Ion Exchange Chromatography and Spectrophotometry An introductory Undergraduate Laboratory Experiment N. Foster, 6. Pestel, and B. Mease Lehigh University, Bethlehem, PA 18015
While most types of chromatography (TLC, HPLC, GLPC, paper, and gel-permeation) are used for the separation of complex organic substances, the forte of ion exchange chromatography is the separation of cations and anions. The isolation of high purity salts of the 2+ transition metals is now accomplished routinely by this method; ion exchange is the only means available for the separation of the 3+ ions of the lanthanide series. Ion exchange chromatography has also proven extremely useful for the separation of amino acid mixtures produced by the degradation of proteins (I ). Ion exchange experiments for introductory chemistry courses often include such activities as the separation and identification of ions (2),the recovery of valuable metals from waste (3),and simplifying the qualitative analysis of ions in mixtures (4). Likewise, experiments in the quantitative use of simple spectrophotometry abound (5-7). A few experiments have been published describing the combination of the ion exchange technique with other analytical methods 18-10), The experiment described herein has been designed for an introductory course in chemical equilibria in aqueous media in which the students have already experienced the quantitative use of spectrophotometry in calculating equilibrium constants for the formation of colored complexes in solution. The main instructional focus of this laboratory exercise is the demonstration of the equilibrium principles involved in the ion exchange process; a secondary goal is the illustration of a semiquantitative application of spectrophotometry. The students use ion exchange chromatography to separate a mixture of chloro complexes of transition metal ions and then use spectrophotometry to define qualitatively the efficiency of the ion exchange columns. The Structure ol the Resin and the Chloro Complexes T o understand the ion exchange process, one must first undcrstnnd the naturr of the staticmar). phase in this system. Svnrhetic ion exchange resins are polymeric mawrink containing a large numb& of ionic functional groups. Resins are available commercially that will selectively bind cations (hence, cation exchange resins) or anions (anion exchange resins). The cationic resins are further divided into strong acid resins, usually containing sulfonic acid groups, and weak acid resins, containing carhoxylic acid moieties. Anion exchange resins of the type used in this experiment contain amine functional groups. Strong base exchangers are commonly quaternary amines; weak base types contain secondary and tertiary amines. Amberlite-IRA 400, the specific resin used herein. is a strone base exchanzer. The mrtal ions to heseparated in thisrxperiment (Ni(IIj, CIIIIIIand Fec11I1,are first converred into their rhloro complexei by treatment with 9 M HCI since the readily available cation exchanee resins do not show sufficient selectivity to separate thesecations. The chloro complexes can be easily This paper was presented by B. Pestel at the 17th Middle Atlantic Regional Meeting of the American Chemical Society on April 8, 1983, in White Haven. PA.
170
Journal of Chemical Education
separated by passage through an anion exchange column. All of the following complexes are possible in HCI solution: N?+ + C1- =NiC1+ CuZ+ + c1- = CuCIC CuClf c1- == CuC12(,, C"C12(,, c1- = CuCIi
+
+
Fe3+ + C1- = FeCI2+ FeCI2++ C1- d FeC1: CI-+ FeCls(,,, FeCl: FeClsc.,) C1- * FeClh
+
+
One may predict that the species present in 9 M HCI solution are NiCl+, CuCli, and FeCL (11). The Function of the Resin: the Ion Exchange Process A typical anion exchange process is illustrated by the following general equation: ~ N ( R ) ~ o H+A"= ~ N ( R ) ~ ] , +*OHA~solid resin in solution solid resin in solution
In a system such as this, the anion associatedwith theresin is called the mobile ion. Its identity is determined by the electrolyte concentration and composition of the solution with which the resin is in contact. The identity of the mobile ion is important because the separation of anions by this procedure depends upon the affinity of the resin for one anion over another. This experiment calls for pretreating the resin with 8 M HC1 to establish the chloride ion as the mobile ion in the column.
+
1-N(R)$OH-H+ CI- == (-N(R)~CI- + H20 When the resin in the chloride form is exposed to solutions containing other anions, exchange equilibria will be estahlished in the column. Descriptions of two extremes of hehavior will serve to illustrate the way the exchange equilibria work in affecting the separation of mixtures of anions. Example (1):Any cationic material will pass through the column rapidly. A cation will not be retained because it does not interact with the functional group on the cationic resin; i t cannot exchange with the chloride ion because it has absolutely no affinity for the quaternary amine group. Example (2): On the other hand, an anion, for example one of the complex ionsgenerated in this experiment (FeCL), will have an affinity for the positively charged group on the resin. Therefore, it can exchange with the C1- and thereby become bound to the stationary phase. I-N(R)$C~-+ FeCl:
d
tN(R)jFeCla
+ Cl-
The extent to which this exchange takes place will determine the rate of the anion's progress through the column. All gradations of hehavior can theoretically occur.-from no affinity and consequent rapid passage to an affinity so great that the anion remains hound to the resin and never elutes.
T h e ability to separate several different anions in a mixture b y ion exchange will depend upon t h e differences in their relative affinities for the cation on the resin and their response t o changing composition of t h e moving phase.
by 30 mL distilled water. Discard this eluant. Remove the resin from the column and store it in an appropriately labeled bottle for future use.
Column Efficiency T h e efficiency of a n y chromatographic column is determined by t h e numher of its theoretical plates. Rather t h a n concentrating o n t h e theoretical, mathematical definition of column efficiencv. we have chosen t o a n ~ l av simnle s ~ e c t r o photometric ass;; of t h e eluant f r a c t i o i t h a t w i i enihle the students to d i s.~ l"a veranhicallv the elution ~ r o f i l e sof the ions. - . F r o m this graph, t h e students can qualitatively assess t h e efficiency of t h e separation of ions offered by t h e resin under t h e conditions employed. Simple spot tests are first done on each fraction of t h e elu a n t t o determine a t which point the ions hegin t o elute. Spectrophotometric assays are t h e n done to determine t h e quantitative distribution of each ion within t h e fractions. From t h e graph of absorbance versus fraction numher t h e efficiency of t h e column can h e judged.
Experimental: Part 11-Analyzing the Eluant Fractions, Spot Tests Initially test each tube for all ions. (Based on the nature of the chloro complexes, students should be able to guess what ion elutes first.) In progressing from fraction 1to 20, however, you may assume that once an ion has given a positive test in a series of fractions and then a negative test in a subsequent tube that the ion has been eluted and will not appear in further fractions. You may then cease testing for it.
Experimental: Part I-Chromatography This experiment can he completed in two 3-h laboratory periods. Part I can be comfortably done in one period, leaving the analysis in Parts I1 and 111 for the following neriod. Important note: Throughout this entire chromatoeranhic .. . sdsivsis. . . neo& let the fluid level i n the t h e resin l i r h ~ shappens, channels rill column drop h e l m rhal dwelq>in the rrrin bed and thprepnratm rffir imq will or vastl) decreased. If the tluid lrwl should drup below the rpiin, the column must be emptied and repacked. The columns used in this procedure were 11mm (id.) X 450 mm glass columns from Ace Glass, Inc., equipped with flow regulator valves. Any column of similar size with a means tostart and stop the flow (far example, a buret) could be used. Preparing the Stock Solution A solution cnntaining 1.5 mg/mL of each ion is prepared in 9 M HCI in advance by the instructor. Ions used in this study are Ni(II), Cu(II), and Fe(lII), dissolved in themedium as their nitrate salts. Eachstudent (or each column setup, as it may be advantageous for the students to work in pairs) will need 0.5 mL of this solution. The solution may conveniently he dispensed from a buret or from a graduated dispenser on a large bottle. Preparing the Column
Place approximately 5 g of the dry resin in a 100-mL beaker. (If you use a graduated beaker, this quantity will reach the 20 m L mark.) Add 20 mL distilled water and swirl the contents. Add to this 5 mL 8 M HCI and pour the slurry into the ealumn slowly but all at once. Rinse the beaker with 2 or 3 mL 8 M HCl and pour this immediately onto the column. Tap the column gently to settle the resin, which should form a column about 15 cm high. During all these operations the valve at the bottom of the column should be open. Insert a small glass wool plug at the top of the column and allow the solution in the column to flow through the resin to within 1cm above the plug. Now activate the column by adding 15 mL more 8 M HCI and draining it to within 1em of the plug. Discard this eluant. The resin at this point may have changed from yellow to brown. Separating the Ions Add 0.5 mL of the solution containing the chloro complexes from Part I directly to the column. Drain the liquid to within 1cm of the plug. Add 1mL 8M HCI to rinse any sample off the walls of the column and again drain to within 1cm of the plug. Add 15mL of 8M HCI and begin to collect eluant fractions of 4 mL each in numbered test tubes at a flow rate of 4 mL per minute (about 1dropls). When the 8M HCI is within 1cm of the plug, add 30 mL 3M HCI and continue collecting fractions of 4 mLeach at thesame flow rate. When this solution is within 1em of the plug, continue the elution with 30 mL distilled water. When this is gone, you should have collected 20 fractions, each with a volume of 4 mL. Reclaiming the Resin Before proceeding with the analysis of the fractions, reclaim the resin by running 10 mL 1M NazSOa through the column, followed
Copper Transfer 6 to 10 drops of the eluant t o a clean tuhe. Add 15Mammonia dropwise until the solution is just barely basic to litmus. Adjust the pH of the solution to slightly acidic to litmus by adding 2 drops of 6 M acetic acid. Add several drops of 10%hydrorylamine hydrochloride solution followed by several drops of euproine reagent (a saturated solution of 2.2'-biquinoline in isoamyl alcohol). The appearance of arose to purple color in the top layer of liquid in the tube confirms the presence of copper (11).Record the tube numbers that give a positive test for copper. lron
pink color indicates a possible trace contamination rather than the true presence of iron. Record the tube numbers which give a positive test for iron. Nickel Transfer 6 to 10 drops of the eluant to a clean test tube. Neutralize the solution by adding drops of 15 M NHa until the solution is just basic to litmus. Add several drops of 1%dimethylglyoxime reagent and mix the contents. The formation of a hriek red precipitate confirms the presenceof nickel. The precipitate may takeas long as 5 min to form. If no precipitate appears after 5 min, add 1 drop of ammonia and wait another 5 mi". Record the tuhe numbers which give a positive test for nickel.
Experimental: Part Ill-Spectrophotometric Analysis of Elution Profiles The soot tests determine which elution fractions contain which ion.
analysis. Prepare each of the following solutions for spectrophotometric analysis using eluant from the first tuhe in which a specific ion was detected and alternate tubes thereafter. Copper Add to 1-mL aliquots of the eluant the following solutions in the indicated order: (1) 2 mL 10%hvdroxvlamine hvdrochloride solution: , .. 121 2 mL 1117 tartarir arid solution, 1.11addiug 6 Af KH, aropwiw adjust the pH to %G using narrou -range pH test paprr, and I 11 add 5 rill. ruprolnr reagmr. Shnke r a h solution tur 1-2 mm. Set sside and allow the layers to separate. Carefully remove the top layer and place it in a cuvet to make the spectrophotometric measurement. Record the absorbance of each solution at 545 nm using isoamyl alcohol as the blank. (Note that while waiting for the lavers to senarate. vou mav begin another analysis.) lron Add to 2-mL aliouots of the eluant the followine" in the order eiven: u (1)1mL 10%hydroxylamine hydrochloride solution; (2) 1mL 0.5% phenanthroline solution, and (31 using 2 M sodium acetate adjust the pH to 3-4 (test with narrow-range pH paper). Allow the solutions to sit for approximately 1h. Dilute the solutions by the addition of 25 mL distilled water. Record the absorbanceof eachsolution at 605nm using distilled water as a blank. (Note again that while waiting for this reaction to go to completion you may proceed with anotherassay.) ~
Volume 62
Number 2
February 1985
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