HPLC 1.Introduction

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INSTUMENTAL ANALYSIS PRACTICAL 213 PHC

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HPLC

What is HPLC ?

H P L C : High : Performance (Pressure) : Liquid : Chromatography

What is HPLC used for ?

1. Separation of mixed components 2. Qualitative analysis / Quantitative analysis 3. Preparation of interest components

HPLC Basic Instrumentation

Solvent Delivery Injector Solvent Reservoirs Pump Sample Injection Column Separation Detector Data Processor

Separation and Analysis

A C B A A B C C C Separation A B C A B C A C C

Qualitative analysis What are components A, B and C ?

Quantitative analysis What is the concentration of components A, B and C ?

Separation and Analysis

DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY

Solvent(mobile or moving phase)

A + B + C

Sample (A+B+C) OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO Column OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO Solid Particles (packing material- OOOOOOOOOO OOOOOOOOOOO stationary phase) OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO Eluant (eluate)

2. Parameters used in HPLC

2. Parameters used in HPLC 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 t R t R t 0 t 0 k ’ = t R - t t 0 0

2. Parameters used in HPLC Retention parameters

2. Parameters used in HPLC Column efficiency

The degree of band broadening (width of the peak).

It is measured in number of theoretical plates: t R W N = 16 ( t R / W ) 2 The height of the theoretical plate H is given by: H = L / N L : Column length

2. Parameters used in HPLC Degree of separation

t R1 k ’ 1 t R2 W 1 W 2 k ’ 2 Selectivity factor: which describes the separation of two species (A and B) on the column Separation factor : = k ’ B k’ A  =

t R2

–

t 0

/

t R1

–

t 0

2. Parameters used in HPLC Degree of separation

Resolution : R s = 2 X (t R2 – t R1) W 1 + W 2

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 ) h x 0.05

h W0.05

S = W 0.05

2f f W 0.05

S = 1 : The peak is completely symmetric.

S > 1 : Tailing S < 1 : fronting

2. Parameters used in HPLC Peak symmetry

2. Parameters used in HPLC Practice Exercise - A chromatogram of a mixture of species A,B,C,D provided the following data:

Non retained A B C D

Retention time (min)

3.1

5.4

13.3

14.1

21.6

Width of peak base

------- 0.41

1.07 1.16

1.72

Calculate:

1-The Capacity factor for A ?

2-The Resolution for C and D ?

2. Parameters used in HPLC Practice Exercise - calculate the resolution and selectivity (



) of the following peaks:

Mobile phase A B 0.34

1.67

1.98

W -- 0.16

0.14

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.