Ion Chromatography. Anion Exchange. Chromatography Ion Exchange Theory. Dr. Shulamit Levin

Transcription

1 Ion Exchange Chromatography Chromatographic Process BA Mobile phase Stationary Phase A Shula Levin Bioforum B Distribution: K = C s/c m B shulal@zahav.net.il A Elution through the Column Chromatogram Ion Exchange Theory Cation Exchange vs Anion Exchange Cation Exchange Anion Exchange Cation exchange columns have a negative charge to attract cations. Anion exchange columns have a positive charge to attract anions SAMPLE IONS IN 1. INJECTION ION EXCHANGE INSIDE A PORE IN THE STATIONARY PHASE 2. ADSORPTION: DISPLACEMENT OF COUNTER IONS COUNTER IONS OUT 3. ELUTION MOBILE PHASE ADDITIVES 1

2 ION EXCHANGER ANION EXCHANGE K XY B Y X B X Y Analysis of Ions Ion Chromatography ION (Mono, Di, Tri Valent) Ionic Functional Group CATION EXCHANGE ANION CATION K M NH A M NH A NH M IMMOBILIZED ON THE STATIONARY PHASE Organic Acids Phenols Inorganic F Cl Organic Amines Inorganic Metals NH 4 Ions can be characterized as: organic or inorganic, anion or cation, mono or polyvalent. Ion Exchange Theory Strong vs. Weak Exchange Materials Cation exchanger Anion exchanger SO 3 NR 3 STRONG COO NH 3 WEAK Strong Exchangers stay ionized as ph varies between 2 and 12. Weak exchangers can lose ionization as a functionof ph. Ion Exchange Bonded Functionalities WEAK STRONG Cation Anion R COO Na N R Cl Carboxylic Acid SO 3 Na Sulfonic Acid H Primary, Secondary or Tertiary Amine R N R Cl R Quaternary Amine Typical chemical functionalities used for commercial exchangers. 2

3 Columns Matrices SilicaBased Polymerbased ionexchangers Hydrous Oxide =MOH =MO H =MOH =M OH Resin Ion Exchange Theory Packing Supports Capacity Swelling Mass Transfer Size Separation Reverse Phase Efficiency ph Range Equilibration Literature SilicaBased Both resin and silica based ion exchangers have advantages and disadvantages which are summarized here. F, OH > OAc HSO 3 > CN ION EXCHANGE > Br ANIONS RETENTION ORDER: Charge/Size Ratio A < A < A When the charge is the same: charge/size determines the retention > H 2 PO 4 > HCO 3 > Cl > NO 3 > I > NO 2 > ION EXCHANGE Li > H > Na > NH 4 > K > Rb > Cs > Ag Be > Mn Transition metals Al Mg CATIONS Retention Order MONOVALENT DIVALENT Zn > Co TRIVALENT > M > Ce > M > Ca transition metals > Sr 3

4 Properties of Mobile phases Compatibility with the detection mode Suppressed or Nonsuppressed (Conductivity Detector). Nature of the competing ion Concentration of the competing ion Mobile phase's ph Buffering capacity of the mobile phase Ability to complex the ionic sample components Organic modifiers Ion capacity The number of functional groups per unit weight of the stationary phase. A typical ionexchange capacity in IC is mequiv/g Conductivity and PDA Detectors in Series Anion Exchange Detection: Direct Conductivity after Suppression Cation Exchange ms Fluoride 1 ppm 2. Chloride 2 ppm 3. Nitrite 4 ppm 4. Bromide 4 ppm 5. Nitrate 4 ppm 6. Phosphate 6 ppm 7. Sulfate 4 ppm Detection:UV (PDA) at 214 nm Minutes 5 Column: Eluent: Flow rate: 1.0 ml/min Injection vol.: 50 µl Waters ICPak Anion HR 1.2 mm Sodium Carbonate/ 1.2 mm Sodium Bicarbonate 4

5 IONIZATION and RETENTION WEAK ACIDS HA H A pka~ 45 At ph >45 the main species is A The Equilibrium Constant HAc H Ac K a = (H ) (Ac) (HAc) ph and pk a HB WEAK BASES H B (H ) = K a (HAc) (Ac) ph = pk a log (HAc) (Ac ) pka ~ 78 At ph < 78 the main species is BH A general understanding of ionization constants, ph, and pk a are useful in understanding ion exchange and buffer phenomena. ANION EXCHANGE ELUTION ORDER IN ION EXCHANGE CATION EXCHANGE Amino Acids Analysis In Plasma Ion Exchange with Ninhydrin detection STRONGER ACID STRONGER BASE 1 1 R E S P O N S E 0 VOID 2 3 R E S P O N S E 0 VOID 2 3 TIME (MIN.) TIME (MIN.) 5

6 Ion Exchange of Proteins Liquid Chromatography Protein Separation Modes Primary, Secondary, Tertiary Structure Protein Structure Net Charge Carbohydrate Groups Hydrophobic Regions Hydrophilic Groups Aromatic Groups Disulfide Linkages Hydrogen Bonding Physical Characteristics of Proteins vs Chromatographic Technique Size in Solution (number of amino acids) Size Exclusion (Gel Filtration) Chromatography Hydrophobicity (hydrophobic amino acids) ReversedPhase Chromatography Hydrophobic Interaction Chromatography Unique Structure and Chemical properties Affinity Chromatography Charge (acidic or basic amino acids) Anion or CationExchange Chromatography Effect of Salt Gradient Duration on Protein Separation Binds at Low Ionic Strength Elute with Step or Continuous Gradients of Increasing Ionic Strength Separations are based on net surface charge on protein with oppositely charged groups on ionexchanger Proteins elute from column using either a gradient of increasing salt concentration (most common) or changing ph (less common) 6

7 Ion Exchange Chromatography: Anion exchange schematic Equilibration with buffer Ion Exchange Chromatography: Contd. Salt gradient continues, bound proteins elute typically highest pi protein Unbound proteins elute, either uncharged or positively charged proteins Salt gradient continues, higher bound proteins elute Salt gradient begins, counter ions compete with bound proteins for binding sites Salt gradient continues reaches highest salt concentration, most tightly bound protein elutes Definitions for Ion Exchange Type of Ion Exchangers Charged Molecules Anions are negatively charges Cations are positively charged Ion exchangers Anion exchanger: covalently bound positively charged groups Cation exchanger: covalently bound negatively charged groups Buffer Weak acid or base that controls ph Counterion Strong or weak ion that competes with analyte for binding to ion exchanger Anion exchangers pka = 10.5 Quaternary ammonium Q strong OCH 2 N (CH 3 ) 3 Diethylaminoethyl DEAE weak OCH 2 CH 2 N H(CH 2 CH 3 ) 2 Diethylaminopropyl ANX weak OCH 2 CHOHCH 2 N H(CH 2 CH 3 ) 2 Cation exchangers pka = 2.3 Sulfopropyl SP strong OCH 2 CHOHCH 2 OCH 2 CH 2 CH 2 SO 3 Methyl sulfonate S strong OCH 2 CHOHCH 2 OCH 2 CHOHCH 2 SO 3 Carboxymethyl CM weak OCH 2 COO pka = 4.9 7

8 Strong versus Weak Ion Exchangers Strong and weak do not indicate how tightly bound Strong exchanger is always ionized, weak exchanger s ionization varies with ph Weak Cation Exchange column pk a Weak Anion Exchange column pk a ~9 Strong exchangers recommended where there is a need to run at extreme ph Ion exchange capacity of a weak ion exchanger varies with ph sample loading (binding) capacity can vary with ph due to loss of charge from the exchanger Advantages Moderate resolution IonExchange Concentrating technique. Can load large volumes of dilute sample Nondenaturing protein elution techniques Preferred first step when costeffective affinity is not available Limitation Separation by charge and charge distribution; insensitive to other properties Fractions may need to be desalted prior to next purification or characterization step (e.g., Mass Spectrometry) Isoelectric Point of a Protein (pi) Isoelectric Point of a Protein (pi) ph higher than protein s pi Cation Exchanger (negative surface attracts molecules cation Support ph is lower than protein s pi 8

9 Control of Ion Exchange Separations Retention is optimized with ionic strength, usually by changing gradient slope Different counterions can give different selectivity Temperature can affect chromatography Changes several variables simultaneously Seldom used for stability considerations Selectivity is most profoundly altered with ph Effect of Gradient Slope Rib A Cyt C Apr Lys 0.2 M NaCl 0.3 M NaCl 0.5 M NaCl Minutes 20mM Sodium Phosphate, ph 6.2, 1 ml/min, 00.x M NaCl in 34 min Control of Ion Exchange Separations Effect of Counterion Retention is optimized with ionic strength, usually by changing gradient slope Different counterions can give different selectivity Temperature can affect chromatography Changes several variables simultaneously Seldom used for stability considerations Selectivity is most profoundly altered with ph Rib A Apr Cyt C NaCl KCl Minutes 20mM Sodium Phosphate, ph 6, 1 ml/min, 00.2 M XCl in 34 min 9

10 Control of Ion Exchange Separations Ion Exchange Selectivity ph effects Retention is optimized with ionic strength, usually by changing gradient slope Different counterions can give different selectivity Temperature can affect chromatography Changes several variables simultaneously Seldom used for stability considerations Selectivity is most profoundly altered with ph ph 6.1 ph 6.9 ph 7.0 ph 7.1 ph Minutes Buffer Selection Preferred Buffers Buffer pka should be near ph at which the user wishes to operate Buffer ions should have same charge as functional groups on packing material (PO 4 for cation, Tris for anion) Should maintain constant ph even when salt concentration is increased Counter ions are typically Na for cation exchange and Cl for anion exchange NOTE: All buffers should always be FILTERED!! Phosphate buffer is preferred for Cation Exchange Chromatography Transparent in UV pka near neutral has a singly and double charged state in anion exchange phosphate charges would separate 2 charged phosphate states and create ph gradient Tris buffer is popular choice for anion exchange chromatography ph of 8 is often preferred because many proteins have pi of

11 Good s Buffer NaCl as Counter Ion Described in the research of Dr. Norman Good et al. in pka value between 6.0 and 8.0 High solubility Non toxic Limited effect on biochemical reactions Very low absorbance between 240 nm and 700 nm Enzymatic and hydrolytic stability Minimal changes due to temperature and concentration Limited effects due to ionic or salt composition of the solution Limited interaction with mineral cations Limited permeability of biological membranes Examples: CAPS, MOPS, TAPS, HEPES NaCl is preferred counter ion Biological fluids are always rich in sodium chloride chaotropic character (i.e. ability to make water less polar) Lowers 'saltingout' effect on hydrophobic molecules Maximizes solubility during elution and improves recovery Other counter ions Varying ionic strength Can improve or alter selectivity Can affect binding capacity of packing material Gradient: Starting point Sample High Equilibration Injection Gradient Salt ReEquilibration Volume Wash unbound proteins 1020 cv ~ 5 cv tightly bound proteins Summary Measure charge variation among proteins ProteinPak HiRes Ion Exchangers Full range of functional groups Stable and reproducible Used with UPLC for best resolution Use ph for most efficient method development Auto Blend Plus Technolgy for easy ph adjustment 510 cv 510 cv Typical linear gradient for ionexchange chromatography 11