ANALYTICAL HPLC METHOD DEVELOPMENT

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

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 • Development approach

• Theoretical
• Empirical
• Theoretical Vs. Empirical

Thinking Experience

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

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 • 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?

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

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 • Sample Detection

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

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

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 • HPLC method development effect of mobile phase in
Reverse Phase HPLC

• Solvent Selectivity
• Change in organic solvent
• Change in pH
• Change in buffer
• Buffer capacity

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 • Solvent Selectivity
• Solvent Selectivity triangle
Basic

Acidic Dipolar
• Solvent strength

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 • 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

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 • 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.
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 • Impact of pH on Acidic & Basic analyte.
• 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:

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 • 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 220
(hydroxymethyl)
aminomethane
8.2-10.2 Borate 210

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

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• Introduction

Silica is heart of HPLC

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

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

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 • Column Length

• Short (30-50mm) - short run times, low backpressure

• Long (250-300mm) - higher resolution, long

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 • 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

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

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 • 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.

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 • 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.

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 • 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.

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 • Column Selection

• 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.

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 • 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.

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• Column behavior at low pH
• 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.

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 • 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.

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• Detector Options

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• Detector Requirements

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QUESTIONS ?

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 • Contacts

• Suchitra Ravan: 9860138162

Sales.mumbai@adhocscientific.com
info@adhocscientific.com

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Thank You

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