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) 0.5 1.0 2.0 4.0 8.0
Absorbance (540nm) 0.094nm 0.274nm 0.688nm 1.073nm 1.866nm
Standard Deviation SD = [N∑X2 – (∑X)2] N(N – 1) = 0.70694
T he Standard Curve of Versus the 540 A Concentration of Bromphenol Blue
Mean: 0.799
8, 1.866
1.8 1.6 1.4 absorbance (nm)
Coefficient of Variation CV = SD Ave. = 0.8845595596
2
1.2 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
co n cen tratio n (u L )
8
10
Student SIM SUDERIO TEOPE TIMBOL UY
Absorbance (540nm) 0.266nm 0.144nm 0.194nm 0.126nm 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)
