Method 5:  Ion exchange chromatography

1.  Introduction

     Ion exchangers are solid water-insoluble "high-molecular" substances ("polyelectrolytes") - we're talking about the solid phase here - that can exchange ions bound to them with other free-in-solution ions of the same charge. From UCanterbury's bchml eng site: Ion exchangers are resins composed of cross-linking polymers that possess electrically active functional groups. These groups are special in that they can exchange either positive or negative ions. Two key factors determine the effectiveness of an exchanger: its affinity for the ion in question and the number of active sites available. This process is stoichiometric and can be reversed by altering conditions on the exchanger. The following illustration shows the basics of ion exchange:

[equations'n'stuff]

     Ion exchange procedures require intermediary agents, along with the ions to be exchanged, in a consistent aqueous buffer solution. The exchanger contains a solvent in its pores; all ionic exchange occurs on the surface of the solid matrix. Since we have to maintain electroneutrality, the exchanged and emigrating ions form an ion pair; the emigrating ions can be washed away with solvent. Thus, the same solid phase can be re-used multiple times, or regenerated in the same experiment when it has been rendered ineffective by loading with exchanged ions. The bound ions can then be eluted by changing the ionic strength or the pH of the mobile phase. The central parameters of these procedures are pK, diffusion, and the law of mass action (aka, ideal law of equilibrium):

[Fig 1. Principle of anionic and cationic exchangers.]

     Biomolecular binding strength depends on solution pH, since it affects the number of ions available for exchange. Proteins can be zwitterions, so you'll need to use either an anionic or cationic exchanger. Whatever the conditions, we have to determine the isoelectric point of the protein at different pHs to see what charges the proteins can have and where it's electroneutral. When pH < pI of a given molecule, it will be positively charged and we'll need a cation exchanger; vice versa when pH > pI. The amphoteric character (ability to react as either acid or base) of proteins allows us to detect ionic interference of other substances and improve our own protein separation. The principle of ion exchange processes will be explained more clearly using an anionic exchange as an example. Today, most people use synthetic organic ion exchangers on a polystyrene base (DOWEX) or natural polymers like cellulose, dextran, or silicate. The macromolecules of the ion exchanger normally make up a 3D network, onto whose surface a huge number of ionizable groups are covalently bonded. Whereas the type of matrix material is generally flow characteristic (the type of ions used and their chemical/mechanical stability are more solid), the groups covalently-bonded to the matrix and the strength of those bonds determine what the exchangable ions can be:  every group gives an exchange of very basic (anion exchanger) for very acidic (cation exchanger) character.

[Fig 2. Principle of an ion exchange process.]

     Typical functional groups in an anion exchanger are quaternary amines such as diethyl aminoethyl groups (DEAE - non-denaturing, sorbents have good loading capacity), while those for cation exchangers include organic and inorganic acids like carboxymethyl groups (CMs) or sulfonates. These groups are covalently coupled to the matrix material (Fig. 3). Since exchange groups are only inserted in their ionic form, it's important to know their pK values. Such values can be found by a simple titration curve, as shown for a CM sephadex in Fig 4. Because so many biologically important substances contain ionizable functional groups (amino acids, proteins, nucleotides, nucleic acids, metabolites, etc), biochemical methods for the isolation and separation of charged compounds are quite valuable - some charged compounds are electrostatically bound to the exchanger and others are not.

[Fig 3. Cellulose-based ion exchanger.]
[Fig 4. Titration curve for CM sephadex.]

2.  Experiment

2.1  Separation of ATP, ADP, and AMP

     Ion exchange chromatography is predominantly done as column chromatography, though everything can be separated using TLC as well - we would use TLC for metabolite analysis after introducing a specific substrate or assessing the purity of the substance. In this experiment, we'll be investigating how AMP, ADP, and ATP (ATP = most negative!)separate by their different migration rates in the prescence of an ion exchanger using TLC; we'll also be concurrently separating a multi-component mixture and the hydrolysis products of ATP.

     Transfer 15mg ATP in 250µL 0.1M HCl (proton donor for the hydrolysis) into an Eppendorf container. Heat the reaction for 20 minuntes at 95°C in a water bath. Make up the required stock solutions from pure ATP, ADP, and AMP - 10 mg adenosine phospate per 1 mL water. (The TA will do this.) For the TLC, we'll be running 2 gels with different solid phases:  a polyethylene-imine proofed cellulose plate (anion exchanger) and a plain cellulose plate. Make 6 spots:  pure AT/D/MP, one each of the 2- and 3-component mixtures, and one for the hydrolysate. Mark them! After drying the spots, run the gels in a mobile phase of 0.5 LiCl in 2M formic acid (Irritant!) - we'll use the same solvent for both TLCs. After the run, observe the phosphoric acid esters by their UV fluorescence on the silica gel plate (UV l = 254 nm); the non-fluorescing spots should already be visible on the plate.

Calculations
- Sketch the chromatogram.
- Determine R_f values.
- Interpret both chromatograms.
- How could a nucleotide analysis be done quantitatively?
Do a column chromatography statt TLC?

2.2  Separation of ATP, UMP, and CMP

     Nucleotides contain both acidic and basic functional groups, so we can separate mixtures with both anionic and cationic exchangers. The column chromatography employing alkali ionic exchange (anionic exchangers, eg DOWEX 1 x 2 in KCl) will be used to fractionate, isolate, and characterize the building blocks of nucleic acids. In our experiment, we'll be using an already-prepared column that has been equilibrated with 10 mM HCl. Continue as follows:

1. Wash the column with about 50mL 10mM HCl. Ignore this step for the 1st column; it's prep for the rest of them.
2. Solvate 10mg CMP and 5mg both UMP and ATP in 1mL water = 3 different solutions! 80µL of each will be used for the column (we're running 1) - pipette it in!
3. Wash the column with 25-27mL 10mM HCl to elute any not-bound substances. You should get 5-6 fractions for every 5mL eluted.
4. The nucleotides will be eluted by a linear increase in ionic strength (5 columnfuls of 0-0.5M NaCl in 10mM HCl each, flow rate = 2 mL/min). (Gradient elution!) Collect the eluate in 5mL fractions.
5. After the end of the elution, wash out the column with 150mL 10mM HCl. Save the first 25mL in 5 fractions for good measure.

Calculations - Nucleotides can be analyzed and identified by their characteristic UV spectra. Take the spectrum of a pure nucleotide between 240 and 350 nm, then measure all the fractions at 260 nm. For the fractions that absorb at 260nm (A₂₆₀), measure again at 280nm. From the quotient of A₂₈₀/A₂₆₀, we can find out which nucleotide was eluted first. Compare your values with the following lit values:

Substance: CMP UMP ATP

A280/A260 (at pH2): 2.09 0.39 0.22

- Discuss the measured values, the spectra taken, and the comparison with the lit values.
Look up stuff about the Warburg-Christian method - it's generally used to measure relative concentrations of proteins; the original intention was as a series of correction factors to account for nucleic acid contamination of protein solutions. Tyr and Trp (and thus, proteins containing them) absorb strongly at 280nm; nucleic acids at 260nm.
- For each substance, make up a chromatogram by putting the absorbance vs. their fraction number (mm paper used). Draw the structural formula and explain this using calculated elution ratio for the NT.

2.3  Ion exchange chromatography of proteins

     The chromatographic behaviour of amphoteric polyelectrolytes in ion exchange are characterized by size and isoelectric point (IEP). In order to bind to an anion exchanger, proteins require a pH value that lies above their IEP; in cationic exchange, we need a pH range below the IEP. For the experiment, we require 2 materials (with code numbers) at our disposal: CM Fractogel and DEAE Fractogel (by Merck and Darmstadt). Our protein mixtures will be separated on these exchangers; the elution ratio we get will tell us which one we need to use for our samples.

• Using a Pasteur pipette, lay down some quatz sand as a frit. Make up a dry silica slurry using 25mM Tris buffer and make up a 1-1.5cm column.
• Equilibrate the gel with 10mL 25mM Tris buffer (pH = 7.5). Take care to not disturb the column.
• Make up the sample mixture, put in as you know how, and let it sink in:

  Cytc: Catalase:

  50µL 10 mg/mL solution (pI = 10.6) 50µL 65 kU/mL solution (pI = 5.6)

• Wash the gel with 4x 500µL Tris buffer. Collect the eluate in a labelled container.
• Elute the rest of the stuff in a 500 µL series (100 µL/dilution) of 0.05, 0.1, 0.2, 0.3, and 0.5 M NaCl in 25 mM Tris (pH = 7.5).
  We'll play with the catalase and (visible) Cytc fractions last. As a qualitative catalase test:  Add 20µL 10% HOOH to each fraction and record the violence of the reaction with ++, +, or -. What's going on? What's the reaction equation?

3.  Questions

1. In the AT/D/MP experiment, what order do you expect to see in the separation on the PEI-proofed cellulose plate (ie, anion exchanger)?
   I would expect ATP to be the lowest on the plate, since it has the highest negative charge and will therefore have a higher affinity for the positive charges in the solid matrix.
2. Why is it possible to detect dark spots on the TLC plate under UV light, if the substances in question are colourless?
   Many TLC plates are treated with a fluorescent indicator that glows a bright green color when placed under UV light; the compounds adsorbed on the plate show up as dark spots against this bright green background.
3. What is an R_f value and how is it calculated?
   It is the ratio of the distance the substance moved to the distance the solvent moved; each component of a solution will have a specific Rf for the same solvent when the chromatography occurs for a specific length of time.
   Rf = Distance traveled by solute/Distance traveled by solvent

4. What reaction is happening in the qualitative catalase test? What's the reaction equation?
     The catalase test is frequently used with bacterial cultures to see if they produce catalase. HOOH is added to your culture/sample; if catalase is present, the two react and O₂ is produced - bubbles indicate a positive test.
   2 H₂O₂ -----> 2 H₂O + O₂
5. What should we be careful for when loading a column chromatography?
   Make sure the level of the gel is even and make sure there are no bubbles - while it wouldn't really matter in deciding whether something will elute or not, it will make a huge difference when you go and try to collect the separate fractions.
6. Quickly explain the principles of ion exchange chromatography.
   See notes below.
7. Write an objective for each of the 3 parts of the experiment.
   - To separate adenosine phosphates based on charge.
   - To separate mono-phosphate bases, familiarizing ourselves with gradient elution in column chromatography.
   - Using anion exchange methods, to exploit characteric IEPs of proteins for separatory purposes.

4.  Literature

• Dorfner, K. (1964). "Ionenaustauscher" Eigenschaften und Anwendungen, 2. Auflage. Verlag de Gruyter: Berlin. (ME30/29)
• Cooper, G.C. (1981). Biochemische Arbeitsmethoden. Verlag de Gruyter: Berlin. (BC10/22)
• Williams, B.L. and K. Wilson (1984). Methoden der Biochemie, 2. bearbeitete und verbesserte Auflage. Thieme Verlag.
• Janson, J.C. and L. Ryden (1989). Protein Purification. VCH: Weinheim.
• Stryer, L.L. (1995). Biochemistry, 4th Edition.  Freeman & Co.: New York. 50
• Hahn-Deinstrop, E. (1988). Dünnschicht-Chromatographie: praktische Durchführung und Fehlervermeidung. Wiley-VCH: Weinheim.
• Kraus, L. (1996). Dünnschichtchromatographie. Springer: Berlin.
• Frey, H.P. and K. Zieloff (1993). Qualitative und quantitative Dünnschichtchromatographie: Grundlagen und Praxis. VCH: Weinheim.
• Karger, B.L. and W.S. Hancock (1996). Methods in Enzymology: High resolution separation and analysis of biological macromolecules, Fundamentals. Academic Press: San Diego. 270, 49-56.

• Jacobs, S. (1966). "Ion Exchange Chromatography of Amino Acids." Methods of Biochemical Analysis. John Wiley and Sons, Inc: London. v.14 p.177-202.

Kyra's notes

     IEC is an LC technique that separates based on charged groups of a molecule - these interact with the opposite charges on molecules making up the stationary phase (ie, ionizable functional groups coupled to an inert matrix). These functional groups on the matrix are already associated with free ions in solution (mobile phase) - "exchangable counterions" if you will. The charged molecules in our sample (ie, AM/D/TP) compete with the solvated ions for association with the groups on the stationary phase - the more charged the sample molecule is, the more it's going to be slowed down in the column. Thus, the sample is separated based on charge. Binding affinity depends on the conditions on of the run (eg, pH, concentration), along with the types and numbers of charged groups we're looking at. Since our sample is a mixture that we established ourselves, pretty much everything is going to bind, instead of the extra debris getting washed away.
     Anion exchange is when the stationary phase possesses a positive charge and the ion in solution is an anion; cation exchange entails the reverse conditions. Since we're looking at ionizable sections of whatever molecule in question, pH, pK, and IEP are going to be issues to be considered. When pH < pI of a given molecule, it will be positively charged and we'll need a cation exchanger; vice versa when pH > pI. It's complicated when you take everything into account. It is however, essentially just the mobile phase that we're messing around with in order to elute what we want, ie by changing pH, ionic strength, etc.
brahms.chem.uic.edu/~chem455/source/download/lab5.pdf

We'll be working with anionic exchangers.

Conditions are quite varied for proteins and peptides, while aa runs are done under more standardized conditions.

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