The solutes that are found in cells are either accumulated from the environment or created within the plant; generally, organic compounds are synthesised while inorganic solutes are acquired from the soil. There is a variety of methods by which inorganic elements can be detected and quantified in plants - in extracts or in plant material that has been vaporised. Analysis broadly depends on one of the following: (a) the optical properties of elements when burning; (b) the mass of the element or its ions; (c) the emission of X-rays (X-ray fluorescence); (d) the use of ion-specific electrodes or (e) the chromatographic separation of ions (ion chromatography).
For some analytical techniques, plant material can be used directly either by vaporising it at high temperature (e.g. see Section 2.2.2) or by freezing it at very low temperature (e.g. see Section 2.6.2). In most cases, however, mineral elements are extracted prior to analysis. This is most simply done by heating a plant or plant part in water or dilute acid (e.g. 100 mM acetic acid) - a couple of hours at 80°C will extract virtually all of any Group 1 cations (e.g. sodium or potassium) present in that tissue. In order to ensure complete dissolution of mineral elements, the plant material can either be heated in a mixture of concentrated nitric acid and sulphuric acid and the extract diluted for analysis or heated alone (dry ashed) at high temperature (550°C) to remove all the organic components before dissolution of the inorganic matter in dilute nitric acid (Humphries, 1956). Some constraints are imposed by the nature of (and impurities in) the acids used to dissolve the samples or by loss of those elements that are volatile below the dry-ashing temperature.
When vaporised (atomised) in a flame, atoms emit light, as electrons that have absorbed energy fall back into lower energy shells; the wavelength of the emitted light is characteristic of the element and the number of photons emitted is in proportion to the elemental concentration. These characteristics of the light emitted when elements are burned form the basis of one of the simplest means of determining the mineral composition of plants. An extract is converted into a fine mist (nebulised) and blown into a flame (e.g. acetylene burning in air). Elements do not need to be separated: all that is needed is a flame, a monochromator (an optical device that selects the wavelength, generally a diffraction grating) and a detector (e.g. a pho-tomultiplier) to measure how much light is emitted at that specific wavelength. For example, any potassium in an extract will be atomised and emit light at a wavelength of 766.5 nm; the amount of light will be determined by the photomultiplier and hence the concentration of potassium can be estimated. This is flame emission spectrometry (FES). Not all elements emit sufficient light for analysis using FES, but they can still be analysed using their absorption of light when atomised in a flame.
In atomic absorption spectrophotometry (AAS), the monochromator and detector are used to determine how much light of a specific wavelength is absorbed after passing through an atomised element. The light is emitted from a lamp that contains the element to be analysed (e.g. potassium), which is heated by a tungsten filament in an atmosphere of an inert gas such as argon. For potassium, the lamp will emit light at wavelengths of 769.9, 766.5, 407.7 and 404.4 nm. The monochromator is used to select one of these wavelengths and the element in the flame will absorb light in proportion to its concentration - and so can be determined quantitatively. Lamps can be obtained for a wide range of elements and although an air/acetylene flame is not hot enough to atomise all elements (e.g. silicon), hotter flames can be obtained by burning acetylene in nitrous oxide.
While conventional FES and AAS use a nebuliser to deliver solution into a flame, it is also possible to atomise elements using a hollow graphite rod that is heated electrically to several thousand degrees. This 'graphite furnace' (GF) can be used to atomise a solution or solid material without the need for extraction: elemental analysis is by AAS (termed GF-AAS). A further possibility is to spray solution into a stream of argon that flows into a 'torch' where the gas stream is heated to about 10 000°C using a radio-frequency generator. At this temperature, a plasma forms where the atoms are present in an ionized state. This is known as an inductively coupled plasma or ICP. Elemental composition can be determined using optical emission spectroscopy.
ICP can also be combined with mass spectrometry, where the analysis depends not on optical emission, but on the determination of the mass of elements ionised in the plasma (ICP-MS). The solute can be introduced to the plasma via a nebuliser or directly, without first making a liquid extract, using a laser (laser ablation ICP-MS).
Unlike AAS or FES, which provides information on a single element for each test, ICP-MS can provide information on many elements in a single analysis.
Elements can also be estimated by their emission of X-rays. Just as light is emitted as electrons return to the ground state after absorbing energy when raised to high temperature, X-rays are emitted when elements are excited in a beam of X-rays (high-energy photons) or high-energy electrons: the process is known as X-ray fluorescence (XRF). This is a very powerful tool for biologists, especially when used in conjunction with an electron microscope (see Section 2.6.2).
The estimation of the concentration of elements using ion-specific electrodes is based on completely different properties of materials than those discussed so far. A membrane is used to separate ions and this leads to a difference in voltage across that membrane. The best known of ion-specific electrodes is the pH electrode. In this electrode, the membrane is made of a glass that is permeable to hydrogen ions, but not to other ions. Hydrogen ions diffuse through the glass and come to equilibrium with the external solution; this leads to a difference of voltage across the membrane, which is measured using a high-impedance voltmeter in conjunction with a standard 'reference' electrode: the potential difference is directly proportional to the logarithm of the ionic concentration in the external solution (according to the Nernst equation; see Section 3.6.4). Apart from hydrogen ions, there are electrodes whose potential is responsive to the concentration of NH4+, Ba2+, Ca2+, Cd2+, Cu2+, Pb2+, Hg2+, K+, Na+, Ag+, Br-, CO32-, Cl-, CN-, F-, I-, NO3-, NO2-, ClO4-, S- and SCN- - none is as specific as the pH electrode and so care has to be taken in the presence of other ions that can also cross the membrane (see also Section 220.127.116.11).
This is a form of liquid chromatography where ions in solution are separated by their interaction with a resin. Generally, the ions are detected by the conductivity of the effluent from the column in which the ions have been separated; ion chromatography can be used to detect and quantify anions as well as cations and each analytical run provides information on all the ions that are separated.
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