The relationship between absorbance and sample concentration was confirmed using Beer and Lambert’s law, once the experimental results had been calculated. The unknown concentration samples were labelled A, B, C, and D, respectively.
About 100μl of each sample was taken and put into other tubes with corresponding labels. To each of these tubes containing the sample was added 1.0ml of enzymes. Thereafter, the contents were incubated for 15 minutes at normal temperature.
The glucose concentrations increased steadily with a constant variation showing direct proportionality by 0.4mM, according to the formula below:
C2 = C1 x V1 / V2; Where V2 equals 1ml, C1 2 mM, and V2 is the value of glucose.
The glucose amount was then obtained from the values of the standard glucose concentration using Beer-Lambert’s law. The formula is usually written thus: Mass (g) = conc. (Mol/L) x vol (L) x Mol. weight (g/mol).
Where Mass refers to the Glucose amount, conc. represents the standard Glucose concentration, and vol is 0.001 L.
The results of the sample glucose with unknown concentrations were recorded in the second table. Calculations were done following the formula discussed below. The Beer-Lambert law calculator was also used as an alternative form of calculation. Therefore, glucose values were taken from the equation of Figure 1 above (y = 0.0019x – 0.0085). In this case, the formula becomes x = y – c ÷ m; Where y is the Spectrophotometer reading. For example, (x = 0.427 – 0.0085 ÷ 0.0019) = 220.26 μg, as mentioned in the table below. In the last column, glucose concentration was taken using the formula: (conc. mM = mass (μg) ÷ M.Wt.) Where mass is the amount of glucose, and M.Wt. is a constant value that equals 180. As an example, (conc. mM = 220.26 ÷ 180 = 1.22 mM).
In table 1 below, the results obtained were recorded from the known concentrations according to the Beer and Lambert’s law.
Discussion
This report has attempted to describe the application of Beer and Lambert’s law. The law is applied in the analysis of both known as well as unknown samples using a spectrophotometer to determine the measurements and subsequent analysis relative to Beer and Lambert’s law. For example, in tables 1, 2 and figure 1, results were calculated using this law and they confirm that there is a relationship between the concentration of the absorbing substance and the absorption coefficient. Therefore, it can be clearly seen that the sample thickness and glucose concentration have a proportional relationship with the absorbing substance. As an example, in table 1, sample 2, the 0.106 spectrophotometer reading is attributed to 0.4mM of the standard glucose concentration and 72μg of glucose amount. On the other hand, the 0.696 reading of sample 6 is attributed to 2.0mM of glucose concentration, and 360μg of glucose amount.
Results for the unknown glucose concentration are given in table 2 and they reflect the same proportionality originally seen with the standard glucose, according to Beer and Lambert’s law. Figure1 reveals a proportional relationship between absorbance values and concentrations.
Finally, this experiment might not get accurate results due to the changing circumstances such as concentrations, the samples’ incubation period, and the sampling time in the device. These circumstances are bound to bring about errors in the experiment and in order to minimise such errors, care must be taken to ensure the realisation of more accurate results.