Principles for HPLC

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Nov 12, 2013 (3 years and 4 months ago)

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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 CH
3
CN, CH
3
OH)


gradient elution


CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

H
2
O

H
2
O

H
2
O

H
2
O

CH
3
CN

CH
3
CN

Reverse Phase

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

H
2
O

H
2
O

H
2
O

CH
3
OH

Reverse Phase

C
6
H
6

C
6
H
6

C
6
H
6

CH
3
OH

Non
-
polar

polar

Polarity?

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

H
2
O

H
2
O

H
2
O

CH
3
OH

Reverse Phase


50/50?

C
6
H
6

C
6
H
6

C
6
H
6

CH
3
OH

Mobile phase

More/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, MgCl
2
, K
3
PO
4
,
NH
4
SO
4
)


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, SO
3
-



Weak cation exchanger (WCX)


carboxylic acid, COO
-

Anion Exchangers


Strong anion exchanger (SAX)


quaternary ammonium, e.g., N(CH
3
)
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


RNH
3
+

= RNH
2

+ 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
-

< NO
3
-

< citrate
3
-

(strong)


Cations:


Li
+

< H
+

< NH
4
+

< K
+

< Mg
2+

< Ca
2+

(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

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

K
+

K
+

K
+

K
+

COO
-

COO
-

COO
-

COO
-

Cyt c

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

K
+

K
+

K
+

K
+

K
+

COO
-

COO
-

COO
-

COO
-

Cyt c

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

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

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

NH
3
+

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

NH
2

NH
2

NH
2

NH
2

NH
2

NH
2

NH
2

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,”
2
nd

ed. John Wiley & Son: New York,
1997.