HPLC By S Mhaske & Dr S Nayak

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AN OPERATIONAL OVERVIEW HPLC INSTRUMENTATION & METHODOLOGY Mr.Sushil Mhaske Assistant Professor Bansal College of Pharmacy, Bhopal. www.bansalpharmacy.com Dr. S. Nayak Principal

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H P igh erformance L iquid C hromatography Bansal College of Pharmacy, Bhopal

Methods in Chromatography :

Methods in Chromatography Adsorption Chromatography ♣ Normal Phase chromatography ♣ Reversed Phase chromatography ♣ Ion Exchange chromatography ♣ Affinity chromatography ♣ Hydrophobic chromatography Bansal College of Pharmacy, Bhopal.

Methods in Chromatography:

Methods in Chromatography Partition Chromatography ♣ Gas chromatography ♣ Liquid Liquid Partition chromatography Size Exclusion Chromatography ♣ Gel Permeation chromatography ♣ Gel chromatography ♣ Gel Filtration Bansal College of Pharmacy, Bhopal.

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Bansal College of Pharmacy, Bhopal. Components of HPLC Mobile Phase Gradient Controller Pump Injector Column Detector

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LOAD The Sample Loop Inject Move the sample loop into the mobile phase flow Bansal College of Pharmacy, Bhopal.

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Bansal College of Pharmacy, Bhopal. Components of HPLC

Elution Techniques:

Elution Techniques Isocratic Gradient Bansal College of Pharmacy, Bhopal. HPLC – Gradient Curves

Isocratic :

Isocratic Same mobile phase throughout Bansal College of Pharmacy, Bhopal.

Gradient:

Gradient lower polarity/elution strength followed by gradually increasing the polarity/elution strength. Bansal College of Pharmacy, Bhopal.

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Bansal College of Pharmacy, Bhopal. Selection of Column & Mode in Chromatography Solubility & Molecular Weight

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Characteristics of Exploratory HPLC Method Method Column Mobile Phase Remarks Reversed Phase C-8, C-18, Phenyl, Cyano Mixture of aqueous organic phase Suitable for neutral or non Ionized compounds soluble in water/Methanol/ ACN Normal Phase Cyano, Diol, Amino, Silica Mixture of organic solvents Lipophilic compounds, insoluble in water Ion Pair C-8, C-18, Cyano Like RP, except addition of buffers to control pH Suitable for ionic or ionizable compounds Bansal College of Pharmacy, Bhopal Beginners start with 15 or 25 cm, 5 μ m

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HPLC Classification based on type and scale Micro LC 0.3-1 mm ID Semi-micro 1-3 mm ID Conventional 4-8 mm ID Semi-Preparative 10-20 mm ID Preparative 20-50 mm ID Process >50 mm Bansal College of Pharmacy, Bhopal.

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Column Length and Mechanical Separating Power [Same Particle Size] Particle Size and Mechanical Separating Power [Same Column Length ] Separation Performance – Resolution Bansal College of Pharmacy, Bhopal

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Bansal College of Pharmacy, Bhopal. Data Processing Integration Digital chromatography raw data peak data Calibration Establishing a calibration curve of the specified analyte from a set of injected calibration standard solutions Report sample reports entire sample and method information, Sample peak result table, chromatogram, additional data such as spectral info from the PDA summary reports sums, averages, and RSD of specific analytes

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Calculation Methods for Quantitation Bansal College of Pharmacy, Bhopal. Normalised Peak area % Area= Area of Peak * 100 / Total area of Peaks External Standard Calibration RF = Standard Peak(Area or Height) / Concentration of Sample Sample Concentration = Sample Peak (Area or Height) / RF

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Internal Standard (IS) Calculation Method Rs = Area of Drug / Area of IS Slope of Rs Vs Concentration of pure drug is response factor Standard Addition (Spiking) Method Negative intercept on X-axis= Amount of sample Calculation Methods for Quantitation: Bansal College of Pharmacy, Bhopal.

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Bansal College of Pharmacy, Bhopal. Applications Identification Quantitation Purification Trace Analysis

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Waters Binary Gradient HPLC system with PDA detector 515 HPLC pump programmable flow rate, max. pressure 6000 psi Rheodyne 77251 600 Gradient Mixer Pump Control Module M-2998 Photodiode Array Detector Prealigned D lamp, Digital & 3D Mode Empower 2 software C-18 column 5U, 4.6mm*250mm Specifications of HPLC Bansal College of Pharmacy, Bhopal.

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T h a n k Y o u Bansal College of Pharmacy, Bhopal. www.bansalpharmacy.com Mr.Sushil Mhaske Assistant Professor Dr. S. Nayak Principal

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