This document outlines the five main steps for developing an analytical HPLC method: 1) selecting the initial HPLC method and conditions, 2) selecting the initial chromatographic conditions, 3) optimizing selectivity, 4) optimizing system parameters, and 5) validating the method. Key aspects of each step are discussed, including selecting the type of chromatography, column, detector, and mobile phase based on the analytes. The goal is to develop a validated method that provides adequate resolution and selectivity within a desired analysis time.

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PRESENTED BY: AMEENA MEHAB
Ist YEAR MPHAR
St. JOSEPH COLLEGE OF PHARM

3. CONTENTS
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 INTRODUCTION
 STEP 1- SELECTION OF HPLC METHOD AND
INITIAL CONDITIONS
 STEP 2: SELECTION OF INITIAL CONDITIONS
 STEP 3: SELECTIVITY OPTIMIZATION
 STEP 4: SYSTEM PARAMETER OPTIMIZATION
 STEP 5: VALIDATION
 CONCLUSION
 REFERENCES

4. INTRODUCTIO
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HPLC is High Performance Liquid
Chromatography.
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Improved
resolution
Faster
separation
Improved
accuracy,
precision and
sensitivity
ADVANTAGES

5. ANALYTICAL METHOD
DEVELOPMENT
•Selecting method requirements
•Deciding instrumentation
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STEPS
Step 1
• Selection of HPLC method and initial system
Step 2
• Selection of optimum conditions
Step 3
• Selectivity optimization
Step 4
• System parameter optimization
Step 5
• Method validation

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7. STEP 1- SELECTION OF HPLC
METHOD AND INITIAL
CONDITIONS
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8. (a) SAMPLE PREPARATION
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9. (b) TYPES OF
CHROMATOGRAPHY
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Reverse
phase
• Majority of
samples
• Peptide and
small protein
analysis
• Reverse
phase ion
suppression-
for weak
acids and
bases
• Reverse
phase ion
pairing- for
strong acids
and bases
Normal phase
• Low polarity
analytes
• Medium
polarity
analytes
Ion exchange
• Inorganic
anion
analysis
• Cation
analysis
Size exclusion
• High
molecular
weight
compounds
Gradient
HPLC
• Complex
samples with
a large
number of
components
(20-30)

10. (c) COLUMN SELECTION
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Interaction of
components
with packing
material
Properties of
packing material
Knowledge
of sample

11. Packing material
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12. COLUMN DIMENSIONS
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• Short (30-50 mm) –short run times, low
backpressure
• Long (250-300mm) –higher resolution, long run times
• Narrow – higher detector sensitivity
• Wide (10-22mm) –high sample loading

13. (d) DETECTOR
SELECTION
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UV DETECTOR
• Detects only substances which absorb light in UV wavelength
range
• Detects all samples which contain chromophores
FLOURESCENCE DETECTOR
• Detects eluted solutes on basis of flourescence
• For trace analysis
ELECTRICAL CONDUCTIVITY DETECTOR
• Used with ion suppressor column
• To allow salts and buffers to be used in mobile phase without
affecting detector output
REFRACTIVE INDEX DETECTOR
• Least sensitive
• Only when other detectors are inappropriate
• Can handle concentration without overloading the detector

14. (e) MOBILE PHASE
SELECTION
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• Organic phase concentration required for mobile
phase can be estimated by gradient elution method.
•Elution strength of mobile phase depends upon its
polarity.
•Ionic samples can be separated if they are present in
undissociated form.
•If retention time is too long, increase in organic phase
concentration is required.
•If tailing or fronting occurs, the mobile phase is not
totally compatible with the solutes.

15. STEP 2: SELECTION OF INITIAL
CONDITIONS
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 This step determines the optimum conditions to
adequately retain all analytes.
o No analyte has a capacity factor of less than 0.5
o No analyte has a capacity factor of greater than 10-
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Determination of initial conditions-
 By performing 2 gradient runs differing in only the run
time
 Binary system based on either acetonitrile/water or
methanol/water should be used.

16. STEP 3: SELECTIVITY
OPTIMIZATION
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 Aim: To achieve adequate selectivity
 Mobile phase and stationary phase compositions
are taken into account
 To select these, the nature of analytes must be
considered
 Once the analyte types are identified, the relevant
optimization parameters may be selected

17. STEP 4: SYSTEM PARAMETER
OPTIMIZATION
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 Used to find the desired balance between
resolution and analysis time.
 Parameters involved include column dimensions,
column packing, particle size, flow rate
 Parameters maybe changed without affecting
capacity factors or selectivity

18. TYPES OF OPTIMIZATION
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• By change in initial mobile phase
composition and slope of gradient
according to chromatogram
obtained in the preliminary run
MANUALLY
• Experimental design
• Multi criteria decision making
USING
SOFTWARES

19. STEP 5: STEP 5: VALIDATION
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 The objective of an analytical procedure is to
demonstrate that it is suitable for its intended
purpose

20. CONCLUSION
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 Best column, best mobile phase, best detection
wavelength, efforts in their selection can make a
world of difference while developing HPLC method
for routine analysis.
 Determining the ideal combination of these factors
assure faster delivery of desired results- a validated
method for separation.

21. REFERENCES
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 Instrumental methods of chemical analysis,
Gurdeep R Chatwal, Sham K Anand, Page no
2.624-2.639
 HPLC- Quantitative analysis of pharmaceutical
formulations, PD Sethi, Page no 11-16

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