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.