What is the essence of the experiment that determines glucose within the sample?
In this experiment, Dinistrosalicylic acid (DNS) colourimetric method was used to determine glucose within the sample quantitatively. DNS testing method is used to determine the presence of reducing sugars that includes glucose molecules. The testing encompasses oxidation of aldehyde functional group within the glucose molecule and simultaneous reduction of DNS (3,5-dinitrosalicylic acid) to 3-amino,5—nitrosalicylic acid, leading to the production of brown colour. Below is an example of the reaction.
The purpose of the experiment was to examine the presence/absence of diabetes or other carbohydrate metabolism ailments within a mock patient plasma sample.
Method: A stock solution of glucose was supplied at a concentration of 20mmol/l and a reference sample at 7.5mmol/l. A set of 5 test sample of unknown concentration taken at 0, 30, 60, 90, and 120 minutes from patient 1 during an OGTT was collected. Prior to test analysis, all samples (including stock glucose, the reference sample and the 0, 30, 60, 90, and 120 mins. patient sample) were diluted 1 in 20, as the absorbance readings would be off the spectrophotometer balance without first diluting the sample. The diluted stock glucose solution was still being referred to as 20mml/l although it was then 1mmol/l and was used in triplicate to prepare a standard carve with ranges from 0 to 20mmol/l. In triplicate, 500µl of each standard, reference and patient samples were transferred to the new set of labelled microfuge tubes and then another 500µl of DNS reagent was added to all tubes and was mixed thoroughly by vortexing and then after they were all incubated at 70ºC for 30 minutes. After incubation samples were placed in the ice bucket for 3 mins to cool down and the absorbance of the standards and unknown samples were measured at 570nm.
The formula C1V1=C2V2 was used to calculate the final volume of stock glucose (ml).
The results for stock 20mmol/l glucose seen in table 2 and figure 1, showed an increase in absorbance as the [glucose] increased. As the concentration of glucose increased the intensity of the sample colour (from yellow to brown) increased as well, this indicates 3,5-dinitrosalicylic acid(yellow) were reduced to 3-amino,5-nitrosalicylic acid (brown) and glucose was oxidised to gluconic acid. The mean absorbance results are quite evenly distributed but not precise.