Students must come to the practical class with written answers to the following QUESTIONS. If NOT completed and submitted before class commences 50% of the total possible marks for the weeks exercise will be deducted. Demonstrators will check these during class and return to students. The Pre-Practical exercise must be submitted with the report’. Both the exercise below and the Assay of Glucose require the preparation of a standard curve. A standard curve, which is determined using a standard solution, relates data for two variables, e.g. absorbance and concentration. These data are connected with a curve of best fit. Consider the data below for a 2.0 mM solution of glucose, which has been diluted to cover a range of concentrations appropriate for a planned experiment.
Tube Number 1 2 3 4 5 6 Volume of 2.0 mM glucose (ml) - 0.2 0.4 0.6 0.8 1.0 Volume of water (ml) 1.0 0.8 0.6 0.4 0.2 - Glucose (µmoles/tube) - 0.4 0.8 1.2 1.6 2.0 Absorbance at 500 nm 0.05 0.30 0.76 1.01 1.23 1.35
A standard curve for a solution consists of a plot of the amount of glucose as the x-axis versus the absorbance at 500 nm as the y-axis. The data are plotted in the figure below. The correct curve of best fit is the unbroken line. The dotted curve simply connects the points and is not a curve of best fit. The curve of best fit is drawn so that the sum of the distances of any points offset from the curve is the same on either side of the curve. Thus, in the simple example shown below, the two offset points are equidistant from the curve.
In this graph, the linear region of the graph where the Beer-Lambert Law is obeyed lies between 0 and 1.2 µmol of glucose per tube. Continued next page Tube Number 1 2 3 4 5 6 Volume of 2.0 mM glucose (ml) - 0.2 0.4 0.6 0.8 1.0 Volume of water (ml) 1.0 0.8 0.6 0.4 0.2 - Glucose (µmoles/tube) - 0.4 0.8 1.2 1.6 2.0 Absorbance at 500 nm 0.05 0.30 0.76 1.01 1.23 1.35 Linear region Pre-Practical Exercise continued The axes of a standard curve need to be labelled, with the units specified, where appropriate.
Thus, in the current example, the x-axis is entitled Glucose and the units are µmoles/tube and the y-axis is entitled Absorbance at 500 nm; no units are specified for the latter as absorbance has no units. Each axis also needs to be marked into divisions (the scale) to allow the data to be plotted and read accurately. These divisions usually involve multiples 1, 2, 5 or 10, or 0.1, 0.2, 0.5 and 1.0, etc. and not 3, 7, etc. which do not allow data to be plotted accurately using the grid of graph paper or read accurately from the graph. Furthermore, the scale should be chosen so that the resulting curve has a gradient of 45° with respect to the x-axis and y-axis; this minimises errors due to scaling.
Exercise The haemoglobin (Hb) content of blood is normally within the following ranges: males 130-180 g per litre females 110-160 g per litre. It can be ascertained by treating a blood sample with a reagent containing an excess of ferricyanide and cyanide, which converts both Hb and oxygenated Hb into cyanomet-Hb. The latter can be determined conveniently from its absorbance at 540 nm by comparison with a standard curve. In order to construct a standard curve Tubes 1-5 (see below) were prepared using a standard solution containing 0.5g Hb per litre. At the same time a 0.01 ml sample of blood was diluted with water and treated with ferricyanide/cyanide reagent (Tube 6).
All tubes had a total volume of 5ml. The absorbance at 540 nm of Tubes 1-6 was measured and is recorded in the protocol below. Tube Number 1 2 3 4 5 6 (sample) Volume of standard Hb solution (ml) - 1.0 2.0 3.0 4.0 - Volume of cyanide reagent (ml) 1.0 1.0 1.0 1.0 1.0 1.0 Volume of water (ml) 4.0 3.0 2.0 1.0 3.99 Volume of blood (ml) - - - - 0.01 Absorbance at 540 nm 0.025 0.090 0.160 0.230 0.290 0.210 Amount of Hb (mg/tube) Determine the amount of Hb in Tubes 1-5 and enter these data in the protocol. Using the data for Tubes 1-5, construct a standard curve by plotting the absorbance at 540 nm versus the amount of Hb (mg/tube). Answer the following questions about the blood sample: The amount of Hb in Tube 6 = . . . . . . . . . mg The amount of Hb in the 0.01 ml blood sample = . . . . . . . . . mg The concentration of Hb in the blood sample = . . . . . . . . . g per litre Is the Hb content normal? . . . . . . . . .