In the Laboratory
filtrates & residues
Analysis of Mouthwash Eleanor Siegrist Hollidaysburg Area High School, Hollidaysburg, PA 16648 Guy Anderson Bald Eagle Area High School, Wingate, PA 16823
The Science in Motion van project has been bringing chemical instrumentation to high schools in central Pennsylvania for the past seven years. The project now includes a biology van in central Pennsylvania and another chemistry van in the Mon Valley region of western Pennsylvania. These vans are staffed by certified chemistry or biology teachers and permit high school students to use equipment not found in most high schools. High school chemistry teachers from the van service areas have developed laboratory experiments for student use. For example, one activity uses the gas chromatograph to analyze a mixture of alcohols. Students determine the number of alcohols in a mixture and calculate the percentage of each component. Students also use visible spectrophotometers to determine the concentration of a colored solution. The mouthwash activity described here was developed as an experiment for second-year students who have completed the separation of alcohols and spectrophotometer experiments. Activities for the determination of alcohol content of alcoholic beverages can be found in textbooks (1) and this Journal (2). While this is an appropriate activity for a college laboratory, this would not be appropriate for high schools. Mouthwashes were chosen as an alternative. Not only do they have varying alcohol contents, but they also contain a variety of FD&C dyes that can be extracted and separated by solid-phase extraction tubes and then identified by use of a visible spectrophotometer. This experiment has been used successfully for the past two years by high school students in central Pennsylvania.
drawn through and placed in test tube 3. Next, 5 mL of 20% isopropyl alcohol is used and the eluate is placed in test tube 4. Finally, 5 mL of 40% isopropyl alcohol is drawn through the column and placed in test tube 5. All volumes of alcohol are kept at 5 mL to simplify the procedure and all the eluates are collected so that a desired sample is not discarded. If a distinct color change is observed during one of the above steps, stop drawing the alcohol solution through the column when the color change occurs. For example, Red No. 40 may still be coming off the column when Blue No. 1 begins to be removed. The Red No. 40 solution may be placed in the previous test tube and the Blue No. 1 solution may be removed and placed in the appropriate test tube. If some dye still remains on the column, rinse the C-18 tube with
Separation of Dyes Before extracting the dyes, most of the alcohol should be removed from the mouthwash. Samples of the mouthwashes (15 to 20 mL) are gently heated on a hot-plate until the volume is reduced to one third of the original volume. The samples should cool before being used. C-18 solid-phase extraction tubes (Supelco 5-7055 or Fisher P479) are used to extract and separate the dyes. Eluents are pulled through the column using a 10-mL syringe and a three-way stopcock (BioRad 732-8107). Both the solid-phase extraction tube and the syringe are connected to the stopcock (see Fig. 1). The C-18 tube is primed by drawing a 5-mL portion of isopropyl alcohol through the column. This is followed with 5 mL of distilled water. A small amount of water should be left on the column. Eluents from these steps may be washed down the drain with a large volume of water. Five test tubes are needed for the separation. Place a 2-mL sample of the concentrated mouthwash in the C-18 tube. Slowly draw the sample onto the column. Place the eluate in test tube 1. The C-18 tube is washed with 5 mL of distilled water. This eluate is placed in test tube 2. Place 5 mL of 10% isopropyl alcohol in the C-18 column. This is
Figure 1. Diagram of apparatus used to extract and separate dyes from mouthwash. Two types of C-18 solid-phase extraction tubes are illustrated.
Vol. 74 No. 5 May 1997 • Journal of Chemical Education
567
In the Laboratory
Table 1. Maximum Wavelengths of Dyes FD&C Dye
Wavelength (nm)
Blue No. 1
630
Blue No. 2
610
Green No. 3
625
Red No. 3
527
Red No. 33
530
Red No. 40
502
Yellow No. 5
428
Yellow No. 6
484
Yellow No. 10
413
Table 2. Student Results, Mouthwash Analysis Dyes Mouthwash
Found
Label
Listerene Mint
Green No. 3
Scope Peppermint TWIW Rinse
Label
Green No. 3
22
21.6
Blue No. 1
Blue No. 1
19.5
16.6
Red No. 40
Red No. 40
8.2
7.5
Act for Kids
Red No. 33
Red No. 33
0
0
Treasury Golden
Yellow No. 5
Yellow No. 5
16
14
TWIW Mint
Blue No. 1 Yellow No. 5
Blue No. 1 Yellow No. 5
19.2
18.9
100 % isopropyl alcohol until the column is clean. The authors have successfully reused the columns with several sections of students. Dye Identification Milton Roy Spectronic 20 spectrophotometers have been used to identify the dye(s) extracted from the mouthwash samples. Most commercial mouthwashes contain one or two dyes. The instruments should be “on” for 15 min before they are to be used. Two or three cuvettes are selected. One is filled with distilled water to serve as a blank. The others hold the dye samples from the separation procedure above. Advise students that the same dye may be found in more than one test tube. Have them select the dye solution that has the most intense color. Students measure the absorbance of each dye every 25 nm from 400 to 650 nm. Additional measurements may be made at smaller wavelength intervals as the absorbance reaches its maximum value. Remind students to rezero the instrument each time the wavelength is changed. A scanning instrument could be used if one is available. Students plot the absorbance of the dye (y-axis) as a function of wavelength (x-axis). If two dyes were found they may be plotted on the same graph. The wavelength of maximum absorbance is then determined. From a list of dyes and the wavelengths of maximum absorbance, dye(s) may be identified. Table 1 is a partial list of FD&C dyes from the Handbook of U.S. Colorants, Foods, Drugs, Cosmetics and Medical Devices (3). Percent Alcohol Determination The gas chromatograph should be “on” before the beginning of this part of the experiment. Leary (2) recommends the preparation of alcohol standards for a procedure of this type. Solutions of 5, 10, 20, and 30% ethyl alcohol and water (volume:volume) are prepared. Each laboratory
568
Percent Alcohol Found
group may be assigned to prepare one of the solutions. Solutions may then be shared by all the laboratory groups. Inject 5-µL samples of each standard and the original mouthwash sample into the gas chromatograph. Determine the area under each peak by measurement or by cutting out the peaks and weighing them. A table should be prepared containing the following information: percent alcohol (standards), area of alcohol peak, area of water peak, total area (water + alcohol), and percentage of area due to alcohol. The percentage due to alcohol (y-axis) is plotted against percent alcohol (x-axis). A best-fit line (curve) is drawn. This calibration graph is used to determine the percent alcohol in the original mouthwash sample. Table 2 contains typical student results. Students do a very good job of identifying the dyes. Accuracy of the alcohol determination depends upon how carefully students measured their peak areas from the gas chromatograph. This activity provides a review of various analytical techniques. Students learn that more than one instrument or technique may be required to analyze a product. Interest is increased by the use of a common product, and safety concerns are minimized because the only reagents used are ethyl alcohol and isopropyl alcohol. Acknowledgment We wish to thank Donald Mitchell and the Science in Motion van project at Juniata College in Huntingdon, PA, for their support and guidance in developing this experiment. Literature Cited 1. Skoog, D. A.; West, D. M.; Holler, F. J. Fundamentals of Analytical Chemistry, 5th ed; Saunders: New York, 1988; pp 805–806. 2. Leary, J. J. J. Chem Educ. 1983, 60, 675. 3. Marmion, D. M. Handbook of U.S. Colorants, Foods, Drugs, Cosmetics, and Medical Devices, 3rd ed.; J. Wiley and Sons: New York, 1991; p 249.
Journal of Chemical Education • Vol. 74 No. 5 May 1997
