Welcome to SpectraSchool
Welcome to the introduction to spectroscopy page. Here you will find an explanation of the principles for a range of spectroscopic techniques including infrared (IR), ultraviolet-visible (UV/Vis) and nuclear magnetic resonance (NMR). Each technique has a drop down box containing clear explanations and descriptions with embedded videos and clickable links to animations (many of which are interactive) to aid your learning.
Spectroscopy is the study of the interaction of electromagnetic radiation in all its forms with matter.
The interaction might give rise to electronic excitations, (e.g. UV), molecular vibrations (e.g. IR) or nuclear spin orientations (e.g. NMR).
When a beam of white light strikes a triangular prism it is separated into its various components (ROYGBIV). This is known as a spectrum.
The optical system which allows production and viewing of the spectrum is called a spectroscope. There are many other forms of light which are not visible to the human eye and spectroscopy is extended to cover all these.
Absorption of infrared radiation brings about changes in molecular vibrations within molecules and 'measurements' of the ways in which bonds vibrate gives rise to infrared spectroscopy. Atom size, bond length and bond strength vary in molecules and so the frequency at which a particular bond absorbs infrared radiation will be different over a range of bonds and modes of vibration.
An organic molecule may contain quite a number of different bonds. All of these bonds will be vibrating, and clearly, different bonds will be vibrating at different frequencies.
There are two basic modes of vibration – ‘stretching’ and ‘bending’.
For a particular covalent bond in a molecule, only a particular set of vibrational frequencies is possible. Suppose a bond is vibrating at a frequency ν1 and its next available frequency is ν2; then, if radiation with a frequency (ν2 - ν1) is incident on the compound containing this bond, some of the radiation is absorbed and the bond vibrates at the higher frequency.
The frequency for a particular bond is more or less independent of other bonds in the compound; therefore, determination of the frequencies in the infrared region which are absorbed by a compound gives information about the types of bonds which are present.
An infrared spectrometer analyses a compound by passing infrared radiation, over a range of different frequencies, through a sample and measuring the absorptions made by each type of bond in the compound. This produces a spectrum, normally a ‘plot’ of % transmittance against wavenumber.
No two organic compounds have the same infrared spectrum and so individual, pure compounds can be identified by examination of their spectra. In the region, 7 - 11 microns (1430-910 cm-1) there are many absorption bands and even pairs of almost identical organic molecules show up differences here. This region is known as the “fingerprint region” and provided that a chemist has a copy of the spectrum, any unknown pure compound can be identified by making a simple comparison.
The region, 2.5 - 7 microns (4000-1430 cm-1) is simpler, and has less absorption bands. This region is used to aid the determination of structures because particular groups can be more easily identified. Bending frequencies tend to be rather numerous and complicated and are not used very much in identification. Stretching frequencies are much more useful and important groups can usually be identified with great certainty. The carbonyl group, for example, is a very strong chromophore, absorbing at approximately 1700 cm-1.
Absorption of infrared radiation brings about changes in molecular vibrations so in studying infrared spectroscopy we are looking at the ways in which bonds in molecules vibrate (see video in the section above).
Atom size, bond length and strength vary in molecules and so the frequency at which a particular bond absorbs infrared radiation will be different over a range of bonds and modes of vibration. Measuring the absorption of infrared radiation by a material provides very useful information about structure.
Since no two organic compounds have the same IR spectrum, a compound can be identified with certainty by comparing its spectrum with that of a known pure compound. If they are identical, then they are one and the same.
The units of spectroscopy are:
c = νλ where,
c = velocity of light (3.00 x 108 m s-1)
ν = frequency (Hz)
λ = wavelength (m)
E = hν where,
E = energy (kJ mol-1)
h = Planck’s constant (6.63 x 10‑34 Js)
The electromagnetic spectrum covers a very wide range of wavelengths, and different units are therefore used in different regions.
For very short wavelengths, nanometres (1 nm = 10-9 m) are preferred.
In the visible and UV regions, wavelengths can also be expressed in millimicrons (mμ)
1 μ = 10-6 m 1 mμ = 10-9 m therefore1 mμ = 1 nm
In the infrared region, wavelengths can be expressed in microns (μ).Wavenumbers are the number of waves per cm and are often referred to as reciprocal centimetres (cm-1)
In a typical spectrum a C - H absorption occurs at 3000 cm-1 whilst a C = O absorption occurs at 1740 cm-1 at a lower wavenumber, higher frequency and higher energy than the C - H stretching vibration.
NB: Organic chemists loosely refer to wavenumbers as “frequency” and so in books and other sources you may see spectra labelled as “frequency (cm-1)”.
Widely used in both research and industry, infrared spectroscopy is a simple and reliable technique used for a variety of measurements and in quality control. It is especially useful in forensic science both in criminal and civil cases. Spectrometers are now small, and can be easily transported, even for use in field trials. With increasing progress in new technology, samples in solution can now be measured accurately (water produces a broad absorbance across the range of interest, and thus renders the spectra unreadable without this new technology).
Some instruments will also automatically tell you what a substance is by referencing it to a store of thousands of spectra held in storage.
By measuring at a specific frequency over time, changes in the character or quantity of a particular bond can be measured. This is especially useful in measuring the degree of polymerisation in polymer manufacture or in identification of polymer degradation for example.
The progress of formation of an epoxy resin being hardened by an amine cross linking agent can be monitored by observing the appearance of a hydroxy group in the spectrum of a polymerising sample (or by the disappearance of an epoxy group).
Modern research instruments can take infrared measurements across the whole range of interest as frequently as 32 times a second. This can be done whilst simultaneous measurements are made using other techniques. This makes the observations of chemical reactions and processes quicker and more accurate. Infrared spectroscopy has been highly successful for applications in both organic and inorganic chemistry.
A second type of IR spectrometer is a dispersive spectrometer. The rotating mirror, M temporarily reflects the reference beam towards the machine optics whilst blocking the sample beam. Reference beam and sample beam are alternately blocked and reflected. The diffraction grating disperses the IR into a ʽspectrumʼ of wavelengths; this series is reflected to the detector. The thermocouple converts the different wavelengths of IR reaching it to a signal which is represented as a spectrum. The difference between reference and sample signals shows which parts of the spectrum have been absorbed by the sample.
Another type of IR spectrometer is a Fourier Transform (FT) spectrometer. In the FT spectrometer, an interferometer is used instead of a diffraction grating. All frequencies (or wavelengths, ν ~ 1/λ) reach the detector at the same time. The spectrum is obtained by a mathematical calculation (a Fourier Transform). The FT spectrometer is more responsive, accurate and precise than a dispersive spectrometer.
Two balls separated by a spring will oscillate (harmonic) if stretched and released. The frequency of the vibrations depends on the strength of the spring ( ≡ bond strength) and the mass of the balls ( ≡ atoms).
The total energy of the vibration is given by the sum of the potential energies of the ‘compressed’ and ‘extended’ positions. This model suggests that the spring will vibrate at any energy dependent on the initial separation of the balls but, this is not true for molecules. The energy is quantised, which means that only certain energy levels are allowed according to the formula:
En = [n + ½]hν where n = 0,1,2,3, etc.
In addition, molecules can only absorb (or emit) energy equal to the spacing between two levels and, for a harmonic oscillation, this can only occur between adjacent levels. However, bonds in real molecules do not vibrate harmonically.
When atoms approach each other closely, they exert a force of repulsion, and beyond a certain separation distance, a bond breaks. Quantisation produces unequal separations of energy levels which add complications to spectra. Finally, molecules will not absorb infrared radiation unless they possess a dipole, thus H2 is transparent to infrared whilst HCl absorbs.
Many molecules possess dipole moments due to non-uniform distributions of positive and negative charges on the various atoms
e.g. Hδ+- Clδ-
These (permanent) dipoles occur when two atoms in a molecule have substantially different electronegativities (see table below).
These values can be obtained from measurement of the dielectric constant. When the symmetry of a molecule cancels out, there is no net dipole moment and the value is therefore 0. The highest dipole moments are in the range of 10 to 11.
Information about the molecular geometry of a molecule can be deduced from the dipole moment. For example the data indicates that carbon dioxide (CO2) is a linear molecule but ozone (O3) is not.
For non-linear molecules, for example pentane, there are a number of vibrations given by (3N – 6). N is the number of atoms in the molecule. Pentane, C5H12, (17 atoms) has 45 different vibrations! Although there are a large number of vibrational modes here, the situation can be simplified by considering that each functional group can be considered independently. So a methyl group for example should have the same normal modes of vibration no matter where it is located in a molecule.
Water (H2O) has three modes of vibration.
Carbon dioxide (CO2) is a linear molecule and produces just two peaks in the spectrum.
This is because the symmetric stretch does not have a dipole and the two bending vibrations (in plane and out of plane) are degenerate (i.e. of equal energy). The reason that only two peaks appear in the carbon dioxide spectrum is because the symmetrical stretching vibration does not have a change in dipole moment as it vibrates. The two bending vibrations are degenerate and vibrate at exactly the same frequency.
Other simple organic molecules produce a large number of peaks which could be considered in terms of those vibrations which correspond to CH3, CH2, benzene ring and the main functional groups e.g. alkene C=C, carbonyl C=O, alcoholic O-H, amine N-H etc. The following link shows the spin rotation of the aspirin molecule.
The approximate value for the frequency (ν cm-1) of absorption of a bond, can be calculated from the following (ν is referred to as wavenumber).
Where c = velocity of light, k = force constant of a bond between atoms of masses M1 and M2.
For a very light atom such as hydrogen, the value for the term, (M1+M2)/M1M2 is approximately unity (~1.0) and has a relatively large value. The frequency for a C-H bond is approximately 3000 cm-1. Compare this value with that calculated for a bond between carbon and chlorine; here, (M1+M2)/M1M2 = 0.11; the frequency for a C-Cl bond is approximately 600-800 cm-1.
v (wavenumber) depends on the strength of the bond between the atoms M1 and M2 as given by K, the force constant.
Values of the force constant for a number of bonds are given in the table below.
|Bond||Force constant / dynes cm-1|
|C-C||4.5 X 105|
|C=C||9.6 X 105|
|C≡C||15.6 X 105|
|C=O||12.1 X 105|
|C≡N||17.73 X 105|
This indicates that a C=C double bond is approximately twice as strong a s a C-C single bond and that a C≡C triple bond is approximately three times as strong as a C-C single bond, although there is no simple correlation between the force constant and the strength of a bond. Notice that the force constant is greater for C=O than C=C and greater for C≡N than C≡C. Clearly the bonds are stronger when the difference in electronegativity increases (and the subsequent dipole moment).
The strength (intensity) of an absorption in the infrared is dependent on the change of dipole moment occurring during the vibration. If there is no change in dipole moment then the radiation cannot interact with the vibration and there is no absorption. Thus the C≡C stretching vibration does not appear in symmetrically substituted dialkylacetylenes.
2-methylbut-2-ene exhibits only a small dipole moment change and therefore absorbs very weakly. Carbonyl and similar highly polar groupings absorb strongly.
Absorption of single bonds to hydrogen e.g. C-H, N-H, O-H occur around 4000-2500 cm-1. For electronegative atoms i.e. oxygen and nitrogen, the peak will be broadened by intra or inter molecular hydrogen bonding. Absorption of triple bonds e.g. C≡C, C≡N (but also X=Y=Z) occur around 2500-2000 cm-1.
Absorption of double bonds e.g. C=O, C=C occur around 2000-1500 cm-1. These values fall to lower frequencies when bonds are conjugated (increasing single bond character). With carbonyl stretching frequencies, values are significantly affected by the inductive effect of an atom and by the mesomeric affect of groups bonded to the carbonyl carbon.
Absorptions occurring around 1500-400 cm-1 are due to other vibrations e.g. rotating, scissoring and some bending. This is referred to as the ‘fingerprint region’
When some atoms are placed in a strong magnetic field, their nuclei behave like tiny bar magnets aligning themselves with the field. Click here to view full animation
Electrons behave like this too, and for this reason both electrons and nuclei are said to possess “spin”, i.e. any spinning electric charge has an associated magnetic field.
Just as electrons with opposite spin pair up with each other, a similar thing happens with protons and neutrons in the nucleus. If a nucleus has an even number of protons and neutrons (e.g. 12C), their magnetic fields cancel each other out and there is no overall magnetic field; however, if the number of protons and neutrons is odd (e.g.13C and 1H ), the nucleus has a magnetic field. If the substance is placed in an external magnetic field, the nuclear magnet lines up with the field, in the same way as a compass needle lines up with a magnetic field. The nuclear magnet can have two alignments, of low energy and high energy.
The Basics - Supplying Energy
To make the nucleus change to the higher energy alignment, energy must be supplied.
The energy absorbed corresponds to radio frequencies. The precise frequency of energy depends on the environment of the nucleus, that is, on the other nuclei and electrons in its neighbourhood.
So, by placing the sample being examined in a strong magnetic field and measuring the frequencies of radiation it absorbs, information can be obtained about the environments of nuclei in the molecule.
NMR is particularly useful in the identification of the positions of hydrogen atoms (1H) in molecules.
The NMR spectrum of ethyl benzene, C6H5CH2CH3, is shown opposite.The frequencies correspond to the absorption of energy by 1H nuclei, which are protons. Notice that there are three major peaks of differing heights.
Each peak corresponds to a hydrogen atom in a different molecular environment. The area under each peak is proportional to the number of that type of hydrogen atom in the molecule.
The largest peak
Corresponds to the five atoms in the benzene ring [C6H5].
The second largest
Corresponds to the three hydrogen atoms in the methyl group [CH3].
The third peak
Corresponds to the two hydrogen atoms in the methylene group [CH2].
The hydrogen atoms in a particular type of environment have similar positions in an NMR spectrum. Normally, this position is measured as a chemical shift from a fixed reference point. The reference point normally used is the absorption of a substance called tetramethylsilane (TMS), which has the formula (CH3)4Si.
The simplified proton NMR spectrum of ethanol enables the hydrogen atoms to be easily identified. Notice also that spectra also show the integration of the peaks (the area under each peak).
Thus in the spectrum opposite, the smallest peak represents the single H in the OH group (integration of 1)
the middle peak represents the H in the CH2 group (integration of 2)
the largest peak represents the H in the CH3 group (integration of 3)
If the spectrum of ethanol is recorded as a high-resolution spectrum, more detail is apparent and the peaks appear as singlets, doublets, triplets, quartets etc.
If the spectrum of ethanol is recorded as a high-resolution spectrum, more detail is apparent and the peaks appear as singlets, doublets, triplets, quartets etc.
The sets of peaks are due to interaction of protons from neighbouring groups. Thus, in the spectrum of ethanol, the CH3 group affects the CH2 group and vice versa. The phenomenon is known as spin-spin coupling and provides essential information for a skilled NMR technician to interpret a spectrum.
In a molecule the nucleus of an atom can induce in the electrons of the chemical bonds attached to it a very weak magnetic moment. This affects the magnetic field at a neighbouring atom’s nucleus.
The interaction is known as coupling and this causes the peaks to be split into a number of smaller ones. Protons can usually interact with other protons that are up to three bonds away.
Protons in the same chemical environment do not show coupling with each other.
View the following animations for a more detailed explanation of spin-spin coupling:
Spin-spin coupling 1: Spinning charges can be regarded as minute (atomic) bar magnets
Spin-spin coupling 2: Atoms giving rise to chemical shifts in the low resolution NMR spectrum of ethanol
Spin-spin coupling 3: High resolution NMR spectrum and an explanation of spin-spin coupling.
The CH3 protons produce a peak at δ 1.8 but, instead of a single peak, a triplet is produced. This is because the CH3 protons couple with the adjacent two CH2 protons.
The CH2 protons produce a peak at δ 3.2 but, instead of a single peak, a quartet is produced. This is because the CH2 protons couple with the adjacent three CH3 protons.
In general, if there are 'n' protons three bonds away from the resonating group, the absorption will be split into a multiplet of n+1 lines.
NMR is one of the most powerful methods for analysis of chemical samples, biological compounds, medicines etc.
- Gives structural information on a range of different elements in molecules including hydrogen, carbon, phosphorus and many others.
- Is Commonly used for structure determination of molecules in solution.
- Gives information regarding motion in molecules, structural flexibility and how they interact during chemical reactions.
- Can be used to determine shapes and structures of large complex molecules, such as how proteins fold, twist and coil.
- Can be used to evaluate the proportions of solid and liquid components in fatty foodstuffs such as margarines and low-fat spreads.
NMR is also used a lot in pharmaceutical sciences and medicine, for example:
- dynamic studies
- diagnosis of tissue abnormalities
- pH control in diabetics
- body scanning by the closely related techniques known as MRI (no side effects)
The human eye and brain together translate light into colour. Light receptors within the eye transmit messages to the brain, which produces the familiar sensations of colour.
Newton stated that the surface of an object reflects some colours and absorbs all the others. We perceive only the reflected colours.
The surface of the red apple is reflecting the wavelengths we see as red and absorbing all the rest. An object appears white when it reflects all wavelengths, and it appears black when it absorbs all wavelengths.
Red, green and blue are the additive primary colours of the colour spectrum. Combining balanced amounts of red, green and blue lights also produces pure white. By varying the amount of red, green and blue light, all of the colours in the visible spectrum can be produced.
Considered to be part of the brain itself, the retina is covered by millions of light-sensitive cells, some shaped like rods and some like cones. These receptors process the light into nerve impulses and pass them along to the cortex of the brain via the optic nerve.
Colour in organic compounds is associated with unsaturation.
A carbon-carbon double bond in ethene, for example, absorbs energy as electrons in π bonds are promoted to higher energy levels. Bonds formed between carbon atoms in ethene are the result of overlap between atomic orbitals, producing bonding and antibonding molecular orbitals.
Orbitals are regions of space in which there is a 90% chance of finding electrons. However, in antibonding orbitals, which are of higher energy, electrons will not be found.
Transitions between orbitals of lower energy and antibonding orbitals occur when electromagnetic radiation of suitable energy is absorbed by the molecule.
In ethene, absorption of radiation of wavelength 171 nm is sufficient to promote electrons to π* anti-bonding orbitals.
In buta-1,3-diene however, radiation of a longer wavelength (222 nm) will promote electrons. The reason for this difference is due to delocalisation of the electrons in the π bonds of buta-1,3-diene. When electrons occupy delocalised π bonds they are spread out over a larger volume and the energy associated with them is lowered. As the extent of delocalisation of electrons increases further, their energy lowers even more. Electrons can consequently be promoted more easily in extensively delocalised molecules and the wavelength of radiation concerned with the transition increases significantly.
Alternating double and single bonds is called conjugation and as the extent of conjugation in a molecule becomes more extensive, the energy required to promote electrons lessens significantly. The spectra of some conjugated hydrocarbons show clearly that, as the extent of conjugation increases, the absorption of UV radiation occurs at longer wavelengths.
Energy, E = hc
As the wavelength (λ) increases the energy needed to promote an electron decreases.
The appearance of several shoulders for a given chromophore is common for highly conjugated systems. It often depends on the solvent used and is due in part to electronic transitions between different vibrational energy levels existing in each electronic energy level.
The spectra of polycyclic aromatic hydrocarbons (arenes) is very interesting. As the number of aromatic rings increase, the extent of hyperconjugation increases and the delocalisation of π electrons becomes extensive.
For some compounds radiation in the visible region of the electromagnetic spectrum is sufficient to promote electrons to an excited state and consequently these molecules are coloured.
Tetracene consists of bright orange crystals because the compound absorbs light from the blue end of the visible spectrum.
Pentacene is deep purple, absorbing light from the red end of the spectrum.
These polycyclic aromatic compounds are organic semiconductors. The delocalisation of the π electrons is extensive and allows for movement of electrons throughout the molecule and conduction of a current.
Beta-carotene absorbs throughout the UV region but particularly strongly in the visible region with a peak at 470 nm. Carotene consists of 11 alternating single and double bonds.
Groups which absorb light are known as chromophores.
Transition elements are found in the d block of the Periodic Table and the most interesting feature of transition metal compounds is that most are highly coloured.
d block elements use s, p and d orbitals in bonding, forming complexes which exhibit a variety of oxidation states and involve other species called ligands.
To explain the reasons behind colour in transition metal complexes we need to briefly examine the nature of d orbitals and the way in which they interact with ligands.
Crystal Field Splitting
The majority of transition metal complexes are octahedral complexes, containing six ligands surrounding the central ion.Tetrahedral or square planar complexes are less common.
The ligand field which forms around the d orbitals causes the energy of the electrons in them to increase, but this increase is not the same for all of the d orbitals. For an octahedral complex, the energy of the orbitals is split into two. Three of the orbitals (t2g) are of lower energy and two have higher energy (eg).
The energy difference (Δo) is caused by the juxtaposition of the ligands and d orbitals.
The dz2 and dx2 – y2 orbitals line up with the ligands, creating greater repulsion and occupy higher energies whereas the remaining dxy, dyz and dxz reside in between the ligands.
Coloured complexes all contain from 1 – 9 d electrons. Complexes that are colourless do not contain metals with this particular electron configuration.
Consider a solution of chromium(III) nitrate; Click here to view full animation
This solution is violet in colour because the solution absorbs yellow light. Yellow is the complimentary colour of violet.
Copper(II) sulfate solution appears blue because it absorbs orange-yellow light.
Cr3+ has three electrons, each singly occupying the t2g orbitals. When light is absorbed by the solution, an electron is promoted from a t2g orbital to a vacant, higher energy eg orbital.
Transition metal compounds are coloured because the energy involved with these transitions occurs in the visible part of the spectrum.
The nature of ligands and the shape of a complex affect the magnitude of the energy difference (Δo, Octahedral; Δt, tetrahedral; Δsp, square planar) between d orbitals split, in a field of ligands. Octahedral, tetrahedral and square planar produce differences in crystal field splitting because of their different geometries and the proximity of ligands to the d orbitals.
Consider some chromium(III) complexes:
- These octahedral complexes absorb radiation in three different regions of the visible spectrum.
- The CN ligand has the largest field strength and the F ligand is about 2/3rds of that value.
- The differences are due to more extensive orbital interactions with ligands.
Energy, Δo = hc
Since hc is constant, the energy difference between eg and t2g orbitals will be inversely proportional to wavelength. Higher values for Δo therefore cause transitions to take place at shorter wavelengths, which approach the ultraviolet region (in the case of ligands) with a high field strength .
The charge on a metal ion has a significant effect on crystal field splitting.
For the same ligand, a metal ion with a higher oxidation number will produce a greater crystal field splitting.
This is because the ligand will be more strongly attracted to the metal ion (which has a higher charge density) and will approach the d orbitals more closely, increasing the extent of repulsion and raising the magnitude of the energy difference (Δo) between the orbitals.
Solutions of iron(III) complexes are yellow or red whilst iron(II) complexes are pale green. This is because as the oxidation state of the metal increases, Δo increases and hence the frequency of the absorption moves towards higher frequency, i.e. for iron (III) the absorption moves towards the blue end (higher energy end) of the spectrum.
The Co(III) hexaammine complex has a Δo value which is about twice that of the Co(II) hexaammine complex.
For a given ligand and oxidation state, the magnitude of crystal field splitting increases down a group. Ions of metals in the first row of the transition series have lower Δo values than ions in the same group below. This is because, as the metal ions get larger the ligands are physically more remote from those orbitals with which they do not directly interact (i.e. t2g) and repel less. Hence the difference in energy between t2g and eg increases.
Essentially, a mass spectrometer performs three functions:
1. Creates positive ions from a neutral sample
2. Separates the ions according to their mass/charge ratio
3. Measures the relative abundances of ions and their relative masses; the information being represented as a mass spectrum.
Ionisation chamber: A vaporised sample is drawn in by virtue of the very low pressure inside the apparatus. Atoms (or molecules) are bombarded by fast moving electrons from a heated filament, maintained at a high p.d. The electrons collide with the atoms or molecules and remove other electrons forming positive ions.
High vacuum: The apparatus is kept at a pressure of 10-7 mm of mercury or less. Atmospheric pressure is about 760 mm Hg and so this value represents a very low pressure indeed. This very low pressure is described as a 'high vacuum', consequently very few particles are present in the spectrometer and this reduces collisions between electrons and residual air molecules.
Acceleration and collimation region: The ions are then accelerated and collimated (made into parallel beams) into a fine beam by an intense electric field; the overall product is a very fine beam of positive ions travelling with a uniform high velocity.
Deflection chamber: The beam of ions then passes into the deflection chamber. Here the poles of an electromagnet are placed astride the bent tube. This produces a high intensity, variable magnetic field within the tube, normal to ion beam. The field causes a change in the direction of movement of these positive ions. The magnitude of this deflection depends entirely of the mass/charge ratio of the ion. Thus the ion beam is split up into a series of separate beams, each of which has particles of one specific mass/charge ratio.
Detector: By adjustment of the intensity of the variable magnetic field, each separate ion beam can be directed in turn through a fine receiving slit at the other end of the tube where it meets a negatively charged collecting plate. Each positive ion accepts electrons and is 'neutralised'. A very small electric current then flows in the collector circuit which is amplified and recorded as a mass spectrum. This produces a 'plot' of intensities of successive ion beams; the height of each peak being proportional to the number of ions of a given mass/charge ratio reaching the collector plate in unit time.
Liquid chromatography/ High Pressure Liquid Chromotography (HPLC)
Chromatography covers a broad range of physical methods used to separate and/or analyse complex mixtures.
The constituents to be separated are distributed between two phases: a stationary phase and a mobile phase, which percolates through the stationary phase.
A mixture is introduced into one end of the stationary phase, which is contained in a column or coated onto a substrate and the contents are flushed through the system.
Each constituent is adsorbed (or dissolved) to a greater or lesser extent as passage through the stationary phase takes place and since each therefore migrates at a different rate, separation of the mixture is achieved.
A simple example is the separation of inks using paper chromatography.
Chromatography has developed into a highly sophisticated and varied procedure which is used in chemical or bio-processing industries; the need to separate and purify a product from a complex mixture is a very necessary and highly important step in the production line. The separation can be achieved with great precision; even very similar compounds, such as proteins that may only vary by a single amino acid, can be separated this way. In fact, chromatography can purify any soluble or volatile substance if the right adsorbent material, carrier material, and operating conditions are employed.
Although there are other types of chromatography e.g. paper chromatography and thin layer chromatography (TLC), most modern applications of chromatography employ a column. The column is where the actual separation takes place. It is usually a glass or metal tube of sufficient strength to withstand the pressures that may be applied across it. The column contains the stationary phase. The mobile phase runs through the column and is adsorbed onto the stationary phase. The column can either be a packed bed or open tubular column.
The mobile phase is comprised of a solvent into which the sample is injected. The solvent and sample flow through the column together; thus the mobile phase is often referred to as the "carrier fluid." The stationary phase is the material in the column for which the components to be separated have varying affinities. The materials which comprise the mobile and stationary phases vary depending on the general type of chromatographic process being performed. The mobile phase in gas chromatography is generally an inert gas. The stationary phase is generally an adsorbent solid or liquid distributed over the surface of a porous, inert support.
- The mobile phase in liquid chromatography is a liquid of low viscosity which flows through the stationary phase bed. This bed is usually an inert solid such as silica gel (SiO2.xH2O) or alumina (Al2O3.xH2O).
- The effectiveness of the separation is controlled by temperature since higher temperatures improve the adsorbing properties of the stationary phase.
- A wide range of solvents can be used in liquid chromatography and the optimum solvent is often chosen by running preliminary TLC experiments.
- Liquid chromatography can be improved by applying pressure above the liquid in the column; nitrogen is often used at a pressure of approximately 2 atm.
High Performance Liquid Chromatography (HPLC)
The efficiency of separation increases as the particles in the stationary phase are made smaller. This is because the solute particles can form an equilibrium with the stationary phase more rapidly.
However, if the particles are made smaller, conductance (or flow) through the column becomes more difficult. HPLC (sometimes called ‘high pressure liquid chromatography’) is operated at pressures in the order of 100 – 150 atmospheres.
The stationary phase normally consists of silica particles of diameter 10-6 m, the surface pores having a diameter of 10-8 to 10-9 m.
The surface area of the solid is therefore very high, and when coated with a liquid which can interact with the solute particles in the mobile phase, separation of a mixture becomes very efficient indeed.
Flow through HPLC columns is very slow (0.5 – 5.0 cm3 min-1) so the column must be very short and its volume will consequently be very small. Micro syringes are used to inject a sample and must be very precise.
In addition sophisticated pumps must be used to produce accurate and reproducible flow rates. Flow rates can be increased up by raising the temperature and by changing the nature or polarity of the mobile phase. Amounts of solute separated by HPLC are normally too small to collect so a UV lamp is used to detect the presence of each component as it passes through the column.
As each solute passes through the detector, the absorption of radiation is recorded as a peak in a chromatogram.
HPLC coupled with mass spectrometry is a powerful analytical process but other methods of detection/analysis can be used which work in conjunction with chromatography. Some of these include IR spectroscopy, UV/Vis spectroscopy and fluorescence spectroscopy.
Gas Chromatography and Mass Spectrometry (GC-Mass spec)
A gas chromatograph utilises a narrow tube known as the column, through which a vaporised mixture flows in a carrier gas stream (this is known as the mobile phase).
The gaseous constituents flow at different rates depending on their various chemical and physical properties and their interaction with the column filling (called the stationary phase).
The function of the stationary phase in the column is to separate different components, causing each one to exit the column at a different time (retention time). The retention time might be altered by the carrier gas flow-rate and the temperature for a particular purpose.
In a GC analysis, a known volume of gas or liquid is injected into the "entrance" of the column, usually using a microsyringe. As the carrier gas sweeps the mixture of molecules through the column, the motion is inhibited by their adsorption, either onto the column walls or onto packing materials within the column.
The rate at which the molecules progress along the column depends on the strength of adsorption, which in turn depends on the type of molecule and on the stationary phase materials. Since each type of molecule has a different rate of progression, the various components of the mixture are separated as they progress along the column and emerge from the end. A detector is used to monitor the outlet stream from the column. The time at which each component reaches the outlet and the amount of that component can then be measured.
Generally, substances are identified (qualitatively) by the order in which they emerge (elute) from the column and by the retention time of the compound in the column.
Further relevant background material and resources for both teachers and students is available in the DVD resources ‘Uncovering Chemical Secrets’ and ‘Uncovering Chemical Secrets II’ (produced by Peter Edwards and Peter Hollamby) which are available from The School of Chemistry, Cardiff University, Cardiff, CF10 3AT (phone 029 20874023). Both DVDs are designed specifically as a resource for GCE A-level. The Royal Society of Chemistry will not be responsible for the content of such DVD resources and shall not be held liable for any claim or loss arising as a result of reliance or use of such DVD resources.