Analytical HPLC method development .pdf

Pharma industry

1. ANALYTICAL HPLCMETHOD DEVELOPMENT
Suchitra Ravan
Ad Hoc Scientific-
A company with a purpose
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2. Overviewof HPLC Method Development Strategy
•Development approach
•Separation goal
•Nature of sample
•Sample pre treatment
•Sample detection
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3. •Development approach
•Theoretical
•Empirical
•Theoretical Vs. Empirical
Thinking Experience
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4. •Resolution
•Peak tailing
•Plate Counts
•Retention Time
•Run time
•Relative Standard deviation
•Separation Goal
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5. •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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6. •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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7. •Sample Detection
•Chromophoric –UV
•Non chromophoric-Refractive index
Evaporative light scattering
Fluorescence
•Characteristics of Universal detectors
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8. •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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9. •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
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10. •Solvent Selectivity
•Solvent Selectivity triangle
Basic
Acidic Dipolar
•Solvent strength
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11. •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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12. •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 pKaof the
compound
3.Extreme cases (8-10.5)
•Harshness will compromise column lifetimes.
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13. •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.
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14. •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
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17. •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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18. •Introduction
Silica is heart of HPLC
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19. •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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20. •Stationary phases
•C18
•C8
•C3,C4
•Phenyl
•Amino(NH2)
•Cyano (CN)
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21. •Column Length
•Short (30-50mm) -short run times, low backpressure
•Long (250-300mm) -higher resolution, long
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22. •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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23. •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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24. •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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25. •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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26. •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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27. •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
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28. •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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29. •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
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30. •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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31. •Detector Options
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32. •Detector Requirements
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33. QUESTIONS ?
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34. •Contacts
•Suchitra Ravan: 9860138162
[email protected]
[email protected]
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35. Thank You
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