HPLC Lab
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SCHA-319
High Performance Liquid Chromatography (HPLC) is a very powerful and commonly used separation method. Its applications range throughout the chemical, biochemical, biological and pharmaceutical sciences. HPLC has two major advantages over gas chromatography. First, for GC the analytes must be volatile (only 15% of all compounds fit this requirement) and second, the mobile phase only serves as a mass transport function, since gasses make very poor solvents. In HPLC we only need compounds that are soluble in our mobile phase and we can use mobile phases with differing solvating powers and thus gain a greater level of control over the separation process.
In this lab we will separate and quantify the amount of caffeine, acetaminophen and aspirin present in Excedrin
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formulations and will compare this to the amount in a generic product. We will also examine a diet cola product and quantify the amount of caffeine present.
The stationary phase used in this lab is the standard packing used most commonly which is octadecylsilane (or C18). The mobile phase we will use is a mixture of methanol and water to which a small amount of acetic acid has been added. The mobile phase is very polar and compounds that are more polar will spend more time in the mobile phase and thus elute more quickly than less polar compounds.
Procedure:
Prepare your tablets and standards in A335.
Note: Filter sufficient amount of the solutions to proceed to the next dilution step.
All samples injected into the HPLC must be filtered.
You should weigh your tablets before you grind them up so you can determine the percent active ingredients in a tablet. Cite this value in your report.
Solution 1. Grind up a single Excedrin and dissolve the powder in 65 mL of methanol.
Transfer this solution to a 100 mL volumetric flask. Fill to the mark with 1% Acetic acid in water. Filter enough of this solution to get 2.00 ml of this solution and dilute up to
25.0 ml in a volumetric. Transfer this solution to an HPLC vial. Cap and label this vial.
Solutions 2 and 3. Also prepare a generic tablet and a Canadian (or other alternate) tablet in the same way as for solution 1.
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Solution 4. Acetaminophen Standard. Dissolve 6.5 mg (known exactly) of
Acetaminophen in ~15 mL of methanol. Transfer this solution to a 25 mL volumetric flask and fill to the mark with the 1% acetic acid in water.
Solution 5. Aspirin Standard. Dissolve 25 mg (known exactly) of Aspirin in ~15 mL of methanol. Transfer this solution to a 25 mL volumetric flask and fill to the mark with the
1% acetic acid in water.
Solution 6. Caffeine Standard. Dissolve 20 mg of caffeine in ~15 ml of methanol.
Transfer this solution to a 25 mL volumetric flask and fill to the mark with the 1% acetic acid in water.
Transfer, with filtration, solutions 4- 6 to labeled HPLC vials.
Bring these solutions to A143 for analysis.
Use the sequence table to automate your analysis to capture the quantitative data. You may start with SCHA319 and modify it. You may use another method if directed by the instructor. Discuss with your lab instructor the various parameters in the method.
Using solution 1 (or an older solution of Excedrin) modify the percentages of the mobile phase components. Comment in your report on the effects you see. Modify the wavelength of detection. What effect does this have?
Each run takes about 4 to 6 minutes. So you have plenty of time to modify things. Select other parameters to modify. What effect do these modifications have?
Set up and start your sequence. Run the various tablet samples and your single compound standards. While the data is being collected for the first several samples you may return to A335 and prepare your standards for the quantification part of the experiment. You sequence may be modified, i.e. added to, on the fly. The method cannot be.
As part of your sequence Run a sample of the Excedrin that has been sitting in solution for a while. Do you see four peaks from a sample that has been sitting around for a while? Why would that be? What is the extra compound?
Quantification samples. (Use pipets with disposable tips for these measurements)
Solution M0. Prepare a solution that has a filtered 0.250 ml of each of the actives
(solutions 4 – 6) diluted up to 10.0 mL of mobile phase in a volumetric.
Solution M1. Prepare a solution that has a filtered 0.500 ml of each of the actives
(solutions 4 – 6) diluted up to 10.0 mL of mobile phase in a volumetric.
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Solution M2. Prepare a solution that has a filtered 1.00 ml of each of the actives
(solutions 4 – 6) diluted up to 10.0 mL of mobile phase in a volumetric.
Solution M3. Prepare a solution that has a filtered 2.00 ml of each of the active
(solutions 4 – 6) diluted to to 10.0 mL of mobile phase in a volumetric.
Solution M4. Prepare a solution that has a filtered 3.00 ml of each of the active
(solutions 4 – 6) diluted to to 10.0 mL of mobile phase in a volumetric.
From the data in solutions M0-M4 and the values obtained from solutions from the three tablets report the amount of the three actives in the three different products. You will need to prepare a calibration curve to do this (use Excel). Pay attention to the dilutions you made and be aware that the FDA will not allow massive deviations from the bottle assay. Comment in your report on what your sources of error might be. I suggest that you plot milligrams of the actives on column and then factor back all you dilutions to get assay results in mg per tablet. Good practice calls for you to do at least duplicate injections for the quantitative standards and the unknowns.
You should also make a blank injection.
Include an injection of a sample of flat diet Pepsi. What is amount of caffeine in a 12 ounce can. How does this compare to the expected amount? (Search the web) What might be the other peaks in this assay – look at the ingredients? Run a suspect or two.
What are the structures of the actives? These should be included in your report.
What column did you use? Stationary Phase?, Length? and bore? Brand name? You should calculate the number plates in the column (use the aspirin peak). Calculate the resolution between the acetaminophen and caffeine. What are the capacity factors for the three active ingredients?
You may depart the lab when you quantitative data collection has started. This is one of the major reasons we have an auto sampler. Your sequence should have a shutdown procedure included. Your instructor will assist you with this.
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