HPLC 1. Introduction 1.Introduction  INSTUMENTAL ANALYSIS  PRACTICAL 213 PHC  HPLC

1. Introduction H: High P : Performance (Pressure) L : Liquid C : Chromatography What is HPLC ?

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

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

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

Separation and Analysis 1. Introduction

2. Parameters used in HPLC

Parameters used in HPLC 2. Parameters used in HPLC Retention parameters Column efficiency parameters parameters 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

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 (2-10)

2. Parameters used in HPLC Retention parameters

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:

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

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

2. Parameters used in HPLC Resolution :

2. Parameters used in HPLC Resolution :

2. Parameters used in HPLC Peak symmetry S : Symmetry factor ( T : Tailing factor ) S = 2f W 0.05 f h x 0.05 h S = 1 : The peak is completely symmetric. S > 1 : Tailing S < 1 : fronting W0.05

2. Parameters used in HPLC Peak symmetry

- A chromatogram of a mixture of species A,B,C,D provided the following data: Calculate: 1-The Capacity factor for A ? 2-The Resolution for C and D ? 2. Parameters used in HPLC Practice Exercise Retention time (min) Width of peak base Non retained A B C D

2. Parameters used in HPLC Practice Exercise W Mobile phase A B calculate the resolution and selectivity (  ) of the following peaks:

2. Parameters used in HPLC Practice Exercise On a given 200cm column the sample required 4.80 min to emerge, and the width of the peak at the base is 12.0 cm : - Calculate N and H.