Analytical HPLC method development .pdf

ANALYTICAL HPLC METHOD DEVELOPMENT
Suchitra Ravan
Ad Hoc Scientific-
Acompanywith apurpose
This presentationis a property of Ad Hoc Scientific Pvt Ltd

Overview of HPLC Method DevelopmentStrategy
• Development approach
• Separation goal
• Nature of sample
• Sample pre treatment
• Sample detection

• Development approach
• Theoretical
• Empirical
• Theoretical Vs. Empirical
Thinking Experience

• Resolution
• Peak tailing
• Plate Counts
• Retention Time
• Run time
• Relative Standard deviation
• Separation Goal

• Nature of Sample
• How many number of components present in a
sample?
• What is the chemical structure?
• What is molecular weight?
• Is compound neutral ? ( no buffer in mobile phase)
• Does it undergo ionization?
• What is pka of compound?
• Does is UV active? How is UV spectra?
• How is the solubility?

• Sample pre-treatment
• It is ready for injection?
• Does it need dilution?
• Does it need buffering/ stabilization?
• Does it need dissolution & Extraction?

• Sample Detection
• Chromophoric – UV
• Non chromophoric- Refractive index
Evaporative light scattering
Fluorescence
• Characteristics of Universal detectors

• HPLC Mode
• Reversed Phase HPLC
• Normal Phase HPLC
• Hydrophilic-Interaction Chromatography [HILIC]
• Hydrophobic-Interaction Chromatography [HIC]
• Ion-Exchange Chromatography [IEC]
• Size-Exclusion Chromatography [SEC]

• Solvent Selectivity
• Change in organic solvent
• Change in pH
• Change in buffer
• Buffer capacity
• HPLC method development effect of mobile phase in
Reverse Phase HPLC

• Solvent Selectivity
• Solvent Selectivity triangle
Basic
Acidic Dipolar
• Solvent strength

• Change in organic solvent
• There are 2 types of organic solvent
1. Protic Solvents
Ex- water, ethanol, methanol, ammonia, acetic acid
2. Aprotic Solvents
Ex- acetone, dimethyl sulfoxide, DMF

• Change in pH
• The pH range most often used for reversed phase
can be divided into
1. low pH (1-4)
• Minimum Peak Tailing
• Rugged methods
• Most recommended
2. intermediate pH (4-8)
• Choose wisely considering the pKa of the
compound
3. Extreme cases (8-10.5)
• Harshness will compromise column lifetimes.

• The mobile phase pH can have a dramatic effect on the
ionization state of analytes
• At a pH equal to its pka, an analyte will be in both ionized &
neutral states, resulting in poor chromatography.
• Effects on a basic compound:
• Impact of pH on Acidic & Basic analyte.

• Buffers for Reverse phase HPLC
pH Range Buffer UV cutoff (nm)
1.1-3.1 Phosphate 210
6.2-8.2 Phosphate 210
11.3-13.3 Phosphate 210
2.1-4.1 Citrate 250
3.7-5.7 Citrate 250
4.4-6.4 Citrate 250
3.8-5.8 Acetate 230
7.3-9.3 Tris
(hydroxymethyl)
aminomethane
220
8.2-10.2 Borate 210

• Selection of HPLC Columns
• Introduction
• Type of Silica
• Stationary phases
• Column length
• Column diameter
• Particle Size
• Pore Size
• Surface area
• Carbon load

• Introduction
Silica is heart of HPLC

• Type of Silica
• Type A
Metal contaminants Ni, Al, Zn, Fe
Asymmetry, tailing, change in RT
• Type B
Highly pure, Less acidic

• Stationary phases
• C18
• C8
• C3,C4
• Phenyl
• Amino(NH2)
• Cyano (CN)

• Column Length
•Short (30-50mm) - short run times, low backpressure
•Long (250-300mm) - higher resolution, long

• Column Diameter
•Short ID (30-50mm) – short run times, low backpressure
•Long ID (250-300mm) – higher resolution, long run times
•Narrow ID ( 2.1mm)- high detector sensitivity
•Wide ID ( 10-22 mm)- high sample loading

• Particle Size
•Smaller particles offer higher efficiency, but also cause
higher backpressure.
•Choose 3µm particles to resolve complex, multi-component
samples.

• Pore Size
•Larger pores allow larger solute molecules to be retained
longer through maximum exposure to the surface area of
the particles.
•Choose a pore size of 150Å or less for sample MW  2000.
•Choose a pore size of 300Å or greater for sample MW >
2000.

• Carbon Load
•Higher carbon loads generally offer greater resolution and
longer run times.
•Low carbon loads shorten run times and many show a
different selectivity.

• Column Selection
• To get a separation you must have round about interaction
with the stationary phase.
• Many carbons: choose stationary phase with carbon
Compound: Hydrophobic mode of interaction.
• Acids & bases can be difficult to separate.
• The “neutral” form is usually retained more on a reverse
phase(C18).
• “ionic” form is not retained as much.

• Phenolic phases can be useful when separating
aromatic, polycyclic & unsaturated species.
• Mode of interaction: - interaction between the
electron rich double bonds within the analyte &
stationary phase phenyl moieties.
• NH2 & CN Phases are suggested for separating polar
organic molecules.
• Column Selection

• Column behavior at high pH
• The pH of the mobile phase also affects the stability &
lifetime of a silica based column.
• Neutral/ Basic pH: mechanism of degradation is
dissolution.
• This will be affected by:
1. The type of bonding
2. The type of silica.
3. Mobile phase parameters like buffer strength, organic
composition & operating temperature.

• Acid hydrolysis of the bonded phase from the silica
surface
• Changes in solute retention overtime.
• Increase in rate of hydrolysis with decreasing pH.
• The buffer strength & organic modifier have less of an
effect at low pH than at high pH.
• Column behavior at low pH

• Detector Selection
Detector selection is based on:
• Chemical nature of analytes
• Potential interferences.
Detector’s response to all compounds in the mixture.
• Limit of detection required.
• Availability & cost of detector.

• Detector Options

• Detector Requirements

QUESTIONS ?

• Contacts
• Suchitra Ravan: 9860138162
Sales.mumbai@adhocscientific.com
info@adhocscientific.com

Thank You

Analytical HPLC method development .pdf

Recommended

Recommended

More Related Content

Similar to Analytical HPLC method development .pdf

Similar to Analytical HPLC method development .pdf (20)

Recently uploaded

Recently uploaded (20)

Analytical HPLC method development .pdf