Experiment # 1

Use of Micropipettor and Spectrophotometer

Submitted by
Group # 5

Group Members:
SIM, Michelle D.
SUDERIO, Gellina Ann R.
TEOPE, Jonnah Kristina C.
TIMBOL, Danica Kaye P.
UY, Regina Celine DG.

Submitted to
Josefino R. Castillo, MS

Date Submitted
July 2, 2009
INTRODUCTION

The study of Cell and Molecular Biology involves a vast number of methods and

techniques as it studies the structural unit of the living system. Laboratory instruments are

devised to achieve accuracy and precision as it deals with up to microscopic level.

The micropipettor is one of the most commonly used instruments in science laboratories.

The micropipette is a Wisconsin invention developed through interactions among several people,

primarily inventor Warren Gilson and Henry Lardy, a professor of biochemistry at the University

of Wisconsin-Madison. Pipettes are used to accurately measure and dispense relatively small

volumes of liquid. A small volume micropipettor has a range of 0.5–10 mL, a mid range

micropipettor can handle 10-100 mL of liquid and a large volume micropipettor can measure up

to 100-1,000 mL. It is a precision instrument and should be handled with utmost care.

A Spectrophotometer is an instrument used to measure the intensity of wavelengths in a

spectrum of light compared with the intensity of light from a standard source. It has varied

applications in the qualitative analysis of sample purity, DNA and protein quantitation, cell

density measurements, and assays involving enzyme – catalyzed reactions. The most common

spectrophotometers are used in the UV(200 – 400nm) and visible regions of the spectrum(400 –

700nm), and some of these instruments also operate into the near-infrared region(700 – 900nm)

as well. The VIS and UV spetrophotometric techniques are used mainly on qualitative

determination of components present at low concentration. The IR measuring technique is

mainly used for qualitation. The spectrophotometer compares the intensity of the trensmitted

light with that of the incident light.

The objective of this experiment is to familiarize the students with the use of the

micropipettor and the spectrophotometer and to some of their applications in scientific research.
MATERIALS AND METHODS

A. MATERIALS
Materials:
• Bromphenol blue (1.25%w/v)
• 5 Test tubes
• Micropipettors and tips

Special equipments:
• Spectrophotometer
• Vortex mixer

B. METHODS
A. Use of micropipettors and spectrophotometer (by group)

The spectrophotometer was warmed up for twenty minutes before using and was set at

540nm. One mL of distilled water was placed in each of five test tubes. With the micropipettors,

successive amounts of bromphenol blue were added. The volumes of the bromphenol blue added

were 0.5, 1.0, 2.0, 4.0 and 8.0µL respectively. The manufacturer’s instructions for use of these

micropipettors were followed scrupulously. Each tube was then vortexed until the dye was in

solution. The spectrophotometer was zeroed with distilled water. Afterwhich the absorbance

were read and the readings were recorded. Finally, the results were graphed. The standard

deviation and the coefficient of variation were calculated, where:

SD= [N∑X2 – (∑X)2] / [N(N-1)]

CV= SD / Ave.

B. Use of micropipettors and spectrophotometer (individual)

One mL of distilled water was placed in the test tube. With the micropipettors, 4.0 µL of

bromphenol blue was added. The tube was then vortexed until the dye was in solution. The

spectrophotometer was zeroed with distilled water. Afterwhich the absorbance was read and the

reading was recorded.

RESULTS AND DISCUSSION

There are two laboratory instruments commonly used in Cell and Molecular Biology

namely the micropipettor and spectrophotometer. In this experiment, the following results were

obtained:

Bromphenol Blue (μL) Absorbance (540nm)

0.5 0.094nm
1.0 0.274nm
2.0 0.688nm
4.0 1.073nm
8.0 1.866nm

Standard Deviation
SD = [N∑X2 – (∑X)2]
N(N – 1)
T he Standard Curve of
540 A
Versus the
= 0.70694
Concentration of Bromphenol Blue

Mean: 0.799 2
8, 1.866
1.8
Coefficient of Variation
CV = SD 1.6

Ave. 1.4
= 0.8845595596
1.2
absorbance (nm)

4, 1.073
1

0.8
2, 0.688
0.6

0.4
1, 0.274
0.2
0.5, 0.094
0
0 2 4 6 8 10
co n cen tratio n (u L )
 Student Absorbance (540nm)
SIM 0.266nm
SUDERIO 0.144nm
TEOPE 0.194nm
TIMBOL 0.126nm
UY 0.348nm

Standard Deviation
SD = [N∑X2 – (∑X)2]
N(N – 1)
= 0.09178

Mean: 0.2156

Coefficient of Variation
CV = SD
Ave.
= 0.425696

This experiment tackles on the familiarization of two commonly used laboratory

equipments: the micropipettor and spectrophotometer.

A specific amount of bromphenol blue was dropped and mixed with each of the five test

tubes. Using the spectrophotometer, the absorbance for 0.5μL of bromphenol blue was 0.094nm,

for 1.0μL bromphenol blue it was 0.274nm, 0.688nm was recorded for 2.0μL of bromphenol

blue, for 4.0μL bromphenol blue, 1.073nm and lastly for 8.0 μL bromphenol blue, 1.866nm.

The results show that there is a direct relationship between the amount of dye in the

solution and the amount of light absorbed by the solution. As the amount of dye increases, the

amount of light absorbed also increases.

Each of the students placed 2.5 micro liters of bromphenol blue in their respective test

tubes and their solutions were mixed using the vortex mixer. The results varied from each

student’s solutions that used the same amount of dye and micropipettor.
 A graph of the amount of bromphenol blue versus the absorbance in the

spectrophotometer should give a straight line. The standard deviation provides a measure of the

reproductibility of the pipetting skills. The smaller the standard deviation, the better the skills of

the student.

Post Lab questions

1.) On what operational procedures does the consistency of micropipettor usage depend?

Ans. There must be consistency on the pickup and dispense of the liquid. There must also be a

smooth action in pressing the button of the micropipettor. One must also not place the tip directly

on the bottom of the tube to avoid inconsistency.

2.) Explain the relationship between absorbance value, optical density, and percent

transmittance.

Ans. There is a logarithmic dependence between the transmission of light through a substance

and the concentration of the substance, and also between the transmission and the length of

material that the light travels through. The percent transmittance multiplied by 100, is usually

converted absorbance.

REFERENCES
[1] Berkley, James. Use of Automatic Digital Micropipettor. USA. CNE. 1993

[2] Bissen, Shirley. How to Use a Micropipettor. USA. Prentice – Hall. 1998.

[3] Harris, Daniel. Exploring Chemical Analysis (4th ed). New York: W.H. Freeman &
Company. 2009.

[4] Pungor, Erno. A Practical Guide to Instrumental Analysis. USA: CRC Press LLC. 1995.

[5] http://www.bioedonline.org/slides/slide01.cfm?tk=4 (Retrieved June 28, 2009)