NAME______
Biology 22 Ion Exchange Chromatography Worksheet
1. List the absorbance values obtained for the Green Dye dilution series for the standard curve.
Standard Curve
Concentration of Green Dye (mg/ml) / Absorbance at 550 nm1
0.5
0.25
0.125
0.0625
0.03125*
0.015625*
*if further dilutions are needed to have
5 values between 0-2 absorbance units
2. Attach a copy of your standard curve for absorbance at varying concentrations of green dye. Be sure to label the axes and draw a best fit line through the points. Include the equation of the trendline. No caption is required.
3. Complete the following table to show the results extrapolated from your standard curve. If you measured the absorbance for each fraction individually, use one row for each fraction. If you combined the fractions and took one absorbance value, report that information in the first row. If you did not dilute the sample, skip columns 3 and 4 and give the undiluted concentration in the last column.
GFP Fraction(order of elution) / Absorbance at 550 nm / Dilution / Absorbance Corrected for Dilution / Concentration
(mg/ml)
1
2
3
4
5
6
7
8
9
10
4. Explain the basis of the separation for Cytochrome C and GFP in this experiment, specifying the ionic interactions between the matrix (bead), protein and eluting buffer(s).
5. What would happen if 0.5 M KOAc had been used first to elute the sample? Why?
6. In our experiment, we separated molecules that had different charges. How can molecules with varying amounts of the same overall charge (i.e. two proteins with different amounts of negative charge) be separated by ion exchange chromatography?
7. What is the total amount (in mg) of GFP recovered in the column fractions? What percentage of the amount of GFP in the sample was recovered in the eluant? [Hint: The sample loaded on the column contained 0.4 mg of GFP]