Lab #3: Ion Exchange Chromatography
Lab #3: Ion Exchange Chromatography
Objective
The purpose of this experiment was to separate proteins on the basis of their net charge at a particular pH. In cation exchange chromatography positively charged molecules are attracted to a negatively charged column. Conversely, in anion exchange chromatography, negatively charged molecules are attracted to a positively charged column. Experimental results could be monitored in a predictable way by controlling running pH, salt concentration, and by selecting the type of ion exchanger.
Procedure: all procedures are listed in the lab manual.
Results
Table 1: Abs 280 Raw Data
A B C D E
Sample
Dilution Factor Measured Abs280 Undiluted Abs 280
(B x C) Graph Bar
HEW 80 0.918 73.44
LOAD 10 0.881 8.81 1
Phos 1 4 0.767 3.068 2
Phos 2 1 0.527 0.527 3
Phos 3 1 0.248 0.248 4
Phos 4 1 0.050 0.05 5 Carb 1 4 0.646 2.584 6
Carb 2 1 0.490 0.490 7
Carb 3 1 0.127 0.127 8
Graph: Abs 280 (Undiluted) Table 2: Biuret Raw Data
A F G H I J
Sample
Dilution before adding Biuret reagent Additional dilution factor (Biuret reagent) Total dilution factor into cuvetter
(F x G) Measured Abs
540 Undiluted Abs
540
(H x I)
HEW 40 5 200 0.127 25.41
LOAD 5 5 25 0.117 2.925
Phos 1 2 5 10 0.102 1.020
Phos 2 1 5 5 0.073 0.365
Phos 3 1 5 5 0.017 0.085
Phos 4 1 5 5 0.002 0.010
~~~~~~~~~~~~ ~~~~~~~~~~~~ ~~~~~~~~~~~~ ~~~~~~~~~~~~ ~~~~~~~~~~~~ ~~~~~~~~~~~~
Carb 1 1 5 5 0.066 0.330
Carb 2 1 5 5 0.015 0.075
Carb 3 1 5 5 0.006 0.030
Table 3: Mg Calculations
A J K L M N O
Sample
Undiluted
Abs 540 Slope from standard curve: Lab 2(abs 540/1mg/ml in cuvette) Undiluted mg/ml
(J / K) ml Mg proteins of all types
(L x M) Subtotals
8x dil HEW mixed with beads
48 Corresponding undiluted original HEW with beads
25.41
0.280
90.750
6
544.284 LOAD 2.925 0.280 10.304 48 398.59
Phos 1 1.020 0.280 3.6428 15 54.355
Phos 2 0.365 0.280 1.304 15 19.56
Phos 3 0.085 0.280
Objective
The purpose of this experiment was to separate proteins on the basis of their net charge at a particular pH. In cation exchange chromatography positively charged molecules are attracted to a negatively charged column. Conversely, in anion exchange chromatography, negatively charged molecules are attracted to a positively charged column. Experimental results could be monitored in a predictable way by controlling running pH, salt concentration, and by selecting the type of ion exchanger.
Procedure: all procedures are listed in the lab manual.
Results
Table 1: Abs 280 Raw Data
A B C D E
Sample
Dilution Factor Measured Abs280 Undiluted Abs 280
(B x C) Graph Bar
HEW 80 0.918 73.44
LOAD 10 0.881 8.81 1
Phos 1 4 0.767 3.068 2
Phos 2 1 0.527 0.527 3
Phos 3 1 0.248 0.248 4
Phos 4 1 0.050 0.05 5 Carb 1 4 0.646 2.584 6
Carb 2 1 0.490 0.490 7
Carb 3 1 0.127 0.127 8
Graph: Abs 280 (Undiluted) Table 2: Biuret Raw Data
A F G H I J
Sample
Dilution before adding Biuret reagent Additional dilution factor (Biuret reagent) Total dilution factor into cuvetter
(F x G) Measured Abs
540 Undiluted Abs
540
(H x I)
HEW 40 5 200 0.127 25.41
LOAD 5 5 25 0.117 2.925
Phos 1 2 5 10 0.102 1.020
Phos 2 1 5 5 0.073 0.365
Phos 3 1 5 5 0.017 0.085
Phos 4 1 5 5 0.002 0.010
~~~~~~~~~~~~ ~~~~~~~~~~~~ ~~~~~~~~~~~~ ~~~~~~~~~~~~ ~~~~~~~~~~~~ ~~~~~~~~~~~~
Carb 1 1 5 5 0.066 0.330
Carb 2 1 5 5 0.015 0.075
Carb 3 1 5 5 0.006 0.030
Table 3: Mg Calculations
A J K L M N O
Sample
Undiluted
Abs 540 Slope from standard curve: Lab 2(abs 540/1mg/ml in cuvette) Undiluted mg/ml
(J / K) ml Mg proteins of all types
(L x M) Subtotals
8x dil HEW mixed with beads
48 Corresponding undiluted original HEW with beads
25.41
0.280
90.750
6
544.284 LOAD 2.925 0.280 10.304 48 398.59
Phos 1 1.020 0.280 3.6428 15 54.355
Phos 2 0.365 0.280 1.304 15 19.56
Phos 3 0.085 0.280