Scientific Practice: The Glass Beads Exercise
Introduction
This exercise is about analytical methods and their Accuracy, Precision and (maybe) Sensitivity.
The exercise developed from a final year project to measure human body volume by the novel application of an old method (the method is called "dilution analysis"). The idea was to put a person (an object of irregular shape and unknown volume) into a large box (an object of known volume) and add a known amount of a marker gas to the air in the box. The degree to which the marker gas was diluted could be used to determine the volume of air in the box which, when subtracted from the volume of the box, would give the estimated volume of the person.
In this exercise, the large box is replaced by a small volumetric flask and the person of unknown volume is replaced by a number of glass beads; your task is to determine the volume of the beads. This simplified experimental model enables us to explore some of the problems associated with ‘doing science’. It should illustrate some of the elements of experimental design, the need for replicate observations, and the role of statistical analysis in scientific discovery.
The Problem (you should work in teams)
You are provided with…
· One 25ml volumetric flask containing a ‘spoonful’ of small glass beads
· A variety of standard laboratory materials and equipment (eg burettes, micropipettes, water, electronic balance, spectrophotometer, measuring cylinder, dye, etc.)
You are asked to…
· Apply methods using the equipment to measure the volume of the glass beads without removing the beads from the flask. [There are four methods to use, one of which is expected to be more accurate than the others (ie it yields a result which is closest to the true value) and can, therefore, be used as a 'reference' method against which to compare the others.]
· Plan how you will compare the other methods with the ‘reference’ method and determine the accuracy, the precision and the sensitivity (though this is less obvious) of each method.
Procedures (you do not have to do these in this order!)
1. Method 1 Indirect Weighing (the ‘Reference’ Method?)
1.1 Weigh the flask plus beads.
1.2 Fill the flask exactly to the mark with distilled water and re-weigh.
1.3 Remove some of the water (you don’t need to remove it all) and refill to the mark. Repeat this procedure to give a total of five measurements of the weight of flask plus beads plus water.
1.4 For later - calculate five estimates of the volume of the beads. [Should you consider the temperature of the water; is it important? Can you correct for any effect it has?]
2. Method 2 Measurement of the Volume by Subtraction
2.1 Starting with the flask (plus beads) filled to the mark, pour the water from the flask into a measuring cylinder, ensuring you do not lose any of the beads! Record the volume of water in the cylinder.
2.2 Refill the flask to the mark and repeat the procedure a further four times.
2.3 For later - from the values, calculate five estimates of the volume of the beads. [Obviously the beads and the cylinder retain some of the water as a surface film; this introduces a 'systematic' error into your observations. Is it large? Is it important? Is it measurable? Will it vary?]
3 Method 3 Measurement of the Volume by Addition
3.1 Empty the water from the flask containing the beads. Using a burette filled to the zero mark, add water to fill the flask to the mark and note how much was added.
3.2 Empty the flask, refill the burette and repeat a further four times.
3.3 For later - from the volumes added from the burette, calculate five estimates of the volume of the beads and record them in the table below. [Again, is water adhesion a problem?]
4 Method 4 Dilution Analysis Method using Methylene Blue
This is a more elaborate procedure. The principles have applications in biology (and elsewhere) where the more direct methods (1, 2 and 3 above) cannot be applied. Methylene blue is used here as a convenient coloured ‘marker’ substance. Other substances could act as markers, including radioactive compounds. Make sure the spectrophotometer is turned on for 20 min before use to allow it to warm up.
4.1 You are provided with a stock solution of methylene blue. Unless told otherwise, determine what approximate volume of stock solution you need to give an absorbance reading (at a suitable wavelength) of roughly 0.4 when diluted out to 25 ml with water (ie put in the flask and made up to the mark).
Note the wavelength ______
Note the volume of stock needed______
4.2 Prepare a 'standard' (aka 'reference' or 'known') solution of methylene blue by adding exactly the above amount of methylene blue stock to the empty 25ml volumetric flask and making it up to 25ml with water. After mixing, decant off some this into a labelled cuvette ready to have its absorbance measured.
4.3 Now use the flask with the beads. Add 10 to 15 ml of water then add the same amount of the methylene blue stock solution noted in 4.1, then fill to the mark with water and mix well. Decant off some of the resultant coloured solution into a labelled cuvette ready to have its absorbance measured.
4.4 Rinse the flask with the beads to remove the methylene blue and repeat step 4.3 a further four times (to give four more ‘unknowns’). [Note if the methylene blue colours the beads; if it does, could this be a source of error, and what can you do about it?]
4.5 Zero the spectrophotometer against a water blank at the chosen wavelength. Measure the absorbance of the 'standard' sample prepared in step 4.2 and the five 'unknown' samples. Record the absorbance values to as many figures as possible.
4.6 For later - from the recorded absorbance values, calculate five estimates of the volume of the beads and record them in the table below. This will require some logical manipulation of the numbers; try to figure out how to do that, bearing in mind that if you double the volume in which you dilute a fixed quantity of dye, then the absorbance of that solution will halve - so, absorbance and volume vary in a simple, inversely proportional way (ie Ab1 x V1 = Ab2 x V2).
5 Sensitivity (if time allows)
We can ask a simple question related to sensitivity; is our reference method able to detect a change in the number of glassbeads present?
5.1 Remove (or add – it’s up to you) one glassbead from (to) the flask.
5.2 Now repeat Method 1.
Data Analysis (do in the lab if you have time)
6.1 Calculate and record the estimated bead volumes from the methods into the tables above. You might want to do this before you leave the laboratory, for if there are any gross errors in your procedures you will have the opportunity to rectify them. Note that CV% is the ‘coefficient of variation’; it is SD/Mean*100
6.2 A computer-based tutorial on the analysis of your data is available, though we will go through the analysis during the next Collaborate session.
6.3 You should also determine whether there were significant differences between the results gained by the different methods (and if the ‘reference’ method was sufficiently sensitive to pick up removal or addition of a single glassbead). Note that in applying a ‘t-test’ (see online tutorial), you should consider if the variances of the two populations are similar. Again, we will go through this part of the analysis during the next Collaborate session.
2