Name ……………………………..…………….

Nutkamol Asirasiriwanich ID…………….……….

6531063730 Group ….……
6 Instructor ……………………………….
Dr.ch alisa

Laboratory Guidelines for Fundamental of Cell and Molecular Biology (3000109)

Basic Techniques in Biochemical Analysis

Biochemical analysis techniques refer to a set of methods, assays, and procedures that
enable scientists to analyze the substances or biomolecules found in living organisms and the
chemical reactions within cells or body fluids in both normal and pathological conditions. The
concept is based on the determinations of chemical, physical and biological properties of
substances. Biochemical analysis is a rapidly expanding field and is a key component of
applications in other biological sciences such as biology, molecular biology, microbiology,
nutrition, etc. Moreover, it is very useful in medicine which can be applied for screening,
diagnosis, treatment and prevention of diseases.
Biochemical analysis can be divided into two major categories:

 Qualitative analysis refers to analyses in which substances are identified or classified by

their chemical or physical or biological properties. For example, the detection of sugar in
the urine sample of diabetic patients by Benedict’s test.
 Quantitative analysis refers to analyses in which the amount or concentration of
substances may be determined (estimated) and expressed as a numerical value in
appropriate units. For example, monitoring of blood sugar level of diabetic patients
analyzed by glucose oxidase reactions.
Quantitative analysis can be measured by several methods including.
 Gravimetry is a set of methods used in analytical chemistry for the quantitative
determination of the substance based on its mass.
 Titrimetry is a common laboratory method of quantitative chemical analysis that is
used to determine the concentration of an identified substance.
 Colorimetry is a method of determining the concentration of a chemical compound
in a solution with the aid of a color reagent.
 Spectrophotometry is a quantitative analysis of molecules depending on how much
light is absorbed by colored compounds. In this session, we focus on this method.
Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Principle of spectrophotometry

The first principle of spectrophotometry is that the intensity of the color is a measure
of the amount of a material in solution. The second principle of spectrophotometry is that
every substance absorbs or transmits certain wavelengths of radiant energy but not other
wavelengths. For example, chlorophyll absorbs red and violet light, while it transmits yellow,
green, and blue wavelengths. The transmitted and reflected wavelengths appear green.

In the spectrophotometric analysis, the intensity of radiation, transmitted by an

absorbing medium placed between the light source and the detector is measured as a
function of wavelength. The graph of the intensity of radiation absorbed and the wavelength
is called the “absorption spectrum” and is the characteristic of the absorbing component.
From the absorption spectrum, the max (the wavelength that provide maximum absorption)
can be determined. The max depends on the structure and derivative of each substance.
For example, oxygenated hemoglobin provides maximum absorption at max 540 and 580
nm, whereas deoxygenated hemoglobin provides maximum absorption at max 560 nm
(figure 1). Thus, the absorption or transmission of specific wavelengths is the characteristic for
a substance, and a spectral analysis serves as a “fingerprint” of the compound.

Figure 1 Absorption spectra for oxygenated and deoxygenated human hemoglobin.

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Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Spectrophotometer

A spectrophotometer is an instrument that can measure a light beam's intensity as a

function of its wavelength. The instrument is fairly easy to operate and widely available.
Important features of spectrophotometers (figure 2) include the following.
1. Light source
 Deuterium lamps provide powerful and stable light in the UV region (190-370 nm)
 Tungsten lamps give stable light in the visible (380-750 nm) and near infra-red areas
(750-1100 nm)
2. Monochromator is the optical device that transmits a mechanically selected band of
wavelengths of light from a wider range of wavelengths available from the light source.
3. Cuvette is an optically clear container for holding liquid samples in a
spectrophotometer.
4. Photometer is a detector for measuring the intensity of light.
5. Display scales can be divided into two units:
 Absorbance (A) or optical density (OD)
 % transmittance (%T)

Figure 2 General schematic of a spectrophotometer.

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Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Laws of photometry

Spectrophotometry is a scientific method based on the absorption of light by a

substance and takes advantage of the two laws of light absorption.

 Lambert’s Law: the proportion of light absorbed by a medium is independent of the

intensity of incident light.

A sample which absorbs 75% (25% transmittance) of the light will always absorb 75%
of the light, no matter the strength of the light source. This allows different
spectrophotometers with different light sources to produce comparable absorption readings
independent of the power of the light source.

 Beer’s Law: the absorbance of light is directly proportional to both the concentration
of the medium and the thickness of the medium. In spectrophotometry, the thickness
of the medium is called the path length.

The absorption and transmission of light through a specimen is used to determine

molar concentration of a substance. The relationships between absorption, transmittance and
concentration are stated using the Beer-Lambert Law.

 Beer-Lambert Law: the concentration of a substance is directly proportional to the

amount of light absorbed by the substance and inversely proportional to the logarithm
of the transmitted light. The mathematical formula showing this relationship is:
I0 100
A = kcl = log = log = log 100 - log %T = 2 – log %T
I %T

k = molar absorption coefficient (constant value for each substance at specific wavelength)
l = pathlength (In general, pathlength of a standard cuvette = 1 cm.)
A = Absorbance or optical density (OD)
%T = %Transmittance c = Concentration of solution
I 0 = Intensity of the incident light I = Intensity of transmitted light

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Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Deviation from Beer-Lambert Law

Ideally, the relationships between absorption, transmittance and concentration are

followed the Beer-Lambert Law when 20-80 %T or 0.1-0.8 OD are measured. If the values out
of this range, the relationships between absorption, transmittance and concentration will
deviate from the Beer-Lambert Law (figure 3).

Figure 3 Relationships between absorption, transmittance and concentration

Approaches for determining the concentration

When performing quantitative analysis (i.e., finding the concentration of an unknown

sample) using spectrophotometry, three different procedures may be used:

1. Calculate directly using absorbance (A) and known molar absorption coefficient of the
substance. [A = kcl]

2. Analyze a standard of known concentration and calculate the unknown concentration:

3. Plot a standard curve of known standard absorbance VS concentration (figure 3).

In recent years spectrophotometric methods have become the most frequently used
and important methods of quantitative analysis. They apply to many industrial and clinical
settings involving the quantitative determination of compounds that are colored or that
react to form a colored product.

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Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

For precision and accuracy of the biochemical analysis, we should also concern about
the suitable handling of basic scientific equipment such as pipettes.

Pipetting Procedure

A pipette is a laboratory tool for measuring liquid in analytical chemistry and general
laboratory. Pipetting procedure and pipette selection effect to the accuracy of the
measurement. There are various types of the pipette in analytical biochemistry. In this lesson,
we focus on four types (figure 4).

Figure 4 Various types of pipettes commonly used in laboratories.

1. Volumetric pipette or bulb pipette

These pipettes have a large bulb with a long narrow portion above with a single
graduation mark as it is calibrated to deliver accurately fixed volume.
2. Pasteur pipette or droppers
This type of pipette is made from glass or plastic and fitted with a rubber bulb at the
top. Pasteur pipettes are used for transferring small quantities of liquids.

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Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

3. Graduated pipette or measuring pipette

This type of pipette is made from glass or plastic with long cylinder shape. It is capable
of measuring multiple volumes. Each size has a volume indicator, varying according to the
resolution of the application such as 0.1, 1.0, 2.0, 5.0 and 10.0 mL. This type of pipette can
be further divided into two types.
 Mohr pipette: the graduation marks end before the tip.
 Serological pipette: the graduation marks continue to the tip.

Pipetting procedure for measuring pipette (figure 5)

1. Use a rubber ball to suck solution into the pipette. Keep the volume of liquid above the
desired volume scale. Use a dry finger to close the top of the pipette. Do not directly use
mouth suction or blowing to measure or transfer reagents to avoid the chemical
consumption.
2. Adjust the volume by slightly moving the finger to let air move into the pipette to adjust
the level of the liquid at the desired volume by looking at the bottom level of the
meniscus in the eye level. Then push the finger to the top of the pipette again firmly.
3. Release the solution into the container. Tap the pipette tip against the inner wall of the
container to remove the excess liquid around the pipette without blowing. Do not touch
the tip of the pipette to prevent chemical contamination and dangers from chemical
exposure.

Figure 5 Pipetting procedure for measuring pipette.

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Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

4. Automatic pipette
Automatic pipette is mechanical pipette that can accurately measure very small
amounts of a liquid from microliter to milliliter scales.
Pipetting procedure for automatic pipette (figure 6)
1. Set the desired volume by turning the volume adjustment knob. Firstly rotate the volume
adjustment knob a little over, then slowly dropping to the desired volume.
2. Put the pipette tip into the tip of the automatic pipette firmly.
3. Press the plunger button to the first step and hold (step 1 in figure 6), then dip the tip into
the solution or liquid at 3-4 mm from the tip.
4. Gently release the plunger button to the resting step, allow the solution or liquid to be
aspirated into the pipette to the desired volume and prevent air bubble in the pipette tip
and solution contamination inside the automatic pipette (step 2 in figure 6).
5. Lift the automatic pipette up from the solution, tap the tip against the inner wall of the
container to get rid of excess fluid that is attached to the outside of pipette tip. Always
hold the automatic pipette vertically when there is solution inside the tip to avoid solution
flow into the automatic pipette.
6. To release the solution (step 3 in figure 6), tilt the test tube or container then tap the
pipette tip against the inner wall of the container. Gently press the plunger button to the
first step and continually press to the second step (step 4 in figure 6) to release all the
solution into the container.
7. Remove the used tip from the automatic pipette by pressing tip ejector button.

Figure 6 Pipetting procedure for automatic pipette

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Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Objectives
Upon completion of this laboratory practice, the student should be able to:
1. Explain the principle of quantitative analysis
2. Use proper technique in pipetting procedure and operating a spectrophotometer.
3. Explain Beer-Lambert law and calculate the concentration of unknown sample based
on spectrophotometry.
4. Determine the accuracy and variance of the analysis

Experiment 1 Principle of spectrophotometry and Beer-Lambert law

Each substance has an absorption property at a specific wavelength. Some colored

substances can absorb visible light (380-750 nm). The maximum absorbed wavelength is called
max which is the unique property of each substance. In this experiment, potassium
permanganate (KMnO4) will be used (max = 525 nm) as shown in figure 7.

Figure 7 Absorption spectrum of an aqueous solution of potassium permanganate

Beer-Lambert Law states that the concentration of a substance is directly proportional

to the amount of light absorbed by the substance. Thus, the relationships between the
absorbance and concentration is a linear equation.

Procedures of experiment 1
1. Dilute the 0.4 mM KMnO4 solution into 0.3, 0.2 and 0.1 mM, respectively. Label the
concentration on each tube.
2. Set the absorption wavelength of the spectrophotometer at 525 nm (max of KMnO4).
9
 Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

3. Use the cuvette with distilled water to set blank (A = 0). Discard distilled water.
4. Add 4 mL of 0.1 mM KMnO4 solution into the cuvette then measure the absorbance.
5. Discard the KMnO4 solution. Wash the cuvette with distilled water 3 times. Wipe the
outside of cuvette with tissue paper.
6. Repeat step 4-5 with the 0.2, 0.3 and 0.4 mM KMnO4 solution, respectively. Record each
absorbance in the table. Draw the graph representing the relationship between
absorbance (A) and solution concentration (c). ( เบาะ G V2
0.4 ✗ 1 i
Czth
The result of Experiment 1
Tube Volume (mL) of Volume (mL) of Calculated concentration Absorbance
standard KMnO4 distilled water of KMnO4 solution (mM) (A)
solution (0.4 mM)
Blank 0 4 0 0.000
Standard 1 1 3 0.1 0.203

Standard 2 2 2 0.2 0.955

Standard 3 3 1 0.3 0.680

Standard 4 4 0 0.4 0.924

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 Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Experiment 2 Molar extinction coefficient of KMnO4 calculation

The molar extinction coefficient (k) is the absorbance of 1 Molar solution when the
thickness of the medium is 1 cm which is the constant of each substance. This coefficient can
be calculated from the slope of the graph between absorbance and concentration of the
substance or calculated by the equation: A = kcl (when l = 1 cm)
Thus, the molar extinction coefficient (k) of KMnO4 can be calculated by using the data
obtained from experiment 1.
Calculation:
อ ความ น
ะ
ะ อ 680
.
.
ะ
0.267
.....................................................................................................................................................................
k

0:3
.....................................................................................................................................................................
.....................................................................................................................................................................
Experiment 3 Determination of concentration of an unknown KMnO4 solution

The max and the molar extinction coefficient of KMnO4 solution can be used with
the spectrophotometry method to determine the concentration of an unknown KMnO 4
solution by using the equation: A = kcl (when l = 1 cm)
Procedures of experiment 3
1. Set absorption wavelength of the spectrophotometer at 525 nm (max of KMnO4).
2. Use the cuvette with distilled water to set blank (A = 0). Discard distilled water.
3. Add 4 mL of unknown KMnO4 solution into the cuvette then measure the absorbance.
4. Calculate the concentration of unknown sample by using the equation A = kcl.
(Use k value obtained from experiment 2)
The result of Experiment 3
No. of unknown sample A525 k (cm-1 mM-1) Concentration (mM)
3 อ } 00
.
2.267 130

Calculation:
A ะ
KCI
.....................................................................................................................................................................
0.3 =
( 2.267) (C) (1)
.....................................................................................................................................................................
C- 0.1 } 2 m M
.....................................................................................................................................................................
-

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Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Experiment 4 Determining the accuracy and variance of the analysis

Verification of the accuracy of biochemical analysis can be performed by repeating the
measurement of the same sample with the same method for 10-20 times under the same
conditions (investigator, reagents and tools). The accuracy of measurement can be calculated
from the coefficient of variation (%CV) by calculating the mean (𝑥̅ ) and standard deviation
(SD) then calculate the %CV by using the following equation.
SD 𝑥 100
%CV =
𝑥̅
This coefficient of variation calculation can be called intra-assay variation or within-
assay variation. In this experiment, the student will analyze the accuracy and variance of
KMnO4 absorbance and concentration measurement. Before starting the experiment, please
make sure that you have studied and understood all the procedures. Use the clean test tubes,
pipettes and all equipment correctly to minimize the variation of the analysis.
Procedures of experiment 4
1. Pipetting the standard KMnO4 solution (concentration 0.4 mM)
 Experiment A: adding 0.5 mL of the solution in each tube for 10 tubes
 Experiment B: adding 1.0 mL of the solution in each tube for 10 tubes
2. Preparing the diluted solution of each tube with distilled water by
 Experiment A: adding 3.5 mL of distilled water in each tube.
 Experiment B: adding 3.0 mL of distilled water in each tube.
3. Set absorption wavelength of the spectrophotometer at 525 nm (max of KMnO4).
4. Use the cuvette with distilled water to set blank (A = 0). Discard distilled water.
5. Add 4 mL of the solution into the cuvette then measure the absorbance.
6. Discard the solution. Wash the cuvette with distilled water 3 times. Wipe the outside
of cuvette with tissue paper.
7. Repeat step 5-6 with the remaining tubes, respectively. Record each absorbance in the
table. Calculate the initial concentration of the solution in each tube. Don’t forget to
include the dilution factor (Exp A: dilution factor = 8, Exp B: dilution factor = 4).

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 Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

8. Calculate of 𝑥̅ , SD and %CV obtained from the experiment A and B by these equations.
SD 𝑥 100
%CV =
𝑥̅

9. Discuss accuracy and precision of the results

 Accurate if the initial concentration equal or close to 0.4 mM
 Precise if the calculated %CV equal or close to 0
10. Discuss laboratory techniques in biochemical analysis
 Good technique: accurate and precise ค เ ม น
.

ด าย C เขา ะ
Cz V2
 Random error: accurate but not precise 0.4 ✗ 0.5 ะ
[ zxt
 Systemic error: precise but not accurate
Cz ะ 0.05
The result of Experiment 4
Tube No.
Experiment A
1 2 3 4 5 6 7 8 9 10
Volume of KMnO4 solution (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Volume of distilled water (mL) 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5
Mix the solution thoroughly and then measure the absorbance at 525 nm
A525 ะ
kcl 0.1010.102 อ เอ 30.098 อ อ 97 อ เออ อ 1140.106 0.1050.098
.
.
.
.

Initial concentration (mM) ( 8) 0.356 0.160 0.165 0 } 46 อ ง 920.3530.80 20.3 79 0.370 0.196
× .
.

Experiment A: 𝑥̅ = ………………
0.3 แ 2
SD = ………………
0.0168 4.651
%CV = ………………
Tube No.
Experiment B
1 2 3 4 5 6 7 8 9 10
Volume of KMnO4 solution (mL) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Volume of distilled water (mL) 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0
Mix the solution thoroughly and then measure the absorbance at 525 nm
A525 =
KCI 0.192 0.2420.297 0.29 } 0.2990.137 0.49 0.440.297 0.238

Initial concentration (mM) 0.4010.401 0.909 0.1030.904 อ 3930.1040.409 0.9090.39

.
e

Experiment B: 0.902
𝑥̅ = ……………… SD = ………………
0.0051 1.2 69
%CV = ………………

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 Course: 3000109 1
Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Conclusions and discussions

Thisexperimentconsists ottwosub experiments Experiment Aandtxperiment
.....................................................................................................................................................................
-

withdifferences in vdumeof k Mn 0µ solution and theamountofdistilledwater

..................................................................................................................................................................... .

E eriment A
Xp Used 0.5 mL of KM n Oesolution and 3.5mL otdistilledwater and
.....................................................................................................................................................................
,

experiment 8 used 1 mL of kmnoesdution and 3mL ofdistilled

........................................................................................................................................... ..........................
water .

ehesteps afterthatareallthesame After obtaining theabsorption

.....................................................................................................................................................................
..

valueofeachtesttube.us thevaluesobtained in the equation A- kclto

.....................................................................................................................................................................
e -

.....................................................................................................................................................................
tindtheralue of C to lhecktheaccuracy Afterrepeating theexperimentfor .

10 Initial loncentrations wereobtaine d. Iakeall 1 อ

........................................................................................................................................... ..........................
valuestofinds.D.to
เอ tubes .

.....................................................................................................................................................................
eheck Precision .

Theresultot experiment 4 is lessaccurate andprecise than etperimentg

.....................................................................................................................................................................
as theinitial concentrationofetperiment A wasfartherfrom 0.4m Mthan............
......................................................................................................................................................... of

experimehtt and % cvotexperiment A wasfarther...............................................

...................................................................................................................... to 0 thanof

.....................................................................................................................................................................
experiment อ .

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References
 Farrell, S.O. and Ranallo, R.T. Experiments in Biochemistry: A hand-on Approach.
Brooks/Cole. Thompson Learning, Inc., 2000
 Spectrophotometry Handbook. GE Healthcare Life Sciences. 2013.
(https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-
Aldrich/General_Information/1/ge-spectrophotometry.pdf)
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 Course: 3000109 Laboratory guideline
Fundamental of Cell and Molecular Biology Basic Techniques in Biochemical Analysis

Quiz
A student obtained the solution with an initial concentration equal to 0.05 mM. The
solution has the maximum absorbance at wavelength 620 nm and Molar extinction coefficient
cm
(k) = 8 x 104. To generate the standard curve between concentration and absorbance, this
student tries to dilute the solution with different concentrations as shown in the table.
k = 8 × 104 cm x ✗
ฑื๊ =
0.8

1. Calculate the final concentration and the absorbance in each tube.

-

Tube No. 1 2 3 4 5 6
Sample solution (mL) 0 0.3 0.6 0.9 1.2 1.5
Distil water (mL) 3.0 2.7 2.4 2.1 1.8 1.5
Final concentration (μM) 0 0.005 0.010 อ อา 5 อ อ 20 . . 0.025

A620 0 0.004 0.008 อ อ 12 0.016 .

อ .
⑦ 20

2. If the student does not set the absorbance of the blank tube as zero before measuring
the absorbance of the other tubes, how is the absorbance of the solution in the
experimental tubes? How is the standard curve between concentration and absorbance?
Theoriginisnotat 10,0 1. Thegraftshifts up
.....................................................................................................................................................................
.

.....................................................................................................................................................................
.....................................................................................................................................................................
.....................................................................................................................................................................
3. If the student uses a cuvette tube with 0.5 cm of the light path in the experiment, how is
the absorbance of the solution in each experimental tubes?
the absorbanawill beredaed byhal f.
.....................................................................................................................................................................
.....................................................................................................................................................................
.....................................................................................................................................................................
........................................................................................................................................... ..........................
4. If the wavelength in the instrument is set as 630 nm, how is the standard curve between
concentration and absorbance of the experimental tube?
Thegraphissteeper
.....................................................................................................................................................................
.

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