RESULTS & CALCULATIONS

Table 1
No. BSA Stock Water (ml) Solution C (ml) Solution D (ml)
Solution (ml)
1 0 6.0 5 0.5
2 0.3 5.7 5 0.5
3 0.6 5.4 5 0.5
4 0.9 5.1 5 0.5
5 1.2 4.8 5 0.5
6 3.0 3.0 5 0.5
7 4.0 2.0 5 0.5
8 5.0 1.0 5 0.5
9 6.0 0 5 0.5
Unknown 1 *5.0 ml of 1.0 5 0.5
unknown 1 (not
BSA solution)
Unknown 2 *1.0 ml of 5.0 5 0.5
unknown 2 (not
BSA solution)

Table 2
No. Absorbance 1 Absorbance 2 Average of Concentration
(test tube) Sample 1 Sample 2 absorbance (mg/ml)
(750nm) (750nm)
1 0 0 0 0
2 0.128 0.132 0.130 0.100
3 0.208 0.220 0.214 0.200
4 0.276 0.343 0.310 0.300
5 0.343 0.358 0.351 0.400
6 0.535 0.540 0.538 1.000
7 0.579 0.581 0.580 1.333
8 0.600 0.584 0.592 1.667
9 0.620 0.611 0.616 2.000
Unknown 1 0.242 0.242 0.242
Unknown 2 0.127 0.121 0.124

1.0 Calculation:
Example for test tube 2:
𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 1+𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 2
Average of Absorbance : 2
0.128+0.132
: 2

: 0.130
*Proceed to all of the test tube (3-unknown 2)
Calculation for concentration:
Concentration of BSA solution : 2mg/ml
Using this formula: M1= Molarity of BSA Solution
V1= Volume of BSA Solution
M1V1 = M2V2 M2= Molarity of solution
V2= Volume of Solution
Example: (Test tube 2)
(2mg/ml) (0.3ml) = (M2) (6ml)
M2 = 0.1 mg/ml
*Proceed to all of the test tube (test tube 3-unknown 2)
*Concentration for unknown 1 & 2 is determined by using calibration curve made
2.0 Graph of absorbance vs concentration
(First trial)

Graph of Absorbance 1 vs Concentration

0.8

0.7

0.6

0.5
Absorbance 1

0.4

0.3

0.2

0.1

0
0 0.5 1 1.5 2 2.5
Concentration (mg/ml)

Diagram 1: Standard calibration curve for protein at Absorbance 1 vs Concentration

(Second trial)

Graph of Absorbance 2 vs Concentration

0.8

0.7

0.6
Absorbance 2

0.5

0.4

0.3

0.2

0.1

0
0 0.5 1 1.5 2 2.5
Concentration (mg/ml)

Diagram 2: Standard calibration curve for protein at Absorbance 2 vs Concentration

Average

Combination of abs 1, abs 2, and average abs VS

Concentration
0.8
0.7
0.6
Absorbance

0.5
0.4
0.3
0.2
0.1
0
0 0.5 1 1.5 2 2.5
Concentration (mg/ml)

Absorbance 1 Absorbance 2
Average Absorbance Linear (Average Absorbance)

Diagram 3: Standard calibration curve for protein at average absorbance VS

concentration
3.0 Estimate the concentration of protein in unknown sample:
The value of concentration for unknown 1 is obtain from the graph made. At absorbance 1=
0.242, so the value of concentration for unknown 1 is 0.580mg/ml for 1 ml. We use 5ml of
unknown 1, so the concentration is 0.580 mg/ml ×5 ml = 2.9 mg/ml.

2.9 mg/ml is for 1⁄100 part of the solution, so the exactly concentration is 2.9 mg/ml × 100 =
290 mg/ml.
Example finding value of concentration of both unknown using graph made:
orange colour is unknown 1, black is unknown 2.
*Proceed to find all the concentration of both unknown needed at all absorbance (abs 1,
2 and average)

Diagram 4: Calibration curve for absorbance 1 VS concentraion

Table 3
No. of unknown 1 2
Conc. at Absorbance 1 (mg/ml) 290 38
Conc. at Absorbance 2 (mg/ml) 340 36
Conc. at Average (mg/ml) 320 37
*All concentration obtained is from graph made
4.0 The amount of proteins that are present in 200ml of sample is:
At absorbance 1 for unknown 1, the value of concentration for 1 ml is 0.58 mg/ml, so for 200
ml, we need to 0.58 mg/ml × 200 ml = 116 mg/ml. The calculated concentration value is for
1⁄
100 part of solution of unknown 1, so 116 mg/ml × 100 = 11600 mg/ml is the exactly
concentration value for unknown 1 at absorbance 1 for 200 ml.
*Repeat the calculation for unknown 1& 2 at each absorbance obtained
Table 4
No. of unknown 1 2
Conc. at Absorbance 1 (mg/ml) 11600 7600
Conc. at Absorbance 2 (mg/ml) 13600 7200
Conc. at Average (mg/ml) 12800 7400

Using this formula:

𝑚𝑜𝑙
molarity = 𝑣𝑜𝑙𝑢𝑚𝑒 (𝐿)

Convert mg/ml to mol/Liter:

𝑚𝑔 1000𝑚𝑙 𝑚𝑔 1𝑔
0.58 × = 0.58000 ×
𝑚𝑙 1𝐿 𝐿 1000𝑚𝑔

𝑔
= 0.58 𝐿 × MW of soya solution; 1⁄920 𝑔/𝑚𝑜𝑙

= 0.000630 mol/ L
𝑚𝑜𝑙
molarity = 𝑣𝑜𝑙𝑢𝑚𝑒 (𝐿) mass of protein in 200 ml:

mol = molarity × volume (L) = mol×MW of soya solution

= 0.000630 mol/L × 0.200 L = 0.000126 mol × 920 g/mol
= 0.000126 mol = 0.116 g of protein
*repeat for both unknown at absorbance 1, absorbance 2, average absorbance
Table 5: Amount of protein in 200m ml of sample
No. of unknown 1 2
Amount of protein at Absorbance 1 0.116 0.076
in 200 ml (g)
Amount of protein at Absorbance 2 0.136 0.076
in 200 ml (g)
Amount of protein at Average 0.128 0.076
absorbance (g)
DATA ANALYSIS
(First trial)

Graph of Absorbance 1 vs Concentration

0.8

0.7

0.6
Absorbance 1

0.5

0.4

0.3

0.2

0.1

0
0 0.5 1 1.5 2 2.5
Concentration (mg/ml)

Diagram 1: Calibration curve for protein at Absorbance 1 vs Concentration

Diagram above show a calibration curve for protein at absorbance 1 vs concentration. The
sample that we took is the first trial sample. From the graph made, we found that almost of the
point is not nearer to the linear line. So to be compared with the theoretical calibration curve
for protein, the points must be nearer to the linear line at least 2 points. After analysing the
graph, we found that the graph produced by us is different from the theoretical calibration curve.
(Second trial)

Graph of Absorbance 2 vs Concentration

0.8

0.7

0.6
Absorbance 2

0.5

0.4

0.3

0.2

0.1

0
0 0.5 1 1.5 2 2.5
Concentration (mg/ml)

Diagram 2: Calibration curve for protein at Absorbance 2 vs Concentration

Diagram above show a calibration curve for protein at absorbance 2 vs concentration. The
sample that we took is the second trial sample. From the graph made, we found that almost of
the point is not nearer to the linear line. So to be compared with the theoretical calibration curve
for protein, the points must be nearer to the linear line at least 2 points. After analysing the
graph, we found that the graph produced by us is different from the theoretical calibration curve.
Average

Combination of abs 1, abs 2, and average abs VS

Concentration
0.8
0.7
0.6
Absorbance

0.5
0.4
0.3
0.2
0.1
0
0 0.5 1 1.5 2 2.5
Concentration (mg/ml)

Absorbance 1 Absorbance 2
Average Absorbance Linear (Average Absorbance)

Diagram 3: Calibration curve for protein at average absorbance VS concentration

Diagram above show a calibration curve for protein at average absorbance vs concentration.
The graph is the combination of absorbance 1, absorbance 2 and average absorbance. Basically,
the graph is focusing to compare the three of the absorbance obtained in the experiment. There
is no slightly different between the points among the absorbance 1, absorbance 2 and average
absorbance. From the graph made, we found that almost of the point is not nearer to the linear
line. So to be compared with the theoretical calibration curve for protein, the points must be
nearer to the linear line at least 2 points. After analysing the graph, we found that the graph
produced by us is different from the theoretical calibration curve.