance (T) and
absorbance (A) value are learnt. The maximum absorbance (A max) of a compound and
how it is determined were also have been focused. The use of protein standard curve to
determine the concentration of unknown compound was managed in this Practical Report 4
of Spectrophotometer.

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 REFERENCES

Jim Clark. (Aug 16, 2020). Chemistry LibreTexts. The Beer-Lambert Law. Retrieved the URL
from:

https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_
Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Electr
onic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law

(Oct 24, 2021). Wikipedia. Standard Curve. Retrieved the URL from:

https://en.wikipedia.org/wiki/Standard_curve

Garrett P. From Math Insight. Inflection points, concavity upward and downward. Retrieved the
URL from:

http://mathinsight.org/inflection_points_concavity_upward_downward_refresher

Nipun. (Aug 3, 2015). Pediaa. Difference Between Absorbance and Transmittance. Retrived the
URL from :

https://pediaa.com/difference-between-absorbance-and-transmittance/

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