AbstractThere are twoparts to this experiment. The first part involves determining error analysiswith the use of micropipettes. Learning about error analysis is crucial indetermining the most important errors and whether they have a significance inthe final results. From the results, it was determined that the P-200 pipetteis preferred for more accurate and precise measurements.
Part two of thisexperiment focuses on preparing two acid buffer solutions to test. Thepreparation of buffers is a common laboratory technique in biochemistry. Thepurpose of using buffers is to hinder any changes in pH when an acid or base isadded in a solution. A buffer solution with the assigned pH value of 8.0 iswhat will be prepared in this experiment to test. The resulting pH values of thetwo buffers measured to be 8.1 and 7.96.
There is a small discrepancy of pHbetween the theoretical and experimental pH values. Introduction Micropipettesare ubiquitous in biochemical laboratory experiments. It is a reliableprecision instrument that allows for taking accurate and precise measurementvolumes of liquids. Despite its apparent importance, the origin of themicropipette should not be taken for granted. The micropipette was firstdiscovered by German physician Heinrich Schnitger at the University of Marburgin 1957 (Klingenberg, 2005). This breakthrough has revolutionized modernbiotechnology and molecular biology with handling of small liquid volumes. However,pipetting errors do exist and failure to accurately pipette will result in anexperiment being irreproducible.
That is why the accuracy of the pipettes relyon the investigator. Pipettes need to be maintained and practiced with goodtechnique with a proper understanding of how they work. The first portion ofthis laboratory experiment will focus on getting acquainted with the use ofmicropipettes by using different microliters to measure out water. This methodwill be useful in performing a statistical analysis for each set of results. Thereare various micropipettes that are used in the laboratory that range in sizesof P20, P200, and P1000. These sizes are denoted at the top of the pipette.
Theyalso dispense liquids between the ranges of 1 and 1000 ?L (Prilliman, 2012).However, in this experiment, the types of micropipettes that will be used rangefrom 0.1 to 2.5 ?L. Before using the micropipette, it is important to masterits use before carrying out any experiment. Liquids are never drawn directlyinto the shaft of the pipette.
Hence, why there are plastic disposable tipsthat attached to the shaft of the micropipette. S for adjusting the volume ofthe 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 boxare generally used for the micropipettes with the size of P20 and P200 whereasthe large, blue tips are used for the P1000 micropipette. Thesecond part of the experiment involves creating a buffer solution. Since mostsolutions drastically alter pH change, buffers serve the purpose of resistingthat change.
Buffers are fundamental in many biological mechanisms; which,require a stable pH range in order to carry out biochemical reactions. This isoften the case when proteins or enzymes are looked at. Take for instance the pHrange of human blood. It is necessary that the range is maintained between pHlevels of 7.
35 to 7.45; otherwise, the hemoglobin will not successfully bind tooxygen which will then result in a homeostatic imbalance (Singer et al., 1948). Fora buffer solution to occur, two species are required. One that can react withhydroxide (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 oneanother. Many buffer solutions are generated by combining a weak acid and itsconjugate or by combining a weak base and its conjugate.
In this experiment, thebase tris and the acid HCl will be used to prepare the buffer solution. Bufferssolutions are generally effective in a pH range of +/- one pH unit on eitherside of the pKa (Stewart et al., 2009). The Henderson-Hasselbalch equation isdenoted below to provide the necessary information on preparing a buffersolution: When generating a buffer solution, it is important tonote that there is a threshold to the amount of acid or base that can be addedto a buffer solution before one of the components is used up.
The limit is bestknown as the buffer capacity and is defined as “the moles of acid or basenecessary to change the pH of one liter of solution by one unit” (Stewart etal., 2009). Experimental Procedure For part one of the experiment, it involvedunderstanding the mechanism of the pipette. For starters, there are sixdifferent measurements to be recorded: 1000 ?l of the P-1000, 100 ?l of theP-1000, 100 ?l of the P-200, 20 ?l of the P-200, 20 ?l of the P-20, and 5 ?l ofthe P-20 respectively. The P-1000 was the first pipette used in this part ofthe procedure. To obtain the desired volume, the pipette was set to 1000 ?l byadjusting the dial. Afterwards, a sterile blue tip was loaded for the P-1000pipette.
Precaution was taken to prevent contaminating the tip. A 100-mL glass beaker was filled with 50 mL ofdeionized water. To load the water into the pipette, the plunger was presseddown slowly to the point of first resistance. While the plunger was still heldat the first stopping point, the tip was inserted into the solution ofdeionized water but not too deep. Then, the plunger was slowly released to drawup the deionized water. The pipette tip was filled to final volume beforeremoving 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 dispensingthe sample of deionized water into the cuvette by pressing the plunger down thepoint of second resistance. At this point was when the measurement in grams ofthe sample was recorded.
The process of loading the deionized water from thebeaker 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 fivetrials. The cuvette was emptied and the tip of the pipette was discarded. The above procedure was repeated for the rest of thefive 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 filledwith the measurements of all the pipettes, a statistical analysis was performedto determine the mean, standard deviation, relative error, and relativestandard deviation. The second portion of the experiment dealt with creatinga buffer solution.
To start, the molarity and the volume of the tris buffersolution to be made was determined. Then the number of moles required wascalculated by multiplying the molar concentration of the buffer by the volumeof the buffer that will be made. Afterwards, the amount of tris in grams wasdetermined by multiplying the number of moles by the molecular weight of thetris. These calculations can be viewed under the calculations section of thelab report. Next, 1.
211 grams of tris was dissolved into 80 mL ofdistilled deionized water. This was 80% of the final volume. 1 M HCl wascollected in a separate beaker.
By using a pipette, the HCl was added into thesolution 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 intoa 100-mL glass beaker. Before taking the pH of the buffer, the pH meter wascalibrated. Once it was finally calibrated, the pH of the 50-mL solution of thetris-HCl was measured. This same process was repeated for another 50-mLtris-HCl solution. Once the solutions were measured and recorded, the tools,glassware, and instruments at the lab station were cleaned and put away. ResultsCalculationsHow many grams of tris: How many moles of tris: Expected pH of buffer: Relative error frompipetting:Trial 1:Trial 2:Trial 3:Trial 4: Trial 5: Relative Standard errorfrom pipetting: DataFigure 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 micropipetting 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 desiredvolume in microliters. This chart represents five different trials for eachpipette and volume. The measurements for each trial are recorded in grams. Atthe end of each of the 5th trials, there is an average and astandard deviation. Figure 2-1Figure 2-1. Graphicalrepresentation of tris-HCl buffer solution. Also in the image is the pH meterthat was used to measure the pH of the buffer. Figure 3-1Figure 3-1.
Graphicalrepresentation of the pH readings of the tris-HCl buffer solution after twotrials. Discussion The reference pipetting method used in each of the trialsin part one was used to determine the precision and accuracy of each pipettingand volume. Based on the data and calculations from this experiment, the P-200is the preferred pipette for accurate measurements. This was determined bycalculating the standard deviation, relative standard deviation, and relativeerror.
The standard deviation for the P-200, 20 microliters was 0.000365 whilethe relative standard deviation and the relative error were 0.93% and 1.0%respectively.
The standard deviation indicates the distribution of valuesaround the average (Thomas, 1996). It is preferred for the standard deviationto be tight around the mean. The relative standard deviation indicates whetherthe regular standard deviation is a small or large value when compared to theaverage of the data set. The relative standard deviation for the P-200, 20microliters 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 istightly clustered around the average. If the percentage were to be a lotbigger, then that would indicate that the values are more spread out. Although the results appeared to have stayedrelatively close to the mean, it does not indicate that errors will not existin the lab even with the highest precautions.
An effective strategy toeliminate any effects in accuracy, precision, and trueness would be to avoidprolonged 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 avoidcontaminating both the sample and the tip. Any contaminations may have anadverse effect on the final reading. Finally, it is required that the plungeris pushed to the correct point of depression otherwise bubbles may form in thetip 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 personalpreferences, background, and training.
These dissimilarities in techniques caneither negatively or positively influence the precision, accuracy, and truenessof results from an experiment. To ensure pipetting accuracy, it is importantthat laboratories adopt standard operating procedures for pipetting strategiesand verify that all operators are professionals and adequately trained. Byincreasing the amount of consistency in results acquired, the level of qualityand credibility of the experiment will be enhanced. The second part of the experiment dealt withgenerating buffer solutions. Buffers consist of a weak acid and its conjugatebase.
In this experiment, the tris was the base that was used and HCl was heacid. This system allowed to absorb either H+ or OH- due to the reversiblenature of the dissociation of the HCl. HCl can release H+ ions to neutralizeOH- and form water.
When generating a buffer, it is important to bemindful of the factors that constitute a good buffer. The first is that thebuffer must have a pkA between 6 and 8. In this experiment, the pKa of thebuffer was 8.1, which is not significantly far from the range.
The mostimportant factor of a buffer is that is must have chemical stability. In otherwords, the buffer must be stable and should not break down under workingconditions. By any means should the buffer oxidize or be influenced by thesystem in which it is being used. Therefore, buffers with metabolites forinstance should be avoided as much as possible. Metabolites are defined asproducts that serve the purpose of breaking down or metabolizing an entity intoa different substance (Berdy, 2005). The results from this experiment show that theexperimental 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 secondtrial. The reason for the decrease in pH after the second trial may have mostlikely been due to human error. HCl was added in a drop-wise fashion into thesecond 50-mL beaker when that was not necessary. This was done in hopes ofreaching the desired pH.
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