Part 1: Readying the HPLC for Operation

1. Ready the HPLC by making sure the solvent reservoirs are full and the waste bottles have at least 1 Liter of volume available to accommodate the waste solvent.

2. Launch Open Lab by clicking HPLC1 (online) (Figure 1.1)

Figure 1.1: HPLC1 (online) desktop icon.

3. Use the Instrument Control Tab. Confirm that all components are green and indicate “ready.” (Figure 1.2)

a. If this is not the case, press the “On” button.

b. The DAD will take a few minutes to warm up; if there is a lightning bolt through the purple lamp, wait until it has gone away, and the ready bar has turned green.

c. The fraction collector will not be used in this experiment.

Figure 1.2: "Instrument Control" tab indicted by top, green arrow. Bottom right, red arrow indicates the DAD window.

Part 2: Separating a Paraben Mixture

Parabens are a class of chemicals widely used as preservatives in the cosmetic and pharmaceutical industries. Parabens are effective preservatives and are primarily used for their bactericidal and fungicidal properties. Parabens can be found in shampoos, shaving gels, personal lubricants, topical/parenteral pharmaceuticals, spray tanning lotion and toothpaste. Parabens can also be used as food additives. Recently, there has been an in-vivo and in-vitro studies have shown that parabens have weak estrogenic activity. Because parabens are endocrine disrupters, paraben concentrations have been mitigated and must not surpass the maximum permitted levels. Paraben mixtures are often used in order to increase the efficiency of the preservation. Take the time to look up general paraben structures to develop an understanding of their chemical structure.

Because parabens are often found in mixtures, HPLC can be used to separate the individual compounds. To determine the components in the mixture, reverse phase HPLC will be performed through a C₁₈ column. In this portion of the experiment, gradient and isocratic elution data will be collected and compared. The paraben mixture 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.

To begin, five isocratic experiments will be performed. The conditions for the five experiments are listed in Table 2.2.

Table 2.2: Isocratic Experiment Conditions