Nathalie E. Dabuet Lemuel D. Lozada Experiment 9 Ion-Exchange Chromoatography I.

Abstract

SAD1 Joanna Del Rosario

Ion exchange chromatography is defined as a vital procedure of the analysis for alkali metal cations and anions. Moreover it is process that determines the distribution of complex species in a given solution. In the following undertaking, the equivalent of the salt is computed through the means of Ion-exchange chromatography, anion exchange in particular with the use of Hydrochloric acid as the eluting agent. The mobile phase which includes the salt (NaCl) was passed into and out of a column of tightly packed anionexchanger resin, the stationary phase. HO, being the anion of the resin is exchanged with Cl-, the anion of the salt. Liberated HO was then titrated to phenolphthalein. The experiment resulted to an endpoint of 6 mL of standardized 0.1018 M HCl and experimentally acquired weight of salt, 253.9456 g. II. Key Words: Ion-exchange chromatography, Resin, Eluting Agent

III. Introduction

chromatography as adjustments are made. Thus, the protein of interest can be separated from many Ion-exchange chromatography separates others. molecules on the basis of their charged groups, which cause the molecules to interact Ion-exchange chromatography is named on the electrostatically with opposite charges on the basis of the exchangeable counter ion. Involves the stationary-phase matrix. Therefore, the procedure interaction of a solid insoluble phase that contains is limited to purification of ionizable molecules. The positively or negatively charged sites (the resin) stationary phase carries ionizable functional groups with the mobile phase (eluent solution) that coupled to an inert matrix material. Because of the contains the counter ion. When the stationary principles of electroneutrality, these immobilized phase bears a positive charge and the charges are electrostatically associated with exchangeable ion is an anion, the process is exchangeable counter ions from solution. Charged referred to as anion-exchange chromatography. molecules to be purified compete with these When the stationary phase bears a negative charge counter-ions for binding to the charged groups on and the exchangeable ion is a cation, it is referred the stationary phase and are thereby retarded on to as cation-exchange chromatography. The ionthe basis of their charge. Different types of exchange equilibrium can be written as: molecules will bind to the matrix with affinities that depend on both the conditions used and the types Cation exchange: B + (aq) + A+ Resin- (s) and number of individual charged groups. These A+(aq) + B+ Resin-(s) differences lead to resolution of various molecule Y-(aq) types by ion-exchange chromatography. In typical Anion exchange: X-(aq) + Res+ Y-(s) + Resin+ X-(s) protein purification using ion exchange chromatography a mixture of many proteins derived An example of one of the most frequently from a bacterial cell is applied to the column. After used resin the is copolymer of styrene and 4-12% molecules that do not bind are washed away, divinylbenzene which has been sulfonated conditions can be gradually adjusted, such as by (acidified) to produce a strong-acid cation increasing the concentration of a simple counter ion exchanger or aminated (basified) to give a strongor by altering the pH, to release the molecule of base anion exchanger. interest from the stationary phase. Molecules with different charges will elute at specific points in the

Chem 27.1 Ion-Exchange Chromatography

The experiment aims to determine the equivalent weight of the unknown salt through the use of Ion-exchange chromatography. IV. Methodology 0.50 +- 0.01 g of an unknown was weighed and transferred to a 250 mL volumetric flask. After which, the weighed substance was dissolved in enough distilled water diluted to the mark.

0.1522g Na2CO3 x = 0.1014 M HCl Trial 1 0.1014 M Trial 2 0.1019 M Trial 3 0.1020 M Average 0.1018 M

Separately, a glass column was plugged with Normality of Titrant cotton and packed with enough resin to fill it. The resin was regenerated with 1 M NaOH. Afterwards, Normality = h x M all traces of NaOH was washed off with water. In order to examine the completeness of the washing, = 1 x 0.1018 M 3 drops of phenolphthalein. If the liquids clears, the = 0.1018 N washing is complete. In case it does not, washing is repeated until expected results occur. Number of Equivalents Moreover, 10 mL of an unknown salt solution was passed through the column, immediately followed by 100 mL distilled water. The final drop of the washing was tested, similar to the aforementioned. The eluent was then titrated and washed with the standard HCl, using phenolphthalein. . From the gathered experimental data, the equivalent weight of the salt was calculated. V. Results 6 mL 0.1018 N 1.5511 g 253.9456 g/equivalents VI. Discussion Macid x Vacid x mol ratio x dilution factor = number of equivalents 0.1018 M x 0.006 L x equivalents Equivalent Weight of Sample Equivalent Weight = = = 253.9456 g/equivalents x = 6.108 x 10
-3

Volume of Standard Base Normality of Standard Base Weight of Sample Equivalent Weight of Sample

Sample Computations: Standardization of Titrant g Na2CO3 x titrant x x = M

Ion exchange is a relies on the interaction of ions held on porous, insoluble solid and ions in a solution which is slowly brought into contact with the solid. The ion solution can be prepared using regular methods while the solid part of the process can be natural clays or zeolites. Due to its widespread application in the industry, synthetic ion exchange resins are now being manufactured. Synthetic ion exchange resins are made of high-molecular-weight, cross-linked polymers which contain large numbers of ionic functional groups per molecule. These functional groups would

Chem 27.1 Ion-Exchange Chromatography

effectively serve as pores wherein ions will attach of anion exchange, hydroxide) ions in the resin and upon contact. Cation-exchange resin contains in the solution are in equilibrium. acidic groups while its anion counterpart has basic + + + + A (aq) + RB RA + B (aq) groups. Based on the solutions ability to acidify or for which an equilibrium constant K is given by alkalize through titration and the effective use of K = ion-exchange resins, certain characteristics about a given salt sample can be determined. The whole + + process of ion-exchange chromatography deals where RA and RB are molar concentrations in the solid phase (in the resin). with the stationary phase and the mobile phase. The stationary phase refers to the resin, which is normally done in a glass column. It is made up of the ion exchange resin, which is the primary source of ions to be exchanged. Hydronium ions are released during cation exchanges and hydroxide when anion exchanges are required. The increased surface area of the molecular structure of the resin makes it efficient for ion exchange. The mobile phase refers to the eluent or the solution applied to the setup. The resin used in this experiment is made up of polystylene and divinyl benzene. In the treatment of the resin, what one is really treating are the benzene groups. The anion exchanger in anion exchange resins are sometimes called quartenary nitrogen ions, which acts as a strong base. Its weaker counterpart contains secondary or tertiary amine groups. The exchangers of strong acid types have sulfonic groups; its weak complement owes their action to carboxylic groups. Note that these functional groups are always ionized; which makes them good ion exchangers. Ion exchange separations are normally performed in conditions wherein on of the ions dominate the other in both species. In the removal of ion A, the concentration of the B ions will be much greater than the previous. As a consequence, B ion concentration is essentially constant in both phases, which would make the equation = K = K

where K is the distribution constant. It represents the affinity of the resin for ion A relative to ion B. In general, when the K for an ion is large, the ions are more attached to the functional groups, making them easier to retain. Lower K values would also mean that the ion currently bound can be discarded easily. Choosing a common reference ion permits the comparison of distribution ratios for different types of ions. In cation exchange resins:
+ + + + + + + + +

Li < H < Na < NH4 < K < Rb < Cs < Ag < Tl 2+ 2+ 2+ 2+ 2+ 2+ 2+ < Mg < Zn <Co < Cu < Cd < Cd < Ni < 2+ 2+ 2+ 2+ Ion exchange reactions are always Ca < Sr < Pb < Ba stoichiometric and reproducible. It can also be reversed. The degree of how the reaction proceeds For anion exchange resins: will depend on the resin preference. Ions with F OH < CH3COO < Cl < NO2 < CN < Br < higher charges are generally preferred over those NO3 < I with lower charges. The selectivity of a resin for a given ion can be measured by the selectivity Note that the affinity of the resin increases with coefficient (K). increasing charges. The selectivity coefficient also varies from depending on changes in solution When a dilute solution is passed through conditions. The whole point of the chromatographic the column, there will come a time when the determination would be lost if the selectivity amounts of unknown and hydronium (or in the case coefficient is disregarded.

Chem 27.1 Ion-Exchange Chromatography

The normal setup would be a glass column or burette with a cotton plug and almost 10 inches of resin. However, we used a beaker with the resin in hopes of making the experiment less time consuming and to reduce errors. The first step would be letting the resin reach its starting point. After choosing the appropriate ion exchanger, it is required to let the setup reach equilibrium before applying the sample. The exchanger groups on this stage are associated with exchangeable counter ions. In regenerating the resin, 1 M NaOH was used since the experiment calls for an anion exchange. Note that high amounts of OH in the setup will lead to the displacement of metal ions. After reaching equilibrium, all traces of NaOH should be washed with enough distilled water before adding the sample, to prevent excess NaOH to react with the salt solution. Incomplete washing of the resin would lead to errors; as it is the washing that will be titrated later on, which would, by then, still have excess OH . Note that the liquid level should never fall below the resin level to prevent the creation of gaps between the beads. It would lead to the increase in the flow rate of the eluent, which may cause some of the anions to be not displaced. Phenolphthalein is used as an indicator for the amount of OH ions in the washing. Once the indicator show that the washing is not basic anymore, one can then proceed to the next step. Since we used a beaker, the flow rate of the eluent would be disregarded as it would be the swirling that will be important. After proper swirling of the beaker, the setup must be allowed to reach equilibrium. After allowing it to settle for some time, we can now proceed to the washing of resin. Note that decarbonized distilled water must be used to prevent error. The washing, which contains the OH displaced by the eluent, would then be titrated with a standardized titrant, which in this case is HCl; the titrant used is still phenolphthalein.

exchanged ions from the sample. The number of hydroxide ions has a 1:1 ratio with the substance. We can determine the molecular weight of the salt by using the determined equivalent weight. VII. Conclusion Chromatography is generally used for the separation, identification and determination of the components of a complex mixture. It is primarily used in the purification of materials used in the production of medicine or other industrial substances. In ion exchange chromatography, substances are separated depending on their charges. The ionic groups of the exchanger columns are covalently bonded to the matrix and to small concentrations of counter ions. When a sample is added, an exchange with the weakly bonded counter ions take place. The affinity of the resin to bond with charged ions generally vary depending on the ions charge, or the condition of the resin itself. The selectivity coefficient can be computed using the concentration of the ions used. The process of washing is a very important part of the procedure, as incomplete washing would lead to terrible results. If the normal setup is used, the flow rate and the state of the glass column should not be neglected as it would lead to more erroneous data. VIII. Recommendation Due to the accumulation of errors caused by even the slightest negligence, it is recommended that the proper way of doing the experiment should be discussed properly in the pre-lab discussion. It is also apparent that the washing of the resin is very time consuming. It is advised to increase the time allotted for the experiment. High amounts of distilled water are also used, which led to some running out of reserves.

The volume of titrant used would be Proper materials should also be used to stoichiometrically equivalent to the amount of displaced OH , which is equivalent to the amount of reduce the percentage of errors. More trials should

Chem 27.1 Ion-Exchange Chromatography

be done in order for the students to properly average the result and not rely on very erroneous data. IX. References

Giecel Dela Cruz, A. P. (2012). Experiment 9: Ion Exchange Chromatography. University of the Philippines - Manila, Manila. Ion Exchange Chromatography. (n.d.). Retrieved October 1, 2012, from Ion Exchange Chromatography: http://www2.chem.uic.edu/min/chem455/docs/Lab_ 5.pdf

I hereby certify that I have given substantial contribution to this report

_______________________ Lozada, Lemuel D.

_______________________ Dabuet, Nathalie E.

Chem 27.1 Ion-Exchange Chromatography

Chem 27.1 Ion-Exchange Chromatography