Adam Griffith Chem 384 March 2002 page 1 Adam Griffith
CHEM 384
HPLC Sunscreen Analysis Introduction
The objective of this lab is to become familiar with the use of HPLC analysis in the determining of chemical compositions of materials through the analysis of sunscreen. The techniques used in this lab can be applied to analysis of most chemicals that absorb in the ultraviolet and visible spectrum, thus making the HPLC a valuable tool in analytical chemistry.
Background
Sun burn and its link to skin cancer are linked together with ultraviolet (UV) radiation. UV radiation is broken down into three categories — UVA, UVB, and UVC. Of these, only UVA and UVB are of any immediate concern. UVC is absorbed by the ozone layer and the atmosphere; however, in future years UVC may become a problem due to changes that we are making in the atmosphere. UVA is considered to be the cause of “immediate sunburn” which diminishes shortly after exposure to sunlight. UVA is generally considered to be in the 320-400 nm wavelength range. UVB (290-320 nm) is the source of “delayed sunburn”, which occurs 12-24 hours after exposure to sunlight, and is also considered to be the primary source of sun-related skin cancer and skin aging. For this reason, most sunscreens are designed to block primarily in the UVB range. They accomplish this task by placing chemicals in sunscreen that absorb UV radiation in this range. Common chemicals found in sunscreen are oxybenzone (benzophenone), octyl salicylate, octyl methoxycinnimate, and octyl dimethyl PABA (Padimate 0). The maximum amounts of these chemicals that may be placed in sunscreen are 6%, 5%, 7.5%, and 8% respectively by weight or volume (depending on phase).
Procedure
The procedure for this experiment was primarily based from the procedure used in (1). The procedure calls for the creation of four standard solutions of the chemicals listed above, with four different dilutions of each (total 16 standards), as well as two solutions of common sunscreens. The two sunscreens used were Banana Boat Sunblock Lotion SPF 15 and Walgreen’s Sunblock Lotion SPF 25.
Sunscreen Solution Procedure
1) Accurately measure 0.2500 g of sunscreen and place in a 50 ml beaker.
2) Add 40 ml 2-Propanol to the beaker and stir (use magnetic stir bar) until sunscreen is dissolved or a homogenous suspension is created.
3) Place the suspension/solution into centrifuge tubes. Use the centrifuge.
4) When particulate material has collected to the bottom of the tubes, draw off the solution with pipettes, making sure not to disturb the particulate. Dilute this solution to 100 ml in a volumetric flask.
5) Repeat steps 1-4 for at least one other sunscreen. Standard Solution Procedure
Adam Griffith Chem 384 March 2002 page 2
Based upon the amount of sunscreen used above and the maximum allowed percentage composition of the chemical being used, standard solutions are to be prepared. What follows are the amounts used for ~0.2500 g of sunscreen dissolved and diluted to 100 ml. All dilutions are with 2-Propanol and are to be done in volumetric flasks.
Padimate 0 (CAS#: 21245-02-3):
1) Dissolve 0.5 ml Padimate 0 into 250 ml 2-Propanol (Solution A) 2) Take a 5 ml aliquot of Solution A and dilute to 50 ml (or 10 ml to 100) 3) Take a 2.5 ml aliquot of Solution A and dilute to 50 ml (or 5 ml to 100) 4) Take a 1 ml aliquot of Solution A and dilute to 50 ml (or 2 ml to 100) 5) Take a 0.5 ml aliquot of Solution A and dilute to 50 ml (or 1 ml to 100)
Octyl Methoxycinnimate (CAS#: 5466-77-3):
1) Dissolve 0.5 ml Octyl Methoxycinnimate into 250 ml 2-Propanol (Solution B) 2) Take a 10 ml aliquot of Solution B and dilute to 100 ml (or 5 ml to 50)
3) Take a 5 ml aliquot of Solution B and dilute to 100 ml (or 2.5 ml to 50) 4) Take a 1 ml aliquot of Solution B and dilute to 50 ml (or 2 ml to 100) 5) Take a 0.5 ml aliquot of Solution B and dilute to 50 ml (or 1 ml to 100)
Benzophenone (CAS#: 131-57-7):
1) Dissolve 0.25 g Benzophenone into 250 ml 2-Propanol (Solution C) (may require mild heating)
2) Take a 15 ml aliquot of Solution C and dilute to 100 ml (or 7.5 ml to 50) 3) Take a 5 ml aliquot of Solution C and dilute to 100 ml (or 2.5 ml to 50) 4) Take a 1 ml aliquot of Solution C and dilute to 50 ml (or 2 ml to 100) 5) Take a 0.5 ml aliquot of Solution C and dilute to 50 ml (or 1 ml to 100)
Octyl Salicylate (CAS#: 118-60-5):
1) Dissolve 0.25 ml Octyl Salicylate into 250 ml 2-Propanol (Solution D) 2) Take a 15 ml aliquot of Solution D and dilute to 100 ml (or 7.5 ml to 50) 3) Take a 5 ml aliquot of Solution D and dilute to 100 ml (or 2.5 ml to 50) 4) Take a 1 ml aliquot of Solution D and dilute to 50 ml (or 2 ml to 100) 5) Take a 0.5 ml aliquot of Solution D and dilute to 50 ml (or 1 ml to 100) HPLC Analysis Procedure
1) Turn on the HPLC and begin pump
2) Clean the HPLC injection syringe using Acetone, DI water, and Acetone and dry glass and metal parts in oven (do NOT put the plastic parts in the oven)
3) Wash the injection coil with 3 20 µl samples of the standard solution being tested or of the sunscreen solution being analyzed
4) Set the method and parameters to be used (310 nm, 50/50 Water/Methanol, 1ml/min flow, 15 min stop time, 400 bar pressure max.)
5) Inject ~30 µl into the coil and move lever to inject. Remove syringe and move lever back.
6) Repeat for all standards and sunscreens. There should be only one peak for each standard and several peaks for each sunscreen.
Adam Griffith Chem 384 March 2002 page 3 Data
Only a few standards could be run before time ran out for this lab. The results from the HPLC showed multiple peaks for one standard chemical, indicating the presence of other chemicals in the test. Another run of the same chemical with the same parameters but with a stronger concentration resulted in peaks at approximately the same location with approximately the same size, which should not have occurred based on the dilutions used. This suggests that either the standard chemical is in too low of a concentration to be detected or the solvent used by the HPLC should be different. Either way, there was not time to prepare a more concentrated solution or to change the solvent before time ran out.
Discussion and Conclusion
The results of this experiment were inconclusive. To devise a proper procedure many more HPLC tests must be run for each chemical with varying parameters such as wavelength measured, solvents used, and solvent ratios. Additionally, running a UV-vis analysis on each of the standards should be done to locate the proper wavelength for analysis. Based on the results, the following changes are recommended:
Recommended Changes to the Procedure Day 1
1) Standard solutions should be made at four dilutions between 1 and 10% (v/v or w/v depending on phase of chemical). Both students each prepare 2 sets of solutions.
2) The sunscreen solution should be made from ~2-2.5 g sunscreen per 100 ml 2-Propanol, instead of 0.25 g per 100 ml. (One person prepares sunscreen while the other performs UV-vis analysis of standards). If possible, prepare a second dilution of each sunscreen.
3) UV-vis analysis should be performed on one dilution of each of the four standard solutions. From this, a single wavelength should be determined where all four chemicals absorb in significant amounts. (One person performs UV-vis analysis while the other prepares the sunscreen solution).
4) Begin trial runs on HPLC to determine proper operating parameters. Use 50/50 Water/2-Propanol for initial mixture, 10-15 minute trials. (This can be done by one person if the other is still preparing the sunscreen solution). Recommend time be reduced to 5 minutes if possible.
5) Begin performing analysis of standards when HPLC parameters have been determined and if time permits.
Day 2
1) Perform HPLC analysis of standards and sunscreen solutions. Begin with one trial for each solution/dilution.
2) If time permits, perform a second trial for each solution/dilution. A third trial may be performed if there is still time.
3) Create calibration curves from the standard results and analyze data for the sunscreens. 4) Report the analyzed concentration of each chemical in each sunscreen solution. Convert this
Adam Griffith Chem 384 March 2002 page 4 Sources
(1) Abney, Scalettar, “Saving Your Students’ Skin. Undergraduate Experiments That Probe UV Protection by Sunscreens and Sunglasses”, Journal of Chemical Education, Vol. 75, No. 6, June 1998.
(2) Reisch, Marc S., “Spotlight on Sunscreens”, Chemical and Engineering News, Dec 3, 2001. (3) Walters, et. al., “The Spectrophotometric Analysis and Modeling of Sunscreens”, Journal of
Chemical Education, Vol. 74, No. 1, Jan 1997.
