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Ion Exchange Chromatography Lecture

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Ion Exchange Chromatography Lecture

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Ion Exchange Chromatography Lecture

  1. 1. Ion-Exchange Chromatography Introduction to Chromatography: General Principles Ion-Exchange Chromatography Principles Ion-Exchange Chromatography Resins Net Charge of Amino Acids, Peptides, and Proteins Integrated and Automated Systems Reading: N & B Ch. 5
  2. 2. Introduction Chromatography literally means “color writing”. Chromatography was invented by the Russian botanist Mikhail Tsvet in 1900. He used it to separate chlorophyll-containing extracts of plants. Key idea is that molecules of interest interact differentially with the stationary phase and a mobile phase, and thus can be separated.
  3. 3. The Basic Principle
  4. 4. Partition Coefficient and Relative Mobility Partition coefficient describes the affinity of a compound for the stationary phase. a or (Kav)= molecules adsorbed on stationary phase molecules in stationary and mobile phase Can have values between 0 and 1. Example, a molecule with a = 0.4 will be 40% adsorbed on the stationary phase. Relative mobility or retention factor (Rf) describes the affinity of a molecule for the mobile phase. Rf = 1 – a (Recall Rf from TLC in Organic Chem)
  5. 5. Ion-Exchange Chromatography Separates molecules on the basis of charge Beads of the resin modified so that they contain a cationic or anionic functional group that can be positively charged, negatively charged, or neutral depending on pH. A solution that contains the species of interest is applied to the column containing the resin, and the sample either binds to the resin or passes through the column. A gradient (e.g., salt or pH) can then be used to elute the desired compound if the compound adhered to the resin.
  6. 6. Ion-Exchange Chromatography Would the resin in this example be considered an anion-exchange resin or a cation-exchange resin?
  7. 7. Ion Exchange Resins Proteins with net negative charges (excess of negative charges) adsorb to anion exchangers, while those with net positive charges (excess of positive charges) adsorb to cation exchangers. The strength of the adsorption increases with increased net charge.
  8. 8. Ion-Exchange Chromatography: a closer look
  9. 9. Electrostatic Potential Map of the Surface of a Protein http://www.expasy.ch/spdbv/images/1YDRsurf.jpg
  10. 10. Desorption Two possibilities exist to desorb sample molecules from the ion exchanger: 1. Reducing the net charge by changing pH. 2. Adding a competing ion to "block" the charges on the ion exchanger.
  11. 11. Principle of Ion-Exchange Chromatography Fig. 5-13 (Ninfa & Ballou)
  12. 12. Principle of Ion-Exchange Chromatography cont. Fig. 5-13 (Ninfa & Ballou)
  13. 13. What is the nature of the functional groups that are covalently linked to the resin? You will be using DE52, which contains DEAE functional groups attached to a cellulose matrix
  14. 14. Depending on the pKa value of the charged ligand, the ion exchangers are divided into strong and weak. Strong ion exchangers are fully charged over the total pH range normally applicable to proteins and peptides. With weak ion exchangers, the charge displayed is a function of the eluent pH.
  15. 15. Examples Strong anion exchangers -CHN+(CH)trimethylaminoethyl TAM 233 -CHN+(CH)triethylaminoethyl TEAE 24253 Weak anion exchangers -CHN+Haminoethyl AE 243 -CHN+(CH)diethylaminoethyl DEAE 24252 Strong cation exchangers -SO- sulpho S 3 -CH2SO3 - sulphomethyl SM Weak cation exchangers -CH2COO- carboxymethyl Cadapted from N & MB Table 5-2
  16. 16. How Do We Know If “Our” Protein Is Going to Bind the Ion-Exchange Resin That We Are Using? – pH, pKa, pI & Buffers Revisited: pH = -log[H+] (not strictly true but a useful, working definition) pH = pKa + log([basic form]/[acidic form]) [HH eq] Isoelectric point (pI) is the pH at which a molecule has a net charge of zero. Buffers useful ±1 (or ±0.5) units above and below their pKa
  17. 17. Deciding on the Charge of Our Protein: We start by considering a simple, weak acid: RCOOH Then, we consider a weak base: RNH2 Then, we will consider a compound that has both of the above functional groups – i.e., an amino acid Then, we will consider small peptides Finally, we will extrapolate to a polypeptide – i.e., a protein This discussion will require some board work
  18. 18. Group pK a N-terminal amino 8.0 C-terminal carboxyl 3.1 Asp, Glu 4.0 Lys 10. 4 Arg 12. 5 The pKas of groups or side chains can and do vary somewhat from what their values are in free amino acids. The values in the table below are meant to be approximate, but on average, fairly representative
  19. 19. Determination of pI for a Protein http://ca.expasy.org/tools/pi_tool.html http://emboss.sourceforge.net/ (and a number of other sites) Or, experimentally determine pI by using isoelectric focusing, a topic we will take up when we discuss SDS PAGE.
  20. 20. Now that we understand the concept of pI (I hope), we are in a better position to consider the choice of ion exchanger Pharmacia handbook
  21. 21. The pH vs. net surface charge curves for three different proteins are shown. Schematic chromatograms for a CM and a DEAE ion exchanger are shown at the top and bottom, respectively.
  22. 22. A Question to Ponder Proteins are usually least soluble and often precipitate at their isoelectric point. WHY?
  23. 23. Considerations Conditions used to purify a protein are often determined empirically. You likely will choose which resin to use on the basis of the pI of the protein (if it is known or can be estimated). Then you need to decide on the buffer, the salt, the steepness of the gradient, etc… You may want to run some pilot experiments. After you decide which resin you want to use, you will then have to: -swell (hydrate) of the resin -load the sample -equilibrate with buffer -elute the sample -pack or pour the column -locate the sample -equilibrate the sample -determine purity
  24. 24. Gradients of a neutral salt are formed by mixing two eluents, one containing a low concentration of the neutral salt (buffer A) and one containing a high concentration of this salt (buffer B). But for their salt contents, the two eluents are identical. Chromatography systems usually control the gradient formation by the use of two pumps, one for buffer A Gradient Elution Amersham Biosciences and one for buffer B
  25. 25. A simple gradient maker: high salt low salt Fig. 5-14 (Ninfa & Ballou) In our ion-exchange chromatography lab, we will not use a gradient. Rather, we will use a step elution in which we go from low salt to high salt in one step
  26. 26. Steep vs. shallow gradient elution
  27. 27. Steep vs. Shallow Gradient Elution – Another View Amersham Biosciences The distance between peaks is controlled by the slope of the gradient
  28. 28. A Bit More On Integrated and Automated Chromatography Systems HPLC (High Performance or High-Pressure Liquid Chromatography) FPLC (Fast Protein or Fine Performance Liquid Chromatography)
  29. 29. Schematic of an automated system (FPLC) Low salt High salt Pumps Sample load Fraction collector Sheehan, David (2003). Fast Protein Liquid Chromatography. 244. pp. 253.

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