Lab #3: Ion Exchange Chromatography

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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
ABCDE
Sample
Dilution FactorMeasured Abs280Undiluted Abs 280
(B x C)Graph Bar
HEW800.91873.44
LOAD100.8818.811
Phos 140.7673.0682
Phos 210.5270.5273
Phos 310.2480.2484
Phos 410.0500.055

Carb 140.6462.5846
Carb 210.4900.4907
Carb 310.1270.1278

Graph: Abs 280 (Undiluted)

Table 2: Biuret Raw Data
AFGHIJ
Sample
Dilution before
adding Biuret
reagentAdditional dilution factor (Biuret reagent)Total dilution factor into cuvetter (F x G)Measured Abs
540Undiluted Abs
540

(H x I)
HEW4052000.12725.41
LOAD55250.1172.925
Phos 125100.1021.020
Phos 21550.0730.365
Phos 31550.0170.085
Phos 41550.0020.010
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Carb 11550.0660.330
Carb 21550.0150.075
Carb 31550.0060.030

Table 3: Mg Calculations
AJKLMNO
Sample

Undiluted
Abs 540Slope from standard curve: Lab 2(abs 540/1mg/ml in cuvette)Undiluted mg/ml (J / K)mlMg 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

LOAD2.9250.28010.30448398.59
Phos 11.0200.2803.64281554.355
Phos 20.3650.2801.3041519.56
Phos 30.0850.2800.304154.56
Phos 40.0100.2800.0357150.535
477.6
Carb 10.3300.2801.1791517.685
Carb 20.0750.2800.268154.02
Carb 30.0300.2800.107151.605
23.31

Table 4: E 1% Calculations
ALPQDR
Sample of proteinCharge at pH 7Undiluted mg/mlUndiluted g (of protein per liter)% of a liter (1000 gm)
Which is proteinUndiluted abs 280E 1% = Abs for a 1 % solutionEstimate of E 1 % HEW90.75090.7509.07573.448.092
LOADNeg10.30410.3041.0338.8108.528
Carb 1pos1.17901.17900.11792.58421.92

Ovomucoid-8.03.6
Ovalbumin-8.06.2
Ovotransferrin+3.010.7
Lysozyme+3.026.0

Lab 0

Same as L100% x
(P / 1000)
Lab 0

Questions:
1.Calculating the concentration of protein of all types in mg/ml in hen egg-white. Total hew mgs used: 544.91 mgs
Total hew ml used : 7 ml
Mg/ml = 544.91mg / 7 ml = 77 mg/ml

2.Comparing the total protein recovered (in mg) from all fractions combined with the total quantity of protein you mixed the beads. Theoretically the total of recovered proteins should be less or sum up to equal the amount first mixed with the beads. According to table 3: Total recovered mgs= 477.6.475 + 23.31 = 500.91 mg

Total % recovered = (500.91/ 544.248) x 100 = 93%

3.Are the data consistent with a hypothesis that the carb#1 fraction has a higher ratio of lysozyme relative to other proteins than the case for the phos #1 fraction? The results in table 3 indicate that Carb #1 has a higher ration of Lysozyme relative to other proteins than the case for the phos #1 fraction, in our case the E1% of the carb 1 was calculated 21.92, showing that the sample was enriched with Lysozyme.

4.Are the data consistent with a hypothesis that the carb 1 fraction contains only lysozyme or do the data instead suggest that other proteins also elute with lysozyme in that fraction? In our case the E 1% was recorded to be 21.92, indicating that the sample for...
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