4.1: HPLC Pre-Lab Assigment

During the in-lab portion of this module, you will use different mobile phase conditions to separate mixtures of compounds using reverse-phase HPLC. Please prepare for the in-lab meeting by reading the entire contents of this Module and completing the tasks described below:

Pre-lab assignment: learn the theory that explains separation of compounds using reverse phase chromatography

Investigate parabens: In Part 1, you will separate a mixture of parabens. Parabens are a class of chemicals widely used as preservatives in the cosmetic and pharmaceutical industries. Take the time to learn about parabens and look up general paraben structures to develop an understanding of their chemical structure and polarity. The paraben mixture that you will work with in lab has already been prepared for you and contains 4-hydroxy benzoic acid, methyl-4-hydroxy benzoate, ethyl-4-hydroxy benzoate, and propyl-4-hydroxy benzoate. Draw these structures and order them according to their relative polarity.

Simulated separation: Go to go to the Multi-Dimensional Separations website (click here): http://www.multidlc.org/hplcsim/hplcsim.html. You will use this website to simulate chromatographic separation of a mixture of parabens. Follow the steps in the box below:

Follow these steps to simulate the HPLC separation of paraben compounds:

1. Open the Multi-Dimensional Separations Website (do not use Safari). Take note of three regions of the screen: (1) The controls are on the left (highlighted with a red box and arrow in the figure below). With the controls, you can add or remove compounds, and change the solvent conditions and gradient parameters. (2) A simulated chromatogram is shown at the right. The simulation uses the van Deemtee equation (discussed previously) along with physical properties of the analytes to predict their elution time and peak width in a standard C18 reverse-phase HPLC column. (3) The list of compounds and their respective parameters are listed at the bottom of the screen.

   

   Figure \(\PageIndex{1}\): The opening page of the HPLC Simulator. The controls panel is highlighted with a red box and arrow.

2. Go to Manage Compounds tab of the control panel (top). Select the available paraben compounds (three of them will be in the paraben mixture you will work with in lab) and deselect the compounds that were pre-seleted by default. Note that each paraben compound has multiple possible names - their common names are used in the control panel, but their IUPAC names are given in this manual.
3. Notice that you can locate each compound in the chromatogram. Examine the chomatogram at the right, and the compounds list table listed under the chromatogram. The retention time (\(t_R\) is given for each compound. The \(y\) axis of the chromatogram corresponds to \(t_R\). Click on each of the compounds in the list (below the chromatogram). The selected compound will appear orange in the Simulated Chromatogram window.
4. Go to Column Properties tab of the control panel (bottom). Notice there is only one option for the column (Agilent SB-C18 column), but there are options to change the dimensions of the column (length, inner diameter, porosity, and particle size). Match the dimensions of the columns in our labs at Duke (column details are in the section on HPLC components at Duke). Take note of the calculated void volume and void time (\(t_0\)).
5. Go to Column Properties tab of the control panel. Change the temperature to \(20^{\circ}C\) or something close to room temperature. You can change the temperature by sliding the blue dot to the left and right - when you do this, the chromatogram will change accordingly. Notice the effect that temperature has on the retention time of all analytes.
6. Go to Mobile Phase Composition.
   • Change solvent B to Methanol and select Isocratic Elution Mode. Change the percent of solvent B to match the five isocratic conditions that you will use in Part 1 of the in-lab portion. Observe how the peak resolution, peak shape, and retention times change with differnt solvent conditions.
   • Select Gradient Elution Mode. The default is a linear gradient from 5% to 95% solvent B over 5 minutes. Play around with the length of time of the gradient, and then try some different gradient conditions and observe how the peak resolution and peak shapes change.

Pre-lab questions:

1. Draw the structures of the compounds that will be used in part 1 of the in-lab portion, as well as any addition parabens that were used in the simulation. Compare your predicted elution order to the simulated elution order. Explain why the compounds elute in this order.
2. One of the components in our real mixture, 4-hydroxy benzoic acid, is not available in the simulated list of compounds. Where do you expect this compound to elute relative to the other parabens? Please consider that this molecule is unique compared to the other parabens due to its carboxylic acid functional group, and explain whether that factor should increase or decrease its retention time.
3. Describe the effect of a gradient elution on resolution and peak shape, compared to an isocratic elution.
4. Make a list of materials needed to conduct calibration of the HPLC and measurement of the [caffeine] in beverage samples.
5. (optional) If you wish to measure [caffeine] in a specific beverage, bring some to lab.