International University - Vietnam National University, HCMC

School of Biotechnology - Department of Applied chemistry

REPORT 1
USE OF PIPETMEN

Course: Methods in Biochemistry Laboratory

Semester 2 (2023 – 2024)
Instructor: MSc. Le Tran Hong Ngoc
TA: Trinh Thi Xuan
Group number: 4
Full name Student ID
Nguyễn Võ Minh Ngọc BTBCIU21082

Lâm Vân Nghi BTBCIU21079

Nguyễn Trần Thiên Ân BTBCIU21065

Trương Hoàn Mỹ BTBCIU21054

Date of submission: 28/03/2024

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 International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry

ABSTRACT
Accurate measurement and transfer of liquid volumes are critical in biochemical experiments for
reliable concentration and absorbance results. This study investigated the efficacy of pipetting
techniques, specifically separate and serial dilutions, in ensuring precise volume transfer.
Micropipettes and fresh tips were used for each transfer to prevent contamination and ensure
accuracy. Results indicate that the serial dilution technique yielded greater accuracy, with a higher
R-squared data value (0.1745 > 0.0093) compared to the separate technique. Analysis of graphs
revealed deviations from the expected linear trend, suggesting potential errors in pipetting
techniques and procedural lapses. Negligent factors such as solution evaporation and inadequate
cleaning procedures may have contributed to unexpected outcomes. Despite imperfections, a clear
correlation between absorption and concentration was observed. These findings emphasize the
need for meticulous experimental execution to minimize errors and enhance reliability.

INTRODUCTION
In the realm of biochemistry, precise measurement of liquid volumes is indispensable for
ensuring accurate experimental outcomes. The ability to transfer small volumes reproducibly is
particularly crucial when dealing with minute quantities of substances, where even minor errors
can lead to significant deviations in results. Among the various techniques available for
measuring liquid volumes, the utilization of pipettes stands as a cornerstone in laboratory
practices.

The experiment outlined here aims to elucidate the accuracy, precision, and calibration of
pipetting devices, specifically focusing on three common types: P20, P200, and P1000 pipettes.
These instruments are essential tools in biochemical research, facilitating the transfer of precise
volumes ranging from sub-microliters to milliliters. By meticulously examining the performance
characteristics of these pipettes, researchers can ensure the reliability and reproducibility of their
experimental data, thus enhancing the validity of their findings.

The significance of this study lies in its contribution to the optimization of experimental
procedures in biochemistry. Accurate pipetting is fundamental to various biochemical assays,
including enzyme kinetics, molecular biology techniques, and drug discovery processes.
Therefore, a thorough understanding of pipette accuracy, precision, and calibration is
indispensable for researchers striving to attain reliable and meaningful results in their
investigations.

The experiment involves acquiring pipettes of various sizes, along with the appropriate size tips,
and conducting multiple pipetting trials to measure the accuracy and precision of volume
delivery. Additionally, the experiment includes procedures for assessing the calibration of pipettes

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 International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
using a standardized protocol. Two key techniques employed in this experiment include separate
dilutions and serial dilutions. These techniques involve precise dilution of solutions using pipettes
and subsequent analysis to determine the concentration of the diluted solutions. By employing
these techniques, researchers can evaluate the performance of pipettes across a range of volumes
and assess their suitability for various biochemical assays. Through systematic data analysis,
researchers can derive meaningful insights into the accuracy, precision, and calibration of
pipetting devices, thereby optimizing experimental procedures in biochemistry.

MATERIALS AND METHOD

Materials
Chemicals Equipment
● Distilled water ● P200, P1000 micropipettes
● Stock cobalt (II) chloride 2M solution ● Ten test tubes
● Cuvettes
● Spectrometer
● Vortex
● Stirring rod
● Marker

Method
1. Separate dilutions
First, five test tubes were marked following the numerical sequence. Then, these tubes
were filled with distilled water and cobalt (II) chloride with specific volumes as described
below. All the tubes needed shaking well by using a stirring rod.

Table 1. The volume of each compound was added to each test tube for separate dilutions.
Tube Distilled water (μL) Cobalt (II) chloride (μL)

1 4000 0

2 3940 60

3 3900 100

4 3200 800

5 2800 1200

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 International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
2. Serial dilutions
Similar to the first technique, five marked tubes were prepared with 4000μL of distilled
water in each initially. Next, 4000μL of stock Cobalt (II) chloride was added to tube two
and shaken well using a vortex. Then, 4000 μL of solution was removed from tube two,
put into tube 3, and mixed well. This step was repeated for the rest of the tubes as
illustrated in the below figure. Importantly, each time of transfer, the liquid mixture had to
be mixed briefly.

Figure 1. The demonstration of serial dilution technique.

After performing the two dilution techniques, the solution from each tube was delivered to each
cuvette and measured the absorbance by the spectrometer at a wavelength of 510nm. Finally, all
the data would have been collected to make the graph as in the result section.

RESULTS
1. Separate dilutions:
Table 2. The concentration and absorbance of each test tube in separate dilutions.

Tubes Concentration (M) Absorbance

1 0 0.1712

2 0.03 3.0675

3 0.05 2.2781

4 0.4 1.3602

5 0.6 0.8068

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 International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry

Concentration of CoCl2 · (H2O)6 in the first dilution:

C1×V1 = C2×V2

<=> 2 (M)× 60 (μL) = C2× 4000 (μL)

<=> C2 = 0.03 (M)

C1: the concentration of stock solution
V1: the volume of Cobalt (II) Chloride
C2: the concentration of the solution after dilution
V2: the total volume of the solution after dilution

Figure 2 - Absorbance of Cobalt (II) Chloride in separate technique.

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 International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry
2. Serial dilutions:

Table 3. The concentration and absorbance of each test tube in serial dilutions.

Tubes Concentration (M) Absorbance

6 0 0.1687

7 1 0.2563

8 0.5 0.3112

9 0.25 1.5322

10 0.125 2.3754
Concentration of CoCl2 · (H2O)6 in the first dilution:

C1×V1 = C2×V2

<=> 2 (M)× 4000 (μL) = C2× 8000 (μL)

<=> C2 = 1 (M)

Figure 3 - Absorbance of Cobalt (II) Chloride in serial technique.

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 International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry

DISCUSSION
Since the accurate concentration and absorbance results were dependent on the preparation of
dilutions in experiments, the procedure for utilizing pipettes in both separate and serial dilutions
was to ensure the liquid volumes were transferred accurately and consistently, preventing volume
deviations in a solution from being transferred. Additionally, to prevent solution contamination, it
was crucial and essential to employ fresh tips for every solution transfer in every distinct dilution
experiment. As a result, upon completion of both experiments, the serial technique provided
better data than the separation technique, determined by the greater R-squared value of the serial
technique (0.1745 > 0.0093). “R2 is a statistical measurement of the proportion of the variance for
a dependent variable that is explained by an independent variable in a regression model.” [2] The
closer to one of this number, the less variance of error than the variance of the dependent variable.
In contrast, both techniques got this value closer to zero, meaning the collected data were not
reliable. This could also be seen in the relationship between the concentration and the absorbance
of samples shown in the line graphs. Although the absorbance should, in theory, be proportionate
to the concentration, the results of the experiment showed that the absorbance was maximum at
low concentrations and steadily reduced as the concentration increased. This unsatisfactory might
have relied on the imperfect procedure performance. Such incorrect volume was transferred by
pipette or incorrect pipetting could have resulted in imprecise concentration. Besides, leaving
bubbles when delivering the solution to the cuvettes could affect or prevent the spectrometer from
giving out clear data as the bubbles occupied the space of cobalt (II) chloride so that the light was
unable to travel through the solution. Another critical reason was the unarranged samples while
conducting the spectrometry method leading to troublous data.
In addition, in terms of the efficiency comparison between the two dilution methods, the serial
one seems to outweigh the separate or parallel one. Beneficially, serial dilution helps yield a
highly diluted solution without using large amounts of chemicals, which is suitable for some lab
fields requiring very low sample concentration such as cell study, and microbiology.[3]

CONCLUSION
As drawn from the result, the serial technique obtained the higher R-squared value, meaning it
was a better method for pipetting than the separate one. However, this R-squared did not seem
perfect as it was so far away from one (R2 = 0.1745), thus not showing a satisfying way of
pipetting. For explanation, it could be due to the group’s unpleasant performance. Generally, it is
crucial to be careful and thoughtful while experimenting.

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 International University - Vietnam National University, HCMC
School of Biotechnology - Department of Applied chemistry

REFERENCES
[1] Experiment 1 Use of Pipetmen. Methods in Biochemistry lab manual.
[2] Fernando, J. (2023, December 13). R-Squared: Definition, Calculation Formula, Uses, and
Limitations. Investopedia. https://www.investopedia.com/terms/r/r-squared.asp

[3] A. (2022, July 11). Dilution - Dilutions of Solutions - Definition; Meaning, Formula, Methods
&amp;amp; Importance of Dilution. BYJUS.
https://byjus.com/chemistry/dilution-definition/#:~:text=Serial%20dilutions%20are%20used
%20in,plated%20to%20an%20agar%20plate.