I.

Objectives
At the end of the exercise, the students should be able to:
1. purify the isolated egg albumin; and
2. determine the molecular weight of albumin.

II. Materials and Methods

A. List of Necessary Chemicals

Name of Compound Physical and Chemical Hazards

Properties

Sephadex G-100 White powder Causes eye irritation

Causes skin irritation

Water, H2O Colorless Liquid Not hazardous

MP: 0 C
BP: 100 C
Density: 1 g/cm3
18.016 g/mol

Blue dextran Powdered solid Causes eye irritation

Causes skin irritation

Sodium hydrogen phosphate white, odorless powder May be harmful if inhaled

Na2HPO4 MW: 141.96 g/mol
⍴: 1.52 g/mL

Sodium dihydrogen white, odorless powder May be harmful if inhaled

phosphate MW: 119.98 g/mol
NaH2PO4 MP: 212°C
⍴: 2.04 g/mL

Sodium azide, NaN3 White powdered solid Causes eye irritation

MP: 275 C Causes skin irritation
BP: 300 C
MW: 65.01 g/mol

B. Materials needed
1. Glassware
a. Stirring rod
b. Pipet
c. 2 50 mL beaker
d. 1 100 mL beaker
e. 20 test tubes
2. Equipment
a. Column (pakicheck neto julie di ko sure kung tinuturing syang equipment)
b. Spectrophotometer
3. Other materials needed
 a. Rubber stopper
b. Tissue
c. Masking tape for labels
C. Schematic diagram
D. Set-ups
1. Column (yung nasa manual)
E. Pre-lab calculations
Equations for postlab analysis
Calculation of the column volume, VT
V = �r2h

Calculation of the amount of buffer needed to equilibrate the column

Vbuffer = 2(VT)

Calculation of the internal volume (VI)

VI = VT - V0
Calculation of the distribution coefficient, Kav
Kav = (Ve - V0) / (VT - V0)

Calculation of the molecular weight

III. Data blanks

Table 3.3.1. Data on the determination of column volume, VT.

Parameter Value

Radius of the gel bed, cm

Height of the gel bed, cm

Column volume, cm3

Table 3.3.2. Data on the determination of the elution volume and absorbance of blue
dextran for void volume, Vo, measurement.
Test tube number Elution volume, mL Absorbance at 500 nm

6
 7

10

11

12

13

14

15

16

17

18

19

20

Table 3.3.3. Data on the determination of the elution volume of the protein standard.
Test tube number Elution volume, mL Absorbance at 280 nm

10

11
 12

13

14

15

16

17

18

19

20

Table 3.3.4. Data on the determination of the protein content of the isolate.

Test tube number Absorbance at 595 nm

10

11

12

13

14

15
16

17

18

19

20