WELCOME

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PRESENTED BY: AMEENA MEHABOOB Ist YEAR MPHARMSt. JOSEPH COLLEGE OF PHARMACYMETHOD DEVELOPMENT IN HPLC

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CONTENTS3INTRODUCTIONSTEP 1- SELECTION OF HPLC METHOD AND INITIAL CONDITIONSSTEP 2: SELECTION OF INITIAL CONDITIONSSTEP 3: SELECTIVITY OPTIMIZATIONSTEP 4: SYSTEM PARAMETER OPTIMIZATIONSTEP 5: VALIDATIONCONCLUSIONREFERENCES

INTRODUCTIONHPLC is High Performance Liquid Chromatography.

4ADVANTAGES

ANALYTICAL METHOD DEVELOPMENTSelecting method requirements Deciding instrumentation

5STEPS

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STEP 1- SELECTION OF HPLC METHOD AND INITIAL CONDITIONS7

(a) SAMPLE PREPARATION8

(b) TYPES OF CHROMATOGRAPHY9

(c) COLUMN SELECTION10

Packing material11

COLUMN DIMENSIONS12 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

(d) DETECTOR SELECTION13

(e) MOBILE PHASE SELECTION14 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.

STEP 2: SELECTION OF INITIAL CONDITIONS15This step determines the optimum conditions to adequately retain all analytes. No analyte has a capacity factor of less than 0.5 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 timeBinary system based on either acetonitrile/water or methanol/water should be used.

STEP 3: SELECTIVITY OPTIMIZATION16Aim: To achieve adequate selectivityMobile phase and stationary phase compositions are taken into accountTo select these, the nature of analytes must be consideredOnce the analyte types are identified, the relevant optimization parameters may be selected

STEP 4: SYSTEM PARAMETER OPTIMIZATION17Used to find the desired balance between resolution and analysis time.Parameters involved include column dimensions, column packing, particle size, flow rateParameters maybe changed without affecting capacity factors or selectivity

TYPES OF OPTIMIZATION18

STEP 5: STEP 5: VALIDATION19The objective of an analytical procedure is to demonstrate that it is suitable for its intended purpose

CONCLUSION20Best 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.

REFERENCES21Instrumental methods of chemical analysis, Gurdeep R Chatwal, Sham K Anand, Page no 2.624-2.639HPLC- Quantitative analysis of pharmaceutical formulations, PD Sethi, Page no 11-16

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