Download - Principles for HPLC Methods Development
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Principles for HPLC Methods Development
Bioanalytical ChemistryLecture Topic 4
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Five Stages Define problem Experiment with key variables Evaluate Optimize Troubleshoot
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Define What is the purpose?
– Analytical– Preparative
What are the molecular characteristics of the analyte and sample?– CHASM
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CHASM Charge
– Positive/negative Hydrophobicity Affinity
– “lock and key” sites Solubility & stability
– pH, ionic strength, organic solvents Molecular weight
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Analytical vs. Preparative Analytical Requirements
– Linearity– Precision– Accuracy– Sensitivity– Assay reproducibility– Robustness
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Analytical vs. Preparative Preparative Requirements Recovery Product purity Capacity Costs
– Scale up– Process throughput– Speed
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Methods Development Select the mode pH map Optimize gradient/elution
– gradient slope– eluent concentration
Loading study– overload: peak width and shape
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Common Modes Reverse phase (RPC)
– Stationary phase hydrophobic and mobile phase hydrophilic
• column: silica, polystyrene covalently modified with alkyl chain 3-18 C’s
– EX: octadecylsilane (ODS) - C18
• mobile phase: buffered water + organic solvent (propanol CH3CN, CH3OH)
• gradient elution
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CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3 H2O
H2O
H2O
H2O
CH3CN
CH3CN
Reverse Phase
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CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
H2O
CH3OH
Reverse PhaseC6H6
C6H6
C6H6
CH3OH
Non-polar polar
Polarity?
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CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
H2O
CH3OH
Reverse Phase – 50/50?
C6H6
C6H6
C6H6
CH3OH
Mobile phaseMore/less polar?
Non-polar polar
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Common Modes Ion-Exchange (IEC)
– Ion exchange interactions between cationic or anionic analyte and stationary phase bearing opposite charge
• stationary phase: polystyrene, silica modified with functional groups such as quaternary amines
• mobile phase: buffer containing increasing concentration of salt (NaCl, MgCl2, K3PO4, NH4SO4)
• gradient elution
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Evaluation Resolution
– degree of separation between analyte and other species present in mixture
– bandspreading– selectivity
Recovery– mass recovery– activity recovery
Capacity
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Developing Your Application
Proteins
Antibodies
Peptides
Nucleic acids
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Proteins All modes can potentially be used
Ion exchange common first step– mobile phase less denaturing
Antibodies – Affinity
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Peptides amino acid chain < 30 residues (5000 MW)
reverse phase most commonly used– historical
ion exchange can be equally effective
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Nucleic Acids gel electrophoresis commonly used
anion exchange predominant chromatographic method
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Ion Exchange Sample must be ionized in order to be retained
on column significantly
Anion exchange (anionic acidic proteins)X- + R+Cl- = X-R+ + Cl-
Cation exchange (protonated basic proteins)X+ + R-K+ = X+R- + K+
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Column Type 4 types: strong/weak cation/anion
Strong - ionization of ionic group does not change over usual pH range– better starting point
Weak - lose charge and sample retention for certain pH ranges
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Cation Exchangers Strong cation exchanger (SCX)
– sulfonic acid, SO3-
Weak cation exchanger (WCX)– carboxylic acid, COO-
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Anion Exchangers Strong anion exchanger (SAX)
– quaternary ammonium, e.g., N(CH3)4+
Weak anion exchanger (WAX)– diethylaminoethyl (DEAE)
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pH Effects Anion exchange
– RCOOH = RCOO- + H+
– INcrease in pH leads to greater sample ionization and retention
Cation exchange– RNH3
+ = RNH2 + H+
– DEcrease in pH leads to greater sample ionization and retention
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Salt/Buffer Effect Mobile phase cations/anions can displace
analyte on column
All salts are NOT equal– Anions:
• F- < OH- < Cl- < NO3- < citrate3- (strong)
– Cations:• Li+ < H+ < NH4
+ < K+ < Mg2+ < Ca2+ (strong)– Polyvalent ions held more strongly by ion
exchange column than monovalent ions
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Salt/Buffer Effect Need to select appropriate pH:
– Anion exchange, pH > 6 used– start: pH 8.5
• protein stable?• extreme end of pH range• binding should be tightest
– Cation exchange, pH < 6 used (pH 4.0)
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Salt/Buffer Effect Select Salt
– 0.5 - 1.0 M
Gradient– 0 - 100 % gradient - to determine relative
retention of sample– long, shallow to start:
• 0 - 1 M NaCl, 50 - 100 CV’s
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Organic Solvent Effect Addition of organic solvents decreases
retention– Be careful! Can denature biomolecules
Can be used to create changes in selectivity
EXS: methanol or acetonitrile– water miscible
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Cytochrome c Function:
Redox protein involved in cell apoptosis and respiration
Structure: heme protein– FW 12,384
(horse)– Basic protein 3CYT: Takano, T., Dickerson, R. E.: Redox conformation
changes in refined tuna cytochrome c. Proc. Natl. Acad. Sci. USA 77 pp. 6371 (1980)
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What mode should we use?
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Cyt cCOO-
COO-
COO-
COO-
K+
K+
K+
K+
K+
K+
K+
K+
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Cyt cCOO-
COO-
COO-
COO-
Cyt c
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
K+
K+
K+
K+
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COO-
COO-
COO-
COO-
Cyt c
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
K+
K+
K+K+
K+
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COO-
COO-
COO-
COO-
Cyt c
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
Na+
Na+
Na+
Na+
Na+
Na+
Na+Na+
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Effect of pH
What Does Cyt c look like at low pH?
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COO-
COO-
COO-
COO-
Cyt c
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
Na+
Na+
Na+
Na+
Na+
Na+
Na+Na+
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Effect of pH
What Does Cyt c look like at high pH?
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COO-
COO-
COO-
COO-
Cyt c
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Na+
Na+
Na+
Na+
Na+
Na+
Na+Na+
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Effect of pH
So low pH more effective for cation exchange than high pH
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Useful References “The Busy Researcher’s Guide to
Biomolecular Chromatography,” Perspective Biosystems, publication date unknown.
Snyder, L.R.; Kirkland, J.J.; Glajch, J.L. “Practical HPLC Method Development,” 2nd ed. John Wiley & Son: New York, 1997.