Gas Chromatography and HPLC

By: Avipsita, Sid and Momina

Introduction

Gas Chromatography

High Performance Liquid Chromatography (HPLC)

Simply Put

Gas Chromatography

Carrier Gas

Injector Port

Column

Detector

Results

Click for more>>>

Back to Introduction

Injector Port

Back to Introduction

Back to Gas Chromatography

The process of gas chromatography involves the injection of tiny amount of the sample into the head of a

chromatography column, usually done by use of a micro syringe.

Carrier Gas

Back to Introduction

Back to Gas Chromatography

Suitable gases include:1. Nitrogen2. Helium3. Argon4. Carbon

dioxide

The sample that is put into the machine via the injector port is vaporised. The vaporised gaseous sample is carried through the column by the flow of an inert (carrier) gas, which acts as

the mobile phase.

The column contains liquid, which is in stationary phase- this liquid is absorbed onto the surface of a inert solid.

The different components of the sample separate as it passes through the column because they move at different rates.

Column

Back to Introduction

Back to Gas Chromatography

Simple Analogy: “Think of a swarm of bees and wasps moving over a field of flowers. The bees would tend to stop to pick up nectar from the flowers, but the wasps would not. Therefore, at the other side of the field the wasps would be detected first, and then the bees.”

Detector

There are many types of detector that can be used but they all identify the separate substances leaving the column.

Different detectors have different ranges of selectivity:1. A non selective detector- responds to all compounds except the carrier gas2. A selective detector- responds to a range of compounds with a common physical

or chemical property3. A specific detector – responds to a single chemical compound.4. Flame ionisation detector

Back to Introduction

Back to Gas Chromatography

Flame Ionisation detector

• The gases leaving the column are mixed with hydrogen and air, and burnt.

• Organic compounds burn in the flame producing ions and electrons that conduct electricity.

• A large electrical potential is applied at the burner tip and a collector electrode is located above the flame.

• The resulting current of the burning organic compound is measured.

Why use this type of method? Back to Introduction

Back to Gas Chromatography

Why use that type of method?

• It is robust• It is relatively easy to use • It can measure the mass of each substance identified.So what is the drawback?• It destroys the sample.

Back to Introduction

Back to Gas Chromatography

Results

• The results are displayed on a monitor or printout.

Back to Introduction

Back to Gas Chromatography

High Performance Liquid Chromatography (HPLC)

i. It involves having a column packed with a solid phase (such as alumina or calcium carbonate). A solution of the plant pigment is added to the top of the column.

ii. Solvent is added to the column to make the mobile phase. For instance, addition of a plant pigment solution

iii. Tap at the bottom is always open allowing liquid to continually flow out.

First you need to know what column chromatography is

Check this out: http://www.chem-ilp.net/labTechniques/ChromatographyAnimation.htm

So what is HPLC? Back to Introduction

What is HPLC?

There are two types: Back to Introduction

Instead of the solvent being allowed to drip through a column under gravity, it is forced through high pressure; making the process much faster. A very small particle size for the column packing material is used. This gives greater surface area for the interactions between stationary phase and the molecules flowing past; allowing better separation of the components.

HLPC is basically highly improved form of column chromatography.

The two types of HPLC:

Normal Phase HPLC

Reversed Phase HPLC

Back to Introduction

Normal Phase HPLC

Back to Introduction

Back to Types of HPLC

Column is filled with silica particles and the solvent is non-polar. So polar molecules passing through the column will

stick for longer to the polar silica than the non polar molecules. This means that the non-polar ones will pass

through the column more quickly.

Reversed Phase HPLC

- In this process the silica is modified to make it non polar by attaching a long hydrocarbon chain to its surface.

- A polar solvent, for example a mixture of water and ethanol, is used.- This means that there will be a strong attraction between the polar molecules

in the test sample. The attraction between the hydrocarbon chains attached to the silica (stationary phase) and the polar molecules in the solution is very weak. Polar molecules spend most of their time moving with the solvent; meaning that the polar molecules will travel through the column more quickly.

Back to Introduction

Back to Types of HPLC

What is Retention Time?

Retention Time

Back to Introduction

Identifying Substances

Different compounds have different times as retention times depend on: • The pressure used-as it affects the rate of flow• The nature of the stationary phase- what the material is and how big the particles are • The exact composition of the solvent• The temperature of the column

It is the time taken for a compound to travel through the column and reach the detector. Time is measured from the point at which the sample is injected to the point at which the display shows a maximum peak height for that compound.

Identifying Substances

Back to Introduction

UV absorption is one way as many organic compounds absorb UV light of various wavelengths. The amount of light absorbed will depend on the

amount of a particular compound that is passing through the light beam at a time.

There are many ways to identify the substance which is passed through the column

For example: