Changing Mode of Separation from Isocratic to Gradient Chromatography Ques

Week #14 Homework


For this homework you will need to make use of the high performance liquid chromatography

(HPLC) simulator available online at: You will be using the simulation to explore how HPLC separations can be manipulated. Specifically you will see how the mode of reversed phase liquid chromatography (RPLC) functions and how changes in the mobile phase influence the resulting chromatograms.


Here are a few terms that you need to be familiar with in completing this assignment.

  • Stationary phase – the surface, and surface chemistry, of the particles that are inside the HPLC column.
  • Mobile phase – the liquid that is being pumped down the HPLC column and that causes the analytes to move down the column.
  • Elution – pertaining to analytes coming off the column.
  • Mobile phase strength (or elution strength) – the relative ability for a given mobile phase to elute analytes from the column (i.e a stronger mobile phase elutes analytes more rapidly).
  • Band or zone – the region within the column that contains a given analyte.
  • Chromatogram – the graphical representation of the separation, with the detector signal strength plotted versus time.
  • Peak – the representation of the
  • Isocratic – typically pertaining to the mobile phase, where the mobile phase remains constant throughout the separations (e.g. 80% water, 20% methanol).
  • Gradient – typically pertaining to the mobile phase, where the mobile phase changes over a specified period of time (e.g. 80% water and 20% methanol to start, but over 15 minutes the mixture changes to 35% water and 65% methanol).
  • Solvent A and B – the solvents are pumped into the HPLC column to elute the mobile phase. Solvent A is typically water, or an aqueous buffer, and solvent B is typically a water soluble organic solvent (e.g. methanol, acetonitrile).
  • Retention factor (k) – a measure of the retention of an analyte that allows for comparisons between different instruments and conditions (this measure accounts for things like, flow rate, tubing length, and other physical parameters).
  • Retention time (tR) – the time at which the analyte passes the detector (measured by the peak maxima).
  • Reversed (stationary) phase – a hydrophobic stationary phase, it is termed reversed as the original chromatography stationary phase was hydrophilic (now termed normal phase).
  • Baseline separation – a separation where the individual peaks for each compound do not overlap, the trace returns to the baseline before rising to the next peak. Question 1

To start this assignment click on “Manage Compounds” tab and identify the compounds that have been selected, and find their chemical structures. The stationary phase used in the simulation is a reversed phase column (with a C18 stationary phase). Looking at the structures, predict the elution order of the compounds. What chemical property determines how strongly retained an analyte might be?

Question 2

For the default mobile phase conditions (isocratic elution, 40% acetonitrile) what is the elution order for the compounds? Are there any compounds that elute on-top of each other (co-elute), if so which ones? In general, what factor about the compounds determines their retention order? (Hint – look a the structures of the compounds.)


In the “Mobile Phase Composition” tab you can alter how the separation takes place. You can change Solvent B (acetonitrile or methanol), you can do an isocratic or gradient separation, and change the amount(s) of solvent B in each of those respective separation modes. Note: When you make any change in the mobile phase composition the chromatogram responds immediately, and will change the time axis to fit the new chromatogram. Pay close attention to the maximum time on the chromatogram, otherwise it may seem like there is little to no change in the chromatogram.

Question 3

You will explore what is known as the linear solvent strength model for chromatographic elution. in brief, the theory is that the elution of analytes can be predicted, in a liner manner, based on the strength of the solvent, and their respective retention factors.

Prepare a spreadsheet where you will record the retention factor (k) for each analyte at three different acetonitrile (solvent B) percentages (20%, 40%, and 60%). Plot ln(k) vs %B include all the components on the graph, and extrapolate the retention factors at different %B values.

From the graph that you prepared, determine and report the optimal percentage of acetonitrile to quickly separate all the compounds. You should verify/refine the percentage selected from the graph with the simulator.

Repeat the above processes but change solvent B to methanol. Plot ln(k) vs %B for methanol, and determine the best mobile phase composition for the separation of the compounds.


Question 4

Change the mode of separation from isocratic to gradient. The default gradient conditions are in the image to the right. The gradient is defined by the values in rows 1 and 2. Row 1 is the starting conditions, in this instance the mobile phase starts at 95% water, and 5% acetonitrile. Row 2 defines how fast the mobile phase changes, in this instance, 5 minutes after the start of the separation the percent acetonitrile will have increased (linearly) to 95% and the water will have decreased to 5%.

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Observe what happens to the chromatogram when when you change the gradient in the manners listed below. In each case, give your best interpretation/understanding for why you are seeing the results you get: (remember to keep an eye on the time on the x axis).

  1. Increase the starting %B to 35% (keep the rest at the default settings).
  2. Set the starting %B back to 5%, and change the final %B to 45%.
  3. Set the final %B back to 95%, but change the time for row 2 to 20 minutes.

Question 5

The best separations are the ones that achieve baseline separation for all the analytes and elute them from the column in the fastest possible time.

Using acetonitrile as the solvent B, determine (and report) the best isocratic and gradient separations that you can achieve (within reason) for these compounds. Repeat the above process but with methanol as solvent B.

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