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HPLC 1. Introduction 1.Introduction CHROMATOGRAPHY Chromatography basically involves the separation of mixtures due to differences in the distribution.

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Presentation on theme: "HPLC 1. Introduction 1.Introduction CHROMATOGRAPHY Chromatography basically involves the separation of mixtures due to differences in the distribution."— Presentation transcript:

1 HPLC 1. Introduction 1.Introduction CHROMATOGRAPHY Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase. Mobile phase: Provides the analyte transport Stationary phase: Immobile phase Analyte : Mixture of components dispersed in the mobile phase

2 HPLC 1. Introduction Chromatography Stationary phase Solid (adsorptio) Liquid (partition) Mobile phase Gas (GC) Liqiud (LC) 1.Introduction

3 H: High P : Performance (Pressure) L : Liquid C : Chromatography GC : Gas chromatography TLC: Thin layer chromatography IC : Ion chromatography What is HPLC ?

4 What is HPLC used for ? 1. Introduction 1. Separation of mixed components 2. Qualitative analysis / Quantitative analysis 3. Preparation of interest components Separation analysis and/or preparation of interest components

5 HPLC Basic Instrumentation Solvent Reservoirs Pump Solvent Delivery Injector Sample Injection Column Separation Detector Data Processor 1. Introduction

6 B A C A A B C C C Separation CCCC BB AAA Separation and Analysis Qualitative analysis What are components A, B and C ? Quantitative analysis What is the concentration of components A, B and C ? 1. Introduction

7 Separation and Analysis

8 A B C Identification Component A elutes the same time as a caffeine peak. Component A is identified as caffeine. What is component A? Caffeine Sample 1. Introduction

9 Determination Peak area (or height) is proportional to the concentration (or amount) of the component. The concentration of component A(caffeine) is determined by comparing the peak area with that of the standard caffeine peak. What is the concentration of component A? A B C Caffeine (1mg/ml) 5ul injection (5ug) 1. Introduction

10 Separation Mechanism Separation is determined by column (packing material) and mobile phase (solvent). A B C time Mobile phase elutes components. Packing materials retain components in the column. CBA Column Packing material ↓ ↓ ↓ ↓ Mobile phase (solvent) C > B > A 1. Introduction

11 modes in HPLC LC ModePacking materials Mobile phase Normal phase chromatography e.g. silica gel N-Decane – N-hexane  Polar stationary phase  Non- polar mobile Reversed phase chromatography Silica-C18(ODS) Water- Methanol  Non-polar stationary phase  Polar mobile phase 1. Introduction

12 HPLC Instrumentation provide a continuous constant flow of the eluent through the HPLC injector, column, and detector Flow rate range: from 0.01 to 10 ml/min Pressure range: from 1 to 5,000 psi Free from puls Use inert gas 1.PUMP

13 2. Parameters used in HPLC The JASCO advanced technology team has again met the challenge and designed a new line of HPLC instruments, The LC-1500series more than satisfies in response to the growing demand for greatly expanded HPLC analyses in the fields of not only biochemistry, pharmaceutical and medical science, but also in the areas of among other organic and inorganic compounds, foods, agricultural sciences, polymeric and natural substances and pollution. The LC-1500 series comprises pumps, detectors, autosamplers, its own column oven and other units each having built-in intelligence and incorporating many features with much higher levels of operability and reliability in addition to multiple functions, higher performance and higher accuracy than before, making them the most advanced instruments available.

14 Parameters used in HPLC 2. Parameters used in HPLC Retention parameters Column efficiency parameters Condition for Separation Retention : When a component in a sample interacts with the stationary phase in the column and a delay in elution occurs. Column efficiency : Goodness of a column

15 2. Parameters used in HPLC Retention parameters t R : retention time (The time between sample injection and an analyte peak reaching a detector at the end of the column) k’ : capacity factor ( is often used to describe the migration rate of an analyte on a column) t 0 : the time required for the component not retained by the column to pass through the column tRtR t R - t 0 t0t0 k’ = t R - t 0 t0t0

16 2. Parameters used in HPLC Retention parameters

17 The degree of band broadening (width of the peak). It is measured in number of theoretical plates: 2. Parameters used in HPLC Column efficiency tRtR W N = 16 ( t R / W ) 2 H = L / N L : Column length The height of the theoretical plate H is given by:

18 2. Parameters used in HPLC Degree of separation t R1 t R2 k’ 1 k’ 2 W1W1 W2W2 Selectivity factor: which describes the separation of two species (A and B) on the column Separation factor : = k’ A k’ B  = t R2 – t 0 / t R1 – t 0

19 2. Parameters used in HPLC Degree of separation R s = W 1 + W 2 2 X (t R2 – t R1) Resolution : R= 1.5

20 2. Parameters used in HPLC Resolution :

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