Scribd
Upload
78 views

Method Development Presentation

This document discusses strategies for developing analytical HPLC methods. It covers theoretical and empirical development approaches, separation goals, sample properties like structure and solubility, sample pretreatment needs, detection methods, HPLC modes, effects of mobile phase composition, column selection factors, and detector selection criteria. The key aspects of HPLC method development include understanding the sample's nature and goals of analysis to optimize separation and detection.

Uploaded by

hassaan haider
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
78 views

Method Development Presentation

This document discusses strategies for developing analytical HPLC methods. It covers theoretical and empirical development approaches, separation goals, sample properties like structure and solubility, sample pretreatment needs, detection methods, HPLC modes, effects of mobile phase composition, column selection factors, and detector selection criteria. The key aspects of HPLC method development include understanding the sample's nature and goals of analysis to optimize separation and detection.

Uploaded by

hassaan haider
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 35

ANALYTICAL HPLC METHOD DEVELOPMENT

Ad Hoc Scientific-
A company with a purpose

This presentation is a property of Ad Hoc Scientific Pvt Ltd Suchitra Ravan


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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• Development approach

• Theoretical
• Empirical
• Theoretical Vs. Empirical

Thinking Experience

This presentation is a property of Ad Hoc Scientific Pvt Ltd


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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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?

This presentation is a property of Ad Hoc Scientific Pvt Ltd


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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• Sample Detection

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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• Solvent Selectivity
• Solvent Selectivity triangle
Basic

Acidic Dipolar
• Solvent strength

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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.
This presentation is a property of Ad Hoc Scientific Pvt Ltd
• 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:

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


This presentation is a property of Ad Hoc Scientific Pvt Ltd
This presentation is a property of Ad Hoc Scientific Pvt Ltd
• Selection of HPLC Columns
• Introduction
• Type of Silica
• Stationary phases
• Column length
• Column diameter
• Particle Size
• Pore Size
• Surface area
• Carbon load

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• Introduction

Silica is heart of HPLC

This presentation is a property of Ad Hoc Scientific Pvt Ltd


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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• Column Length

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


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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


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

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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.

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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.

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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.

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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.

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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.

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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.

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• 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.

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• Detector Options

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• Detector Requirements

This presentation is a property of Ad Hoc Scientific Pvt Ltd


QUESTIONS ?

This presentation is a property of Ad Hoc Scientific Pvt Ltd


• Contacts

• Suchitra Ravan: 9860138162


[email protected]
[email protected]

This presentation is a property of Ad Hoc Scientific Pvt Ltd


Thank You

This presentation is a property of Ad Hoc Scientific Pvt Ltd

You might also like