In the Laboratory
Weak Acid pKa Determination Using Capillary Zone Electrophoresis
W
Mike Solow Department of Chemistry, City College of San Francisco, San Francisco, CA 94112;
[email protected] Capillary electrophoresis (CE) has rapidly become an important analytical technique in the fields of chemistry and biology. Uses include sequencing the human genome (1), chiral separation of pharmaceuticals (2), food analysis (3), silicon wafer purity analysis (4), myriad forensic analyses (5), and many others (6). This Journal has described many undergraduate experiments involving CE in recent years (7). With a few notable exceptions (8), most involve the use of CE to separate, quantify, or identify the components of solutions. This article describes a general chemistry experiment designed to measure the pKa of a weak acid, specifically benzoic acid. Students planning to major in biochemistry or molecular biology, who constitute a large fraction of general chemistry students at many institutions today, will run countless electrophoresis experiments while undergraduates. Despite the fact that modern biology relies so heavily on this analytical technique, students get very little if any explanation of the principles of electrophoresis in many biology labs. This laboratory exercise is intended to introduce undergraduate students to capillary electrophoresis while stressing the role of acid–base chemistry. While there are certainly easier ways to find the pKa of an acid (such as titration) this method requires only nanograms of the weak acid and is particularly well suited to analytes such as pharmaceuticals, herbicides, and pesticides that have low water solubility (9). In this experiment, students use capillary zone electrophoresis (CZE) to determine the effective mobility of benzoic acid at two different hydrogen ion concentrations. A plot of these data yields the pKa of benzoic acid. Theory Observed mobility, µobs, is the analyte velocity, v, divided by the electric field strength, E : v (1) E The effective mobility, µeff, of a species is the difference between the observed mobility and the mobility of the electroosmotic flow (EOF): µ obs =
(2)
µ eff = µ obs − µ EOF
For a weak acid (HA) the effective mobility is a function of the extent to which the weak acid is ionized (in the form of A᎑). That is, the effective mobility is equal to the mobility of the conjugate base ion times the fraction of the weak acid that is dissociated: µ eff = µ ion
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A− HA + A −
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(3)
Rearrangement of eq 3 and introduction of the acid dissociation constant, Ka, yields 1 = µ eff
1 K a µ ion
H+
+
1 µ ion
(4)
A plot of 1兾µeff versus [H+] yields a straight line with a slope of 1兾Kaµion and a y intercept equal to 1兾µion. Students generate a plot of this type and use it to determine the Ka (and subsequently the pKa) of benzoic acid. There is an assumption that each of the species involved here has an activity coefficient of unity. This assumption produces a minimal degree of error in the final calculation (10). Experimental Depending on the size of the class, the students are divided into groups of 2–4 students. Students are given minimal guidance in preparing two buffers, a 0.06 M acetate buffer (pH = ∼4.7) and a 0.06 M phosphate buffer (pH = ∼7.2). They are also given a dilute solution containing both benzoic acid (0.074 mM) and a neutral standard, mesityl oxide, (0.22 mM) that is used to determine the µEOF. The students perform a total of four electrophoretic runs, two with each buffer system, on a BioFocus 3000 capillary electrophoresis instrument. The uncoated, fused silica column has a total length of 24.1 cm (19.5 cm to detector). Prior to injection (5 psi s) of each sample, the column is washed with 0.1 M NaOH (20 s), then distilled water (60 s), then filled with the appropriate buffer (60 s). A constant voltage of 20 kV is applied for 4 minutes and detection is done at 200 nm. Two laboratory sessions are allocated for this experiment, buffer and sample preparation is done on the first day and the analysis of the data is done on the second day. Results A sample plot of (1兾µeff) versus [H+] is shown in Figure 1. The average pKa value for benzoic acid in a lab section of 28 students offered the fall semester of 2004 was 4.23 (RSD = 8.5%) (compared to a literature value of 4.18). Hazards Students should be instructed about necessary precautions to be taken when working with concentrated acids and bases, such as 6 M phosphoric acid and 6 M sodium hydroxide. Both benzoic acid and mesityl oxide will irritate the skin after prolonged contact. Students should be advised to immediately wash skin or clothing that comes in contact with the sample solution containing benzoic acid and mesityl oxide.
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In the Laboratory
Literature Cited
−3000 7
3
1 µeff
(V s mⴚ2)
y = (−5.4065 × 10 )x − 3.7416 × 10
1. 2. 3. 4.
R 2 = 0.70154 −4000
−5000
5. −6000 0.0
0.5
1.0
ⴙ
[H ] / (10
1.5
ⴚ5
2.0
2.5
mol/L)
6. 7.
Figure 1. A sample plot of (1/µeff) versus [H+] for benzoic acid in two different buffer solutions. Data bars represent the range of data generated by all the student groups.
Conclusion The determination of the pKa of a weak acid by CZE provides a nice way of incorporating modern instrumentation into the general chemistry lab curriculum. Students appreciate the opportunity to better understand electrophoresis, a technique that is frequently used in their biology lab classes. Acknowledgments This project was supported in part by the Division of Undergraduate Education at the National Science Foundation, grant number DUE-9553786 to the San Francisco Bay Area Collaborative for Excellence in Teacher Preparation, known locally as MASTEP (Math and Science Teacher Education Program). The National Science Foundation DUEILI program provided City College of San Francisco with funding for the purchase of the computers. WSupplemental
9.
Material
Instructions for the students including questions are available in this issue of JCE Online.
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10.
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