Download Principles for HPLC Methods Development

Principles for HPLC
Methods Development
Bioanalytical Chemistry
Lecture Topic 4
Five Stages
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Define problem
Experiment with key variables
Evaluate
Optimize
Troubleshoot
Define
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What is the purpose?
– Analytical
– Preparative
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What are the molecular characteristics of
the analyte and sample?
– CHASM
CHASM
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Charge
– Positive/negative
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Hydrophobicity
Affinity
– “lock and key” sites
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Solubility & stability
– pH, ionic strength, organic solvents
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Molecular weight
Analytical vs. Preparative
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Analytical Requirements
–
–
–
–
–
–
Linearity
Precision
Accuracy
Sensitivity
Assay reproducibility
Robustness
Analytical vs. Preparative
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Preparative Requirements
Recovery
Product purity
Capacity
Costs
– Scale up
– Process throughput
– Speed
Methods Development
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Select the mode
pH map
Optimize gradient/elution
– gradient slope
– eluent concentration
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Loading study
– overload: peak width and shape
Common Modes
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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
Reverse Phase
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH3CN
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
CH3CN
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
Reverse Phase
Polarity?
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
Non-polar
H2O
C6H6
H2O
polar
Reverse Phase – 50/50?
Mobile phase
More/less polar?
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
CH2CH2CH2CH2CH2CH2CH2CH3
Non-polar
H2O
C6H6
H2O
polar
Common Modes
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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
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Resolution
– degree of separation between analyte and other
species present in mixture
– bandspreading
– selectivity
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Recovery
– mass recovery
– activity recovery
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Capacity
Developing Your Application
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Proteins
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Antibodies
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Peptides
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Nucleic acids
Proteins
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All modes can potentially be used
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Ion exchange common first step
– mobile phase less denaturing
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Antibodies
– Affinity
Peptides
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amino acid chain < 30 residues (5000 MW)
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reverse phase most commonly used
– historical
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ion exchange can be equally effective
Nucleic Acids
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gel electrophoresis commonly used
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anion exchange predominant
chromatographic method
Ion Exchange
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Sample must be ionized in order to be
retained on column significantly
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Anion exchange (anionic acidic proteins)
X- + R+Cl- = X-R+ + Cl-
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Cation exchange (protonated basic proteins)
X+ + R-K+ = X+R- + K+
Column Type
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4 types: strong/weak cation/anion
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Strong - ionization of ionic group does not
change over usual pH range
– better starting point
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Weak - lose charge and sample retention for
certain pH ranges
Cation Exchangers
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Strong cation exchanger (SCX)
– sulfonic acid, SO3-
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Weak cation exchanger (WCX)
– carboxylic acid, COO-
Anion Exchangers
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Strong anion exchanger (SAX)
– quaternary ammonium, e.g., N(CH3)4+
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Weak anion exchanger (WAX)
– diethylaminoethyl (DEAE)
pH Effects
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Anion exchange
– RCOOH = RCOO- + H+
– INcrease in pH leads to greater sample
ionization and retention
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Cation exchange
– RNH3+ = RNH2 + H+
– DEcrease in pH leads to greater sample
ionization and retention
Salt/Buffer Effect
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Mobile phase cations/anions can displace
analyte on column
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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
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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
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Select Salt
– 0.5 - 1.0 M
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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
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Addition of organic solvents decreases
retention
– Be careful! Can denature biomolecules
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Can be used to create changes in selectivity
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EXS: methanol or acetonitrile
– water miscible
Cytochrome c
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Function:
Redox protein
involved in cell
apoptosis and
respiration
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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 c
COO- K+
K+
COO- K+
COO- K+
COO-K+
K+
K+
K+
Cyt c
COO- K+
NH3+
COO- K+
NH3
+
Cyt c
COO-
K+
NH3+
NH3+
COO- K+
NH3+
NH3+
NH3+
NH3+
COO-
NH3
NH3+
+
NH3+
Cyt c
COO-
NH3+
NH3+
NH3+
COO- K+
K+
COO- K+
K+
K+
NH3+
COO-
NH3
Na+
+
NH3+
NH3+
Cyt c
COO- Na+
NH3+
NH3+
COO- Na+
Na+
COO- Na+
NH3+
Na+
Na+
Na+
Effect of pH
What Does Cyt c look like at low pH?
NH3+
COO-
NH3
Na+
+
NH3+
NH3+
Cyt c
COO- Na+
NH3+
NH3+
COO- Na+
Na+
COO- Na+
NH3+
Na+
Na+
Na+
Effect of pH
What Does Cyt c look like at high
pH?
NH2
NH2
COO- Na+
NH2
NH2
Cyt c
COO- Na+
NH2
NH2
NH2
COO- Na+
Na+
COO- Na+
Na+
Na+
Na+
Effect of pH
So low pH more effective for cation
exchange than high pH
Useful References
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“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.