Top Banner
CAPE Chemistry - Unit 2 Lecture Notes   Separation Techniques 1 Copyright © 2016 Mark December  Chromatographic Methods of Separation Chromatography is the analytical technique used to separate mixtures of substances into their components. These components become partitioned (unequally distributed/divided) between two (2)  phases, the stationary phase and the mobile phase. The stationary phase is made of an absorbent material such as paper or gel such as silica gel whereas the mobile phase is a suitable liquid solvent, mixture of solvents or gas that moves across the stationary phase carrying the components of the mixture with it. The substances in the mixture travel at different rates depending on their solubility in the mobile phase and the different forces of adsorption, present in the stationary phase. Therefore chromatography can be either partition chromatography  or adsorption chromatography . In partition chromatography, the components of the mixture moves at different rates between the two phases and are divided. In adsorption chromatography, components form bonds of different strength with the stationary phase and are separated from the mixture. The more soluble a component is, the longer it will travel in the mobile phase. Some chromatographic methods include:  Paper Chromatography  Thin-layer Chromatography (TLC)  Column Chromatography  High-performance Liquid Chromatography (HPLC)  Gas-Liquid Chromatography (GLC) Paper Chromatography In paper chromatography, the stationary phase is a v ery uniform absorbent paper what has water absorbed unto it. The mobile phase is a suitable liquid solvent or mixture of solvents. Paper is made of cellulose which contain hydroxyl groups in its structure. Water molecules are able to form hydrogen bonds with these hydroxyl groups and adhere to the cellulose of the paper. The paper is suspended in a container with a shallow layer of a suitable solve nt or mixture of solvents in it as shown below. The mixture to be separated along with the suspected components in the mixture is spotted on a line drawn approximately 1-2 cm from the bottom the paper. This line is known as the base line.
7

Chromatographic Methods of Separation

Jul 06, 2018

Download

Documents

Yogi Ganeshram
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Page 1: Chromatographic Methods of Separation

8/17/2019 Chromatographic Methods of Separation

http://slidepdf.com/reader/full/chromatographic-methods-of-separation 1/7

CAPE Chemistry - Unit 2 Lecture Notes –  Separation Techniques

1 Copyright © 2016 Mark December  

Chromatographic Methods of Separation

Chromatography is the analytical technique used to separate mixtures of substances into their

components. These components become partitioned (unequally distributed/divided) between two (2)

 phases, the stationary phase and the mobile phase. The stationary phase is made of an absorbent material

such as paper or gel such as silica gel whereas the mobile phase is a suitable liquid solvent, mixture of

solvents or gas that moves across the stationary phase carrying the components of the mixture with it.

The substances in the mixture travel at different rates depending on their solubility in the mobile phase

and the different forces of adsorption, present in the stationary phase. Therefore chromatography can be

either partition chromatography  or adsorption chromatography. In partition chromatography, the

components of the mixture moves at different rates between the two phases and are divided. In adsorption

chromatography, components form bonds of different strength with the stationary phase and are separated

from the mixture. The more soluble a component is, the longer it will travel in the mobile phase. Some

chromatographic methods include:

 

Paper Chromatography  Thin-layer Chromatography (TLC)

  Column Chromatography

  High-performance Liquid Chromatography (HPLC)

  Gas-Liquid Chromatography (GLC)

Paper Chromatography

In paper chromatography, the stationary phase is a very uniform absorbent paper what has water absorbed

unto it. The mobile phase is a suitable liquid solvent or mixture of solvents. Paper is made of cellulose

which contain hydroxyl groups in its structure. Water molecules are able to form hydrogen bonds with

these hydroxyl groups and adhere to the cellulose of the paper. The paper is suspended in a container

with a shallow layer of a suitable solvent or mixture of solvents in it as shown below.

The mixture to be separated along with the suspected components in the mixture is spotted on a line

drawn approximately 1-2 cm from the bottom the paper. This line is known as the base line.

Page 2: Chromatographic Methods of Separation

8/17/2019 Chromatographic Methods of Separation

http://slidepdf.com/reader/full/chromatographic-methods-of-separation 2/7

CAPE Chemistry - Unit 2 Lecture Notes –  Separation Techniques

2 Copyright © 2016 Mark December  

Covering the container ensures that the atmosphere in the beaker is saturated with solvent vapour.

Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the

 paper. As the solvent rises up the paper, the components of the mixture that are soluble rises with the

solvent (mobile phase) as well and travels at different rates so the mixtures are separated into different

coloured spots. When the solvent stops moving, the solvent front forms and a chromatograph develops.

Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base

line. The distance travelled relative to the solvent is a constant for a particular compound as long as allother factors are held constant, that is the type of paper and the exact composition of the solvent. The

distance travelled relative to the solvent is called the R f  value. For each compound it can be worked out

using the formula:

Rf  =Distance travelled by compound

Distance travelled by solvent

Question: If one component of a mixture travelled 9.6 cm from the base line while the solvent hadtravelled 12.0 cm, what is the R f  value for that component?

Rf  =Distance travelled by compound

Distance travelled by solvent=

9.6 cm

12 cm= 0.8

In some cases, the separated components are colourless and thus a visualizing agent is needed. Thevisualizing agent functions to convert the colourless components into coloured spots. Amino acids may

Page 3: Chromatographic Methods of Separation

8/17/2019 Chromatographic Methods of Separation

http://slidepdf.com/reader/full/chromatographic-methods-of-separation 3/7

CAPE Chemistry - Unit 2 Lecture Notes –  Separation Techniques

3 Copyright © 2016 Mark December  

 be identified in this way. A small drop of a solution of the amino acid mixture is placed on the base line

of the paper, and similar spots of the known amino acids are placed alongside it. The paper is then stoodin a suitable solvent and left to develop. The position of the solvent front is marked in pencil and the

chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with

amino acids to give coloured compounds, mainly brown or purple. Paper chromatography is from of

 partition chromatography.

Thin-layer Chromatography

In thin-layer chromatography the stationary phase solid and is usually a thin, uniform layer of silica gel(silicon dioxide, SiO2) or alumina (aluminium oxide, Al2O3) coated onto a piece of glass, metal or rigid

 plastic. The mobile phase is a suitable liquid solvent or mixture of solvents. The procedure is similar to

 paper chromatography.

In the case of components of the mixture being colourless, the stationary phase is made of a substance

that fluoresces (glows) in UV light. When UV light is shun on the chromatogram, dark spots will beshown to indicate the position of the component on the chromatograph. In the case of amino acids, the

chromatogram is sprayed with a solution of ninhydrin, as in paper chromatography.

Page 4: Chromatographic Methods of Separation

8/17/2019 Chromatographic Methods of Separation

http://slidepdf.com/reader/full/chromatographic-methods-of-separation 4/7

CAPE Chemistry - Unit 2 Lecture Notes –  Separation Techniques

4 Copyright © 2016 Mark December  

Column Chromatography

This type chromatography is used to separate mixtures with the intention of retrieving one component of

the mixture for further use and not just for identification. In column chromatography, the stationary phase

is packed into a vertical column (burettes may be substituted). The stationary phase is silica gel or alumina

saturated with solvent.

The excess solvent is then drained and the mixture to be separated is then places at the top of the column.

The tap of the column is then opened so that the mixture is absorbed into the stationary phase.

Page 5: Chromatographic Methods of Separation

8/17/2019 Chromatographic Methods of Separation

http://slidepdf.com/reader/full/chromatographic-methods-of-separation 5/7

CAPE Chemistry - Unit 2 Lecture Notes –  Separation Techniques

5 Copyright © 2016 Mark December  

More solvent is then added to the column and allowed to drain through the stationary phase under gravity

 by opening the tap at the base of the column.

The components of the mixture adsorbs to the surface if the stationary phase to different extents. This

 process of washing a compound through a column using a solvent is called elution and the solvent iscalled an eluting solvent or eluent. The time taken a component to emerge at the bottom of the column is

called the retention time. This type of chromatography is most commonly used to:

  Extract useful compounds from plant material

  Isolate intermediates in organic reactions

  Purification of biomolecules  Identify and isolate amino acids, peptides and nucleotides

High-performance Liquid Chromatography (HPLC)

This technique is similar to column chromatography but used a high pressure (400 atmosphere) toincrease the rate at which the solvent (mobile phase) passes through the stationary phase unlike columnchromatography that flows due to gravity. HPLC allows for the use of a much smaller particle size for

the column packing material which gives a much greater surface area for interactions between the

stationary phase and the molecules flowing past it. This allows a much better separation of thecomponents of the mixture. The components, once separated is analysed using UV spectroscopy.

The retention time for HPLC will vary depending on:

Page 6: Chromatographic Methods of Separation

8/17/2019 Chromatographic Methods of Separation

http://slidepdf.com/reader/full/chromatographic-methods-of-separation 6/7

CAPE Chemistry - Unit 2 Lecture Notes –  Separation Techniques

6 Copyright © 2016 Mark December  

  the pressure used (because that affects the flow rate of the solvent)

  the nature of the stationary phase (not only what material it is made of, but also particle size)

  the exact composition of the solvent

  the temperature of the column

HPLC may be used commercially for the identification of the stimulants theobromine and caffeine.Calibration allows for the concentrations of theobromine and caffeine to be found.

Gas-liquid Chromatography

In gas-liquid chromatography, the mobile phase is a gas such as helium or nitrogen and the stationary

 phase is a high boiling point liquid adsorbed onto a solid. The sample is injected into a a heated entrance port using a small syringe, where vaporization occurs. The vapour is carried into the column by the

mobile phase (carrier gas). The syringe needle passes through a thick rubber disc (known as a septum)which reseals itself again when the syringe is pulled out.The injector is contained in an oven whose

temperature can be controlled. It is hot enough so that all the sample boils and is carried into the column

as a gas by the helium (or other carrier gas).

In GLC Different compounds have different retention times. For a particular compound, the retentiontime will vary depending on:

  the boiling point of the compound. A compound which boils at a temperature higher than the

column temperature is going to spend nearly all of its time condensed as a liquid at the beginningof the column. So high boiling point means a long retention time.

  the solubility in the liquid phase. The more soluble a compound is in the liquid phase, the lesstime it will spend being carried along by the gas. High solubility in the liquid phase means a high

retention time.

Page 7: Chromatographic Methods of Separation

8/17/2019 Chromatographic Methods of Separation

http://slidepdf.com/reader/full/chromatographic-methods-of-separation 7/7

CAPE Chemistry - Unit 2 Lecture Notes –  Separation Techniques

7 Copyright © 2016 Mark December  

  the temperature of the column. A higher temperature will tend to excite molecules into the gas

 phase - either because they evaporate more readily, or because they are so energetic that theattractions of the liquid no longer hold them. A high column temperature shortens retention times

for everything in the column.