FE 315 Instrumental Analysis
Atomic Absorption Spectroscopy
Instructor: Prof. Dr. Şenol İBANOĞLU
Atomic spectroscopy requires that atoms of the element of interest be in the atomic state (not combined with other elements in a compound) and that they be well separated in space. In foods, virtually all elements are present as compounds or complexes and, therefore, must be converted to neutral atoms (atomized) before atomic absorption or emission measurements can be made. Atomization involves separating particles into individual molecules (vaporization) and breaking molecules into atoms. It is usually accomplished by exposing the analyte (the substance being measured) to high temperatures in a ﬂame or plasma although other methods may be used. A solution containing the analyte is introduced into the ﬂame or
plasma as a ﬁne mist. The solvent quickly evaporates, leaving solid particles of the analyte that vaporize and decompose to atoms that may absorb radiation (atomic absorption) or become excited and subsequently emit radiation (atomic emission).
Atomic Absorption Spectroscopy (AAS)
AAS is an analytical method based on the absorption of ultraviolet or visible radiation by free atoms in the gaseous state. It is a relatively simple method and was the most widely used form of atomic spectroscopy in food analysis for many years. It is being gradually replaced by the more versatile inductively coupled plasma (ICP) spectroscopy and inductively coupled plasma-mass spectrometry. Two types of atomization are commonly used in AAS: ﬂame atomization and electrothermal (graphite furnace) atomization.
Principles of Flame Atomic Absorption Spectroscopy
In ﬂame AAS, a nebulizer–burner system is used to convert a solution of the sample into an atomic vapor. It is important to note that the sample must be in solution (usually an aqueous solution) before it can be analyzed by ﬂame AAS. The sample solution is nebulized (dispersed into tiny droplets), mixed with fuel and an oxidant, and burned in a ﬂame produced by oxidation of the fuel by the oxidant. Atoms and ions are formed within the ﬂame as analyte compounds are decomposed by the high temperatures. The ﬂame itself serves as the sample compartment. The temperature of the ﬂame is important because it will affect the efﬁciency of converting compounds to atoms and ions and because it inﬂuences the distribution between atoms and ions in the ﬂame. Atoms and ions of the same element produce different spectra so they absorb radiation of different wavelengths. Therefore, it is desirable to choose a ﬂame temperature that will maximize atomization and minimize ionization because the radiation coming from the lamp has emission lines speciﬁc to the corresponding atoms, not ions. This
means that absorption efﬁciency will be decreased when atoms become ionized. Both atomization efﬁciency and ionization increase with increasing ﬂame temperature, so choice of the optimal ﬂame is not a simple matter. Flame characteristics may be manipulated by choice of oxidant and fuel and by adjustment of the oxidant/fuel ratio. The most common oxidant-fuel combinations are air-acetylene and nitrous oxide-acetylene. Also, adding cesium, an element with low ionization energy, to the sample will suppress ionization of other elements in the sample. The instrument instruction manual or the literature should be consulted for recommended ﬂame characteristics.
Once the sample is atomized in the ﬂame, its quantity is measured by determining the attenuation of a beam of radiation passing through the ﬂame. For the measurement to be speciﬁc for a given element, the radiation source is chosen so that the emitted radiation contains an emission line that corresponds to one of the most intense lines in the atomic spectrum of the element being measured. This is accomplished by fabricating lamps in which the element to be determined serves as the cathode. Thus, the radiation emitted from the lamp is the emission spectrum of the element.
The emission line of interest is isolated by passing the beam through a monochromator so that only radiation of a very narrow bandwidth reaches the detector. Usually, one of the strongest spectral lines is chosen; for example, for sodium the monochromator is set to pass radiation with a wavelength of 589.0 nm. Note that the intensity of the radiation leaving the ﬂame is less than the intensity of radiation coming from the source. This is because sample atoms in the ﬂame absorb some of the radiation. Note also that the line width of the radiation from the source is narrower than the corresponding line width in the absorption spectrum. This is because the higher temperature of the ﬂame causes a broadening of the line width. The amount of radiation absorbed by the sample is given by Beer’s law:
Principles of Electrothermal Atomic Absorption Spectroscopy (Graphite Furnace AAS)
Electrothermal AAS is identical to ﬂame AAS except for the atomization process. Electrothermal atomization involves heating the sample to a temperature (2000–3000◦C) that produces volatilization and atomization. This is accomplished in a tube or cup positioned in the light path of the instrument so that absorbance is determined in the space directly above the surface where the sample is heated. The advantages of electrothermal atomization are that it can accommodate smaller samples than are required for ﬂame atomic absorption and that limits of detection are lower. Disadvantages are the added expense of the electrothermal furnace, lower sample throughput, more difﬁcult operation, and lower precision. In addition, matrix interferences are more of a problem with electrothermal atomization.
Instrumentation for Atomic Absorption Spectroscopy
Atomic absorption spectrometers consist of the following components :
1. Radiation source, a hollow cathode lamp (HCL) or an electrode-less discharge lamp (EDL)
2. Atomizer, usually a nebulizer–burner system or an electrothermal furnace
3. Monochromator, usually an ultraviolet-visible (UV-Vis) grating monochromator
4. Detector, a photomultiplier tube (PMT) or a solid-state detector (SSD)
In double-beaminstruments, which are by far the most common, the beam from the light source is split by a rotating mirrored chopper into a reference beam and a sample beam. The reference beam is diverted around the sample compartment (ﬂame or furnace) and recombined before passing into the monochromator. The electronics are designed to produce a ratio of the reference and sample beams. This way, ﬂuctuations in the radiation source and the detector are canceled out, yielding a more stable signal.
The radiation source in atomic absorption spectrometers may be either a HCL or an EDL. Hollow cathode lamps consist of a hollow tube ﬁlled with argon or neon, an anode made of tungsten, and a cathode madeof the metallic form of the element being measured. When voltage is applied across the electrodes, the lamp emits radiation characteristic of the metal in the cathode; if the cathode is made of iron, an iron spectrum is emitted. When this radiation passes through a ﬂame containing the sample, iron atoms in the ﬂame will absorb some of it because it contains radiation of exactly the right energy for exciting iron atoms. This makes sense when we remember that for a given electronic transition, either up or down in energy, the energy of an emitted photon is exactly the same as the energy of an absorbed photon. Of course, this means that it is necessary to use a different lamp for each element analyzed (there are a limited number of multielement lamps available that contain cathodes made of more than one element). HCLs for about 60 metallic elements may be purchased from commercial sources, which means that atomic absorption may be used for the analysis of up to 60 elements.
Several types of atomizers are used in AAS: These include ﬂame, electrothermal, cold vapor technique for mercury, and hydride generation. The ﬂame atomizer consists of a nebulizer and a burner. The nebulizer is designed to convert the sample solution into a ﬁne mist or aerosol. This is accomplished by aspirating the sample through a capillary into a chamber through which oxidant and fuel are ﬂowing. The chamber contains bafﬂes which remove larger droplets, leaving a very ﬁne mist. Only about 1% of the total sample is carried into the ﬂame by the oxidant–fuel mixture. The larger droplets fall to the bottom of the mixing chamber and are collected as waste. The burner head contains a long, narrow slot that produces a ﬂame that may be 5–10 cm in length. This gives a long path length that increases the sensitivity of the measurement.
Flame characteristics may be manipulated by adjusting oxidant/fuel ratios and by choice of oxidant and fuel. Air-acetylene and nitrous oxide-acetylene are the most commonly used oxidant–fuel mixtures although other oxidants and fuels may be used for some elements. Electrothermal atomizers are typically cylindrical graphite tubes connected to an electrical power supply. They are commonly referred to as graphite furnaces. The sample is introduced into the tube through a small hole using a microliter syringe (sample volumes normally range from 0.5 to 10 μL). During operation, the system is ﬂushed with an inert gas to prevent the tube from burning and to exclude air from the sample compartment. The tube is heated electrically. Through a stepwise increase in temperature, ﬁrst the sample solvent is evaporated, then the sample is ashed, and ﬁnally the temperature is rapidly increased to 2,000–3,000◦C to quickly vaporize and atomize the sample.
The monochromator is positioned in the optical path between the ﬂame or furnace and the detector. Its purpose is to isolate the resonance line of interest from the rest of the radiation coming from the ﬂame or furnace and the lamp so that only radiation of the desired wavelength reaches the detector. Typically, monochromators of the grating type are used.
Two types of detectors are used in AA spectrometers, photomultiplier tubes and solid-state detectors. Detectors convert the radiant energy reaching it into an electrical signal. This signal is processed to produce either an analog or a digital readout. Modern instruments are interfaced with computers for data collection, manipulation, and storage.
According to Beer’s law, absorbance is directly related to concentration. However, a plot of absorbance vs.concentration will deviate from linearitywhen concentration exceeds a certain level. Therefore, it is always necessary to calibrate the instrument using appropriate standards that closely resemble the sample. This may be done by running a series of standards and plotting absorbance vs. concentration or, in the case of most modern instruments, programming the instrument to read in units of concentration.
Selection of Standards:
The ﬁrst step in calibration is to select the number and concentrations of standards to use. It is best to use standards that are at least 99.999% pure when preparing multielement standards. When operating in the linear range, only one standard is needed. The linear range may be deter mined by running a series of standards of increasing concentration and plotting absorbance vs. concentration. Operating manuals should contain values for linear ranges. The concentration of the standard should be higher than that of the most concentrated sample. If the range of concentration exceeds the linear range, multiple standards must be used or the sample diluted. Again, the concentration of the most concentrated standard should exceed the concentration of the most concentrated sample.
Interferences in Atomic AbsorptionSpectroscopy
With any analytical technique, it is important to be on the lookout for possible interferences. Atomic spectroscopy techniques are powerful partly because measurements of individual elements can usually be made without laborious separations. There are two main reasons for this. First, as mentioned previously, a single narrow emission line is used for the measurement. Second, these are relative techniques; that is, quantitative results for an unknown sample are possible only through comparison with a standard of known concentration. If there are matrix-effect problems, they can often be overcome by using the same matrix for the standard or by employing the method of additions approach.
The following is a brief discussion of common interference problems in AAS. Two types of interferences are encountered in AAS: spectral interference and nonspectral interference.
Absorption of Source Radiation: An element in the sample other than the element of interest may absorb at the wavelength of the spectral band being used. Such interference is rare because emission lines from HCLs are so narrow that only the element of interest is capable of absorbing the radiation. One example where this problem does occur is with the interference of iron in zinc determinations. Zinc has an emission line at 213.856 nm, which overlaps the iron line at 213.859 nm. The problem may be solved by choosing an alternative emission line formeasuring zinc or by narrowing the monochromator slit width.
Background Absorption of Source: Radiation particulates present as a result of incomplete atomization may scatter source radiation, thereby attenuating the radiation reaching the detector. This problem may be overcome by going to a higher ﬂame temperature to ensure complete atomization of the sample. Some instruments are equipped with automatic background correction devices.
Transport Interferences: These result when something in the sample solution affects the rate of aspiration, nebulization, or transport into the ﬂame. Transport interferences are rarely a problem with graphite furnace instruments but may cause substantial errors in ﬂame AAS. Such factors as viscosity, surface tension, vapor pressure, and density of the sample solution can inﬂuence the rate of transport of sample into the ﬂame. Transport interferences often can be overcome by matching as closely as possible the physical properties of the sample and the standards. For example, use the same solvent for the sample and the standard, or add the interferant in the sample (e.g., sugar) to the standard. The method of additions also may be used to overcome transport interferences.
Solute Volatilization Interferences: Matrix interferences result from the reduction or enhancement of the emitted signal due to differences between the composition of the standard and the composition of the sample. Flame atomic absorption is prone to solute vaporization interferences, which are changes in the lateral migration of an analyte due to the matrix. For example it has been observed in ﬂame absorption and emission that alkaline elements are depressed by elevated levels of aluminum and phosphorus. Chemical interferences occur in ﬂames when calcium is determined in a matrix that varies in phosphate concentration. This is because phosphate reduces the number of free calcium atoms in the ﬂame by converting calcium phosphate to calcium pyrophosphate, which is not decomposed by the ﬂame.
Ionization Interference: Ionization of analyte atoms in the ﬂame may cause a signiﬁcant interference. Easily ionized elements (EIE) like K, Na, Li, Cs, produce large changes in emission intensities in the ﬂame. (Remember that absorption and emission lines of atoms and ions of the same element are different and that atomic absorption spectrometers are tuned to measure atomic absorption, not ionic absorption. Therefore, any factor that reduces the concentration of atoms in the ﬂame will lower the absorbance reading. Ionization increases with increasing ﬂame temperature and normally is not a problem in air-acetylene ﬂames because the temperature is not high enough. It can be a problem in nitrous oxide-acetylene ﬂames with elements that have ionization potentials of 7.5 eV or less. Ionization is suppressed by the presence of EIE, such as potassium, through mass action. When potassium ionizes, it increases the concentration of electrons in the ﬂame and shifts the above equilibrium to the left. Reagents added to reduce ionization are called ionization suppressors.
APPLICATIONS OF ATOMIC ABSORPTION SPECTROSCOPY IN THE FOOD SECTOR
Atomic absorption and emission spectroscopy are widely used for the quantitative measurement of mineral elements in foods. In principle, any food may be analyzed with any of the atomic spectroscopy methods discussed. In most cases, it is necessary to ash the food to destroy organic matter and to dissolve the ash in a suitable solvent (usually water or dilute acid) prior to analysis. Proper ashing is critical to accuracy. Some elements may be volatile at temperatures used in dry ashing procedures. Volatilization is less of a problem in wet ashing, except for the determination of boron, which is recovered better using a dry ashing method. However, ashing reagents may be contaminated with the analyte. It is therefore wise to carry blanks throughout the ashing procedure.
Some liquid products may be analyzed without ashing, provided appropriate precautions are taken to avoid interferences. For example, vegetable oils may be analyzed by dissolving the oil in an organic solvent such as acetone or ethanol and aspirating the solution directly into a ﬂame atomic absorption spectrometer. Milk samples may be treated with trichloroacetic acid to precipitate the protein; the resulting supernatant is analyzed directly. A disadvantage of this approach is that the sample is diluted in the process and the analyte can become entrapped or complexed to the precipitated proteins. This may be a problem when analytes are present in low concentrations. An alternative approach is to use a graphite furnace for atomization. For example, an aliquot of an oil may be introduced directly into a graphite furnace for atom ization. The choice of method will depend on several factors, including instrument availability, cost, precision/sensitivity, and operator skill.
Reagents: Since concentrations of many mineral elements in foods are at the trace level, it is essential to use highly pure chemical reagents and water for preparation of samples and standard solutions. Only reagent grade chemicals should be used. Water may be puriﬁed by distillation, deionization, or a combination of the two. Reagent blanks should always be carried through the analysis. The purest reagents are prepared by sub-boiling distillation.
Standards: Quantitative atomic spectroscopy depends on comparison of the sample measurement with appropriate standards. Ideally, standards should contain the analyte metal in known concentrations in a solution that closely approximates the sample solution in composition and physical properties. If multielement standards are used the individual elements that compose the multielement standard must be checked for other elements that may be present as contaminants, and the ﬁnal concentration needs to include all contaminants. A series of standards of varying concentrations should be run to generate a calibration curve. Because many factors can affect the measurement, such as ﬂame temperature, aspiration rate, and the like, it is essential to run standards frequently, preferably right before and/or right after running the sample. Standard solutions may be purchased from commercial sources, or they may be prepared by the analyst. Obviously, standards must be prepared with extreme care since the accuracy of the analyte determination depends on the accuracy of the standard. Perhaps the best way to check the accuracy of a given assay procedure is to analyze a reference material of known composition and similar matrix.
Labware: Vessels used for sample preparation and storage must be clean and free of the elements of interest. Plastic containers are preferable because glass has a greater tendency to adsorb and later leach metal ions. All labware should be thoroughly washed with a detergent, carefully rinsed with distilled or deionizedwater, soaked in an acid solution (1 N HCl is sufﬁcient for most applications), and rinsed again with distilled or deionized water.
1. Turn the lamp current control knob to the off position.
2. Install the required lamp in the lamp compartment.
3. Turn on main power and power to lamp. Set lamp current to the current shown on the
4. Select required slit width and wavelength and align light beam with the optical system.
5. Ignite ﬂame and adjust oxidant and fuel ﬂow rates.
6. Aspirate distilled water. Aspirate blank and zero instrument.
7. Aspirate standards and sample.
8. Aspirate distilled water.
9. Shut down instrument.
Calibration curve for cupper