separation techniques notes

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CHM1313 Separation Techniques used in Analytical Chemistry

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Separation Techniques used in Analytical Chemistry

MRI and X-rays in medicine

Disadvantages of Medical use of X-rays in medicine.

Medical X-rays show up bones because bones are more opaque to X-rays than softtissue, but are not so good at identifying soft tissue.

Medical X-rays are harmful (causing tissue damage) if over-used.Although MRI (Magnetic Resonance Imaging) scans rely on the same sort of energy gaps

between the two magnetic states of protons in hydrogen nuclei.

Advantages of MRI in medicine

Unlike X-rays, MRI doesn't cause any tissue damage. Unlike X-rays, MRI isn't obscured by bones. MRI picks up particular concentrations of hydrogen nuclei in different tissues - for

example, in water or fat molecules.

MRI can be fine-tuned to pick out particular tissue types which can be shown on a scanas different shades of grey. This includes the ability to distinguish between cancerous or

normal tissue.

X-ray Diffraction

This is a non-destructive analytical techniques to determine the structure of macromolecules and

understanding of their function. X-rays are very short wavelength electromagnetic radiation. The

wavelengths are similar to the interatomic distances in solids. If rays of x-ray of a single wavelength are

directed at a pure crystal, some of the x-rays are diffracted by the planes of atoms in the crystal.


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The working principle is based on diffraction or scattering behavior of atoms when x-ray interacts with

electrons in atoms. Atoms with many electrons are more efficient at scattering than those with fewer

electrons. Hence, hydrogen atom has very poor diffraction behavior since it has only an electron. The

scattering of x-ray by a rotating crystal produce a pattern of intensities that show up as a map of the

centers of electron density. A molecular model is then built to fit the electron density map.

Using this technique, bond lengths and bond angles in molecules can be measured. It also shows the

geometry of the arrangement of atoms and explain how enzymes works based on the structural shape

of the active site.

Introduction to Partition

In general substances dissolve when the energy of the solute-solvent system is lower when the solute is

dissolved than when the solute is not dissolved. This is usually the case if the interactions between the

solute and the solvent molecules are similar to those between the solvent molecules themselves. For

example, ethanol and water are miscible as they shared similar hydrogen-bond interactions between

water-water, ethanol-ethanol and water-ethanol systems.

Molecules can attract one another in a variety of ways:

- Ionic attractions- Ion-dipole attractions- Hydrogen bonding- Van der Waals forces

As a rule polar solvents(e.g water) are more likely to dissolve ions, substances that form hydrogen

bonds(ethanol) and molecules with dipoles(e.g HCl). Non-polar solvents(hexane) dissolve solutes whose

molecules are attracted to each other by only van der Waals forces( e.g iodine).

Solvent Extraction

At equilibrium the rates of movements between two immiscible solvent are equal

The solute molecules distribute themselves in a definite ratio between the two liquids

The ratio depends on the relative solubility of the solute in the two liquids


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The solute is partitioned between the two liquids

Solvent extraction is commonly used in organic chemistry for the separation of a liquid or solid from an

aqueous suspension or solution.

It is usually carried out in the laboratory by shaking the aqueous mixture in a separating funnel with an

organic solvent which is immiscible with water, and then allowing the liquid layers to separate out. The

components of the mixture distribute themselves between the two solvents in concentrations which

are proportional to their relative solubilities.

At any given temperature the ratio of these concentrations for a given solute is known as the

distribution coefficient.

Inorganic salts which may be present as impurities will appear almost exclusively in the aqueous layer.

On the other hand, hydrocarbons and their halides, which do not form hydrogen bonds, are virtuallyinsoluble in water but readily soluble in most organic extraction solvents. Compounds of this type can

often be extracted effectively in a single operation.

Hydrogen bonded substances such as ketones, aldehydes, esters, alcohols, acids, and amines are less

easy to extract. The mixture must be repeatedly treated with a solvent in which the organic solute is

considerably more soluble than in water.

With a given volume of solvent, the effectiveness of the separation achieved increases with the number

of extractions performed. It is usual therefore to carry out three or four extractions with small volumes

of solvent rather than a single extraction with a larger amount.

At 291 K, the partition coefficient of butanoic acid between ether and water is 3.5. Calculate themass of butanoic acid extracted by shaking 100 cm3 of water containing 10 g of butanoic acidwith 100 cm3 of ether.


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For the same aqueous solution of butanoic acid, calculate the mass of butanoic acid extracted iftwo portions of 50 cm3 ether are used.

After separation from the aqueous layer the extracts containing the organic solute are dried by shaking

with a suitable drying agent. The solvent is then distilled off, and the residue purified by crystallization,

or if the extract is a liquid, by distillation.

The liquid most commonly used for extraction purposes is probably ethoxyethane. Apart from its

powerful solvent properties, it has a low boiling point (35 C) which simplifies its removal after the

extraction has been carried out. The fire hazard involved in the use of ethoxyethane can be considerably

reduced by careful handling and sensible precautions such as extinguishing flames in the vicinity of the

separation. Other extraction solvents of importance are petroleum ether, benzene,

trichloromethane,tetrachloromethane and chloromethane.


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Partition Chromatography

Chromatography (Gk. chroma, colour, graphe, a writing) describes processes which allow the resolution

and identification of mixtures by the separation of their components into concentration zones. This is

done by transporting the mixture across a stationary adsorbent using a moving liquid or gaseous phase.

The mechanism of chromatographic separation lies in the repeated adsorption and desorption of thecomponents of a mixture as it passes through a chromatographic system. This effectively multiplies any

differences in the partitioning behavior of the components and enables even complex mixtures to be

resolved without difficulty.

In partition chromatography separation depends upon the distribution of the components of a mixture

between a moving solvent and water bound to a stationary phase such as silica gel. More recently gas

partition chromatography has been developed in which partition of a gaseous mixture occurs between

an inert carrier gas such as nitrogen and a column of non-adsorbing material moistened with a suitable

liquid.

Example of Partition Chromatography- Gas-Liquid Chromatography

The time it takes for the solutes to reach the detector once they have been injected into the column is

known as retention times and will vary depending on the following:

1 the flow rate of carrier gas;2 the temperature of the column;3 the length and diameter of the column;4 the nature of and interactions between the solute and the stationary and mobile phases; and5 the volatility of the solute.


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Question


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(c)


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Example of Partition Chromatography- Paper Chromatography

Paper chromatography works by the partition of solutes between water in the paper fibres(stationary

phase) and the solvent(mobile phase).

As each solute distributes itself(equilibrates) between the stationary and the mobile phase, the distance

a solute moves is always the same fraction of the distance moved by the solvent. The fraction is called

the retardation factor, Rf

If the components do not separate properly, a two way chromatography is carried out.


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Question


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Since amino acids are colorless a locating agent such as ninhydrin is needed to identify the positions of

the amino acids in the chromatogram.


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Adsorption chromatography

Thin-Layer Chromatography

A thin layer of the adsorbent acts as the stationary phase.

A sheet of glass is coated with a thin layer of the adsorbent mixed with a binder such as calcium(II)

sulphate. This is done by mixing the dry powder with a suitable liquid, usually water, to form a thick

slurry, which is applied to the glass by spreading or dipping.

After drying the plate, a little of the mixture is spotted just above one edge, which is then dipped into a

shallow pool of solvent. The solvent is drawn up the adsorbent layer by capillary action, separation- of

the components of the mixture occurring as in a column chromatogram.


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The popularity of thin layer chromatography (TLC) lies in the fact that it combines the convenience and

simplicity of paper chromatography with the choice of a number of inorganic substrates normally used

for column separation. Also TLC lends itself to the resolution of minute quantities of mixtures. The

chromatograms are remarkably free from distortion as the fine state of division of the adsorbent

prevents lateral diffusion. The quantities of adsorbent and solvent involved are very small, and the

plates can be produced easily and quickly.

These advantages have made thin layer techniques attractive to the biochemist and forensic scientist.

For example, minute traces of poison can be detected in a corpse, or the presence of additives in food

confirmed. A recent application is the analysis of steroid metabolites in urine as the basis of an early

pregnancy test

A more efficient adsorption separation is High Performance liquid chromatography(HPLC). This is due to

the smaller particles of the stationary phase coated with inert liquid.

High Performance Liquid Chromatography(HPLC)

This technique is similar to GLC. The main differences are

- the mobile phase is a liquid rather than a gas- the liquid is forced through the column a pressures up to 400 atm- the column is much shorter (10-30 cm)- the components are usually detected by measuring the absorbance of UV radiation through a

cell at the end of the column.

Uses of HPLC

- Separation of peptides and proteins- Analyse urine of athletes for banned substances such as anabolic steroids.- Monitoring pollutants in the atmosphere and in rivers, e.g pesticides- Check the accuracy of data of food labels


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Schematic representation of an HPLC system

Most compounds separated by HPLC absorb UV light.

Summary

Type Stationary phase Mobile phase Visualising agentPC Water coated on

celluloseOrganic or

aqueous solventIodine crystal and UV adsorbance( aromatic

rings),ninhydrin(amino acids)

TLC Aluminium oxide orsilica

Organic or

aqueous solventSimilar to PC

GLC High bp liquid on silica Gaseous N2,He, Ar Not applicable

HPLC Silica or coated withnon-polar group

non-polar solvent Adsorbance of UV radiation


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DNA fingerprinting

This is a high-profile analytical technique due largely to its use in forensic science to screen crime

suspects. It uses the same technique as electrophoresis but rather than relying on filter paper as the

medium, a gel is used. In recent years the technique has been refined so that only a tiny amount of DNA

is needed. This can be extracted from material such as blood, hair, cheek cells, semen or skin.

The DNA is treated with restriction enzymes to break it into fragments that can be analysed by

electrophesis. If the amount is very small, a technique called the polymerase chain reaction(PCR) can be

used to produce more copies of the DNA. It is possible to start the analysis with as little as 0.2 ng of

DNA. Samples of the DNA fragments are put into small wells in the gel near the cathode. The

phosphate groups on the DNA fragments are negatively charged and are attracted to the anode, with

smaller fragments moving faster than the larger ones. The results can be stained to help them show up

or if the DNA fragments are treated with radioactive phosphorus, a photographic print can be obtained.


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Uses of DNA fingerprinting

- establishing the father of a child in a paternity case- making links within a family from samples obtained from both living and deceased relatives- establishing links between archaeological samples of biological origin such as animal skins- medical applications where the presence of a particular polypeptide or protein can be an early

diagnosis of genetic diseases of a problem without symptoms, for instance in newborn babies.

Applications in chemistry and society

Two examples of modern analytical techniques and the ways in which they are used.

Monitoring of PCBs in the atmosphere

Polychlorinated biphenyls are a group of man-made chemicals that have been used in a variety of

manufactured goods since the late 1920s. They can persist in the environment and accumulate in the

food chain. They can be found around waste sites, particularly incinerators, where they can be

converted into similarly dangerous dioxins.

Humans may be exposed to PCBs by consuming contaminated food, drinking contaminated water or

breathing contaminated air. Mother exposed to PCBs may transmit them to their unborn child or to

breast-feeding infants. Effects of PCBs include an increased risk of some forms of cancer, reduced

fertility in men and some neurological effects.

PCBs can be monitored by taking air samples remotely, separating the chemicals found using GLC and

then analyzing them by mass spectrometry. They can also be monitored in water and in soil samples.


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Measuring isotope ratios in ice cores.

Oxygen is one of the key markers when studying past climates. Naturally occurring oxygen exists as two

main isotopes, O-16 and O-18.

The ratio of these isotopes of oxygen in water changes with the climate.

Evaporation and condensation are the two processes tha t most influence the ratio of O-16 and O-18 in

the oceans. Water molecules containing O-16 evaporate slightly more readily than water molecules

containing O-18 due to the higher kinetic energy of O-16 compared to O-18. Similarly, water vapour

molecules containing O-18 condense more readily due to lower kinetic energy of O-18 compared to O-

16.

Thus O-18 water vapour molecules are more likely to condense , forming cloud and then rain over the

ocean region that lead to an increase the concentration of O-18 water in the ocean. Unlike O-18, O-16

water molecules are more likely to evaporate, travel and condense further inland and then rain over the

Antarctic region that lead to an increase in the concentration of O-16 in the ice formed. The exactoxygen ratios can indicate how much ice covered the Earth. This provides pointers to any current change

in climate by examining the isotopic ratio in recent ice cores.

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