Introduction Pipetting Lab Report PDF

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Pipetting Lab in biochemistry to properly use micropipettes ...

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Ashley Nguyen Fall

Experiment 1. Pipetting Lab

August 24

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Data and Observations Table 1. Theoretical Density and Mass of Water and Dish Soap

Water Dish Soap

Density Actual Mass of 750 µL (mg/µL) Sample (mg) 750 1.00 1.01 758

Actual Mass of 75 µL Sample (mg) 75.0 75.8

Actual Mass of 7.5 µL Sample (mg) 7.50 7.58

The listed densities of water and soap are the literature values. These densities were used to determine the actual mass of water and soap for the corresponding volumes. These values do not reflect the theoretical results found in the lab. Table 2. Average Mass of Water Delivered from Micropipettes

Average Mass of 750 µL Average Mass of 75 µL Average Mass of 7.5 µL

Micropipette Capacity 1000 µL Micropipette 200 µL Micropipette 932.3 ± 4.3 mg --262.5 ± 11.8 mg 147.3 ± 3.8 mg --75.5 ± 4.0 mg

20 µL Micropipette ----10.1 ± 0.9 mg

Each listed mass for water is the average of three trials. The standard deviation (uncertainty) for each set of trials was found and included above. Table 3. Average Mass of Dish Soap Delivered from Micropipettes

Average Mass of 750 µL Average Mass of 75 µL Average Mass of 7.5 µL

Micropipette Capacity 1000 µL Micropipette 200 µL Micropipette 791.6 ± 7.2 mg --233.1 ± 16.0 mg 98.4 ± 2.5 mg --64.9 ± 5.9 mg

20 µL Micropipette ----8.4 ± 0.9 mg

Each listed mass for soap is the average of three trials. The standard deviation (uncertainty) for each set of trials was found and included above. Table 4. Theoretical Density of Water and Dish Soap Water Dish Soap

Density (mg/µL) 1.35 ± 0.12 1.12 ± 0.12

The densities listed above were determined using the data recorded for mass of 7.5 µL of water and soap delivered by a 20 µL micropipette. The standard deviation (uncertainty) for this set of data was calculated and reported above

Figure 1. Mass of 750/75 µL of Water and Soap Delivered from 1000 µL Micropipette

1000 µL Micropipette: Mass of 750/75 µL of Water and Soap 1000

750 Microliter

928.0 - 936.6

950

75 Microliter

900

Average Mass (milligrams)

850

784.4 - 798.8

800 750 700 650 600 550 500 450 400 350 300

250.7 - 274.3 217.1 - 249.1

250

200 Water

Soap Type of Substance

Graph Analysis As shown in “Figure 1,” the result from pipetting 750 µL with a P-1000 micropipette was better than the results from pipetting 75 µL for both water and dish soap. The standard deviation of the data recorded for 750 µL was lower. This means that these values were closer together than the values recorded for 75 µL. Therefore, it was more precise to pipette 750µL than 75µL from the P-1000. Note, the volume that yielded the more precise answer was within the range of volumes recommended by the micropipette, and there was more deviation in the measurements for dish soap than there was for water.

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Figure 2. Mass of 75/7.5 µL of Water and Soap Delivered from 200 µL Micropipette

Average Mass (milligrams)

200 µL Micropipette: Mass of 75/7.5 µL of Water and Soap 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50

143.5 - 151.1

75 Microliter 7.5 Microliter

95.9 - 100.9

71.5 - 79.5 59.0 - 70.8

Water

Type of Substance

Soap

Graph Analysis Just as “Figure 1” displayed, “Figure 2” also demonstrated that pipetting a volume within the range of volume on the micropipette yields the more precise data. In both substances, there was less deviation pipetting 75 µL than there was pipetting 7.5 µL from the P-200. While the standard deviation was not low for 75 µL trials, it was a smaller value than the standard deviation found for the 7.5 µL trials. This was more evident in the results for dish soap.

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Figure 3. Mass of 75 µL of Water and Soap: 1000 µL vs. 200 µL Micropipette

Average Mass (milligrams)

Mass of 75 µL of Water and Soap from 1000/200 µL Micropipette 290 280 270 260 250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80

1000 Micropipette 200 Micropipette

250.7 - 274.3

217.1 - 249.1

143.5 -151.1

95.9 -100.9

Water

Soap Type of Substance

Graph Analysis “Figure 3” compares the results of pipetting the same volume of a substance, 75 µL of water and soap, from two different micropipettes, the P-1000 and P-200. The standard deviation of the data recorded from using the P-1000 was significantly higher than the standard deviation found for the data recorded using the P-200. This developed from the scattered values recorded for the P-1000 trials. For both water and dish soap, pipetting 75 µL from the P-200 gave the more precise results. As observed in Figures 1 and 2, the more precise mass was found when the pipetted volume was within the volume range listed on the micropipette.

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Figure 4. Mass of 7.5 µL of Water and Soap: 200 µL vs. 20 µL Micropipette

Mass of 7.5 µL of Water and Soap from 200/20 µL Micropipette 90 85 80

200 Micropipette 20 Micropipette

71.5 - 79.5

75

59.0 - 70.8

Average Mass (milligrams)

70 65 60 55 50 45 40 35 30 25 20 15 10

9.2 - 11.0

7.5 - 9.3

5 Water

Soap Type of Substance

Graph Analysis “Figure 4” revealed the substantial difference between using a P-200 and a P-20 to pipette 7.5 µL. The standard deviation for the data recorded from using the P-20 was significantly lower than the standard deviation found for the data recorded from the P-200 trials. The values from the trials using the P-200 was much more inconsistent. For both water and dish soap, pipetting 7.5 µL from the P-20 gave the more precise results. As observed in graphs 1, 2, and 3, pipetting a volume within the range on the micropipette led to the most precise results.

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Results and Discussion The Pipetting Lab allowed students to practice proper use of lab equipment such as micropipettes and analytical scales. The performed experiment also provided a foundation for students to begin thinking scientifically by formulating valuable experimental observations and analyzing pertinent theoretical data to make logical conclusions. In this experiment, it was learned that micropipette size has an effect on volume uptake. In all cases, shown in Figures 1-4, the volume of the substance that yielded the more precise mass was within the volume range on the micropipette. This indicates the importance of obeying the recommended range on each micropipette and selecting the appropriate micropipette to deliver a specific volume of liquid. For example, the P-1000’s volume uptake range was 200-1000 µL; thus, as seen in the standard deviation observed in Figure 1, for water and soap, the mass recorded when 750 µL was pipetted was more precise than the mass recorded when 75 µL was pipetted. The same was noted with the P-200 when 75 µL and 7.5 µL of water and soap were weighed (Figure 2). The mass found for 75 µL of substance had less deviation, and therefore was more precise. Figures 3 and 4 also help confirm this indication. In Figure 3, P-1000 and P-200 were used to deliver 75 µL of liquid. For both water and soap, the results were more precise when the P-200 was used. In Figure 3, P-200 and P-20 were used to deliver 7.5 µL of liquid. As expected, the results were more precise when the P-20 was used. In both cases, the more precise results presented with less deviation. Thus, it was concluded that in order to increase precision while using a micropipette, the volume pipetted must stay within the recommended volume range for that specific micropipette. Straying from this range increases pipetting error, decreases precision, and skews the final result. A second factor that contributes to the precision of the results from pipetting is the viscosity of the substance being pipetted. Viscous liquids such as dish soap have more restricted movement than water. This constraint not only decreases the uptake and release rate of the substance but also increases the chances of getting air bubbles in the plastic tip of the micropipette. This would cause the micropipette to contain a lower volume than what is desired. Also, because dish soap moves so slowly, it was possible that some soap remained in the pipette. In either case, the volume of the soap actually massed would be less than the designated amount. This suggests that the volume of soap actually measured from trial to trial could differ significantly. Table 1 and 2 confirm that, overall, the average mass of soap had more deviation and uncertainty than water. This has a drastic negative affect on the precision of the results. Two other factors that may affect pipetting error include the angle at which the liquid is being drawn into or released from the pipette and the depth at which the plastic tip on the pipette is pushed into the liquid. It is possible that the angle in which the experimenter holds the pipette is not consistent from one trial to the next. This could result in skewed data. Secondly, with viscous liquids like dish soap, pushing the plastic tip on the pipette deeper into the substance would allow the soap to coat the external layer of the tip. This coat could be transferred and massed with the volume inside the pipette. In order to minimize the potential errors in the experiment caused by pipetting, it is essential to know the proper techniques of using a micropipette. It is important to stay inside the volume range given on each individual micropipette. When dealing with a viscous liquid, it is most effective to aspirate slowly. It is also necessary to allow the pipette tip to be completely filled with the liquid before removing it. This will help avoid the formation of air bubbles and increase the likelihood of obtaining an accurate volume. It is also important to maintain a consistent method and angle while pipetting to decrease the chances of making more systematic error. Lastly, to prevent getting too much volume of the liquid on the outside of the pipette Nguyen 6

tip, do not plunge the tip into the solution. Place the tip far enough into the solution to pull up the substance while not exposing the entry on the tip to the air. After analyzing the data, the average masses of 7.5 µL of water and soap pipetted from the P-20 micropipette were used to determine their densities. This decision was made because the pipetted volume is support by the P-20 micropipette and because this set of data had the lowest standard deviation (uncertainty). The density of water was found to be 1.35 ± 0.12 and the density of the soap was calculated to equal 1.12 ± 0.12. While these values are from the most precise set of data, they are not very accurate. Compared to the literature values (Table 1), the percent error for the found densities of water and soap are 35.0% and 10.9%, respectively.

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