Cedric Kanyinda

Laboratory Experiment #3 –

Spectrophotometer and Standard Curves

Determination of Absorbance Spectra

1. How can you determine at a glance whether or not a particular compound can
be spectrophotometrically analyzed in the visible portion of the electromagnetic
spectrum? (1 point)
By observing the color of the compound

2. How would you determine the concentration of a solution of a compound

whose absorbance of light is beyond the scale of the spectrophotometer? (1
point)
I would dilute

3. If you wished to determine the concentration of a solution of compound Q and

the concentration of a separate solution of compound T, which wavelengths
could you use for greatest sensitivity? (0.5 point)

For the greatest sensitivity I will use the wavelength at the highest pick which is
the one for compound T, and I could also use the one for Q and also the one for
Q&T

4. If you had a solution of both compounds together, what wavelengths could

you use to determine the concentrations of the compounds in the solution?

I will use the one with the highest pick which is located in the Q & T figure point
(397, 1295)

5. In the mixture of solution Q and T, why is an intermediate peak not observed?

6. After isolating DNA from a cell, it is common practice to conduct a

wavelength scan (200-300nm) with an aliquot of the DNA sample. Describe two
reasons for conducting the wavelength scan. (1 point)
p-nitrophenol standard curve

7. Construct a table showing your data. (1 point)

Table 1: Absorbance of p-nitrophenol Standards

Sample Concentration Absorbance
Blank - -
1 0.00 [Tris tube] 0
2 0.005 mM 0.07
3 0.010 mM 0.125
4 0.025 mM 0.29
5 0.050 mM 0.59
6 0.075 mM 0.89
7 0.100 mM 1.1

8. Plot the absorbances of the standard solutions on the ordinate of your graph
paper vs. their concentrations (on the abscissa) and put a reasonable line
through the points (remember that at zero concentration there is zero
absorbance). (2.5 points)

9. Determine the concentrations of p-nitrophenol in your unknowns using the

standard curve you constructed in (3). Then calculate the actual amounts of p-
nitrophenol in each 3ml of your unknowns. (2 points)
Table 2: Absorbance of p-nitrophenol UNKNOWNS
C= A/k
Sample Absorbance Calculated Calculated amount
Concentrattion in 3ml
E 1.1 0.098
J 0.3 0.027

10. Determine the value of k for p-nitrophenol from your standard curve. [Hint:
what does k represent in equation (3)?] Be certain to include the correct units in
all calculations. (2 points)

From the graph k = 11.2 cm^-1 M^-1

 A = kC

k= 0.29/0.025= 11.6
k=1.1/0.1 =11

Average k of both points= (11.6 + 11)/2= 11.3 cm^-1 M^-1

11. Discuss your results and draw conclusions about the unknown. (2 points)

The graph shows a linear increase in absorbance when the concentration

increase, so I can from those data that the absorbance is directly proportional to
the concentration which explain the linear progression.
Well about my unknown, from the table and all data collected I can fairly say
that the higher the concentration, the higher the absorbance and vice versa.

Protein standard curve

12. Construct a table to show your data (1 point)

Table 3: Protein Assays

Tube BSA Solution ( Water Bradford Absorbance
1mg/ml) Reagent 595
1 0 ul 100 ul 5 ml -0.001
2 10 ul 90 ul 5ml 0.050
3 20 ul 80 ul 5ml 0.115
4 30 ul 70 ul 5 ml 0.125
5 40 ul 60 ul 5 ml 0.135
6 50 ul 50 ul 5 ml 0.152
7 60 ul 40 ul 5 ml 0.166
8 70 ul 30 ul 5 ml 0.177
9 80 ul 20 ul 5ml 0.132
10 90 ul 10 ul 5ml 0.183
Unknowns Unknown Water Bradford Absorbance
protein Reagent 595
11 (II) 10 ul 90 ul 5 ml 0.014
12 (I) 10 ul 90 ul 5ml 0.032
13. Construct a standard curve by plotting absorbance (ordinate) against
amount of protein (in micrograms) and use it to determine the amounts and
concentrations of protein in the unknown samples. (2.5 points)

Figure 2. Absorbance vs. amount of porein

14. Discuss your results and draw conclusions about the unknown. (2 points

Inside every tube we had the volume of Bradford reagent, in tube 1 we had 100
ul and there on a decrease of 10 ul in every tubes until in tube 10 which had 10
ul and the amount of BSA was set in decreasing order. So when we ran the tube
through, and then plotted the a graph of absorbance vs. the amount of protein,
from those data I observed that in tube where they where no protein at all the
absorbance was 0 (that no light was absorbed), and as the amount of protein
was increasing so was the absorbance as water was deceased in every tube.
Then I observed the data on the unknown, we were given two unknown protein
0f 10 ul in which we the same volume of water and Bradford reagent but the
absorbance was different in the two unknown. Unknown I produce an
absorbance of 0.014, and unknown II had an absorbance of 0.032 therefore, it
was safe to conclude that the two unknown are different, and are made different
molecules.