Resolution with HPLC Teaching Assistant, an Excel spreadsheet that simulates high performance liquid chromatography separations. Download the Excel file from SaplingPlus, macmillanlearning.com, or the link in reference 27,27. and open the file. You will see a menu page with Resolution at the top left. If not, click the blue home button in the top left corner. Click on Resolution for this problem. Adjust the image size so that you see three graphs along the bottom showing the relationships between resolution and retention factor, separation factor (called selectivity), and plate number (efficiency) similar to those generated in Problem 23-28.
a. Use the top slide bar at the top left to adjust the retention factor. Keep selectivity at 1.10 1.10 and efficiency at 10 000. 10 000. What is the resolution for k1=0, 1, 2, 5, k₁=0, 1, 2, 5, and 10? What is the retention time of the second peak at each retention factor? Tip: Excel shows x' and y2 values for a data point in a graph when you position your cursor over the point.
b. With a retention factor of 11 and efficiency of 10 000,10 000, adjust selectivity to 1.00, 1.00, and 1.20. 1.20. What is the resolution and retention time of the second peak at each value for α?α?
c. With a retention factor of 11 and selectivity 1.10,1.10, adjust the plate number to 2 000, 2 000, 4 000, 4000, 6 000, 6 000, 8 000,8 000, 10 000,10 000, 12 000,12 000, and 17 000.17000. What is the resolution and retention time of the second peak at each value of N?N?
d. Repeat (c). What is the maximum absorbance for peak 11 for plate numbers of 2 000, 2 000, 6 000, 6000 and 17 000? 17 000? Subtract the baseline signal from peak heights.

Problem 23-28

In chromatography, resolution is governed by (a) the plate number, (b) separation factor, and (c) retention factor. Construct a set of graphs to show the dependence of resolution on each of these three parameters. Comment on the nature of the dependence of resolution on each of these three parameters.