The aim of the practical is to test the following hypothesis:
1) 5 urine samples of unknown concentration numbered 1, 5, 6, 7 ad 8.
2) Picric Acid
3) 0.75M Sodium Hydroxide
4) P1000 Gilson Pipette
5) 1cm3, 5cm3 and 10cm3 glass pipettes
6) Vortex Mixer
9) Test Tubes
Prior to the beginning of the experiment students were give data for the standard curve of creatinine concentration ranging from 0.05-0.5mM. Students were also told that creatinine concentrations in a 24-hour sample are usually between 5-20mM. Therefore the five samples would have to be diluted.
Dilution factors of x10, x20, x40, x80 and x10 diluted sample 1, 5, 6, 7 and 8 respectively.
The x10 dilution was prepared by taking 1cm3 of sample 1 and adding this to 9cm3 of distilled water in a clean test tube, this was called the primary procedure. The test tube was labeled 1d. The x20 dilution factor was achieved by initially carrying out the primary procedure, however, sample 1 is substituted with sample 5. From this x10 dilution of sample five 5cm3 is removed and added to 5cm3 of water in a clean test tube this gave a x20 dilution the test tube was labeled 5d. The x40 dilution was made by initially repeating the primary procedure, however, sample 1 is substituted with sample 6. From this x10 dilution of sample 6 3cm3 is extracted and added to 1cm3 distilled water in a clean test tube, this made the x40 dilution the test tube was labeled 6d.
First carrying out the primary procedure made the x80 dilution factor, however, sample 1 was substituted with sample 7. From this x10 dilution of sample seven 7cm3 is removed and added to 1cm3 of distilled water. This made the x80 dilution the test tube was labeled 7d. The x100 dilution was prepared by initially executing the primary procedure, however, sample 1 is substituted with sample 8, to give a x10 dilution of sample 8. 1cm3 of this x10 dilution of sample 8 is removed and added to 9cm3 of water in a clean test tube, this gave the x100 dilution the test tube was labeled 8d. The above dilutions were carried out using the glass pipettes ensuring a clean set was used for each dilution.
The diluted samples were assayed by taking 2cm3 of the sample using a p1000 gilson pipette and placing this in a test tube, then 1cm3 of picric acid was added to the test followed by 1cm3 0.75m sodium hydroxide. These were then mixed gently but well using the vortex mixer and left to stand for 25 minutes to allow coloured complex to form. All samples were assayed in duplicate therefore the above procedure had to be repeated for each dilution. Along with setting up the above samples a reagent blank had to be prepared this was done in the exact same way as described above however, the 2cm3 sample was replaced by 2cm3 distilled water. This was also left for 25 minutes.
The reagent blank was used to set the reference on the spectrophotometer at 510nm. Then the absorbance of all the samples was measured at 510nm.
Once this was completed a calibration graph was drawn using the data for the standard creatinine concentration, 0
To calculate the standard deviation the equation below has to be used.
The standard deviation % can now be found by dividing the standard deviation value by the mean. After this has been worked out the total amount of creatinine excreted by each individual (in mM) can be calculated, by multiplying the total 24-hour urine volume (dm3) by the mean concentration of the eight samples.
SAMPLE Mean creatinine concentration S.Deviation %S.Deviation Concentration of Creatinine (mmol)
1 6.23 1.73 27.8 10.28
2 14.26 5.35 37.5 10.84
3 15.66 4.24 27.1 13.47
4 16.00 4.29 26.8 19.68
5 11.52 2.35 20.4 9.91
6 14.36 4.89 34.1 20.68
7 19.43 5.81 29.9 18.46
8 9.55 1.87 19.6 13.66
This is supported by graph 1, which shows a linear relationship between creatinine concentration and body mass Graph 1 also shows the correlation coefficient as r=0.928, which is very close to 1.00. Therefore it is reasonable to say there is a high positive correlation between body mass and creatinine concentration.
The second graph displays the standard error bars due to variation; these variations could be due to a number of variables. Firstly the amount of urine supplied could be due to environmental factors, such as the weather if it were a hot the body would retain more water and thus there would be less urine produced, and vice versa for a cold day. The actual weight of the person is also a factor, which can cause variations in the data. The individuals may have been weighed with or without clothes, the scales used to measure the individual may have been inaccurate, or they may have been weighed on soft flooring etc. Another possibility is that the actual sample may not have been a 24-hour urine sample.
Further errors can be attributed to the student who carried out the experiment. The student may have been inaccurate with their pipetting, or the gilson pipette itself may have been faulty/uncalibrated. The student may have made errors in the calculations regarding the dilutions; this is seen in the large variations in the pooled results.
Experimentational error had to of occurred due to the fact that graph 2 shows a large standard error i.e. the bars are very long, stating a lot of variation.
As variations and errors occurred it is necessary to give remedies or solutions if the experiment were to be carried out again. Firstly the sample set should all be from within the same age group, also when weighing is being carried all members of the sample set should be weighed at the same place using the same scales. To reduce variation that is caused by the weather all members of the sample set should give samples on the same day so the effect of the weather will be the same on everyone.
The hypothesis has been proven i.e. the creatinine concentration found in 24-hour sample of urine is directly related to the mass of a person.
BSc BIOLOGICAL Sc. 1st YEAR