Outcome 3Area of Study 1: Chemical AnalysisChromatography, Spectroscopy

ChromatographyChromatography is an analytical technique that is used to separate the

substances present in a mixture. It is also widely used to determine the identity of a substance.

The components of a mixture are separated using the principle of adsorption (onto). All types of chromatography have an adsorbant material (often a solid) which forms a stationary phase and a mobile liquid (or gas) called the mobile phase.

The mixture passes over the adsorbent material carried along by a mobile phase.

As the mixture passes over the stationary phase the different molecules within the mixture are adsorbed onto the surface at different rates and with different strengths (according to attraction to surface). Molecules, which adsorb more strongly onto the stationary phase move slower and those that adsorb weakly move faster.

Paper chromatographyStationary phase: adsorbent on paper fibresMobile phase: liquid solvent

The solvent and sample move up the paper by capillary actionSeparation depends on how strongly the components of the mixture

adsorbThe origin is where the drop of sample is

Thin-layer chromatographyQualitative analysisStationary phase: Thin layer of adsorbant (a fine powder e.g. alumina/aluminium oxide (Al2O3) spread on a glass or plastic plate/sheet.Mobile Phase: Liquid solvent (Generally, the stationary phase is polar, and the mobile phase is non polar)

To prepare thin layer chromatography1. Dissolve the pure sample in soluble solution. This is the standard solution2. Dissolve the sample in soluble solution. This is the sample solution.3. Place a small spot of the sample solution near the bottom of a thin-layer

plate. Place a spot of the standard solution next to it, at the same distance from the bottom of the plate

4. When the spots are dry, place the plate in a container with a small volume of solvent (e.g. organic solvent). The lower edge of the plate, but not the spots, should be immersed

5. Allow the solvent to rise until it almost reaches the top of the plate and remove the plate from the container

6. Let the plate dry and examine it under ultraviolet light. If a spot from the sample appears at the same distance from the origin as the spot from the standard solution, the sample contains the standard compound.

A chromatogram is the pattern of bands or spots formed on the plate in thin-layer chromatography or on the paper in paper chromatography

The identity of the chemicals in the mixture can be identified in two ways1. Running standards of known chemicals on the same chromatogram as the

unknown sample2. Measuring Rf values

The relative distance moved by the components of a sample compared to the solvent front (where the solvent reaches) is called the Rf value.

Rf = Distance travelled by component from the origin (x)Distance travelled by solvent from the origin (y)

Rf values will always be less than one The component most strongly adsorbed onto the stationary phase

moves the shortest distance and has the lowest Rf value

A comparison of paper and thin-layer chromatographyPaper chromatography Thin-layer chromatographyCheap FasterLittle preparation Detects smaller amountsMore efficient for polar and water-soluble compounds

Better separation of less polar compounds

Easy to handle and store Corrosive materials can be usedA wide range of stationary phases is available

Column chromatographyStationary phase: Solid or a solid that has been thinly coated in a viscous liquidMobile phase: Liquid

The stationary phase is packed into a glass column. The sample is applied carefully to the top of the packing and the mobile phase, is dripped slowly onto the column from a reservoir above. A tap at the bottom of the column allows the solvent, which is called the eluent, to leave the column at the same rate as it enters it at the other end.

High performance liquid chromatographyMobile phase: Liquid solventStationary phase: Solid adsorbant inside a rigid metal column

Used to analyse large molar mass organic compounds, which are liquids or solids

Extremely sensitive Faster Can allow for better resolution (distinct

separation) of components Wide range of compounds Can separate compounds with relative

molecular masses of 1000 or moreSmall Particles

The very small size of the solid particles allows for more frequent adsorption and desorption of the components, giving much better separation of similar compounds

The small particle size creates a considerable resistance to the flow of the mobile phase and so the solvent is pumped through under high pressure – up to about 14000 kPa

In HPLC, the components are usually detected by passing the eluent stream through a beam of UV light. Many organic compounds absorb UV light, so when an organic compound passes in front of the beam of light, a reduced signal is picked up by a detector. The amount of light received by the detector is recorded on a chart that moves slowly at a constant speed. The resulting trace is called a chromatogram.The detector measures

1. The retention time, Rt, which is the time taken for a component to pass through the column. The retention times are used to identify the components associated with the peaks on a chromatogram.

2. The relative amounts of each component in a mixture may be determined by comparing the areas under each peak with areas under peaks for standard samples.

The ProcessSolid samples are dissolved in a suitable solvent. The liquid sample is injected into the top of an HPLC column. The stationary and mobile phases are chosen to achieve a good separation of the components in the sample. The sample components alternately adsorb onto the stationary phase and then desorb into the solvent as they are swept forward. The time taken to exit the column increases if the component strongly absorbs onto the stationary phase and has a low solubility in the mobile phase.

Gas chromatography The most sensitive of the

chromatographic techniques ideal for the analysis of trace contaminants in samples

Capable of detecting as little as 10-12g of a compound

Limited to gases or compounds that can be readily vaporized without decomposing, these compounds usually have relative molecular masses less than 300

More accurate than HPLC as it can separate components of similar volatilities

Gas chromatography has the following features A small amount of sample is injected into the top of the column through

an injection port The injection port is heated to a temperature sufficient to instantly

vaporize the sample, which is then swept into the column by the carrier gas

The column is a loop, or series of loops, of glass that has an internal diameter of about 4 mm and is 2-3 m long in total.

The column is mounted in an oven and heated The components of the sample repeatedly pass into and out of solution

with the stationary phase. The least soluble are swept out first by the gas into the detector

Qualitative analysis The time taken for a component to pass through the column of a GLC

instrument is called the retention time, Rt. The Rt is characteristic of the component under the operating conditions in use and can be used to identify substances by comparing the Rt of unknown components with those of known substances.

Quantitative analysis The relative amount of a component in a mixture may be determined by

measuring the area under the peak of the component. This area is compared with the areas under peaks for samples containing the component in different known concentrations.

Gas-liquid chromatographyMobile phase: Inert gas (generally nitrogen) called the carrier gasStationary phase: High boiling liquid hydrocarbon or ester

In gas-liquid chromatography, the column is packed with a porous (cork, sponge) solid that has been coated with a liquid hydrocarbon or ester with a high boiling point. This liquid acts as a liquid stationary phase

The column is packed with very fine particles because fine particles increase the surface area, which is coated with the stationary phase. The greater the surface area of stationary phase, the more particles that can be adsorbed at any instant and the better the separation of components.

The injection port is heated to turn the sample components and solvent into a gas.

The components of the mixture pass in and out of solution with the stationary phase. The least soluble move faster and reach the detector first

Gas-solid chromatographyMobile phase: Inert gas (generally nitrogen) called the carrier gasGas-solid chromatography stationary phase: solid

In gas-solid chromatography, the column is packed with an adsorbant solid such as silica gel or alumina. The solid acts as a solid stationary phase

SpectroscopyThe electromagnetic spectrum

Different techniques utilize the effects of radiation from different parts of the electromagnetic spectrum on atoms or molecules to provide us with information about the structure and composition of substancesCan provide us with information about:

The type of atom or molecule that is present How much of a particular atom or molecule is present The structure and bonding within the molecule

Spectroscopic techniques utilize the fact that:

Atoms or molecules absorb and emit electromagnetic radiation of specific energies

Atoms and molecules undergo a change when they absorb electromagnetic radiation

Different parts of the electromagnetic spectrum affect different parts of the atom or molecule

Radiation from each portion of the electromagnetic spectrum has a specific frequency, wavelength and energy associated with it

The energy of the radiation determines which part of an atom or molecule is affected

The Electromagnetic SpectrumElectromagnetic Radiation is light energy which radiates from the sun in the form of vibrating electrical and magnetic fields

As the wavelength increases the energy of the wave decreases As the frequency of the wave increases so does the energy of the wave

Flame Tests Quantitative

How it works:An electron can jump to a higher energy level if it absorbs energy that

corresponds exactly to the difference in energy between the lower energy level and the higher energy level

Higher energy levels are unstable so the excited electrons quickly return to lower energy levels, emitting the energy they had absorbed if the energy emitted falls within the band if energies visible to the eye, it can be seen as coloured light

The energy of a photon of light emitted by an excited electron has a fixed value, equal to the difference in energy between the higher energy level and the lower energy level to which it returns. Limitations:

Most of the electrons in most metals are not excited by the flame of the Bunsen burner, or if they are, the radiation emitted is not in the visible region

Difficulties in distinguishing between two similar flame colours One salt may mask the colour produced by another salt

Atomic emission spectroscopy Quantitative Limited to metals (Group I and II)

Two modification greatly improve the usefulness of the technique Using a hotter flame, so that sufficient energy is available to excite

electrons in a wider range of elements Passing the light through a prism. The different energies in the light

emitted by a heated sample are separated into a series of coloured lines, called an emission spectrum

No two elements will therefore have energy levels of exactly the same energy, so a spectrum is characteristic of a particular element.

Atomic absorption spectroscopy Qualitative Very versatile, being capable of detecting over 70 elements Extremely sensitive, detecting concentrations of elements at parts per

million (ppm) levels or, in some cases, parts per billion (ppb) levelsHow AAS works

To start with the sample must be dissolved in acid so that the metal is converted into ions. The ions are then sprayed into a flame and are converted into atoms.

Each element has a unique absorption spectrum, each element to be analysed require its own light source that will emit light of the correct wavelength. The light is provided by a special lamp called a hollow source cathode lamp, which is made of the same metal as the metal to be determined. The electrons in the atoms of the cathode will emit electromagnetic radiation that will be absorbed by the metal atoms in the sample since it is made of the same metal

A solution of sample to be analysed is sprayed into a flame where it is converted into anatomic vapour light containing the chosen wavelength is passed through the flame. Atoms of the element being analysed that are present in the flame absorb some of the radiation. The light beam is then passed through a filter (monochromator) to select the light of the chosen wavelength, and its intensity is measured by an electronic detector. The amount of light absorbed indicates the quantity of the element present in the original sample.How the element is identified

The light that has not been absorbed by the sample is then passed through a prism and is separated into the individual wavelengths.

As a consequence, certain energies seem to be absent from the beam of light emerging from the sample

Since atoms of different elements have different energy levels, the absorption spectrum can be used to identify the element that produced it

UV-Visible Spectroscopy Quantitative and qualitative analysis Larger range than AAS (visible and UV)

UV-Visible Spectra arise because photons in this region of the electromagnetic spectrum have sufficient energy to promote electrons from low energy levels to higher energy levels

When a substance absorbs visible light, it appears coloured. The colour observed is the complement of the absorbed colour because this is what remains to reach our eyes

How it worksThe radiation source provides ultraviolet and visible light of all

wavelengths. The monochromator selects a particular wavelength from those emitted by the source, and the detector measures the intensity of the light that passes through the sample.

The substance under investigation in solution is placed in the spectrophometer in a special cell made of a quartz or fused silica transparent to ultraviolet and visible light. With a simple spectrophometer, a reference reading is first taken with a cell containing only pure solvent. This is used to compensate for any reflection, scattering or absorbance of the light by the cell and the solvent.

The reference cell is then replaced with a cell containing a solution of the sample. The absorbance by the sample is found by comparing the two readings. By measuring the absorbance at various wavelengths a graph, or spectrum, for the sample can be obtained.

Infrared SpectroscopyInfrared spectroscopy can be used to

Identify functional groups Analyse whether a compound contains single, double or triple carbon

bondsHow Infrared Spectroscopy works

The energy from infrared radiation is not enough to promote electrons to a higher energy level, but it’s enough to bend and stretch the molecule until the atoms in the molecule change position and starts vibrating.

Each particular bond within the molecule will absorb from the radiation the precise amount of energy that corresponds to the same frequency as its natural vibration

This increases the amplitude of vibration of the bond The molecules will then move to a higher vibrational energy

Features of Infrared Spectroscopy Infrared radiation is lower in energy and of a longer wavelength than

visible and ultraviolet light. All molecules will absorb infrared radiation except homonuclear

molecules such as O2 and N2

For a molecule to absorb infrared radiation, the bending or stretching vibrations must change the overall dipole moment of the molecule.

Uses of Infrared SpectroscopyQualitative Analysis

Each frequency absorbed by a molecule corresponds to a particular vibration. Thus we can work out from the IR spectrum the types of vibration occurring and therefore the types of bonds present in the molecule

The energy or radiation absorbed depends on:- The kind of bonds present in the molecule- The environment or other bonds in the molecule

Quantitative Analysis The absorbance of infrared radiation increases as the concentration of the

molecule in a sample increases

A calibration curve of absorbance vs concentration can be constructed (Absorption of a series of standard solutions is measured at the frequency of shark peaks in the spectrum chosen by the monochromator)

Mass SpectrometryAn instrument used to determine the accurate relative isotopic masses

and hence the relative atomic masses of an element. The peak with the highest intensity is known as the base peak and is

assigned an intensity of 100% The intensity of all other peaks is measure relative to the base peak The base peak may or may not be the parent molecular ion The relative intensities of the ions depends on

- The energy of the bombarding electrons- The stability of the ion fragment formed- The ease which ions can lose atoms

Ionisation A sample enters the main body of the mass spectrometer as a gas. (Liquid

or solid samples are vaporized using lasers or other methods) The gaseous sample is admitted through a small inlet into the ionization

chamber, which is at low pressure Here the sample is bombarded with electrons, forming positive ions usually

with a single charge. (Even true for species usually in form of negative ions)

Mass spectrometers always work with positive ionsAcceleration

The positive ions are accelerated to high speeds by an electric field and it is ensure that they all have the same kinetic energy

Deflection The ions enter a region where there is a magnetic field which is at right

angles to the beam of ions This causes the ions to move in a curved path The curved paths have a radius which is dependent on the mass/charge

ratio of the ions The lighter and more positively charged, the greater the deflection

How a spectrum is formed Mass spectroscopy can be used to do more than measure the relative

atom mass of elements It can be used to find out molecular structures When a molecular structure is placed in a mass spectrometer it can give a

range of peaks Two factors cause the many peaks in the spectrum- The fragmentation of the molecules into a large number of positive ions- The occurrence of different isotopes of the atoms that make up the

moleculesFragmentation

High energy electron beams can knock just one electron from the molecule to form a positive ion

The parent molecular ion is known as a radical with one unpaired electron

It is chemically unstable and so will often break into smaller fragments consisting of more positive ions and uncharged free radicals

When an ion fragments into smaller parts, one species will retain the electron to become the uncharged free radical and the other forms a positively charged ion

Note that only the positive ions reach the detector and are shown on the mass spectrum. The uncharged radicals are evacuated by the vacuum pump

Nuclear Magnetic Resonance (NMR) Spectroscopy Uses energy in the radio frequency range of the electromagnetic

spectrum The energy of the radiation is too low to cause electronic, vibrational or

rotational transitions, but it can cause a change in the spin of the particles in the nucleus

How it works All nuclei possess charge and mass Those with an odd mas number also possess spin In the absence of an external magnetic field the two orientations of spin

have the same energy and the spin is orientated randomly When a sample of a compound containing nuclei that have a spin is placed

in a large magnetic field, a small majority of the nuclei will line up in the same direction as the magnetic field and are said to have a parallel spin

The remaining nuclei will line up the opposite direction and have an anti-parallel spin

The nuclei aligned parallel to the magnetic field are at lower energy to those anti-parallel (anti-parallel need more energy for against current)

The difference in energy is of the same frequency as electromagnetic radiation in the radio-frequency range

By subjecting the sample to a pulse of radio-frequency radiation, some of the nuclei will flip from parallel to the anti-parallel alignment

The nuclei will then release that energy and move back to spinning in the parallel direction

The frequency of radiation require to produce the spin-flip is known as the resonance frequency

GlossaryAdsorption – The attraction of one substance to the surface of anotherDesoption – The breaking of the bonds between a substance and the surface to which the substance is adsorbedMobile phase – The phase that moves over the stationary phase in chromatographyStationary phase – A solid, or a solid that is coated in a viscous liquid, used in chromatography. The components of a mixture undergo adsorption to this phase as they are carried along by the mobile phaseEluent – A liquid used as the mobile phase in chromatographyRetention time – The time taken for a component to pass through a chromatography columnCarrier gas – The gas used as the mobile phase in gas chromatography

Theory QuestionsWhy must the level of the solvent be lower than the origin where spots of the mixture are originally placed?

If the solvent were above the level of the origin, the compounds under test would dissolve and disperse throughout the solvent

What is the advantage of a two way chromatogram?A two way chromatogram produces better separation of componenets of

complex mixtures, permitting easier isolation and identification

If two substances do not have very good separation, what would happened to the Rf

values if the separation was carried out for a longer period so that the solvent front moved further?

The Rf values would not change. The movement of the component and solvent front both increase by the same proportion so there is no change in the Rf.

How is a substance separated form other substances through chromatography?As the mobile phase travels up the TLC plate it will absorb the substances

and sweep them forward over the stationary phase. The substances are also attracted to the stationary phase and will tend to ‘stick’ or adsorb onto it. This adsorption and desorption process will happen many times. The substances have different solubilities and adsorption strengths so they undergo this process to different degrees, travel up the plate at different rates and separate.

How can a standard with no substance in it still have a small peak? Water used for dilution of standards or glassware may have been

contaminated with substance Small quantities of the substance may be leached off the column by

solvent Small quantities of other substances with similar retention times to the

tested substance may be present

Why does decreased solubility of a chemical in the stationary phase decrease the retention time?

A compound which is relatively insoluble in the liquid stationary phase tends to spend little time dissolved in the liquid and therefore stationary, but is swept along the column by the gas and reaches the detector quickly. The retention time is therefore low.

Why do the peaks become broader with increased retention time?Compounds that are relatively insoluble in the stationary phase will tend to have all molecules swept along with very little ‘stationary’ time, so retention times of all molecules will be very similar, producing a narrow peak. More soluble compounds will have molecules that move in and out of solution in the stationary phase. More movements in and out of the stationary phase will increase the retention time in the column. The longer the time on the column, the greater the possible variation and the broader the peak.