Principles for HPLC Methods Development

1
Principles for HPLC Methods Development

• Bioanalytical Chemistry
• Lecture Topic 4

2
Five Stages

• Define problem
• Experiment with key variables
• Evaluate
• Optimize
• Troubleshoot

3
Define

• What is the purpose?
• Analytical
• Preparative
• What are the molecular characteristics of the
  analyte and sample?
• CHASM

4
CHASM

• Charge
• Positive/negative
• Hydrophobicity
• Affinity
• lock and key sites
• Solubility stability
• pH, ionic strength, organic solvents
• Molecular weight

5
Analytical vs. Preparative

• Analytical Requirements
• Linearity
• Precision
• Accuracy
• Sensitivity
• Assay reproducibility
• Robustness

6
Analytical vs. Preparative

• Preparative Requirements
• Recovery
• Product purity
• Capacity
• Costs
• Scale up
• Process throughput
• Speed

7
Methods Development

• Select the mode
• pH map
• Optimize gradient/elution
• gradient slope
• eluent concentration
• Loading study
• overload peak width and shape

8
Common Modes

• Reverse phase (RPC)
• Stationary phase hydrophobic and mobile phase
  hydrophilic
• column silica, polystyrene covalently modified
  with alkyl chain 3-18 Cs
• EX octadecylsilane (ODS) - C18
• mobile phase buffered water organic solvent
  (propanol CH3CN, CH3OH)
• gradient elution

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Reverse Phase
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH3CN
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
CH3CN
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
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Reverse Phase
C6H6
Polarity?
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
polar
Non-polar
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Reverse Phase 50/50?
Mobile phase More/less polar?
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
polar
Non-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

14
Developing Your Application

• Proteins
• Antibodies
• Peptides
• Nucleic acids

15
Proteins

• All modes can potentially be used
• Ion exchange common first step
• mobile phase less denaturing
• Antibodies
• Affinity

16
Peptides

• amino acid chain lt 30 residues (5000 MW)
• reverse phase most commonly used
• historical
• ion exchange can be equally effective

17
Nucleic Acids

• gel electrophoresis commonly used
• anion exchange predominant chromatographic method

18
Ion Exchange

• Sample must be ionized in order to be retained on
  column significantly
• Anion exchange (anionic acidic proteins)X-
  RCl- X-R Cl-
• Cation exchange (protonated basic proteins)X
  R-K XR- 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-

21
Anion Exchangers

• Strong anion exchanger (SAX)
• quaternary ammonium, e.g., N(CH3)4
• Weak anion exchanger (WAX)
• diethylaminoethyl (DEAE)

22
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

23
Salt/Buffer Effect

• Mobile phase cations/anions can displace analyte
  on column
• All salts are NOT equal
• Anions
• F- lt OH- lt Cl- lt NO3- lt citrate3- (strong)
• Cations
• Li lt H lt NH4 lt K lt Mg2 lt 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 gt 6 used
• start pH 8.5
• protein stable?
• extreme end of pH range
• binding should be tightest
• Cation exchange, pH lt 6 used (pH 4.0)

25
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 CVs

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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 c
COO-
K
K
COO-
K
K
COO-
K
K
K
COO-
K
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Cyt c
COO-
K
NH3
NH3
NH3
COO-
Cyt c
K
NH3
NH3
COO-
K
NH3
NH3
COO-
K
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NH3
NH3
NH3
COO-
Cyt c
NH3
COO-
NH3
NH3
NH3
COO-
K
K
K
COO-
K
K
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NH3
NH3
NH3
Na
COO-
Cyt c
NH3
COO-
Na
NH3
NH3
NH3
COO-
Na
Na
Na
Na
Na
COO-
Na
33
Effect of pH

• What Does Cyt c look like at low pH?

34
NH3
NH3
NH3
Na
COO-
Cyt c
NH3
COO-
Na
NH3
NH3
NH3
COO-
Na
Na
Na
Na
Na
COO-
Na
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Effect of pH

• What Does Cyt c look like at high pH?

36
NH2
NH2
NH2
Na
COO-
Cyt c
NH2
COO-
Na
NH2
NH2
NH2
COO-
Na
Na
Na
Na
Na
COO-
Na
37
Effect of pH

• So low pH more effective for cation exchange than
  high pH

38
Useful References

• The Busy Researchers 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.