CHROMATOGRAPHY (PART 3)

High Performance Liquid Chromatography (HPLC)

- One of the most commonly used chromatographic technique to determine drugs in pharmaceutical preparations and biological materials.
- A small volume of liquid analyte is injected into a column packed with porous particles (stationary phase).
- The mobile phase is a liquid, forced through a column by high pressure through a delivery pump.
- The outlet of the column is connected to a detector where the eluted substances are detected.

Solvent Reservoir
Pump
Syringe
Column
Detector
Computer System

Components of HPLC SET-UP

Solvent Reservoir

Components of HPLC SET-UP
- Usually a glass bottle that holds the mobile phase (the solvent used in the chromatography process).
- This is connected to the delivery pump.

Delivery Pump

Components of HPLC SET-UP
Pumps the mobile phase through the column at high pressure.

Syringe

Components of HPLC SET-UP
- Is used to transfer the analyte (the substance to be analyzed) into the HPLC system.
- It is used to inject the analyte into the column.

Column

Components of HPLC SET-UP
- Typically made of metal (sometimes glass is used).
- Contains the stationary phase, which is usually made of porous particles like:
- Silica gel
- Silica alumina
- Sometimes cellulose-based stationary phases.

Detector

Components of HPLC SET-UP
- Identifies which substances are eluted (separated) from the column, and in which order (first, second, third, etc.).
- It detects the presence of substances based on the compound's interaction with the stationary phase.

Computer System

Components of HPLC SET-UP
- Collects and records the data detected by the detector.
- This data can be used for analysis and interpretation of the sample’s composition.

0.5–2.0 ml/min

In a standard analytical HPLC system the typical flow rate of the mobile phase through the column is?

Stainless Steel or Glass

High Performance Liquid Chromatography (HPLC)
The column commonly used is?

Pre- Column
Analytical Column

High Performance Liquid Chromatography (HPLC)
The column is made up of two parts:

Pre- Column

High Performance Liquid Chromatography (HPLC)
The column is made up of two parts:
Helps prepare the sample before it enters the main analytical column.

Analytical Column

High Performance Liquid Chromatography (HPLC)
The column is made up of two parts:
This is where the actual separation process takes place.

Gas Chromatography

- A separation technique used for the analysis of volatile substances or substances that can be made volatile.
- The mobile phase is a carrier gas under pressure flowing through the heated column containing the stationary phase.
- The stationary phase is placed on the inner surface of the column or coated onto particles packed into the column.

Sample Injector
Carrier Gas
Column
Column Oven
Detectors

COMPONENT OF GS SET UP

Sample Injector

COMPONENT OF GS SET UP
Necessary for introducing the sample at the head of the column.

Carrier Gas

COMPONENT OF GS SET UP
Acts as the mobile phase. Must be dry, free of oxygen and chemically inert mobile phase.

Helium
Hydrogen

Example of Carrier Gas

Column

COMPONENT OF GS SET UP
- Heart of the system, where the separation of sample components takes place.
- The stationary phase is placed on the inner surface or coated onto particles packed into the this.

Wall Coated Open Tubular (WCOT)
Porous Layer Open Tubular (PLOT)
Surface Coated Open Tubular (SCOT)

Three types of capillary columns are commonly used in gas chromatography:

Wall Coated Open Tubular (WCOT)

Three types of capillary columns are commonly used in gas chromatography:
- Internal wall of capillary column is coated with a very fine film of liquid stationary phase.

Porous Layer Open Tubular (PLOT)

Three types of capillary columns are commonly used in gas chromatography:
The inner surface of the walls of capillary columns is coated with a layer of porous solid material.

Surface Coated Open Tubular (SCOT)

Three types of capillary columns are commonly used in gas chromatography:
Capillary tube wall is lined with a thin layer of porous solid material on to which liquid phase is adsorbed.

- It is safer compared to hydrogen.
- It is comparable to hydrogen in terms of efficiency.
- It has a larger range of flow rates and is compatible with most detectors used in GC.

Helium is the most commonly used carrier gas because:

Surface Coated Open Tubular (SCOT)

Is more efficient than WCOT columns due to the increased surface area of the stationary phase coating inside the column walls.

Column Oven

COMPONENT OF GS SET UP
Serves to control the temperature of the column.

Isothermal Programming
Temperature Programming

GAS CHROMATOGRAPHY
The oven can be operated in two manners:

Isothermal Programming

GAS CHROMATOGRAPHY
The oven can be operated in two manners:
The temperature of the column is held constant throughout the entire separation.

Temperature Programming

GAS CHROMATOGRAPHY
The oven can be operated in two manners:
The column temperature is either increased continuously or in steps as the separation progresses.

Detectors

COMPONENT OF GS SET UP
Device located at the end of the column which provides a quantitative measurement of the components of the mixture as they elute in combination with the carrier gas

Flame Ionization Detector (FID)
Mass Spectrometer

Commonly used Detectors in Gas Chromatography

Order of Elution
Retention Time
Relative Amount

GC CHROMATOGRAM CONSISTS OF:

Order or Elution

GC CHROMATOGRAM CONSISTS OF:
- Order of components coming off the column related too boiling points and polarities of the substances in the mixture