Principles for HPLC Methods Development
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Transcript of Principles for HPLC Methods Development
Principles for HPLC Methods Development
Bioanalytical ChemistryLecture Topic 4
Five Stages Define problem Experiment with key variables Evaluate Optimize Troubleshoot
Define What is the purpose?
– Analytical– Preparative
What are the molecular characteristics of the analyte and sample?– CHASM
CHASM Charge
– Positive/negative Hydrophobicity Affinity
– “lock and key” sites Solubility & stability
– pH, ionic strength, organic solvents Molecular weight
Analytical vs. Preparative Analytical Requirements
– Linearity– Precision– Accuracy– Sensitivity– Assay reproducibility– Robustness
Analytical vs. Preparative Preparative Requirements Recovery Product purity Capacity Costs
– Scale up– Process throughput– Speed
Methods Development Select the mode pH map Optimize gradient/elution
– gradient slope– eluent concentration
Loading study– overload: peak width and shape
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
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3 H2O
H2O
H2O
H2O
CH3CN
CH3CN
Reverse Phase
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
H2O
CH3OH
Reverse PhaseC6H6
C6H6
C6H6
CH3OH
Non-polar polar
Polarity?
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
H2O
CH3OH
Reverse Phase – 50/50?
C6H6
C6H6
C6H6
CH3OH
Mobile phaseMore/less polar?
Non-polar polar
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
Evaluation Resolution
– degree of separation between analyte and other species present in mixture
– bandspreading– selectivity
Recovery– mass recovery– activity recovery
Capacity
Developing Your Application
Proteins
Antibodies
Peptides
Nucleic acids
Proteins All modes can potentially be used
Ion exchange common first step– mobile phase less denaturing
Antibodies – Affinity
Peptides amino acid chain < 30 residues (5000 MW)
reverse phase most commonly used– historical
ion exchange can be equally effective
Nucleic Acids gel electrophoresis commonly used
anion exchange predominant chromatographic method
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+
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
Cation Exchangers Strong cation exchanger (SCX)
– sulfonic acid, SO3-
Weak cation exchanger (WCX)– carboxylic acid, COO-
Anion Exchangers Strong anion exchanger (SAX)
– quaternary ammonium, e.g., N(CH3)4+
Weak anion exchanger (WAX)– diethylaminoethyl (DEAE)
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
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
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)
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
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
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)
What mode should we use?
Cyt cCOO-
COO-
COO-
COO-
K+
K+
K+
K+
K+
K+
K+
K+
Cyt cCOO-
COO-
COO-
COO-
Cyt c
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
K+
K+
K+
K+
COO-
COO-
COO-
COO-
Cyt c
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
K+
K+
K+K+
K+
COO-
COO-
COO-
COO-
Cyt c
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
Na+
Na+
Na+
Na+
Na+
Na+
Na+Na+
Effect of pH
What Does Cyt c look like at low pH?
COO-
COO-
COO-
COO-
Cyt c
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
NH3+
Na+
Na+
Na+
Na+
Na+
Na+
Na+Na+
Effect of pH
What Does Cyt c look like at high pH?
COO-
COO-
COO-
COO-
Cyt c
NH2
NH2
NH2
NH2
NH2
NH2
NH2
Na+
Na+
Na+
Na+
Na+
Na+
Na+Na+
Effect of pH
So low pH more effective for cation exchange than high pH
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.