Posted: February 2nd, 2015
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Objective
At the end of this practical you should be able to:
Accuracy
A measurement is accurate if it gives the true value. If you attempt to pipette 1.00 mL of water, which should weigh 1.00 g, and the balance reads 1.00 g after you dispense the solution, your pipetting was accurate. Percentage accuracy can be calculated as follows: [(True value – your value)/true value] X 100. In the example below [(1.0 -0.7)/1.0]x100 = 30% inaccurate even though it was 0% imprecise.
Precision
Measurements are precise if the same measurement can be made again and again. For example if you try to dispense the 1.00 ml with the pipette ten times, and you dispense 0.70 ml ten times in a row, your pipetting was very precise, but it was inaccurate. Among the factors which affect precision and accuracy are:
Automatic pipettes
These come in two types: air displacement and positive displacement. You will use an air displacement pipette. An automatic pipette must be fitted with the correct disposable tip before use.
Correct procedure for using an automatic pipette
Select an automatic pipette that operates over the appropriate range. Never attempt to set the volume above the maximum limit. Set the volume to be delivered. Fit a new disposable tip to the end of the barrel by pressing the tip on firmly using a slight twisting motion. Never, ever try to use an automatic pipette without its disposable tip. Draw up the appropriate volume as follows: hold the pipette vertically; press down on the plunger until a resistance is met (first stop). Immerse the tip into the sample liquid to a depth of 2 to 4 mm. Keeping your thumb on the plunger, release the pressure slowly and evenly: check that no air bubbles are present. Wait 1 to 2 seconds, then withdraw the end of the tip from the liquid. Place the tip end against the inside wall of the receiving vessel at a slight angle and depress the plunger slowly to the first stop. Wait 2 to 3 seconds, then depress the plunger to the second stop to achieve final blow-out. Withdraw the pipette from the vessel carefully with the tip sliding along the inside wall of the vessel. Allow the plunger to return to the up position. Depress the tip ejector button to discard the tip. You may use the same tip for replicates of the same sample.
Method
In this part of the experiment you will check the precision and accuracy of your pipetting.
You will also compare different pipettes for delivering the same volume of liquid.
Column 1: 100 ml to each well using the 20 – 200 ml pipette
Column 2: same as column 1
Column 3: 200 ml to each well using the 20 – 200 ml pipette
Column 4: 250 ml to each well using the 20 – 200 ml pipette; set to 150 ml and put 2 X 100 ml into each well.
Column 5: Set the 20 – 200 ml pipette to 50 ml. Deliver 2 X 50 ml (to give 100 ml) to each well
Column 6: 250 ml to each well using the 100 – 1000 ml pipette (set to 250 ml)
Column 7: 100 ml to each well using the 100 – 1000 ml pipette (set to 100 ml)
Results 1a
|
C1 100 ml (P200) | C2 100 ml (P200) | C3 200 ml (P200) | C4 300 ml (P200 x2) | C5 100 ml (P200 x2) | C6 300 ml (P1000) | C7 100 ml (P1000) |
Mean | |||||||
SD | |||||||
% CV |
To calculate standard deviation, use the stats function on your calculator. Please familiarise yourself with this before the practical.
Mean is the average, i.e. add up all the values in a column and divide by the number of values (8 in this case).
Sample standard deviation is calculated as follows:
But don’t worry, your calculator will do it for you. Note that it is sample standard deviation, not population standard deviation, that you need.
Coefficient of variation: This is the (standard deviation / mean ) X 100. It indicates the level of precision. %CV of less than 1% is precise.
Discussion
Use the tabulated results to compare:
How good were your replicates?
Was your pipetting precise? How do you know?
What factors may have contributed to imprecise pipetting?
Using the balance
Method & Results
Take 1000 ml automatic pipette, fit a suitable (blue) tip and calibrate the pipette as follows:
If you are using the pipette correctly, your CV should be less than 1%. (Results 1b)
Discussion
How accurate and precise were your results?
Was there any difference in the accuracy and precision achieved at 100 ml and 1 ml?
Was there any difference between the accuracy and precision using this method and 1a? If so what factors may have contributed to these differences?
Questions
Which of the following pipettes would you use to dispense the volumes below?
Pipettes:
P5000 (range 1-5 ml)
P1000 (range 100-1000 ml)
P200 (range 20-200 ml)
P20 (range 5-20 ml)
Volumes:
Objective
At the end of this practical you should be able to:
Preparing Solutions
Solutions are usually prepared with respect to their molar concentrations (e.g. mmol l-1) or mass concentrations (e.g. g l-1). Both can be regarded as an amount per unit volume, where concentration = amount/volume.
For molar concentrations you will need the relative molecular mass of the compound, so that you can determine the mass of substance required. To avoid waste think carefully about the volume of solution you require, but err on the high side to leave room for error. Use distilled water to make up aqueous solutions and stir to make sure all the chemical is dissolved.
Stock Solutions
Stock solutions are valuable when making up a range of solutions containing different concentrations of a reagent. The stock should always be made up in a volumetric flask. This is the most accurate way of making solutions. For routine work further dilutions may be made up in Eppendorf tubes or test tubes etc., but remember this is not as accurate.
Method
% Solutions are the easiest to calculate, because they do not depend on knowledge of the molecular weight. % w/v means percent weight to volume and has units of grams/100 ml. Therefore a 0.1% solution has 0.1 g of solute in a total of 100 ml of solution (not solute).
Pre-Lab questions
0.1% = ? mg/100 ml?
0.1% = ? mg/ml?
What is the concentration of your stock in mg/ml?
What dilution of your stock do you need to get the first standard (100 mg/ml)?
Many assays for drugs or biochemicals do not give an absolute value, but instead require standard solutions, containing known appropriate concentrations of the drug or biochemical, to be assayed at the same time. These standards are used to construct a ‘standard curve’ from which the concentrations of the samples are estimated.
Conc required (mg/ml) | Dilution | ml stock soln (A) | ml dist H2O (B) | Total volume
(A+B) |
100 | 1/? | |||
80 | ||||
60 | ||||
40 | ||||
20 | ||||
0 |
The numbers in the table refer to the standard concentrations not volumes.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
A | 100 | 100 | 100 | 100 | ||||||||
B | 80 | 80 | 80 | 80 | ||||||||
C | 60 | 60 | 60 | 60 | ||||||||
D | 40 | 40 | 40 | 40 | ||||||||
E | 20 | 20 | 20 | 20 | ||||||||
F | 0 | 0 | 0 | 0 | ||||||||
G | Sample | Sample | Sample | Sample | ||||||||
H |
Drawing graphs
A graph is a plot of two quantities. One is something that you control and is called the independent variable. The other is the quantity that changes as you change the independent variable. It is called the dependent variable.
Discussion
Did you get a linear standard curve? Were any points ‘out’, was any dilution ‘out’?
Was your pipetting precise and accurate (precision shown by CV, accuracy by linearity of standard curve)?
What value did you get for your unknown? How did this compare to the actual value? (You will be given this later).
If the standard curve did not work out can you say where you went wrong?
Questions
Molarity means molar concentration, expressed as moles of solute per litre volume of solution (mol l-1). One molar (M; mol l-1) = the molecular weight in grams per litre.
What is the molarity of each of your standards?
What is the concentration of your unknown in moles l-1?
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