HPLC method development
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- 2. 2 PRESENTED BY: AMEENA MEHAB Ist YEAR MPHAR St. JOSEPH COLLEGE OF PHARM
- 3. CONTENTS 3 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 N HPLC is High Performance Liquid Chromatography. 4 Improved resolution Faster separation Improved accuracy, precision and sensitivity ADVANTAGES
- 5. ANALYTICAL METHOD DEVELOPMENT •Selecting method requirements •Deciding instrumentation 5 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 7
- 9. (b) TYPES OF CHROMATOGRAPHY 9 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 10 Interaction of components with packing material Properties of packing material Knowledge of sample
- 12. COLUMN DIMENSIONS 12 • 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 13 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 14 • 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 15 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- 15 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 16 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 17 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 18 • 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 19 The objective of an analytical procedure is to demonstrate that it is suitable for its intended purpose
- 20. CONCLUSION 20 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 21 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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