General Instructions. Read each question carefully. Show your setup for all calculation problems so I can give partial credit if necessary. Include units of measurement on all answers. If you need a calculator, pencil, eraser, more paper, etc., ask.

1. The following description represents an idealized chromatography experiment. 3.00 g of Sephadex G-75 (exclusion limit 75,000 Da) was swelled in 25 mL of 0.1 M NaCl and packed into a column 1.0 cm diameter x 50.0 cm long. After an equilibration time of 30 min, the bed height was 48.0 cm. A sample containing blue dextran and vitamin B₁₂ was added to the column and eluted at a flow rate of 0.10 mL/min. The blue dextran came off in a single fraction corresponding to a volume of 6.0 mL. The B₁₂ came off in two fractions (yeah, right) corresponding to an average volume of 36.0 mL. Address the following points. (10 points)
        a. What was the void volume of the column? (Vo)
        b. What was the packed bed volume? (Vt)
        c. What is the Kave for an egg white protein (ovalbumin) which elutes in a subsequent run at 24.2 mL?

2. Calculate the molar extinction coefficient of NADH at 340 nm from the following information. NADH has a molar mass of 664.45. I weighed out 1.1 mg of NADH and dissolved it in 100.00 mL dH₂O. Using a 1.00 cm cuvette, the following absorption spectrum was obtained. (15 points)

3. From the attached nomogram, determine the speed at which a homogenate should be centrifuged to collect lysosomes (30,000g). The rotor being used has a 4.5 inch radius. (5 points)

4. Plot the following data and determine the protein concentration of the unknown protein in µg/mL. (15 points)
     Tube                 Contents                     %T (read)         A_{650 nm} (converted)
                 0.2 mg/mL
                    BSA                     water
        1         0.0 mL                 1.0 mL             100.0             0.00
        2         0.2 mL                 0.8 mL               68.2             0.166
        3         0.4 mL                 0.6 mL               45.5             0.342
        4         0.6 mL                 0.4 mL               32.1             0.493
        5         0.8 mL                 0.2 mL               10.5             0.979
        6         1.0 mL                 0.0 mL               14.8             0.830
        7         1.0 mL unknown    0.0 mL             35.5             0.450
        8         1.0 mL unknown    0.0 mL             34.6             0.461
        9         1.0 mL unknown    0.0 mL             12.1             0.917
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

5. For each of the following reagents, provide a brief statement of their purpose in the experiments we performed. (5 points)

a. Ammonium persulfate
 

b. Blue dextran
 

c. 0.25 M sucrose
 

d. 0.20 mg/mL (200 µg/mL) bovine serum albumin