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1 (reference)
2
3
4
5
Unknown 1
Unknown 1/2
Unknown 1/10
Unknown 1/100
Concentration of
protein (mg/ml)
0
2
5
8
10
Absorbance
at 550nm
(AU)
0.1787
0.2868
0.5787
0.6816
0.8383
1.4625
1.4438
0.736
0.2819
Corrected
Absorbance
(AU)
0.0000
0.1081
0.4000
0.5029
0.6596
1.2838
1.2651
0.5573
0.1032
mg/ml
mg/ml
Discussion
Proteins are polymers made up of amino acids joined by peptide bonds. These peptide
bonds have partial double bonds character and absorb light. These feature is also
important in measurement of absorbency. Biuret reagent contains Cu 2+ ion which form
complex with peptide bonds of the protein. The Biuret method is not highly sensitive but
it is the most linear because its colour depends on a direct complex between Cu 2+ and
peptide bonds of the proteins. Thus, the colour may slightly different depending on the
concentrations of the proteins.
A standard curve (Figure 1) was graphed using data from the five samples of known
absorbance and concentration. Unknown solution 1/10 and unknown solution 1/100 have
absorbance of 0.5573 and 0.1032 respectively, when ran through the spectrometer. These
points are then plotted on the y-axis of the graph and a line is drawn to the curve. The
intersection of both points show the protein concentration of unknown 1/10 as
mg/ml
and unknown 1/100 as
mg/ml. However, concentrations of unknown 1 and unknown
1/2 could not be determined using this standard curve because the absorbance for both
unknowns are more than the range of recorded absorbance of five samples of BSA.
Conclusion
From the standard curve plotted using the data recorded in this experiment, it can be
concluded that the amount of absorbance is directly proportional to the concentration of
proteins. In other words, the higher the concentration of protein, the higher the level of
absorption. Also, it is dependant on the amount of water in the solution. When the amount
of water increases, the concentration will decrease, thus the absorbance will also
decrease. By using the standard curve from data of this experiment, it is easier to
determine the concentration of unknown protein solution by first determining its level of
absorption.
Reference
1. http://www.iscid.org/encyclopedia/Protein_Assay
2. http://www.freeonlineresearchpapers.com/node/257
3. Lab Manual