Laboratory Experiment pubs.acs.org/jchemeduc
Using Ion Exchange Chromatography To Separate and Quantify Complex Ions Brian J. Johnson* Department of Chemistry, College of St. Benedict and St. John’s University, St. Joseph, Minnesota 56374, United States S Supporting Information *
ABSTRACT: Ion exchange chromatography is an important technique in the separation of charged species, particularly in biological, inorganic, and environmental samples. In this experiment, students are supplied with a mixture of two substitution-inert complex ions. They separate the complexes by ion exchange chromatography using a “flash” technique. After taking a visible spectrum of the eluted complexes, the unknowns are identified by comparison with a table of supplied λmax values and quantified using Beer’s law. This experiment can be completed in under 4 h and is suitable for introductory and intermediate chemistry students.
KEYWORDS: First-Year Undergraduate/General, Second-Year Undergraduate, Laboratory Instruction, Inorganic Chemistry, Hands-On Learning/Manipulatives, UV−vis Spectroscopy, Chromatography, Coordination Compounds, Ion Exchange, Quantitative Analysis
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ions present in the mixture, whereas in others, students separate a mixture of complexes supplied by the instructor.20−22 In all of these cases, students are working with identical samples or ones which differ only in the relative amounts of two or three components. One older paper published in this Journal described an experiment in which unknowns containing two Co(III) complexes (selected from a group) with +2 and +3 charges were separated by ion exchange chromatography and precipitated with sodium perchlorate. The list of unknowns was not supplied.23 In this paper, we report an ion exchange experiment that differs in that it is performed on the microscale, contains a complete list of unknowns, involves an introduction to flash chromatography, uses Beer’s law to quantify the eluted components, and can be completed in less than 4 h.
on exchange chromatography allows the separation of ionic species through differential interaction of ions with a charged stationary phase.1,2 For example, a strong acid cationic exchange resin consists of a completely ionized anionic stationary phase such as sulfonate ions (RSO3−). Cationic metal complexes or metal ions are attracted to it and are retained on the column. Those with high positive charge are retained more tightly and require higher cation concentrations in the eluting solvent in order to displace the metal species from the column. This is the operating principle behind water softening and water deionization, and ion chromatography can be used to analyze water samples for trace metal ions. Several ion exchange chromatography experiments have found their way into commercial laboratory manuals or laboratory experiments reported in this Journal, including separation or purification of biochemical molecules3−6 or analysis of trace inorganic ions.7−11 In the nontrace analysis of labile inorganic ions, those with the same charge (for example, aqueous Ni2+ and Cu2+) may be separated by elution with varying concentrations of HCl or other sources of chloride;12−14 however, in this case the chemical identity and charge of the metal species depends on the strength with which the metal ion binds chloride ions. In some other experiments, slow ligand exchange in a complex generates a mixture to be separated15,16 or a mixture of oxidation states of a metal is generated and separated.17−19 There are few examples of separation of stable coordination complexes by ion chromatography. In some cases, a synthetic target compound is separated from an impurity due to the difference in charge of the complex © 2014 American Chemical Society and Division of Chemical Education, Inc.
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EDUCATIONAL OBJECTIVES The experiment described in this paper is performed by students in the second foundation laboratory course. All of the experiments in this course involve chromatography, with examples spanning organic, inorganic, and biochemistry. However, it is also suitable for a lab course in which an introduction to chromatography, transition metal complexes, or quantitation by Beer’s law is desired. We further aim to enhance student independence in the laboratory. Each student selects the order in which they will perform their experiments. This requires that they come prepared because they cannot just mimic what the other Published: July 18, 2014 1212
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complex is an anion and not retained on the column at all. Third, for maximum convenience, complexes should be commercially available or easily synthesized by teaching assistants (TA) or stockroom workers. Alternatively, complexes prepared in advanced inorganic or general chemistry laboratories might also be a source of unknowns. Seven compounds meet these criteria, including K3[Cr(ox)3]·3H2O, [Co(NH3)6]Cl3, K[Co(EDTA]·2H2O, trans[Co(en)2Cl2]Cl, [Co(NH3)4CO3]NO3, [Co(NH3)5NO2]Cl2, and [Co(en)3]I3. (The student handout also lists some additional complexes that serve as “distractors”.) As each unknown contains two complexes, combinations are eliminated in which both complex ions have the same charge because they are not separable on the column. No unknown combinations are given in which the two components have similar colors. Although such complexes can actually be separated, it is a much more challenging experiment for a student doing ion exchange chromatography for the first time. This leaves 17 combinations, so that it is unlikely that two students in a 16-person lab section are working on the same unknowns.
students are doingthey may be the only one performing a particular experiment that day. There is also the further advantage that student demand for instrumentation and equipment is spread out more evenly over the course of the semester. Finally, for each experiment, students are given different unknowns so that they must analyze their own data. This is both a technique and puzzle-solving experiment that can serve as an alternative to cookbook-style laboratories.24,25 There are a number of specific learning goals for this ionexchange experiment. In addition to teaching the general principles of this type of chromatography, students gain an introduction to flash chromatography, use visible spectroscopy to identify unknowns, prepare solutions with volumetric glassware, and use Beer’s law to quantify their unknowns.
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EXPERIMENTAL OVERVIEW A detailed student handout and instructor preparation notes are provided in the Supporting Information. Students are given approximately 60 mg of a sample that contains a 50/50 mixture of two different coordination complexes. The unknown is dissolved in a small volume of water and loaded onto a short (approximately 3 cm) microscale column containing DOWEX 50Wx4 (100−200 mesh) cation exchange resin in its Na+ form. Students then add water, 0.1 M NaCl, 0.5 M NaCl, 2 M NaCl, and 5 M NaCl in turn until both components elute from the column, as evidenced by the movement of colored bands due to the complex ions. To identify their unknown, they obtain a 400−700 nm spectrum of each band to compare the spectra against given λmax values of the possible unknowns. Although students collect all of each colored fraction, they are advised to separately collect approximately 3 mL of the most concentrated portion of the chromatography band for spectroscopic analysis, as it produces the strongest absorbance and thus allows the differentiation of compounds that absorb at similar wavelengths. Students combine fractions of the same component into the smallest volumetric flask that will hold them (10, 25, or 50 mL) and dilute to the mark with deionized water. They then obtain the visible spectrum of each of these solutions. From the measured absorbances and given molar absorptivities for the unknowns, they are able to calculate the concentration of each unknown from Beer’s law. Using the volume of the solution measured and the molar mass of their unknown, they can calculate the mass of each component present. Because our emphasis is on introducing techniques, students only run one trial of each unknown mixture. In this way, they obtain semiquantitative information about their unknown composition, in addition to identifying each component.
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Columns
Elution from the column is generally straightforward. If one of the unknown components is an anionic complex ion, it is not retained on the cation exchange resin and elutes with the water used to load the column. The cationic complex ions elute with the NaCl solutions of varying concentrations. At the NaCl concentrations employed, the complex ions tend to either move relatively rapidly or not move at all, making it fairly easy for the students to decide whether to add more of the NaCl solution they are using or to go to the next most concentrated one. There are no problems with overlapping bands or twocomponent solutions. We also use this experiment as a way to teach flash chromatography using a setup similar to that described by Horowitz.26 A short piece of Tygon tubing is used to attach a plastic Y tube to the top of a microscale column, and one side of the Y is attached to house N2. A finger or thumb can be used to block the other branch of the Y to create pressure. A further advantage of this system is that additional eluting solvent can be added directly through the open branch of the Y. If house nitrogen is not available, compressed air should also work, and other means of creating pressure for flash chromatography may be adapted.27−30 Alternatively, attaching a piece of tubing to the stopcock at the bottom of the column and using a large syringe to pull solvents though the column is also successful. Any of these methods significantly decrease elution time compared to a gravity flow column. Instruments
EXPERIMENTAL CONSIDERATIONS
Any spectrometer capable of scanning the 400−700 nm range should suffice for this experiment. Typically, one Cary 50 Bio UV Vis spectrophotometer and one Ocean Optics Red Tide USB650 spectrophotometer or two Ocean Optics instruments are available per 16 person lab section. Each student requires four scanstwo of the most concentrated fractions of each unknown used to identify the unknowns and two containing the total volumes of each fraction for the quantitative work.
Unknowns
A complete list of unknowns employed appears in the Instructor Notes supplied as Supporting Information. The coordination compounds used as unknowns were selected based on a number of considerations. First, they should be inert to substitution or isomerization in water or high chloride concentrations, at least over the time frame of a normal laboratory period. In this way, students can make predictions of the order of elution of different unknowns. Second, in order to have multiple unknown combinations, there should be several examples of coordination complexes with different positive charges, as the column is a cation exchange resin. In addition, some complexes are included in which the coordination
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HAZARDS Gloves and goggles should be worn during the course of this experiment. The Dowex 50Wx4 (100−200 mesh) cation exchange resin is a sulfonic acid resin sold in the H+ form. The MSDS lists it as a strong acid with very hazardous dust 1213
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(particularly to the eyes). Students use it in the Na+ form, so stockroom personnel handle the resin in the hood, treat it with 1 M NaOH, and the resulting mixture is supplied to the students. This solution is corrosive. The metal complexes are harmful if inhaled, swallowed, or absorbed through the skin. Flash chromatography involves a pressurized system; care should be taken to avoid overpressurization of the glassware.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This experiment was developed in conjunction with National Science Foundation TUES grant (NSF-DUE 1043566). The author thanks colleagues for helpful suggestions to improve the experiment and Chris Schaller for illustration of the column setup found in the student handout.
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RESULTS AND DISCUSSION This experiment has been used for four semesters. Presently, students identify approximately 75% of the unknown components correctly. Use of the most concentrated fraction (rather than the more dilute solution with the accurately known volume) to obtain the visible spectrum has allowed more accurate determination of wavelength and increased this percentage from the first semester. Most of the remaining errors are probably due to measured λmax values falling in between those of two possible unknowns and selecting the wrong one. The relative broadness of the absorbances, the relatively low molar absorptivities for transition metal complexes, and inaccuracies in either manual or automated peak-picking methods may also play a role. It is important that students obtain spectroscopic data on the same day as they run the column, as some complexes isomerize, undergo aquation, or precipitate in the high salt concentrations over longer periods of time. Students typically report 70−90% recoveries for each unknown. Because unknown combinations are prepared in batches and we do not go to great lengths to ensure they are uniformly mixed, grading emphasis is on the accuracy of the calculation rather than the actual amount recovered. It might be possible to increase both the accuracy of unknown identification and the percent recovered by increasing the amount of unknown the students use; however, we have chosen not to do this to minimize the amount of chemicals used. As most students taking this laboratory course are in their second semester of college, we have also chosen to assign most of the points associated with the experiment to supplying the data that they gather, data analysis, an ACS style Experimental section, and other issues (see the grading rubric supplied with the student handout in the Supporting Information).
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CONCLUSION This experiment introduces students to the concepts of ion exchange chromatography and quantitative analysis via Beer’s law. It is included as one of a semester of experiments that demonstrates chromatography as a technique relevant to organic, inorganic, and biochemists. However, given the relatively clean separation and the ease of visualization of the bands, it could be used in any course where an introduction to chromatography or quantitation is desired.
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ASSOCIATED CONTENT
S Supporting Information *
Student handout and instructor and stockroom preparation notes. This material is available via the Internet at http://pubs. acs.org.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 1214
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