In the Laboratory edited by
Topics in Chemical Instrumentation
David Treichel Nebraska Wesleyan University Lincoln, NE 68504
Capillary Electrophoresis Analysis of Cations in Water Samples
W
An Experiment for the Introductory Laboratory Christopher J. Pursell, Bert Chandler, and Michelle M. Bushey* Department of Chemistry, Trinity University, San Antonio, TX 78212-7200; *
[email protected] Trinity University is the recipient of a NSF–CCLI A&I grant, DUE-995227, which allowed for the procurement of fully automated HPLC and capillary electrophoresis (CE) systems. We are developing an approach where students encounter experiments utilizing these instruments throughout the chemistry curriculum. By repeatedly exposing students to these instruments we hope that they develop a more sophisticated understanding and a deeper appreciation of the limitations and applicability of these two separation methods (1). Experimental Overview The experiment described here is designed for the firstsemester laboratory. CE is used to separate, identify, and quantitate cations (calcium, lithium, magnesium, and sodium) in water samples. While the experiment alone presents an incomplete picture of CE, in conjunction with the experiments encountered later in their studies, it gives the students a foundation upon which to build a more complete understanding of CE. In addition, this experiment is used to demonstrate a number of important skills and concepts to lower-division students. The idea that a separation method must be employed to detect analytes producing identical signals is explained. This is in contrast to other analyses where multiple analytes produce unique signals and a pre-analysis separation step is not necessary, such as is the case with atomic absorption and ion selective electrodes. A number of other fundamental separation and analysis concepts are included. Students determine the identity of cations based on the elution times of standards and construct calibration curves based on the corrected areas. Standard deviations are calculated for elution times and corrected areas. Standards are analyzed three times and water samples are run multiple times to generate data that are treated statistically. Students gain experience with spreadsheets and figures of merit. The result is that students use a modern and sophisticated method of analysis to learn fundamental concepts. The use of more artificial data sets, such as pennies, can be avoided (2, 3). The experimental tasks in this lab can be expanded so that students gain experience with dilutions in producing calibration curves; more freedom can be given in the area of experimental design so that this lab could be adapted to upper-division courses by requiring the students to set their own calibration scales, mix buffer solutions, and choose their own samples. Alternatively, since the majority of the sample preparation is simple dilution and filtration, the experimental tasks www.JCE.DivCHED.org
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can be reduced or eliminated so that the lab can be used as a “dry lab” during weeks when other lab tasks dominate student time, such as on check-in day, or during the “down time” of more extensive experiments. The data can also be produced for the students in advance and the material presented as an in-class exercise. The experiment has been run two ways: as a full laboratory with each lab section generating its own data set, and as a paper lab, where students are given a data set to interpret and discuss. The decision to move to a paper lab was primarily so that the students would gain experience early in the semester with spreadsheets, graphing, and figures of merit. The material was presented on check-in day, which in prior years did not include an experiment. It is presented here as an experiment to provide information on the experimental details. Experimental Considerations Incorporating CE experiments in a lower-division course is challenging. Since the first reported undergraduate experiment involving capillary electrophoresis in this Journal (4), there have been a growing number of such reports. Some of these reports have included particularly nice explanations of the technique (5–11). The vast majority of the reported experiments, however, are geared towards instrumental analysis (12–15) or other advanced courses (16–21) where students have more experience and course enrollments are typically small. A number of these experiments have focused on small ion separations (5, 6, 8, 18, 22). Only one experiment was explicitly designed for the first- or second-year course sequence (23). Our first-semester laboratory accompanies a one-semester general chemistry lecture course. There are typically 6–8 laboratory sections, each enrolling 24–30 students, and each meeting for three hours per week. A single laboratory section meets for a three-hour period including a “prelab” lecture of 20–30 minutes. It is important that experiments work reliably and easily because there is often no other opportunity to revisit a particular experiment should it fail. For parttime instructors and teaching assistants, it is important that procedures are reasonably uncumbersome and instrument instructions are readily understood. Experiments and instruments need to be as fail-proof as possible, characteristics that are not normally associated with CE. Consequently, nearly all previously reported CE experiments are for advanced courses.
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In the Laboratory
Some accommodations are made to successfully incorporate a CE experiment into the first-semester laboratory. Of key importance is access to a high-end instrument with an autosampler and software capable of running batched samples. In situations where course enrollments are low, such automation may not be necessary. The software should be user-friendly. Run times are kept short, under five minutes per analysis, so that a large number of samples can be analyzed quickly. Our procedures are based on those published elsewhere (24). A low pH buffer is used to eliminate electroosmotic flow, so the explanation of the separation focuses only on the mobility of the analytes. This is particularly advantageous in the lower-division courses where the complicating factor of electroosmotic flow can cloud the more important issues. No complexing reagent such as crown ethers (6, 22) is needed if the target analytes are few and the samples are fairly simple in composition. This further simplifies the explanation for the lower-division students. Indirect detection is used and the explanation of this concept is kept simple. For the first two consecutive years the lab was a full laboratory, with each lab section analyzing a full data set and producing their own calibration curves from standards. Each of four standards contains the four test ions at various concentrations. Concentrations are adjusted so that students determine the identity of the ions by determining which peaks increase or decrease in area from run to run. Peaks are not identified by name. Students prepare samples and standards, the teaching assistant loads the sample trays into the instrument and analysis is completed overnight. Data sets can be distributed to students the next day or at the next lab meeting. For the third year, the laboratory was moved to the first week of the semester during the lab check-in procedures. Students were given a complete data set for the series of four standards and one of several water samples. As time permits, lab instructors can also bring groups of students to the instrument. Students then work in groups or individually to decipher the data set. While there are obvious disadvantages to a “paper lab” relative to “hands-on” lab, either method of presenting the lab works well and a “paper or dry lab” may fit in better with existing schedules.
Table 1. Concentrations of Cations in the Standards Standard
Li+ (ppm)
Na+ (ppm)
Mg2+ (ppm)
1 2
030
060
010
100
060
100
030
010
3
100
010
060
030
4
010
030
100
060
Ca2+ (ppm)
Table 2. Average Corrected Area at Each Retention Time for Each Standard Standard
Average Corrected Area/(arb units) 3.0 min
3.3 min
3.5 min
3.6 min
1
3325
6992
01484
07521
2
5436
0712
03613
15144
3
0188
1778
07441
25418
4
1462
4509
12802
02508
Figure 1. Electropherogram of standard 1 with analyte concentrations as reported in Table 1: peak 1 at 1.65 minutes is a system peak common with indirect detection, peak 2 at 2.99 minutes is sodium, peak 3 at 3.33 minutes is calcium, peak 4 at 3.47 minutes is magnesium, and peak 5 at 3.62 minutes is lithium. Run conditions are given in the experimental section.
Experimental Procedures
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Average Corrected Area / 1000
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Each standard sample contains the four cations. The total concentration of ions is kept constant at 200 ppm for all standards. The standard compositions are shown in Table 1. The ions are not identified by name in the printouts given to students. Retention time and corrected area (area兾retention time) are provided. These data are shown in Table 2. Students correlate the expected increase or decrease in corrected area to the known concentration levels to identify each analyte by elution time. Unknown samples can be made available or students can prepare their own. Samples can include pool, tap, river, aquarium, fountain, or sea water. It is recommended that sea or salt water aquarium samples be diluted to 1% before analysis. Samples and standards should be filtered prior to analysis to prevent clogging of the capillary. A Beckman Coulter P/ACE MDQ was used. The capillary dimensions were 50 µm i.d., 40 cm from injection to
25
sodium
calcium
magnesium
lithium
20
15
10
5
0 0
20
40
60
80
Concentration / ppm Figure 2. Calibration curve for the four analytes.
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In the Laboratory Table 3. RSD and R2 Data of Standards RSDa (%) Cation Elution Time
Area for 30 ppm
Area for 60 ppm
Area for 100 ppm
Na+
1.6
38
19
10
05
0.998
Ca2+
2.7
11
10
19
11
0.992
Mg2+
0.7
24
07
06
02
0.998
3.6
04
02
05
02
1.000
Li a
R2 b
Area for 10 ppm
+
n = 4.
b
Calculated by Excel.
detector, and 50 cm in total length. The applied running voltage was 20 kV. The running buffer was 4.0 mM copper(II) sulfate and 4.0 mM formic acid, adjusted to a pH of 3.0. Indirect detection was performed at 214 nm. These conditions are based on a published application (24) with the exception that a complexing agent is not used. Discussion A typical electropherogram of one standard is shown in Figure 1. Table 2 summarizes data on a series of standards that is given to students. Each student also receives print outs for their particular water sample. By comparing their electropherogram data to the standard set, students are able to identify each peak present in their water sample. Students are asked to produce a calibration curve (Figure 2) and determine the concentrations of the cations present in their sample. Tables 3 and 4 summarize the relative standard deviations (RSD) and R2 values for the standards and the student samples. The data are available in the Supplemental Material.W Peak shape improves as the mobility of the analyte more closely matches the indirect detection agent so lithium has the best peak shape. This aids in the calculation of peak area. The RSD values for the areas of lithium are thus the lowest of the four analytes. RSD values for sodium are particularly high at the low end of the calibration curve, and this slope is the smallest of the four analytes. Nevertheless, when all the average values of each analyte at each concentration level are plotted, high correlation coefficients are obtained. RSD values on elution times are small enough so that peak identity is readily determined. RSD values for student data are shown in Table 4. The variation in RSD values is due to a number of factors. The
Table 4. Area RSD of Student Samples Type of Water
Na
Ca
2+
2+
Mg
River
18
18
15
Sea
04
20
14
Lake
12
11
04
Swimming Pool
07
05
32
NOTE: n = 4.
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Hazards CE requires the use of high voltages. Safety measures should be taken to avoid accidental shock. Commercially available equipment, such as was described in this report, typically incorporates such safety features. Acknowledgments This work was supported by NSF-CCLI A&I program under grant DUE-995227. Matching support was also received from Trinity University and Beckman Coulter. Linsday Sondergeld is gratefully acknowledged for obtaining the data set presented in this report and used for the fall 2002 course. Frank Walmsley is thanked for his helpful comments on the preparation of this manuscript. WSupplemental
Material
The student handout, including prelab problems and the report form, electropherograms of the water samples and standards, and the instructor handout and notes are available in this issue of JCE Online. Literature Cited
Area RSD (%) +
most obvious problem comes from sample ion concentrations that are at the low end of the calibration curve such as in the determination of sodium in river water and calcium in 1% sea water. The other obvious problem comes when the concentration of one analyte is so large that the resolution between peaks becomes compromised and the area measurement of the smaller peak thus becomes more uncertain. This is seen with the very high calcium concentrations in the pool water, which then make reproducible measurement of the small magnesium peak difficult. A less obvious cause is poor filtering of samples that can alter the flow in the fused silica capillary. Including this type of detailed data analysis would make this experiment more suited to upperdivision courses.
1. Bushey, M. M. Capillary Electrophoresis and High Performance Liquid Chromatography Experiments Throughout the Undergraduate Curriculum. http://www.trinity.edu/mbushey/ Ceopenframe.htm (accessed Aug 2004). 2. Bushey, M. M. J. Chem. Educ. 1994, 71, A90–A91. 3. Richardson, J. J. Chem. Educ. 1991, 68, 310–311. 4. Weber, P. L.; Buck, D. R. J. Chem. Educ. 1994, 71, 609–612.
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In the Laboratory 5. Demay, S.; Martin-Girardeau, A.; Gonnord, M. J. Chem. Educ. 1999, 76, 812–815. 6. Boyce, M. J. Chem. Educ. 1999, 76, 815–819. 7. Strein, T. G.; Poechmann, J. L.; Prudenti, M. J. Chem. Educ. 1999, 76, 820–825. 8. Janusa, M. A.; Andermann, L. J.; Kliebert, N. M.; Nannie, M. H. J. Chem. Educ. 1998, 75, 1463–1465. 9. Thompson, L.; Veening, H.; Strein, T. G. J. Chem. Educ. 1997, 74, 1117–1121. 10. Copper, C. L. J. Chem. Educ. 1998, 75, 343–347. 11. Copper, C. L.; Whitaker, K. W. J. Chem. Educ. 1998, 75, 347– 351. 12. Herman, H. B.; Jezorek, J. R.; Tang, Z. J. Chem. Educ. 2000, 77, 743–744. 13. Palmer, C. P. J. Chem. Educ. 1999, 76, 1542–1543. 14. McDevitt, V. L.; Rodriguez, A.; Williams, K. R. J. Chem. Educ. 1998, 75, 625–629. 15. Vogt, C.; Contradi, S.; Rohde, E. J. Chem. Educ. 1997, 74, 1126–1130.
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16. Gardner, W. P.; Girard, J. E. J. Chem. Educ. 2000, 77, 1335– 1338. 17. Boyce, M.; Spickett, E. J. Chem. Educ. 2000, 77, 740–742. 18. Hage, D. S.; Chattopadhyay, A.; Wolfe, C. A. C.; Grundman, J.; Kelter, P. B. J. Chem. Educ. 1998, 75, 1588–1590. 19. Valenzuela, F. A.; Green, T. K.; Dahl, D. B. J. Chem. Educ. 1998, 75, 1590–1592. 20. Conte, E. D.; Barry, E. F., Rubinstein, H. J. Chem. Educ. 1996, 73, 1169–1170. 21. Contradi, S.; Vogt, C.; Rohde E. J. Chem. Educ. 1997, 74, 1122–1125. 22. Gruenhagen, J. A.; Delaware, D.; Ma, Y. J. Chem. Educ. 2000, 77, 1613–1616. 23. Welder, F.; Colyer, C. L. J. Chem. Educ. 2001, 78, 1525– 1527. 24. Rivello, J. M.; Harrold, M. B. In Capillary Electrophoresis Procedures Manual: A Laboratory Users Aid for Quick Starts; Jackim, E., Jackim, L. W., Eds.; Applied Science Communications, Inc.: Peace Dale, RI, 1996; p 267.
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