Abstract

There are two
parts to this experiment. The first part involves determining error analysis
with the use of micropipettes. Learning about error analysis is crucial in
determining the most important errors and whether they have a significance in
the final results. From the results, it was determined that the P-200 pipette
is preferred for more accurate and precise measurements. Part two of this
experiment focuses on preparing two acid buffer solutions to test. The
preparation of buffers is a common laboratory technique in biochemistry. The
purpose of using buffers is to hinder any changes in pH when an acid or base is
added in a solution. A buffer solution with the assigned pH value of 8.0 is
what will be prepared in this experiment to test. The resulting pH values of the
two buffers measured to be 8.1 and 7.96. There is a small discrepancy of pH
between the theoretical and experimental pH values.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introduction

                Micropipettes
are ubiquitous in biochemical laboratory experiments. It is a reliable
precision instrument that allows for taking accurate and precise measurement
volumes of liquids. Despite its apparent importance, the origin of the
micropipette should not be taken for granted. The micropipette was first
discovered by German physician Heinrich Schnitger at the University of Marburg
in 1957 (Klingenberg, 2005). This breakthrough has revolutionized modern
biotechnology and molecular biology with handling of small liquid volumes. However,
pipetting errors do exist and failure to accurately pipette will result in an
experiment being irreproducible. That is why the accuracy of the pipettes rely
on the investigator. Pipettes need to be maintained and practiced with good
technique with a proper understanding of how they work. The first portion of
this laboratory experiment will focus on getting acquainted with the use of
micropipettes by using different microliters to measure out water. This method
will be useful in performing a statistical analysis for each set of results.

                There
are various micropipettes that are used in the laboratory that range in sizes
of P20, P200, and P1000. These sizes are denoted at the top of the pipette. They
also dispense liquids between the ranges of 1 and 1000 ?L (Prilliman, 2012).
However, in this experiment, the types of micropipettes that will be used range
from 0.1 to 2.5 ?L. Before using the micropipette, it is important to master
its use before carrying out any experiment. Liquids are never drawn directly
into the shaft of the pipette. Hence, why there are plastic disposable tips
that attached to the shaft of the micropipette. S for adjusting the volume of
the liquid, there is a volume adjustment dial near the very top of the pipette.
There are two separate sizes of tips. The small, clear tips in the yellow box
are generally used for the micropipettes with the size of P20 and P200 whereas
the large, blue tips are used for the P1000 micropipette.

                The
second part of the experiment involves creating a buffer solution. Since most
solutions drastically alter pH change, buffers serve the purpose of resisting
that change. Buffers are fundamental in many biological mechanisms; which,
require a stable pH range in order to carry out biochemical reactions. This is
often the case when proteins or enzymes are looked at. Take for instance the pH
range of human blood. It is necessary that the range is maintained between pH
levels of 7.35 to 7.45; otherwise, the hemoglobin will not successfully bind to
oxygen which will then result in a homeostatic imbalance (Singer et al., 1948).

                For
a buffer solution to occur, two species are required. One that can react with
hydroxide (OH-) ions and the other that can react with hydronium (H3O+) ions.
It is important to note that these two species should not react with one
another. Many buffer solutions are generated by combining a weak acid and its
conjugate or by combining a weak base and its conjugate. In this experiment, the
base tris and the acid HCl will be used to prepare the buffer solution. Buffers
solutions are generally effective in a pH range of +/- one pH unit on either
side of the pKa (Stewart et al., 2009). The Henderson-Hasselbalch equation is
denoted below to provide the necessary information on preparing a buffer
solution:

                When generating a buffer solution, it is important to
note that there is a threshold to the amount of acid or base that can be added
to a buffer solution before one of the components is used up. The limit is best
known as the buffer capacity and is defined as “the moles of acid or base
necessary to change the pH of one liter of solution by one unit” (Stewart et
al., 2009).

 

 

Experimental Procedure

                For part one of the experiment, it involved
understanding the mechanism of the pipette. For starters, there are six
different measurements to be recorded: 1000 ?l of the P-1000, 100 ?l of the
P-1000, 100 ?l of the P-200, 20 ?l of the P-200, 20 ?l of the P-20, and 5 ?l of
the P-20 respectively. The P-1000 was the first pipette used in this part of
the procedure. To obtain the desired volume, the pipette was set to 1000 ?l by
adjusting the dial. Afterwards, a sterile blue tip was loaded for the P-1000
pipette. Precaution was taken to prevent contaminating the tip.

                A 100-mL glass beaker was filled with 50 mL of
deionized water. To load the water into the pipette, the plunger was pressed
down slowly to the point of first resistance. While the plunger was still held
at the first stopping point, the tip was inserted into the solution of
deionized water but not too deep. Then, the plunger was slowly released to draw
up the deionized water. The pipette tip was filled to final volume before
removing it from the solution to avoid any bubbles from building up in the tip.
The scale in which the cuvette was placed inside of was tared before dispensing
the sample of deionized water into the cuvette by pressing the plunger down the
point of second resistance. At this point was when the measurement in grams of
the sample was recorded. The process of loading the deionized water from the
beaker and placing it inside the cuvette was repeated for another four times.
The measurements in grams were recorded in the data table for each of the five
trials. The cuvette was emptied and the tip of the pipette was discarded.

                The above procedure was repeated for the rest of the
five pipettes. The tip was changed only when the volume changed. The small,
yellow tips were used for the P-20 and the P-200. After the table was filled
with the measurements of all the pipettes, a statistical analysis was performed
to determine the mean, standard deviation, relative error, and relative
standard deviation.

                The second portion of the experiment dealt with creating
a buffer solution. To start, the molarity and the volume of the tris buffer
solution to be made was determined. Then the number of moles required was
calculated by multiplying the molar concentration of the buffer by the volume
of the buffer that will be made. Afterwards, the amount of tris in grams was
determined by multiplying the number of moles by the molecular weight of the
tris. These calculations can be viewed under the calculations section of the
lab report.

                Next, 1.211 grams of tris was dissolved into 80 mL of
distilled deionized water. This was 80% of the final volume. 1 M HCl was
collected in a separate beaker. By using a pipette, the HCl was added into the
solution in a dropwise manner until the solution reached to a pH close to 8.0.
Once the desired pH was reached, 50 mL of the Tris-HCl solution was added into
a 100-mL glass beaker. Before taking the pH of the buffer, the pH meter was
calibrated. Once it was finally calibrated, the pH of the 50-mL solution of the
tris-HCl was measured. This same process was repeated for another 50-mL
tris-HCl solution. Once the solutions were measured and recorded, the tools,
glassware, and instruments at the lab station were cleaned and put away.

 

Results

Calculations

How many grams of tris:

 

How many moles of tris:

 

Expected pH of buffer:

 

Relative error from
pipetting:

Trial 1:

Trial 2:

Trial 3:

Trial 4:

Trial 5:

 

Relative Standard error
from pipetting:

 

Data

Figure 1-1

                                                    
          Micropipette Procedure

Pipette

vol. ?l

Trial 1                   

Trial 2

Trial 3

Trial 4

Trial 5

Avg

SD

P-1000

1000

0.9994 g

0.9977 g

0.9955 g

0.9949 g

0.9943 g

0.99636 g

0.00213 g

P-1000

100

0.1104 g

0.1035 g

0.1171 g

0.1174 g

0.1042 g

0.11052 g

0.006706 g

P-200

100

0.0969 g

0.0991 g

0.0993 g

0.0992 g

0.1005 g

0.099 g

0.001304 g

P-200

20

0.0199 g

0.0194 g

0.0192 g

0.0201 g

0.0197 g

0.01966 g

0.000365 g

P-20

20

0.018 g

0.0198 g

0.019 g

0.019 g

0.0193 g

0.01902 g

0.000657 g

P-20

5

0.0054 g

0.0046 g

0.0059 g

0.0056 g

0.0055 g

0.0054 g

0.000485 g

AVG:

0.208327 g

0.001941 g

Figure 1-1. Results from the micro
pipetting procedure. One the far-left axis, there are two of each pipette size:
The P-1000, P-200, and P-20. For each of the pipette sizes, there is a desired
volume in microliters. This chart represents five different trials for each
pipette and volume. The measurements for each trial are recorded in grams. At
the end of each of the 5th trials, there is an average and a
standard deviation.

 

Figure 2-1

Figure 2-1. Graphical
representation of tris-HCl buffer solution. Also in the image is the pH meter
that was used to measure the pH of the buffer.

Figure 3-1

Figure 3-1. Graphical
representation of the pH readings of the tris-HCl buffer solution after two
trials.

 

Discussion

                The reference pipetting method used in each of the trials
in part one was used to determine the precision and accuracy of each pipetting
and volume. Based on the data and calculations from this experiment, the P-200
is the preferred pipette for accurate measurements. This was determined by
calculating the standard deviation, relative standard deviation, and relative
error. The standard deviation for the P-200, 20 microliters was 0.000365 while
the relative standard deviation and the relative error were 0.93% and 1.0%
respectively. The standard deviation indicates the distribution of values
around the average (Thomas, 1996). It is preferred for the standard deviation
to be tight around the mean. The relative standard deviation indicates whether
the regular standard deviation is a small or large value when compared to the
average of the data set. The relative standard deviation for the P-200, 20
microliters was 0.93%. This demonstrates that the standard deviation is 0.93%
of the mean 0.208327- which is small. In other words, the data or values is
tightly clustered around the average. If the percentage were to be a lot
bigger, then that would indicate that the values are more spread out.

                Although the results appeared to have stayed
relatively close to the mean, it does not indicate that errors will not exist
in the lab even with the highest precautions. An effective strategy to
eliminate any effects in accuracy, precision, and trueness would be to avoid
prolonged delay in between aspiration and removal of the tip from the sample.
Another technique that can be used to avoid any inaccuracies would be to avoid
contaminating both the sample and the tip. Any contaminations may have an
adverse effect on the final reading. Finally, it is required that the plunger
is pushed to the correct point of depression otherwise bubbles may form in the
tip or it may also result in not dispensing all the sample into the container.
Individually, none of these factors resulted in a very high error. 

                Strategies among pipette users differ with personal
preferences, background, and training. These dissimilarities in techniques can
either negatively or positively influence the precision, accuracy, and trueness
of results from an experiment. To ensure pipetting accuracy, it is important
that laboratories adopt standard operating procedures for pipetting strategies
and verify that all operators are professionals and adequately trained. By
increasing the amount of consistency in results acquired, the level of quality
and credibility of the experiment will be enhanced.

                The second part of the experiment dealt with
generating buffer solutions. Buffers consist of a weak acid and its conjugate
base. In this experiment, the tris was the base that was used and HCl was he
acid. This system allowed to absorb either H+ or OH- due to the reversible
nature of the dissociation of the HCl. HCl can release H+ ions to neutralize
OH- and form water.

                When generating a buffer, it is important to be
mindful of the factors that constitute a good buffer. The first is that the
buffer must have a pkA between 6 and 8. In this experiment, the pKa of the
buffer was 8.1, which is not significantly far from the range. The most
important factor of a buffer is that is must have chemical stability. In other
words, the buffer must be stable and should not break down under working
conditions. By any means should the buffer oxidize or be influenced by the
system in which it is being used. Therefore, buffers with metabolites for
instance should be avoided as much as possible. Metabolites are defined as
products that serve the purpose of breaking down or metabolizing an entity into
a different substance (Berdy, 2005).

                The results from this experiment show that the
experimental pH values from the two trials were close to the actual pH of 8.0.
The first trial, the pH was 8.1. However, the pH decreased after the second
trial. The reason for the decrease in pH after the second trial may have most
likely been due to human error. HCl was added in a drop-wise fashion into the
second 50-mL beaker when that was not necessary. This was done in hopes of
reaching the desired pH. That is why it is imperative to follow all laboratory
instructions carefully.

 

 

 

 

 

 

 

 

 

 

 

Bibliography

 

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Hastings, A. B. (1948). An Improved Clinical Method For The Estimation Of
Disturbances

Of
The Acid-Base Balance Of Human Blood. Medicine,27(2), 223.
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G. (n.d.). The
Gilson guide to pipetting. Villiers-le-Bel, Franc?a: Gilson.

Klingenberg, M. (2005).
When a common problem meets an ingenious mind. EMBO reports,6(9),
797-

800.
doi:10.1038/sj.embor.7400520

Stewart, P. A., Kellum,
J. A., & Elbers, P. W. (2009). Stewarts textbook of acid-base.
Amsterdam:

AcidBase.org.

Prilliman, S. G.
(2012). An Inquiry-Based Density Laboratory for Teaching Experimental
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Chemical
Education,89(10), 1305-1307. doi:10.1021/ed2006339

Thomas, L., &
Juanes, F. (1996). The importance of statistical power analysis: an example
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Behaviour. Animal
Behaviour,52(4), 856-859. doi:10.1006/anbe.1996.0232

Bérdy, J. (2005).
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doi:10.1038/ja.2005.1

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