1: Gravimetric Calibration of Micropipettes
- Page ID
- 169726
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Ⅰ.Introduction
To ensure the accuracy of the experiment, it is essential that the measuring devices used in the experiments be accurate and reliable. That is, it has to be calibrated at the beginning of the experiment. Micropipette is one of the most important measuring devices used in modern biochemistry laboratory. Today‘s experiment is the gravimetric calibration of volumetric pipette.
Ⅱ.Materials and Methods
A. Materials and Equipment:
1. Deionized water
2. 50 ml beaker
3. Micropipette and tips
4. Electronic analytical balance
B. Procedure:
1. Select one of the Micropipettes (20, 200 or 1000 ml).
2. Bring some deionized water with a clean beaker (50-100 ml), allow it to reach room temperature (record the temperature).
3. Put a weighing dish on the electronic analytical balance and zero.
4. Pipette 10, 100 or 500 ml of water, respectively with 20, 200 and 1000 ml pipette and transfer the water content to the weighing dish. Record the weight.
5. Repeats step 3 and 4 for a total of 10 times.
6. Reset pipette to its max. volume setting after use.
Tips for Pipette Calibration
The process of pipette calibration can be very involved and complicated if the correct steps are not followed. Follow the few guidelines listed below to make the process a little less tedious.
1. Prior to performing any type of pipette calibration, clean and inspect the pipette, both internally and externally. A clogged nosecone can cause the pipette to deliver inconsistent volumes, even though the calibration on the pipette might be correct.
2. If any parts are worn or damaged, replace them prior to starting the calibration process. If a seal or O-ring is worn, it could cause the pipette to leak and deliver inconsistent volumes, but the calibration on the pipette could be fine.
3. Use the manufacturer’s recommended pipette tips. Many times the pipette tip can be the reason why the pipette is not delivering the correct volumes.
When beginning the process of gravimetrically testing pipettes, the following conditions must be fulfilled:
1. Evaporation protection for the weighing chamber. This can be done with the aid of moist blotting paper or an evaporation trap.
2. Testing area should be free of any drafts, direct sunlight or heat.
3. Distilled and degassed water should be used as a sample solution.
4. The pipette, the water, and the room must be maintained at a constant temperature of 20-25 oC ± 0.5 oC.
5. When working with volumes £ 20 ml water should be removed from the weighing dish. When working with volumes ³ 20 ml liquid should be added to the weighing dish.
Proper Pipetting Technique
Proper pipetting technique, in addition to a calibrated pipette will ensure that the correct volume is aspirated and dispensed.
Correct Tip Immersion
Pipette tip is immersed only a few millimeters in solution and is held at a 90o angle from the solution.
Incorrect Tip Immersion
Increasing the angle of the pipette or the depth at which the tip is immersed can increase the hydrostatic pressure and there fore increase the volume aspirated. A 30o incline can increase the liquid column by 0.15%, resulting in improper volumes.
III. Results
- Reorganize your data in the following table.
Room temperature on the day of the experiment: __________℃
Density of water at this temperature (unit): __________
** Please record to the next decimal place(請紀錄至小數點下一位)
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P1000 |
P200 |
P20 |
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Pipette number |
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Set Volume (ul) |
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Measured weight (mg)
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1 |
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2 |
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3 |
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4 |
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5 |
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6 |
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7 |
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8 |
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9 |
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10 |
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Mean (mg) |
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SD (mg) |
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CV% |
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Measured Vol. (ul) |
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% Error |
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- Calculate the mean, standard deviation (SD = {[Sx2 - (Sx)2/n)]/(n-1)}1/2 ) and % coefficient of variation (% CV = [SD/mean] x 100 ).
- Calculate the percent error by first converting the weight of water to volume: % Error = [(Experimental value - expected value) / Expected value] x 100.
Ⅳ.Question and discussion
1. 為什麼本實驗需要知道實驗時的水溫是多少?
如果水溫改變，會影響測量的「準確度」還是「精確度」?
Why it is important to know the temperature of water in the conversion of weight to volume? If the temperature changes, "accuracy" or "precision" of the measurement will be affected?
2. 請寫出下列名詞的定義及在本實驗中代表的意義: SD, CV%, and Error%。
Please define SD, CV%, and Error%, and explain their meanings in this exercise.
3. 請比較你和旁邊兩組的P1000數據，哪一組的P1000吸量管最不準確? 請說明你的理由。
Which P1000 pipette, among yours and your neighbor groups’, is the least reliable one?
Please describe your reasoning based on the values of mean, SD, or CV%.
4. 根據你得到的P1000、P200和P20三支吸量管的數據判斷，哪一支吸量管最不準確?
請說明你的理由。
Which pipette, among the ones you tested, is the least reliable one?
Please describe your reasoning based on the values of mean, SD, or CV%.
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