principles for hplc methods development bioanalytical chemistry lecture topic 4
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Principles for HPLC Methods Development
Bioanalytical Chemistry
Lecture 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 Phase
C6H6
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 c
COO-
COO-
COO-
COO-
K+
K+
K+
K+
K+
K+
K+
K+
Cyt c
COO-
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.