Ion Chromatography
Application of Ion Chromatography to the Investigation of Real-World Samples
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Rebecca J. Whelan,*† Theresa E. Hannon, and Richard N. Zare Department of Chemistry, Stanford University, Stanford, CA 94305-5080; *
[email protected] David J. Rakestraw Eksigent Technologies, 2021 Las Positas Court, Suite 161, Livermore, CA 94550
The undergraduate analytical laboratory provides an excellent venue in which to solidify a student’s understanding of concepts learned in the classroom by applying those concepts to interesting and relevant problems. In addition, carefully chosen projects in this laboratory can introduce students to the thrill of discovering new information about real-world samples. With an appropriately designed research plan, many of the analytical techniques commonly used in quantitative teaching laboratories can give high-quality information about the chemical composition of household items, food and beverages, and samples of environmental interest. The laboratory experience can be made more challenging and interesting by charging the students with the responsibility for selecting the system, generating a plan for the investigation, and gathering and preparing samples for analysis. Ion chromatography (IC) is particularly useful for this kind of creative investigation because of its relatively simple operation and ability to analyze many components of a complex sample simultaneously. In addition, a single IC apparatus can be tailored for different classes of analytes by the selection of different column–eluent combinations. In this article we describe the use of IC as a means to teach important analytical concepts while giving the students a valuable opportunity to identify and investigate a real-world system of interest to them. The experiments described here comprise the culminating laboratory experience in the undergraduate lecture and laboratory course in quantitative analysis at Stanford University. They have been tested and refined in the teaching laboratory over a period of four years. The students are given a total of six 3.5-hour periods in the laboratory to complete the activities described below. After using the IC system in a directed manner to develop familiarity with the system and its capabilities, the students are given considerable freedom to select any real-world system that is amenable to analysis by IC. Students have demonstrated impressive creativity and ambition in choosing their real-world systems, two of which are described in detail. Information about the principles and practical considerations of IC can be found in analytical textbooks (1, 2) and specialized texts (3, 4). Applications of commercial IC instruments in the teaching lab for the analysis of cola beverages (5), pond waters (6), and a few other specific systems (7–10) have appeared in this Journal. Reports describing student-directed or project-based experiments for the quantitative analysis laboratory using other instrumental approaches have also been described in this Journal (11–14). † Address after January 2005: Department of Chemistry, Oberlin College, Oberlin, OH 44074.
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The Ion Chromatography System We use two different ion chromatography systems in the undergraduate analytical laboratory, both of which are based on the LC20 chromatography enclosure from Dionex (Sunnyvale, CA) with a GP50 gradient pump. One system is configured for the detection of transition-metal ions. This system uses an oxalic acid eluent, a mixed cation兾anion stationary phase (CG5A guard column and CS5A separator column), a post-column mixer where the analytes are chemically complexed with the chromophore 4-(2-pyridylazo)resorcinol (PAR), and an absorbance detector cell. The absorbance of the transition metal–PAR complex is monitored at 530 nm. The other system has an anion exchange column (AG12A guard column and AS12A separator column) followed by a suppressor and a conductivity detection cell and uses a carbonate兾bicarbonate eluent system. Cations can be separated instead by replacing the anion exchange column with a cation exchange column (CS12A) and changing the eluent. For the transition-metal separation (with the CS5A column), the mobile phase was 80 mM oxalic acid兾100 mM tetramethylammonium hydroxide兾50 mM potassium hydroxide in deionized water, pH 4.7. For anion separation (with the AS12A column), the mobile phase was 2.7 mM Na2CO3兾0.3 mM NaHCO3 in deionized water, and for cation detection (with the CS12A column), the eluent was 20 mM methanesulfonic acid in deionized water. Injection volume for both instruments is 25 µL. Data collection is controlled by PeakNet software developed by Dionex. Detailed information about the instrumentation and reagents can be found in the Supplemental MaterialW for this article and on the Dionex Web site (15). Student Teams Enrollment in the undergraduate analytical laboratory is usually 20 students, and for the IC experiment the class is divided into teams. The teams are usually composed of three students. In our four years of teaching this laboratory experiment, we have tried different ways of forming the teams. Sometimes the students have been allowed to form their own teams, with the only restriction that they choose someone with whom they have not been teamed up earlier in the course. The purpose of forming new groups is that the students gain experience working with different people—in groups with different working dynamics and not only with their friends. In other years, the teaching staff has assigned the teams. In these assignments, there is some effort to form groups that seem balanced and likely to work well together. Within the groups, the division of labor is not dictated.
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Students are encouraged to vary their work roles within the group so that each student gains experience with the different aspects of the experiment, but there is no formal assignment or alternation of roles. The use of teams is motivated in part by the scarcity of equipment (we only have two IC setups) and also to give the students more experience working collaboratively. The final writeups are done collaboratively and are written following the structure and conventions of the chemical literature. Performing the data evaluation and writeup in a group is intended to familiarize the students with the manner in which research publications are normally prepared. Investigation of an Instructor-Prepared Unknown The ion chromatography laboratory experience begins with several directed activities. These activities are intended to familiarize the students with the IC system and to help them develop analytical skills, including identification of unknowns, preparation and use of a calibration curve, determination of detection limits, and evaluation of the accuracy and precision of an experimental method. Two or three students work together in teams. The teams are assigned to either the anion or the transition-metal IC system. The experimental procedure and data analysis are virtually identical for the two systems, so that the students gain similar experience using either instrument. The experiment, which is to be completed in two laboratory periods, consists of four parts: 1. Measurement of the retention times of seven anions (Br−, Cl−, F−, NO3−, NO2−, PO43−, and SO42−) or six transition-metal cations (Cd2+, Co2+, Cu2+, Ni2+, Pb2+, Zn2+) 2. Determination of approximate detection limits for these ions 3. Identification of the components of an unknown sample prepared by the teaching staff 4. Creation of a calibration curve and determination of concentration of ions in the unknown sample using chromatogram peak areas
The detailed instructions given to the students can be found in the Supplemental Material.W Ionic concentrations as low as approximately 1 mg兾L are routinely detected during these investigations. Measuring Contributions to Band Broadening The students spend one laboratory period investigating the dependence of separation efficiency on flow velocity. Using a standard solution containing several analytes, the students collect one chromatogram at six different flow velocities ranging from 1.0 mL兾min to 1.6 mL兾min. From each chromatogram, plate height for each analyte can be determined by measuring the peak width and retention time. Plotting plate height as a function of flow rate, the students create a van Deemter plot from which they determine the optimal flow rate. Using flow rates between 1.0 mL兾min and 1.6 mL兾min, the students usually find that the lowest flow rate gives the most efficient separations.
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Investigation of a Real-World Unknown For the analysis of a real-world unknown, the teams define an analytical problem that is of interest to them and that can be investigated using the IC apparatus. The students have virtually unlimited range in the system they choose. To help them generate ideas, the students are encouraged to read the application notes from Dionex, survey the current analytical literature, or discuss ideas with members of the teaching staff. Teams have found ideas from product labels, news items, and things they see around them every day. Once the students have identified a system of interest, they are asked to develop a detailed plan for several important steps in the analysis: gathering samples, preparing samples for analysis, calibrating the system, running the samples on the ion chromatograph, and analyzing and interpreting the data. The research proposal, which may or may not include a hypothesis to be tested, is presented informally, in conversation with a member of the teaching staff. Students usually spend two lab periods (3.5 hours each) planning their real-world sampling strategy and doing sample preparation. Another two lab periods are spent running the samples on the IC instruments. Students usually collect samples on their own time, and they are given containers appropriate to their projects in which to collect samples. The groups are encouraged to choose investigations that are realistic for the allotted time period. They typically design studies that require few sample preparation steps. In addition, if their sample chromatograms contain a large number of peaks, they are required to identify and quantify no more than three of the ionic species present. The students must get their research plan approved by the instructor or head teaching assistant, and they must demonstrate a good understanding of any hazards or special safety precautions necessary. Once the experiment is complete, the students work collaboratively to prepare a lengthy, journal-style report of their findings. The reports tend to be between 15 and 20 typewritten pages, including tables, graphs, and references. The teaching staff grades the reports, and the same grade is assigned to all the student authors. Owing to the complexities of real-world sample analysis, grading tends not to emphasize the quality of the data. Instead, points are awarded for clear presentation and reasonable discussion of the data, as well as for a demonstrated understanding of the principles behind the technique. One modification to the report format we have recently implemented is having the students report the results for the instructor-prepared unknown and band-broadening experiments on a worksheet, leaving the formal extended writeup to discuss exclusively the investigation of the real-world sample. We have only tried this organization once, but it seems to help the students focus their writing efforts on the real-world experiments. In the four years we have used this laboratory experience in our class, we have never seen a real-world sample analysis completely fail. The students have always found at least one peak in the samples they chose.
Example 1: Lead Content in Hair Dyes One real-world system, chosen by a student team, was the lead content of progressive hair dyes such as Grecian For-
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mula and Youthair. Lead acetate is the active ingredient in progressive hair dyes; the coloring effect results from the reaction of lead with sulfur (present in hair in small quantities) to produce lead sulfide, a black solid (16). When applied over a period of weeks, these products coat the hair shaft and give a gradual darkening to graying hair. The toxicity of lead absorbed by the body is well-known and has motivated the FDA to set a limit of 0.6% (weight兾volume) for lead in hair dyes (17). The students used IC to determine the lead content of two progressive hair dyes, Grecian Formula and Youthair, and a third brand of hair dye, ColorSpa, that does not specify lead as an ingredient. Sample preparation was straightforward, consisting of diluting the dyes in distilled deionized water to concentrations that gave good signal-to-noise ratios without overloading the column (between 1:10 and 1:100 dilution). The samples were examined on a UV-vis absorbance detector to confirm that any pigments present in the dyes would not interfere with the detection at 530 nm of PAR-complexed lead in the IC detection cell. A calibration curve was prepared by running seven standard Pb2+ solutions of known concentration in the IC and recording their absorbance. The flow rate, 1.1 mL兾min, had been previously determined to be optimal from the van Deemter plot. Each dye sample was run at least four times to evaluate the precision of the method. Calibration curves and unknown samples were measured on the same day to minimize the effect of day-to-day variation in the system. The students found no lead content in ColorSpa, as expected. At a 95% confidence interval, Grecian Formula was found to contain 0.157 ± 0.007% Pb2+, and Youthair was found to contain 0.35 ± 0.09% Pb2+, indicating that both lead-containing dyes were under the FDA limit of 0.6%.
Example 2: Anion Content in Fresh and Canned Juices Another real-world system recently investigated by a student team is the anion content of liquid from canned mandarin oranges and from fresh oranges. Elevated levels of nitrate and nitrite have been reported in some brands of canned vegetables and juices (18), making the determination of these anions in canned food important. In addition, the fluoridation of many water supplies results in high fluoride levels in produce, and the use of phosphate-containing fertilizers raises concern about the levels of phosphate in foods (19). For this investigation, the students used three brands of canned mandarin oranges, Safeway, Geisha, and Dole, and compared their anion levels to each other and to the levels found in the juice from a fresh orange. The juice was collected from the canned oranges by straining, and then further filtered by vacuum filtration. The orange was squeezed, and its juice was separated from the pulp by filtration. The juices were then injected onto the column. A standard solution of anions was run to determine their retention times. The presence of the anions in the different juice samples was determined by spiking a known quantity of each anion into the juice. The spiking experiments indicated the presence of chloride, fluoride, and phosphate in all four samples, whereas nitrate and nitrite were not detected. A calibration curve was prepared for chloride, fluoride, and phosphate by measuring
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the response of the IC to various known concentrations of each anion. The calibration curve was then used to determine the concentration of each anion in the unknown samples. Overall, fresh orange juice contained the highest concentration of phosphate (3.7 mM) and the lowest concentration of fluoride (2.2 mM) and chloride (2.6 mM). Safeway brand mandarin oranges had the highest concentrations of fluoride (2.9 mM) and chloride (3.2 mM).
Other Examples In the past four years that we have done the real-world IC experiment in our quantitative analysis lab, the student teams have successfully investigated many other interesting systems. Some examples include: lead in Chinese herbal medicines, cations in beer, transition metals in water from San Francisco Bay, fluoride in toothpastes and mouthwashes, anions in performance drinks, and transition metals in old batteries. The most interesting investigations have been those in which the students presented a clear hypothesis about the system that the IC data would help them to test. Hazards Safe laboratory practices—including the use of protective clothing, eyewear, and gloves when handling chemicals —should be followed while performing these experiments. In particular, lead and cadmium are extremely toxic and should be handled with gloves and properly disposed of. Solutions of transition-metal ions are usually prepared in dilute nitric acid or hydrochloric acid, creating an additional hazard. The PAR solution contains 2-dimethylaminoethanol, which is corrosive, toxic, and a lachrymator, and ammonium hydroxide, which is corrosive. PAR itself produces an obnoxious smell. Solutions of PAR should be made and handled in a fume hood using gloves and eye protection. The eluent used with transition metals contains tetramethylammonium hydroxide and potassium hydroxide, both of which are corrosive and should always be handled with gloves. IC is a form of HPLC, which means that high pressure is applied to the column. The system should be verified to be correctly assembled and leak free. The hazards associated with the realworld sample vary depending on the system the students choose. The students and teaching staff should work together to identify sources of risk and decide on appropriate precautions before the students begin work in the lab. Conclusions The response from the students about the real-world sample analysis consistently has been enthusiastic. Most of the students do outside reading and research about their topic, and the instructor and teaching staff grading the final reports often learn something new. In contrast with more “cookbook” laboratory experiences that often involve performing measurements that have been done in the same way by previous generations of students, the results in these investigations are truly novel. For many students, this experience is their first encounter with the creative and independent mode of thought required in scientific research. It helps to impress upon them the relevance and importance of concepts they encounter in their textbooks.
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Acknowledgments The funds to purchase the IC system came from a Bing Award for Undergraduate Teaching. The lead in hair dye experiment was designed and performed by Philip Lee, Andres Martinez, and Aaron Staple. The orange juice experiment was designed and performed by Fredysha McDaniel, Jodie Prud’homme, and Leah Barrera. We thank all the Stanford University undergraduates who have taken Chemistry 134 and the graduate students who have been the teaching staff for their contributions to the development of this experiment, especially Walker Wu, Li Zheng, Kim Nguyen, Cheol Keun Chung, and Mary Campbell. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Skoog, D. A.; West, D. M.; Holler, F. J. Fundamentals of Analytical Chemistry, 7th ed.; Saunders College Publishing: New York, 1996. 2. Harris, D. C. Quantitative Chemical Analysis, 5th ed.; W. H. Freeman and Company: New York, 1999. 3. Small, H. Ion Chromatography; Plenum Press: New York, 1989.
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4. Haddad, P. R.; Jackson, P. E. Ion Chromatography: Principles and Applications; Elsevier: New York, 1990. 5. Bello, M. A.; Gonzalez, A. G. J. Chem. Educ. 1996, 73, 1174– 1175. 6. Sinniah, K.; Piers, K. J. Chem. Educ. 2001, 78, 358–362. 7. Graham, R. C.; Robertson, J. K.; Loehle, W. D. J. Chem. Educ. 1982, 59, 340. 8. Koubek, E.; Stewart, A. E. J. Chem. Educ. 1992, 69, A146. 9. Kieber, R. J.; Jones, S. B. J. Chem. Educ. 1994, 71, A218. 10. Dahl, D. B.; Riley, J. T.; Green, T. K. J. Chem. Educ. 1998, 75, 1209. 11. Higginbotham, C.; Pike, C. F.; Rice, J. K. J. Chem. Educ. 1998, 75, 461. 12. Eierman, R. J. J. Chem. Educ. 1998, 75, 869. 13. Dunn, J. G.; Phillips, D. N. J. Chem. Educ. 1998, 75, 866. 14. Henderleiter, J.; Pringle, D. L. J. Chem. Educ. 1999, 76, 100. 15. Dionex Home Page. http://www.dionex.com/ (accessed Jun 2004). 16. Lansdown, A. B. G. Int. J. of Cosmetic Sci. 2000, 22, 167–168. 17. U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Cosmetics and Colors Fact Sheet, Title 21, section 73.2396. http://vm.cfsan.fda.gov/~dms/ cos-lead.html (accessed Jun 2004). 18. Tsang, C. F.; Tsang, C. W. Food Additives and Contaminants 1998, 15, 753–758. 19. Buldini, P.; Cavalli, S.; Trifiro, A. J. Chromatogr. A 1997, 789, 529–548.
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