2. Molecular characteristics of the sample.
Selection of Stationary phase & Mobile Phase
Troubleshooting & Optimization
Data Analysis: Qualitative & Quantitative.
Steps for HPLC method development
3. What are the molecular characteristics
of the analyte or sample?
CHASM
1. Charge
Positive/negative
2. Hydrophobicity
3. Affinity
“lock and key” sites
4. Solubility & stability
pH, ionic strength, organic solvents
5. Molecular weight
4. Bulk (SiO2)x Bulk (SiO2)xSurface Surface
Selection of Stationary Phase
Normal Phase Reverse Phase
SurfaceBulk (SiO2)x
Ion Exchange
5. Normal Phase Reverse Phase Ion
Exchange
Stationary Phase Polar Non polar Silica having
functional
group
Mobile Phase Non polar
(hexane,heptan)
& slightly polar
(isopropanol,
ethylacetate)
Polar (water,
methanol,
acetonitrile,
tetrahydrofuran)
Depending
on analyte,
may salt
gradient or
pH gradient
Sample
Movement
Non polar
fastest
Polar fastest More
cation/anion
Separation based
on
Different
polarities
(functionality)
Different
hydrocarbon
content
Ion
exchange.
Selection of Mobile Phase