In the Laboratory
Capillary Electrophoresis Analysis of Substituted Benzoic Acids
W
An Experiment for the Organic Synthesis Laboratory Nancy S. Mills, John D. Spence, and Michelle M. Bushey* Department of Chemistry, Trinity University, San Antonio, TX 78212-7200; *
[email protected] Capillary electrophoresis (CE), as it is currently practiced, was introduced in the early 1980s (1). Its growth since that time has been extensive. Following the development of commercially-available instrumentation, CE is gradually working its way into the undergraduate laboratory curriculum. There have been a number of recent articles in this Journal that outline such experiments (2–4). However, these experiments are typically worked into analytical or instrumental analysis courses. Only three experiments have been proposed for courses departing significantly from the analytical or instrumental offerings (5–7). It can be argued that for an analytical technique to reach maturity, it should show demonstrated utility in a variety of applications, including those beyond what is found in a typical analytical laboratory. Trinity University is the recipient of a NSF-CCLI A&I grant, DUE-995227, which allowed for the procurement of a fully automated HPLC system and a CE system. A major thrust of the proposal was that through repeated exposure to these techniques students would gain a better and fuller understanding of these methods and their utility (8). In many undergraduate curricula, students may only encounter a particular instrumental technique once. They may only have experience with a single sample type, and their understanding
Table 1. Compound Information for Benzoic Acid and Substituted Benzoic Acids (SBA) Elution Time/min
Melting Point/ °C
S BA
SBA
Benzoic Difference Acid
pKac
a
4.38
5.35
0.97
4.5
3-OCH3
a
110
6.55
5.75
᎑0.80
4.1
4-CH3
182a
4.73
5.78
1.05
4.4
3-CH3
a
111–113
5.15
5.75
0.60
4.3
2-CH3
107–108a
6.18
5.66
᎑0.52
3.9
243
a
5.91
5.62
᎑0.29
4.0
3-Cl
158
a
6.66
5.82
᎑0.84
3.8
4-F
185a
5.77
5.88
0.11
4.1
3-F
a
185
4-OCH3
4-Cl
124
4-CF3
219–222b
3-CF3
104–106b
a
b
7.00
5.81
᎑1.19
3.9
6.03
5.43
᎑0.60
-----d
6.24
5.79
᎑0.45
-----d
Experimental Procedures Students first prepare their compounds via a Grignard reaction with the appropriately substituted bromobenzene using a variation of the procedures outlined in their laboratory manual (10). The reaction is shown in Scheme I. A variety of starting materials is used in each laboratory section to give a range of SBAs (Table1). While the synthesis of ben-
1. Mg, ether 2. CO2 Br
CO2H
ⴙ
3. H3O Z
Z
c
Ref 11. Ref 12. pKa value of benzoic acid is 4.20. Other pKa values taken from ref 13. dValues not available in sufficiently similar solvent.
1226
of the applicability of the technique can be limited by such experiences. We are introducing labs throughout our curriculum in which students utilize these instruments in a variety of modes, in a variety of applications. This laboratory, designed for our second-semester organic chemistry students, is one such example of how we are incorporating CE into the laboratory courses. This is the second time students in our laboratory sequence encounter CE (9). In the first experiment, placed in the first-semester laboratory, students separate cations in water samples. Separation is based upon electrophoretic mobility and the use of calibration curves is stressed. Little to no electroosmotic flow is present as the running buffer is set to a pH of 3.0. In this laboratory, electroosmotic flow is present and that concept is addressed in the student handout. The effects of pH on the separation are addressed, and so this lab in the organic chemistry laboratory builds upon and expands the students’ previous understanding of capillary electrophoresis. At the same time, the lab is well integrated into the more traditional focus of an organic chemistry course as it addresses the electron-donating and electron-withdrawing characteristics of different substituents. In this experiment, students prepare a series of substituted benzoic acids (SBAs). The pKa shift, a result of the electron-withdrawing or electron-donating characteristics of the substituent, is then examined in reference to the electrophoretic migration behavior of benzoic acid. Students typically take this course in the fall of their sophomore year.
Journal of Chemical Education
•
Scheme I. Reaction scheme for Grignard synthesis (Z = 3-OCH3, 4-OCH3, 2-CH3, 3-CH3, 4-CH3, 3-Cl, 4-Cl, 3-F, 4-F, 3-CF3, 4-CF3).
Vol. 82 No. 8 August 2005
•
www.JCE.DivCHED.org
In the Laboratory
zoic acid through carbonylation of phenyl magnesium bromide is found in a number of laboratory texts, no texts that we found included the preparation of SBAs. The electron-donating and electron-withdrawing effects of substituents can play an important role in the ease of formation of the Grignard reagent. It is instructive for students to observe these differences in reactivity and try to understand them. In addition, because the product of each reaction is relatively unique, depending on the size of the lab section, students spend less time “cookbooking” the lab. The elution times for the SBAs are shown in Table 1. Samples are prepared for subsequent CE analysis by dissolving 5 mg of the student-prepared product with 2 mL of methanol. Five drops of this solution are then added to the sampling vials used on the CE instrument. Two drops of a benzoic acid stock solution (1 mg兾1mL MeOH) are added and the vial is then filled to its appropriate level with the buffer for the electrophoresis run (vials hold approximately 1.5 mL). Students label their vials and the instrument is run in a batch mode. A Beckman Coulter P兾ACE MDQ was used for all electrophoretic runs. The capillary dimensions were 50-µm i.d., 40 cm from injection to detector, and 50 cm in total length. The fused silica capillary was conditioned prior to each laboratory section by rinsing with 0.1 N NaOH for eight minutes, water for five minutes, and buffer for ten minutes. Between each analysis the capillary was rinsed with buffer for one minute. All rinses were done at 15 psi. Injections were performed under pressure, at 0.5 psi for 4.0 seconds. The applied running voltage was 25 kV. The running buffer was 5 mM NaOAc兾AcOH buffer adjusted to a pH of 4.2, the pKa of benzoic acid. Detection was at 214 nm. The determination of ionization constants by CE and the optimization of separations by adjustment of buffer pH have been discussed in the literature (14–18). This laboratory course typically enrolls about 160 students. Run times on the CE are kept short, about 9.0 minutes per analysis so that all student samples can be run in a timely manner. The presence of an autosampler and the ability to run the software in a batch mode are essential to be able to use this instrument in a course with a large enrollment.
Discussion Yields of the SBAs are generally good and all students obtain enough product for characterization and electrophoresis. A nice feature of the synthesis involves the isolation of the carboxylic acid by first converting it to the sodium salt, removing it from the organic byproducts through extraction, and then re-isolating the product by acidification. Because this is not the typical isolation product for an organic compound, it helps reinforce the value of understanding acid– base behavior of organic compounds. Laboratory section results are available the next day and distributed to students. A typical electropherogram is shown in Figure 1. The methanol, used to solubilize the analytes, can act as an electroosmotic flow marker. Electroosmotic flow is a consequence of the negatively charged fused-silica capillary-wall surface. Cationic buffer ions associate near this surface. Their electrophoretic migration towards the cathode creates a bulk flow in capillary, the electroosmotic flow. A neutral compound, injected with the analytes sample will reveal the velocity of this flow. By comparing their electropherograms to those belonging to other students, and, by comparing to a few standard runs containing only benzoic acid, students are able to determine which peak is benzoic acid and which is their derivative. In addition, because the benzoic acid was present in lower concentration, students could support their identification of that peak on the basis of relative sizes. Students are also able to compare the effect that the position of their substituent on the aromatic ring has on the pKa value to those prepared by other students. In the context of electrophilic aromatic substitution our students will have previously studied the activating and deactivating properties of various substituents on a benzene ring. In this laboratory they are asked to extend that reasoning to predict the product’s relative pKa value (compared to benzoic acid) by considering the electron-donating and electronwithdrawing properties of the substituent and its position relative to the carboxylic acid. For example, meta- and paramethylbenzoic acid contain an electron-donating substituent and will destabilize the benzoate anion, resulting in an increase in pKa relative to benzoic acid. A similar effect will be seen for para-methoxybenzoic acid where the substituent can donate electron density to the carboxylic acid ipso car-
Hazards Diethyl ether, used in the Grignard synthesis is extremely flammable. Care must be taken that no flames or sparks are present in the laboratory when this reagent is being used. This reagent is also highly volatile and the reagent bottle should not be left open. Care should be taken to ensure that ground glass joints do not allow diethyl ether vapors to escape during the synthesis. While diethyl ether is customarily used in Grignard reactions, tbutyl methyl ether could be considered as an alternative, less volatile solvent. CE procedures require the use of high voltages. Commercially available equipment is typically equipped with safety devices to reduce the likelihood of accidental electrical shock. Methanol is toxic and should be handled with care.
www.JCE.DivCHED.org
•
Figure 1. Electropherogram of student sample with benzoic acid internal standard. A small methanol peak (marking the electroosmotic flow) appears at approximately 3.3 minutes. Benzoic acid appears at 5.66 minutes and 2-methylbenzoic acid appears at 6.18 minutes. Conditions are as described in the text.
Vol. 82 No. 8 August 2005
•
Journal of Chemical Education
1227
In the Laboratory
bon. For meta-methoxybenzoic acid, however, no such resonance is possible and a decrease in pKa relative to benzoic acid is seen due to the inductive electron-withdrawing properties of the methoxy substituent. All other substituents in our sample group are electron-withdrawing substituents and would be expected to increase the acidity of the SBA. The representative student results shown in Table 1 support these trends. The only anomalous result is for the p-fluorobenzoic acid, which suggests that the substituent acts to destabilize the benzoate anion; however, the difference in elution time from the benzoic acid standard is the smallest of the systems examined and may be within experimental error. The magnitude of the elution-time differences do not always follow the magnitude of the pKa changes found in the literature. The primary reason for this discrepancy is due to the nonlinear relationship between pKa values and mobilities. Determination of pKa values by CE is most effectively done in several buffer systems over a pH range (18). As we have not asked our students to comment on the extent of the pKa change we do not see this as a serious problem. If time permitted, it would also be possible to determine the pKa values of the student products by repeating the electrophoretic analysis in several buffers of slightly different pH values (15, 17, 18). It should also be noted that electrophoretic mobility is a function of both size and charge. Thus students should not expect compounds with identical pKa values to necessarily have identical electrophoretic mobilities. Such compounds may well exhibit different mobilities as is the case with several examples in Table 1. There can be some variation from batch to batch with respect to electroosmotic flow time. If student samples are dirty and particulates are introduced into the capillary, run times can shift. This can be minimized by having students filter all samples before filling vials. If students follow directions and add the appropriate quantities of benzoic acid and synthesized material, the peak heights are further confirmation of peak identity as the benzoic acid peak should appear smaller. Occasionally it becomes apparent that a student did not prepare a sample as directed. These cases can be discerned if several students in the lab section prepare the same compound. The student handout explains the occurrence of electroosmotic flow and how that should be accounted for when the students analyze their results. Peaks eluting after benzoic acid, which is 50% ionized under these buffer conditions, are more ionized than benzoic acid. Hence, the pKa values of such compounds are shifted to lower values than that of benzoic acid and the substituent can be classified as an electron-withdrawing group. The opposite is true for compounds eluting before benzoic acid. Students are then able to assess the electron-donating and electron-withdrawing characteristics of the substituents on the various derivatives prepared by the entire class. The effect of electron-donating and electron-withdrawing substituents on the pKa values of SBAs or phenols is commonly covered in organic chemistry texts and, as a result, this experiment reinforces material covered in the corresponding lecture course. In addition, this technique allows students to determine the electron-donating or electron-withdrawing properties of substituents that are not typically covered in their text (i.e., CF3) based on their relative pKa value. We believe this to be a unique example of how CE techniques can be incorporated into the undergraduate curricu1228
Journal of Chemical Education
•
lum beyond the usual analytical chemistry courses. The experiment forces students to think through the relationships between electron donating–withdrawing effects and pKa values, the effects of pH on ionization state, and the effects of ionization state on electrophoretic elution order. 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. Frank Walmsley is thanked for his many helpful suggestions during the preparation of this manuscript. W
Supplemental Material
The student handout, modified for use at other institutions, and mass spectra, 1H NMR and IR spectra of various compounds, are available in this issue of JCE Online. Literature Cited 1. Jorgenson, J. W.; Lukas, K. D. Anal. Chem. 1981, 53, 1298– 1302. 2. Gruenhagen, J. A.; Delaware, D.; Ma, Y. J. Chem. Educ. 2000, 77, 1613–1616. 3. Gardner, W. P.; Girard, J. E. J. Chem. Educ. 2000, 77, 1335– 1338. 4. Harman, H. B.; Jezorek, J. R.; Tang, Z. J. Chem. Educ. 2000, 77, 743–744. 5. Welder, F.; Colyer, C. L.; J. Chem. Educ. 2001, 78, 1525–1527. 6. Valenzuela, F. A.; Green, T. K.; Dahl, D. B. J. Chem. Educ. 1998, 75, 1590–1592. 7. Weber, P. L.; Buck, D. R. J. Chem. Educ. 1994, 71, 609–612. 8. Bushey, M. M. Capillary Electrophoresis and High Performance Liquid Chromatography Experiments Throughout the Undergraduate Curriculum. http://www.trinity.edu/mbushey/ ceopenframe.htm (accessed April, 2005). 9. Pursell, C. J.; Chandler, B.; Bushey, M. M. J. Chem. Educ. 2004, 81, 1783–1786. 10. Gilbert, J. C.; Martin, S. F. Miniscale Preparation of Grignard Reagents. In Experimental Organic Chemistry, 3rd ed.; Sanders College Publishing: Fort Worth, TX, 2002; pp 594– 596. Gilbert, J. C.; Martin, S. F. Miniscale Preparation of Benzoic Acid. In Experimental Organic Chemistry, 3rd ed.; Sanders College Publishing: Fort Worth, TX, 2002; pp 606–607. 11. CRC Handbook of Chemistry and Physics, 58th ed.; Weast, Robert C., Ed.; CRC Press, Inc: West Palm Beach, FL, 1978. 12. Catalog of Organics and Fine Chemicals; Fisher Scientific International, Inc.: Leicestershire, United Kingdom, 2004/05. 13. Carey, F. A. Organic Chemistry, 4th ed.; McGraw Hill: Boston, 2000; p 748. 14. Terabe, S.; Yashima, T.; Tanaka, N.; Araki, M. Anal. Chem. 1988, 60, 1673–1677. 15. Smith, S. C.; Khaledi, M. G. Anal. Chem. 1993, 65, 193– 198. 16. Friedl, W.; Kenndler, E. Anal. Chem. 1993, 65, 2003–2009. 17. Cai, J.; Smith, S. C.; Khaledi, M. G. J. High Res. Chromatogra. 1992, 15, 30–32. 18. Gluck, S. J.; Cleveland, J. A., Jr. J. Chromatogra. A. 1994, 680, 49–56.
Vol. 82 No. 8 August 2005
•
www.JCE.DivCHED.org
