Chromatography & instrumentation in Organic Chemistry

What is Chromatography?

Chromatography is a technique for separating mixtures into their

components in order to analyze, identify, purify, and/or quantify the

mixture or components.

Types of Chromatography

TYPE STATIONARY

PHASE

MOBILE PHASE

Paper solid (filter paper) Liquid

Thin Layer

Chromatography (tlc)

solid (silica) Liquid

Column solid (silica) Liquid

High Pressure Liquid

Chromatography (hplc)

solid (silica) Liquid

Gas Liquid

Chromatography (glc)

solid or liquid gas

Uses for Chromatography

(a) Analyse – examine a mixture, its components, and their

relations to one another

(b) Identify – determine the identity of a mixture or components

based on known components

(c) Purify – separate components in order to isolate one of interest

for further study

(d) Quantify – determine the amount of the a mixture and/or the

components present in the sample

Uses for Chromatography

(a) Pharmaceutical Company – determine amount of each chemical

found in new product

(b) Hospital – detect blood or alcohol levels in a patient’s blood

stream

(c) Law Enforcement – to compare a sample found at a crime scene

to samples from suspects

(d) Environmental Agency – determine the level of pollutants in the

water supply

(e) Manufacturing Plant – to purify a chemical needed to make a

product

Definition of Chromatography

Detailed Definition:

Chromatography is a laboratory technique that separates components

within a mixture by using the differential affinities of the components for

a mobile medium and for a stationary adsorbing medium through which

they pass.

Terminology:

• Differential – showing a difference, distinctive

• Affinity – natural attraction or force between things

• Mobile Medium – gas or liquid that carries the components

(mobile phase)

• Stationary Medium – the part of the apparatus that does not

move with the sample (stationary phase)

Definition of Chromatography

Simplified Definition:

Chromatography separates the components of a mixture by their

distinctive attraction to the mobile phase and the stationary phase.

Explanation:

• Compound is placed on stationary phase (usually paper or silica)

• Mobile phase (suitable solvent) passes through the stationary

phase

• Mobile phase solubilises the components

• Mobile phase carries the individual components a certain distance

through the stationary phase, depending on their attraction to

both of the phases

Illustration of Paper Chromatography

Rf value

Under similar conditions, a component should always travel at the same

speed.

Its identity can be found by comparing the distance it moves relative to

the solvent.

Comparison can be a problem if components have similar Rf values

The unknown substance is new and there is no previous chemical to

compare it with

Types of Chromatography

(a) Paper Chromatography – separates dried liquid samples with a

liquid solvent (mobile phase) and a paper strip (stationary phase)

(b) Thin-Layer Chromatography – separates dried liquid samples with

a liquid solvent (mobile phase) and a glass plate covered with a

thin layer of alumina or silica gel (stationary phase)

(c) Column Liquid Chromatography – separates liquid samples with a

liquid solvent (mobile phase) and a column composed of solid

beads (stationary phase)

(d) Gas Chromatography (GLC & HPLC) – separates vaporized

samples with a carrier gas (mobile phase) and a column

composed of a Liquid or of solid beads (stationary phase)

Paper Chromatography

Stationary phase

Paper (Filter or Chromatography)

Mobile phase

Suitable organic solvent (water /Alcohol)

Thin Layer Chromatography

Here the mobile phase is a liquid

Flowing past a thin layer of powder on a solid support (silica).

Substances that are less attracted to the solid or are more soluble in the

liquid move faster.

And so move further up the plate by the time that the process has been

stopped by taking the plate out of the liqiud. - larger Rf

The surface of the plate consists of a very thin layer of silica on a plastic

or aluminum backing. The silica is very polar. This is the stationary

phase. Spot the material at the origin (bottom) of the TLC plate.

Place the plate into a glass jar with a small amount of a solvent in the

glass jar. This solvent acts as the moving phase.

Remove the plate from the bottle when the solvent is close to the top of

the plate.

Non-polar compounds will be less strongly attracted to the plate and will

spend more time in the moving phase. This compound will move faster

and will appear closer to the top of the plate.

Polar compounds will be more strongly attracted to the plate and will

spend less time in the moving phase and appear lower on the plate.

Be sure to remove the TLC plate when it appears that the solvent front

isn’t moving!

Reason: the solvent is evaporating as it moves up the plate.

Results: If you don’t remove the plate all of the spots will appear near

the top of the plate!!!!! This isn’t a pretty sight and makes it difficult to

get good Rf values!

Visualization Method

Some plates shows colored spots. Most of the time, the spots won’t show

unless they are visualized! Vizualization is a method that is used to

render the TLC spots visible.

A visualization method can be:

(a) Ultraviolet light

(b) Iodine vapors to stain spots

(c) Colored reagents to stain spots

(d) Reagents that selectively stain spots while leaving others

unaffected.

Thin-Layer Chromatography: Determination of Rf Values

Column Chromatography

Solid Phase extraction column

Vary in size and polarity

There are two ways to run a column:

1. Allow gravity to draw samples and buffers through the column resin.

2. Use pumps to push a sample and buffers through a column.

Stationary phase

silica

Mobile phase

suitable organic solvent

Separation

components interact with the stationary phase to different extents

Method

(a) a chromatography column is filled with solvent

and silica

(b) drops of the mixture are placed on top of the

silica – A

(c) the tap is opened to allow the solvent to flow

out

(d) additional solvent is added on top to replace

that leaving

(e) components travel through at different rates and

separate – B

(f) batches of solvent are collected at intervals – C

(g) the solvent in each batch is evaporated to obtain

components

Gas Liquid Chromatography

Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing

through a tube.

And the stationary phase is an non-volatile liquid held on particles of a

solid support.

Suitable for separating volatile mixtures

In practice the Column is contained in a thermostatic oven. (Why ?)

About 1μL of liquid is injected into one end of the column.

As each component reaches the other end it is detected and registered

on a chart recorder.

The Retention Time is characteristic of a particular substance. (for the

same column, temperature, gas flow etc.)

The area under each peak indicates the relative quantities.

Uses of Gas Chromatogephy

(a) Drug Testing

(b) Blood Alcohol Tests

(c) Used in conjunction with a Mass Spectrometer

(d) GLC separates and the MS analyses each part separately

High Performance Liquid Chromatography

HPLC is suitable for the separation of the components of non volatile

mixtures

Mobile phase is a solvent and the stationary phase is a fine silica powder

contained in a slim column

The solvent has to be pushed through the silica using high pressure

Separation is based on the retention of the mixture components by the

silica based column

A better form of column chromatography. Instead of draining down

through the stationary phase, the solvent is forced through under high

pressure.

Stationary phase

silica

Mobile phase

suitable solvent

Separation

similar to column chromatography

Method

1. A sample is injected

2. Solvent and sample are pushed through under pressure

3. Different compounds have different retention times

4. Output can be detected by compounds absorbing UV

5. Can be connected to a mass spectrometer

Uses

Used where mixtures are less volatile and so cannot be separated using

GC

Used to analyse vitamins in food and growth promoters in meat

Spectroscopy

Spectroscopy is analysis of the interaction between electromagnetic

radiation and matter.

Different types of radiation interact in characteristic ways with different

samples of matter

The interaction is often unique and serves as a diagnostic "fingerprint" for

the presence of a particular material in a sample

Spectroscopy is also a sensitive quantitative technique that can

determine trace concentrations of substances.

Mass Spectrometry

Used to calculate the relative atomic mass of an element

Separates positive ions according to their mass to charge ratio

Lighter ions are deflected more by a magnetic field

Uses

Along with measuring relative atomic mass of elements

It measures the relative molecular mass of substances

Can be used to identify unknown components of substances

Molecules are ionised and assessed according to their relative masses

with different molecules having different mass spectra

Used to assess pollutants

Ultra Violet Spectrometry

Involves the absorption of Ultra violet radiation

Used to identify the amount of substance in a sample (quantitative)

A sample and blank is placed between a detector and a UV source and

the amount of light absorbed by the solution is detected

A spectra is obtained which is unique to each element

The technique is quantitative and is used in drug analysis

Infra Red Absorption Spectometry

Each organic compound has a unique infra red absorption spectra

IR is used to detect organic compounds (plastics and drugs)

The detector measures light intensity following passage through a sample

Atomic Absorption Spectrometry

Colorimetry

A colorimeter used to test the concentration of a solution by measuring

its absorbance of a specific wavelength of light.

Different solutions are made, and a control (a mix of distilled water and

another solution) is filled into a cuvette and placed inside a colorimeter to

calibrate the machine. After calibration you find the densities and/or

concentrations of other solutions.