University Of Massachusetts, Lowell
Biochemistry Techniques, Lab1
Students: John Gibson, Caelin Davidson, Nilab Mohmand
Spring 2024
Quantitative Comparison Of Micropipette Use Between First Stop And Second Stop When Withdrawing A Fluid
Abstract
Proper and improper uses of a micropipette are performed in this experiment for training purposes. Specifically, the proper use of pressing a micropipette’s plunger to the “first stop” and the improper use of pressing the plunger to the “second stop” for withdrawing a fluid is tested. The resulting pipetting volumes are compared and contrasted to give a quantitative perspective of the importance of using a micropipette according to standard usage instructions.
Introduction
Pipetting is essential for transferring fluids between containers in any chemistry laboratory. Larger scale pipetting, such as between 10mL and 50mL, can be accomplished with bulb pipettes. The bulb of a bulb pipette is first deflated to give it a suction capacity; then, a release knob allows the fluid to be withdrawn from a container. A medium-scale glass pipettor with a sliding plunger can pipette between 1mL and 10mL. Micropipettes are essential in molecular biology laboratories for pipetting below 1000 μL. Micropipettes are available in different sizes, such as P20, P200, and P2000, for volumes of 20 μL, 200 μL, and 2000 μL, respectively.
Most laboratory workers, scientists, and students are instructed to withdraw a specific volume by pressing the thumb plunger of a micropipette to its “first stop” and then suctioning the volume of a liquid. It is commonly known that the “first stop” gives the precise suction volume.
In contrast, the “second stop” of the plunger is used to expel the fluid because the “second stop” displaces more fluid that may be retained in the micropipette tip by capillary effects and other intermolecular forces. However, the exact extra volume displacement by the “second stop” is not always quantized in all laboratories. This study hypothesizes that the “first stop” technique is proper and should yield more accurate volume dispensing than stopping the plunger at the second stop when withdrawing fluid.
The purpose and aim of this study is to quantitatively categorize the difference in dispensing between using the “first stop” and “second stop” to withdraw a fluid in a laboratory. Two advantages can be obtained with quantitative categorization of proper and improper pipetting techniques. Firstly, a quantitative characterization of a micropipette educates laboratory workers on the importance of adhering to the standard operating procedure because a significant, non-negligible error volume quantity of using the “second stop” of the plunger to withdraw a fluid may be illustrated to the laboratory personnel. Secondly, if a laboratory worker does make a mistake by withdrawing a fluid with the “second stop,” the error percentage of an experiment can be estimated and documented.
A buffer is a solution that resists pH change when an acid or base is added to the solution due to chemical reagent reactions in the solution or is added from outside the solution. Due to the sensitivity of biological reactions to pH levels, a buffer is commonly used in living organisms and molecular biology laboratories to create an aqueous solution suitable for biological reactions or to suspend a molecule with a stable pH level. Laboratory buffers are most commonly prepared by mixing a strong base with a weak acid or mixing a strong acid with a weak base. In a Tris buffer preparation, Tris is titrated with strong acid HCl to near the half-equivalence titration point, where HCL concentration is near half the concentration of the weak base. The half-equivalence titration point affords the buffering capacity according to the following equation.
WeakBase+ H++Cl-WeakBaseH++Cl-
Near the half-equivalence point, the weak base, such as Tris, absorbs most added H+ ions so the pH level doesn’t decrease. This absorption applies to the HCL added during and after the preparation phase. On the other hand, if a strong base is added to the buffer solution after the preparation phase at the half-equivalence point, the conjugate acid salt of the base absorbs the strong base, as shown in the following equation, absorbing the added OH- ions so the pH level doesn’t rise.
WeakBaseH++Cl-+Na++OH-WeakBase + H2O+Cl-+Na+
The theoretical pH value of a buffer solution at the half-equivalence titration point is, by definition, the pKa value of the weak acid in a weak acid-strong base titration because a strong acid has 100% ionization by definition, and 50% of the base is ionized to combine with H+ from the strong acid. For a weak base-strong acid titration, such as a Tris buffer, the pOH value at the half-equivalence titration point is the pKb value of the weak base. The pH value can be obtained by the equation, pH=14-pOH.
Material And Methods
With water as the test fluid, a P200 micropipette was used to draw 100 μL of the liquid and then place the liquid on a weight boat. Transferring 100 μL water was performed 3 times with the technique of withdrawing fluid with the plunger at the “first stop” and another 3 times with the technique of withdrawing fluid with the plunger at the “second stop.” The weight gains of the weight boat were recorded for each of the 3 transfers with the “first stop” technique and 3 transfers with the “second stop” technique. In all 6 trials, the fluid was expelled to the weight boat by pressing the plunger to the second stop.
With a P20 micropipette, the same water liquid transfer exercise was performed to transfer 10 μL of water 3 times with the “first stop” technique and 3 times with the “second stop” technique. Each transfer’s weight gain of the weight boat was also recorded for another 6 trials, totaling 12 trials with water as test fluid.
Furthermore, the experiment used 50% glycerol as the test fluid to perform the same procedure as water as the test fluid with the same set of P200 and P20 micropipettes. 12 trials with glycerol as the test fluid. Overall, 24 trials of fluid pipetting and weighing were performed.
The recorded pipetting weight percent difference from the expected weight for each trial was calculated using the following formula,
% difference = experiment weight - expected weight / expected weight100%
100 mM concentration Tris solution was prepared at pH 7.9; 500 mM of KCl solution was prepared; 25 g/L concentration of Lysogeny Broth was prepared at pH 7.5
Results
The P200 pipettor test results were recorded in the first half of Table 1 below. The weights of 100 μL of water ranged from 0.0995 grams to 0.1002 grams with the fluid withdrawal technique stopping the plunger at the first stop when withdrawing, with an average 0.267% difference between expected and experiment weight; the weighs of 100 μL of water ranged from 0.1432 grams to 0.2899 grams with the “second stop” fluid withdrawal technique, with an average 93.3% difference between expected and experiment weight. In the second half of the table with the P20 pipettor, the weights of 10 μL of water ranged from 0.0096 grams to 0.0099 grams with the “first stop” fluid withdrawal technique, with an average 2.0% difference between expected and experiment weight; the weighs of 10 μL of water ranged from 0.0142 grams to 0.0148 grams with the “second stop” fluid withdrawal technique, with an average 44.33% difference between expected and experiment weight.
Table 1. Result data
Legend: The first column is the micropipette and test fluid used. The Volume column is the micropipette settings during the experiment. For each volume setting for a given test fluid, 3 trials were performed, as recorded in the 3 split rows of the Experiment Weight column. The percent difference is calculated based on the expected weight of water given the density of 1.0 g/mL and the weight of 50% glycerol given the density of 1.13 g/mL from Umassonline.net by Dr. E. Zagriadskaia [1].
Also shown in Table 1, with 50% glycerol, the weights of 100 μL of glycerol ranged from 0.1162 grams to 0.1166 grams with the fluid withdrawal technique stopping the plunger at the first stop when withdrawing, with an average 3.00% difference between expected and experiment weight; the weights of 100 μL of glycerol ranged from 0.1628 grams to 0.3375 grams with the “second stop” fluid withdrawal technique, with an average 97.7% difference between expected and experiment weight. The weights of 10 μL of glycerol ranged from 0.0111 grams to 0.0113 grams with the “first stop” fluid withdrawal technique, with an average 0.885% difference between expected and experiment weight; the weights of 10 μL of glycerol ranged from 0.0164 grams to 0.0274 grams with the “second stop” fluid withdrawal technique, with an average 79.1% difference between expected and experiment weight.
In Figure 1 below, with the P200 pipette dispensing 100 μL of fluid, the average percentage error of pipetting weights with the first stop when withdrawing fluids was compared side-by-side with the average percentage error of pipetting weights with the second stop when withdrawing fluid. For the P20 pipette dispensing 10 μL of fluid, in Figure 2 below, the average percentage error of pipetting weights with the first stop when withdrawing fluids was compared with the second stop when withdrawing fluid.
Discussion
As shown in Figure 1 above with the P200 pipette, the average pipette fluid weight error was much smaller, between 0.267% and 3%, when using the “first stop” technique when withdrawing a fluid. This was true for both water and glycerol test subjects. The large percentage error when pipetting with the “second stop” during withdrawal, between 93% and 97%, was likely due to the larger extra volume of the second stop afforded to dispense extra air to expel any fluid stuck on the wall of the pipette tip. This meant that the second stop should not be used when withdrawing a fluid using a micropipette, and the proper technique was to press the plunger at the first stop and then withdraw a fluid. Also, as shown in Figure 2 with the P20, the first-stop technique yielded a much smaller error of pipetted fluid weights, between 0.9% and 2%; the second-stop technique yielded a much larger error of pipetted fluid weights, between 44% and 79%. All the evidence in both figures pointed to the first stop as the proper technique for withdrawing a fluid with a micropipette in a molecular biology laboratory. The accuracy when using the proper technique in dispensing fluids was much higher when stopping the plunger at the first stop when withdrawing a fluid.
Looking at the error percentages of pipetting weights by P200 and P20 pipette units with the first stop technique, the percentage errors were all quite small, at 0.267% and 2%, respectively, for the water test fluid. However, the absolute error with the P20 pipette was smaller when factoring in pipetting volumes according to the following calculations,
P200: weight error=percent/100 weight =0.267%/1000.1g=0.000267g
P20: weight error=percent/100 weight =2.00%/1000.01g=0.000200g
. This meant that the P20 pipette had higher accuracy in dispensing a small volume. This difference implied that the smaller unit pipette should be chosen to perform a fluid transfer whenever possible. The larger unit pipette should only be used when a smaller pipette couldn't hold the needed volume quantity. For example, dispensing 15 μL of a fluid should be performed with P20, not P200; dispensing 150 μL of a fluid should be performed with P200, not P2000.
Conclusion
The proper use technique of micropipettes improves volume dispensing accuracy, as hypothesized at the beginning of this study. Specifically, the technique of stopping the plunger at the first stop during fluid withdrawal significantly reduces errors in dispensing volumes, as shown in the discussion. Furthermore, when choices among different micropipettes to dispense a fluid are available, the smallest possible micropipette that can hold the volume should be used instead of an overly large micropipette because the smaller pipette generally has higher accuracy and precision. Higher accuracy and precision improve an experiment’s contribution.
References
[1] Zagriadskaia, E. (2024). BIOCHEMISTRY TECHNIQUES BIOL 4210L/5210L Spring 2024. Umassonline.net. https://lowell.umassonline.net/bbcswebdav/pid-2666023-dt-content-rid-28390199_1/xid-28390199_1
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