ANALYTICAL HPLC METHOD DEVELOPMENT
Ad Hoc Scientific-
A company with a purpose
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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
[email protected] [email protected] This presentation is a property of Ad Hoc Scientific Pvt Ltd
Thank You
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