Posted: February 2nd, 2015

Calculate the mean, standard deviation and coefficient of variation (%CV) for each column, and record the results in a table as follows:

Paper, Order, or Assignment Requirements

 

Experiment 1: The use of pipettes; precision and accuracy

 

Objective

At the end of this practical you should be able to:

  • Pipette volumes from 50 ml to 1 ml with precision and accuracy
  • Know the difference between precision and accuracy and be able to quantify each
  • Calibrate a pipette by weighing
  • Choose the appropriate pipette to use for delivery of a given volume

 

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:

 

  1. Personal factors related to the training, skill, care, and experience of the experimenter.
  2. Variant methods that result in greater error than others. This should be considered in the choice of any method.
  3. Instrumental errors that will vary with the precision and conditions of a particular instrument. Proper selection, care, and calibration of an instrument will help to minimise such errors.
  4. Quantity of material that is being assayed. As a general rule, the lower the quantity of material assayed, the greater the percentage error.

 

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

1a Pipetting small volumes into a microtitre plate using automatic pipettes

 

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.

  1. Use one half of a microtitre plate. Note the layout of the plate. There are 12 ‘columns’ labelled 1 to 12. There are 8 ‘rows’ labelled A to H.
  2. Pipette compound X (red) into the wells of the microtitre plate as follows, repeat each measurement 8 times

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)

  1. Read the plate at 450 nm.       Collect the printout.

 

Results 1a

  1. Label the columns of your printout and stick it into your results section.
  2. Circle the highest and lowest value of each set of 8. Calculate the range by subtraction and write in at the bottom of the column.
  3. Calculate the mean, standard deviation and coefficient of variation (%CV) for each column, and record the results in a table as follows:

 

 

 

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:

  1. C1 vs C2; which has higher CV and SD? Why?
  2. the 1000 ml and the 200 ml pipettes for pipetting 100 ml (C2 vs C7). Which is better?
  3. 2 x 150 ml using the 200 ml pipette with 1 x 300 ml using the 1000 ml pipette (C4 vs C6). Which is better?
  4. 2 x 50 ml using the P200 and 1 x 100 ml using the P200 (C5 vs C2). Which is better?
  5. Is there any difference in precision as you increase volumes dispensed using the same pipette (C2 vs C3; and C7 vs C6)?

How good were your replicates?

Was your pipetting precise? How do you know?

What factors may have contributed to imprecise pipetting?

 

1b Calibration of a pipette by weighing

Using the balance

  1. Check that it is level (is the bubble in the spirit level at the back of the machine centred?) If not ask a demonstrator to centre it.
  2. Place an empty vessel on the balance pan and allow the reading to stabilize. Press the tare bar to bring the reading to zero.
  3. Place the chemical in the vessel. (Dispense powdered chemicals with a suitably sized clean spatula).
  4. Allow the reading to stabilize and make a note of the value.
  5. If you add excess chemical, take great care when removing it. Never replace excess chemicals in the original container. Ask your demonstrator.

 

Method & Results

Take 1000 ml automatic pipette, fit a suitable (blue) tip and calibrate the pipette as follows:

  1. Place a square-base plastic beaker on the top pan balance and press the tare button to give a zero display. Close the side doors and open the top door of the balance.
  2. Dispense 1 ml of distilled water into the container. Record the weight in a table.
  3. Zero the balance using the tare button.
  4. Repeat steps 2 and 3 until you have 10 weights.
  5. Using your calculator get the mean, standard deviation and coefficient of variation of your results.
  6. Record values for individual weights, mean, SD and %CV (precision).
  7. Repeat steps 1-6 for 100 ml still using the 1000 ml pipette
  8. Calculate % accuracy for each volume

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:

  1. 9 ml
  2. 5 ml
  3. 200 ml
  4. 0 ml
  5. 350 ml
  6. 20 ml
  7. 28 ml

Experiment 2: Dilutions and Standards

 

Objective

At the end of this practical you should be able to:

  • Make a stock solution from solid compound
  • Make a series of dilutions from a stock solution, with precision and accuracy.
  • Construct a standard curve from these dilutions
  • Calculate the concentration of an unknown from your standard curve

 

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

2a Making a 0.1% stock solution of Tartrazine

% 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).

  1. Place a clean magnetic flea in a 150-200 ml container.
  2. Add 80 ml distilled water.       (Always add ~80% of final volume of water first. Add the chemical to the water, not the other way round).
  3. Weigh out 0.1 g tartrazine.
  4. Add to beaker, place on magnetic stirrer. Start slowly and gradually increase the speed to get sufficient mixing, without splashing.
  5. When fully dissolved, retrieve the magnetic flea from the beaker by carefully pulling it up the side of the beaker with a second flea on the outside. Ask your demonstrator!
  6. Place a funnel in the 100 ml volumetric flask. Carefully pour the contents of the beaker into the flask.
  7. Add a small amount of distilled water (~10 ml) to the beaker to rinse and pour this into the flask also.
  8. Check the volume on the flask.       Carefully add distilled water, dropwise to the flask until the bottom of the meniscus is up to the mark.
  9. Place the lid on the flask, and mix by inverting several times.

 

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)?

2b Making working dilutions (standards) from a stock solution

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.

  1. Prepare 1000 ml of each standard in 1.5 ml Eppendorf tubes (small, conical, plastic tubes with lid attached) as per table. Fill in the blanks in the table before the practical.

 

Conc required (mg/ml) Dilution ml stock soln (A) ml dist H2O (B) Total volume

(A+B)

100 1/?
80
60
40
20
0

 

  1. You are also provided with a solution of tartrazine at unknown concentration (sample). Pipette 100 ml of each standard in quadruplicate into a 96 well plate (each 100 ml into a separate well). See diagram below.
  2. Pipette 100 ml of the sample in quadruplicate into separate wells of the same plate.

 

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

 

  1. Read the absorbance at 450 nm on the plate reader. Show the results to your demonstrator.
  2. If there are large pipetting errors, repeat the pipetting step. If there are dilution errors, repeat your dilutions and pipetting.
  3. Construct a standard curve by plotting concentration (mg/ml) on the x axis (abscissa) vs absorbance on the y axis (ordinate). Paste your labelled printout into your copy also.
  4. Calculate the mean, standard deviation and CV for absorbance of standards and sample.
  5. Determine the concentration of the unknown in mg/ml.

 

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.

  • Always plot the independent variable on the x axis and the dependent variable on the y axis.
  • When plotting standard curves, always plot individual points not averages. If one estimation is grossly in error then this will be clear when drawing the curve, but would not be obvious from plotting the average value.
  • Every graph should be self-explanatory and should be correctly labelled with a title, scales, and legends.
  • Label each axis appropriately with the writing placed so as to read from left to right and from bottom to top. The quantity of the major divisions should be expressed as integers multiplied to the appropriate power of ten. Include the units!
  • Select a scale that places data over most of the graph rather than on just a portion. Try to have the graph take up most of the page, give equal space to equal differences in value. For a standard curve draw the line of best fit through the points.
  • Make the graph as simple as possible; do not include too many lines or symbols.
  • When more than one curve is plotted on a single graph, label each curve clearly and shape the points differently for each curve. Allowable symbols are open circles, filled circles, open and filled triangles, open and filled squares, x’s, plus signs, and a few others. Different curves may also be distinguished by solid lines, dotted lines, and various forms of broken lines.

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.

 

  1. The molecular weight of tartrazine is 534.4. What is the molarity of your stock solution? (1 M tartrazine = 534.4 g/L)

What is the molarity of each of your standards?

What is the concentration of your unknown in moles l-1?

 

  1. You are asked to make up 100 mL of a 2.5 mM solution of tartrazine. How much tartrazine    would you need?

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