Experiment 1: Use of Micropipettor and Spectrophotometer Laboratory Report

Group 7 B.S. Biology 4th Year Biology-5 John Gabriel Rosario, Neil Michael Sese, Mark Anthony Sanchez, Bea Rosalini Santiago, Elaine Roxas

Submitted to: Josefino R. Castillo, M.Sc. Edward A. Quinto, M.Sc.

July 6, 2011

Introduction

Micropipettors are the standard laboratory equipment used to measure and transfer small volumes of liquids. The said equipment is essentially a precision pump fitted with a disposable tip. Depressing the plunger displaces the specified volume of air from the piston which releases the plunger creating a vacuum, which draws an equal volume of fluid into the tip (1).

The volume range of digital micropippetors to be used in laboratories were 0.5-10 microliters, 10-100 microliters and 100-1,000 microliters. Each micropipettor contains a digital readouts that display the specified volume (1).

On the other hand, A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector (3).

The beam of light consists of a stream of photons, represented by the purple balls in the simulation shown below. When a photon encounters an analyte molecule (the analyte is the molecule being studied), there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam (3).

The objective of this experiment is to test the accuracy and precision in using of a micropipettor and a spectrophotometer.

Materials and Methods

Materials

The materials used for the both activity were bromphenol blue (1.25% w/v), microcentrifuge tubes, micropipettors and tips, GENESYS 10 uv-vis spcetrophotometer, vortex mixer, semimicro cuvettes and distilled water

Methods Activity 1: Testing accruacy of operators

The experiment started by warming the spectrophotometer at 540 nm. One millilter of distilled water was placed in each of five microcentifuge tubes. Using micropipettor, the following amounts of bromphenol blue was added to each successive tube: 0.5, 1.0, 1.5, 2.0, and 2.5 microliters. After adding bromphenol blue, each tube was placed in a vortex until the dyes in in the solution.

To test its accuracy, the spectrophotometer was set to zero withi a distilled water and the absorance of the dye solutions was read starting from the least concentrated to the most concentrated.

Activity 2: Testing precision in using micropipettor

One milliters of distilled water was placed in each microcentrifuge tubes. Each member of the group pipetted 2.5 microliters in individual tubes then individual tubes was placed in a vortex and run into a spectrophotometer with the same settings as in activity 1. The absorance of each tubes was noted and standard deviation and coefficient of variation was computed.

Results and Discussion

Table 1: Absorbance of the Dyes in solution starting with the least concentrated to the most concentrated.

Microliters of Bromphenol blue 0.5 1.0 1.5 2.0 2.5

Absorbance 0.004 0.011 0.019 0.024 0.032

Correlation Coefficient: 0.998 Equation of the line: y=0.0138x-0.0027

Figure 1 Line graph of the Absorbance of the series of Bromphenol blue

Microliters of Bromphenol blue

Table 2. Measurement of the absorbance of each tube of each member of the group

Group Member Absorbance

Santiago 0.030

Sese 0.03 4

Sanche z 0.021

Rosario Roxas Standard Deviation: 0.00602 0.037 0.031 Coefficient of Variation: 0.194

Accuracy is a measure of how close a number is to the actual value. In activity 1, linear regression was used with the given absorbance values to show the accuracy in the use of micropipettors. Linear regression is an approach to modeling the relationship between an explanatory variable which is X and response variable denoted by Y. Correlation is a term used to describe how strong the relationship between the two variables appears to be (4). In the results of the said activity, since 0.998 is near 1, the microliters of bromphenol blue has a strong

Absorbance

relationship with its absorbance, resulting to an accurate use of micropipettor.

In activity 2, standard deviation of the said absorbance of each tube was done in order to find how far or close the given set of numbers with the average number. The average deviation were 0.00602. It shows that the one absorbance values is very close to other absorbance values in this set of data. On the other hand, coefficient of variation was also calculated in the said activity. Coefficient of variation is a statistical measure of the dispersion of data points in a data series around the mean. It is needed to be calculated in order to be use in comparing the degree of variation with other data sets. The coefficient of variation of the said activity is 0.194 which is a good value (4).

Conclusion

Absorbance

The experiment is about how to use a micropipettor and spectrophotometer. Specific tests were done in order to test the accuracy and precision of the students in using a micropipettor with the help of the spectrophotometer. With the correlation coefficent of 0.998, standard deviation of 0.00602 and coefficient of variation of 0.194, the group is accurate and precise in using a micropipettor and spectrophotometer effectively.

References
1. Blauch, D. (2009). Spectrophotometer. Retrieved from

http://www.chm.davidson.edu/vce/spectrophotometry/Spectrophotometry.html
2. Karp (2010). Cell Biology (6th Ed.). Singapore: John Wiley and Sons Pte Ltd. 3. Micropipettor. Univeristy of Wisconsin Biotechnology Center. Retrieved from

http://www.biotech.wisc.edu/outreach/pipettestory.html 4. Mendenhall, William (2010) Probability and Statistics Fundamentals. Singapore: Cengage Learning.