Principles for HPLC Methods Development - PowerPoint PPT Presentation

Loading...

PPT – Principles for HPLC Methods Development PowerPoint presentation | free to view - id: 76f01-ODk2N



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Principles for HPLC Methods Development

Description:

Stationary phase hydrophobic and mobile phase hydrophilic ... Dickerson, R. E.: Redox conformation changes in refined tuna cytochrome c. Proc. ... – PowerPoint PPT presentation

Number of Views:2813
Avg rating:3.0/5.0
Slides: 39
Provided by: patriciaa6
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: 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

9
Reverse Phase
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH3CN
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
CH3CN
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
10
Reverse Phase
C6H6
Polarity?
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
polar
Non-polar
11
Reverse Phase 50/50?
Mobile phase More/less polar?
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
CH3OH
CH2CH2CH2CH2CH2CH2CH2CH3
H2O
H2O
CH2CH2CH2CH2CH2CH2CH2CH3
C6H6
polar
Non-polar
12
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

13
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

19
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

20
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

24
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

26
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

27
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)
28
What mode should we use?
29
Cyt c
COO-
K
K
COO-
K
K
COO-
K
K
K
COO-
K
30
Cyt c
COO-
K
NH3
NH3
NH3
COO-
Cyt c
K
NH3
NH3
COO-
K
NH3
NH3
COO-
K
31
NH3
NH3
NH3
COO-
Cyt c
NH3
COO-
NH3
NH3
NH3
COO-
K
K
K
COO-
K
K
32
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
35
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.
About PowerShow.com