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

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

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

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

5. 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

6. Separation and Analysis 1. Introduction

7. 2. Parameters used in HPLC

8. 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

9. 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)

10. 2. Parameters used in HPLC Retention parameters

11. 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:

12. 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

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

14. 2. Parameters used in HPLC Resolution :

15. 2. Parameters used in HPLC Resolution :

16. 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

17. 2. Parameters used in HPLC Peak symmetry

18. - 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 3.1 -------- A 5.4 0.41 B 13.3 1.07 C 14.1 1.16 D 21.6 1.72

19. 2. Parameters used in HPLC Practice Exercise W Mobile phase0.34--- A1.670.16 B1.980.14 - calculate the resolution and selectivity (  ) of the following peaks:

20. 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.