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2.3 ATOMIC SPECTROMETRY: EMISSION AND ABSORPTION

          These techniques are used to determine the concentration of certain metallic ions by measuring the intensity of emission or absorption of light at a particular wavelength by the atomic vapour of the element generated from the substance, for example, by introducing a solution of the substance into a flame.

FOR ATOMIC EMISSION SPECTROMETRY

           Atomic emission is a process that occurs when electromagnetic radiation is emitted by excited atoms or ions. In atomic emission spectrometry the sample is subjected to temperatures high enough to cause not only dissociation into atoms, but also to cause significant amounts of collisional excitation and ionization of the sample atoms to take place.  Once the atoms and ions are in the excited states, they can decay to lower states through thermal or radiative (emission) energy transitions and electromagnetic radiation is emitted. An emission spectrum of an element contains several more lines than the corresponding absorption spectrum. Atomic emission spectrometry is a technique for determining the concentration of an element in a sample by measuring the intensity of one of the emission lines of the atomic vapour of the element generated from the sample. The determination is carried out at the wavelength corresponding to this emission line.

Apparatus

The apparatus essentially consists of the following:
(a) a sample introduction and nebulization system;
(b) a flame to generate the atoms to be determined;
(c) a monochromator;
(d) a detector;
(e) a data-acquisition unit.

           Oxygen, air and a combustible gas such as hydrogen, acetylene, propane or butane may be used in flames. The atomization source is critical, since it must provide sufficient energy to excite and atomize the atoms. The atomic spectra emitted from flames have the advantage of being simpler than those emitted from other sources, the main limitation being that the flames are not powerful enough to cause emission for many elements allowing their determination. Acidified water is the solvent of choice for preparing test and standard preparations, although organic solvents may also be used if precautions are taken to ensure that the solvent does not interfere with the stability of the flame.

Interferences

           Spectral interference is reduced or eliminated by choosing an appropriate emission line for measurement or by adjusting the slit for spectral band-width. Physical interference is corrected by diluting the sample preparation, by matching the matrix or by using the method of standard additions.  Chemical interference is reduced by using chemical modifiers or ionization buffers.

Memory effect

            The memory effect caused by deposit of analyte in the apparatus may be limited by thoroughly rinsing between runs, diluting the solutions to be measured if possible and thus reducing their salt content, and by aspirating the solutions through as swiftly as possible.

Method

           (Note Evaluate and select the type of material of construction, pretreatment, and cleaning of analytical labware used in the Atomic Absorption Spectrophotometric analyses. The material must be inert and, depending on the specific application, resistant to caustics, acids, and/or organic solvents, i.e., plastic.)

           Operate an atomic emission spectrometer in accordance with the manufacturer’s instructions at the prescribed wavelength. Optimize the experimental conditions (flame temperature, burner adjustment, use of an ionic buffer, concentration of solutions) for the specific element to be analyzed and in respect of the sample matrix. Introduce a blank solution into the atomic generator and adjust the instrument reading to zero or to its blank value. Introduce the most concentrated standard preparation and adjust the sensitivity to obtain a suitable reading. It is preferable to use concentrations which fall within the linear part of the calibration curve. If this is not possible, the calibration plots may also be curved and are then to be applied with appropriate calibration software.
            Determinations are made by comparison with standard preparations with known concentrations of the element to be determined either by the method of direct calibration (Method I) or the method of standard additions (Method II).

METHOD I: METHOD OF DIRECT CALIBRATION

           For routine measurements three standard preparations of the element to be determined and a blank are prepared and examined. Prepare the solution of the substance to be examined (test preparation) as prescribed in the monograph.  Prepare not fewer than three standard preparations of the element to be determined, the concentrations of which span the expected value in the test preparation.  For assay purposes, optimal calibration levels are between 0.7 and 1.3 times the expected content of the element to be determined or the limit prescribed in the monograph. For purity determination, calibration levels are between the limit of detection and 1.2 times the limit specified for the element to be determined. Any reagents used in the preparation of the test preparation are added to the standard preparations and to the blank solution at the same concentration. Introduce each of the solutions into the instrument using the same number of replicates for each solution, to obtain a steady reading. 

           Calculation Prepare a calibration curve from the mean of the readings obtained with the standard preparation by plotting the means as a function of concentration. Determine the concentration of the element in the test preparation from the curve obtained.

METHOD II: METHOD OF STANDARD ADDITIONS

            Add to at least three similar volumetric flasks equal volumes of the solution of the substance to be examined (test preparation) prepared as prescribed.  Add to all but one of the flasks progressively larger volumes of a standard preparation containing a known concentration of the element to be determined to produce a series of solutions containing steadily increasing concentrations of that element known to give responses in the linear part of the curve, if at all possible. Dilute the contents of each flask to volume with solvent. Introduce each of the solutions into the instrument using the same number of replicates for each solution, to obtain a steady reading.

            Calculation Calculate the linear equation of thegraph using a least-squares fit, and derive from it the concentration of the element to be determined in the test preparation.  Validation of the method Satisfactory performance of methods prescribed in monographs is verified at suitable time intervals.

LINEARITY

           Prepare and analyze not fewer than four standardpreparations over the calibration range and a blank solution. Perform not fewer than five replicates. The calibration curve is calculated by least-square regression from all measured data. The regression curve, the means, the measured data and the confidence interval of the calibration curve are plotted.
           The operating method is valid when:
           — the correlation coefficient is at least 0.99,
           — the residuals of each calibration level are randomly distributed around the calibration curve.

          Calculate the mean and relative standard deviation for the lowest and highest calibration level.  When the ratio of the estimated standard deviation of the lowest and the highest calibration level is less than 0.5 or greater than 2.0, a more precise estimation of the calibration curve may be obtained using weighted linear regression. Both linear and quadratic weighting functions are applied to the data to find the most appropriate weighting function to be employed. If the means compared to the calibration curve show a deviation from linearity, two-dimensional linear regression is used.

ACCURACY

           Verify the accuracy preferably by using a certified standard material. Where this is not possible, perform a test for recovery.

RECOVERY

           For assay determinations a recovery of 90 per cent to 110 per cent is to be obtained. For other determinations, for example for trace element determination, the test is not valid if recovery is outside of the range 80 per cent to 120 per cent at the theoretical value. Recovery may be determined on a suitable standard preparation (matrix solution) which is spiked with a known quantity of analyte (middle concentration of the calibration range).

REPEATABILITY

           The repeatability is not greater than 3 per cent for an assay and not greater than 5 per cent for an impurity test.

LIMIT OF QUANTIFICATION

           The limit of quantification can be estimated by calculating the standard deviation of not less than six replicate measurements of a blank solution, divided by the slope of a standard curve, and multiplying by 10. If validating a procedure using the method of standard additions, the slope of standards applied to a solution of the substance being examined (test preparation) is used. Other suitable approaches can be used.

FOR ATOMIC ABSORPTION SPECTROMETRY

            Atomic absorption is a process that occurs when a ground state-atom absorbs electromagnetic radiation of a specific wavelength and is elevated to an excited state. The atoms in the ground state absorb energy at their resonant frequency and the  electromagnetic radiation is attenuated due to resonance absorption.  The energy absorption is virtually a direct function of the number of atoms present.

            This appendix provides general information and defines the procedures used in element determinations by atomic absorption spectrometry, either atomization by flame, by electrothermal vaporization in a graphite furnace, by hydride generation or by cold vapour technique for mercury.

            Atomic absorption spectrometry is a technique for determining the concentration of an element in a sample by measuring the absorption of electromagnetic radiation by the atomic vapour of the element generated from the sample. The determination is carried out at the wavelength of one of the absorption (resonance) lines of the element concerned. The amount of radiation absorbed is, according to the Beer’s law, proportional to the element concentration.

Apparatus

           The apparatus consists essentially of the following:
           (a) a source of radiation;
           (b) a sample introduction device;
           (c) a sample atomizer;
           (d) a monochromator or polychromator;
           (e) a detector;
           (f) a data-acquisition unit.
           The apparatus is usually equipped with a background correction system. Hollow-cathode lamps and electrodeless discharge lamps (EDL) are used as radiation source. The emission of such lamps consists of a spectrum showing very narrow lines with half-width of about 0.002 nm of the element being determined.
           There are three types of sample atomizers:

FLAME TECHNIQUE

            A flame atomizer is composed of a nebulization system with a pneumatic aerosol production accessory, a gas-flow regulation and a burner. Although other flame types have been documented, the most commonly used flame is an air–acetylene flame. Because the temperature of the air–acetylene flame is not sufficient to destroy oxides that might form or are present, a nitrous oxide–acetylene flame often is used, depending on the analyte and nature of the sample. The air–acetylene flame burns within a temperature range of 2125° to 2400°, but the nitrous oxide–acetylene flame burns within a temperature range of 2650° to 2800°. The configuration of the burner is adapted to the gases used and the gas flow is adjustable. Samples are nebulized, acidified water being the solvent of choice for preparing test and standard preparations. Organic solvents may also be used if precautions are taken to ensure that the solvent does not interfere with the stability of the flame.

ELECTROTHERMAL ATOMIZATION TECHNIQUE

           An electrothermal atomizer is generally composed of a graphite tube furnace and an electric power source. Electrothermal atomization in a graphite tube furnace atomizes the entire sample and retains the atomic vapour in the light path for an extended period. This improves the detection limit. Samples, liquid as well as solid, are introduced directly into the graphite tube furnace, which is heated in a programmed series of steps to dry the sample and remove major matrix components by pyrolysis and to then atomize all of the analyte. The furnace is cleaned using a final temperature higher than the atomization temperature. The flow of an inert gas during the pyrolysis step in the graphite tube furnace allows a better performance of the subsequent atomization process.

COLD VAPOUR AND HYDRIDE GENERATION TECHNIQUE

           The atomic vapour may also be generated outside the spectrometer. This is notably the case for the cold-vapour method for mercury or for certain hydride-forming elements such as arsenic, antimony, bismuth, selenium and tin. For mercury, atoms are generated by chemical reduction with stannous chloride or sodium borohydride and the atomic vapour is swept by a stream of an inert gas into a cold quartz cell mounted in the optical path of the instrument. Hydrides thus generated are swept by an inert gas into a heated cell in which they are dissociated into atoms. Both cold vapour and hydride generation techniques are very sensitive and have detection limits in the part per billion (ppb) or part per trillion (ppt) range.

Interference

           Chemical, physical, ionization, and spectral interferences are encountered in atomic absorption measurements. Chemical interference is compensated by addition of matrix modifiers, of releasing agents or by using high temperature produced by a nitrous
oxide-acetylene flame; the use of specific ionization buffers (for example, lanthanum and caesium) compensates for ionization interference; by dilution of the sample, through the method of standard additions or by matrix matching, physical interference due to high salt content or viscosity is eliminated.  Spectral interference results from the overlapping of resonance lines and can be avoided by using a different resonance line. The use of Zeeman background correction also compensates for spectral interference and interferences from molecular absorption, especially when using the electrothermal atomization technique. The use of multi-element hollow-cathode lamps may also cause spectral interference. Specific or non-specific absorption is measured in a spectral range defined by the bandwidth selected by the monochromator (0.2 to 2 nm).

Background correction

           Scatter and background in the flame or the electrothermal atomization technique increase the measured absorbance values. Background absorption covers a large range of wavelengths, whereas atomic absorption takes place in a very narrow wavelength range of about 0.005 to 0.02 nm. Background absorption can in principle be corrected by using a blank solution of exactly the same composition as the sample, but without the specific element to be determined, although this method is frequently impracticable. With the electrothermal atomization technique the pyrolysis temperature is to be optimized to eliminate the matrix decomposition products causing background absorption. Background correction can also be made by using two different light sources, the hollow-cathode lamp that measures the total absorption (element and background) and a deuterium lamp with a continuum emission from which the background absorption is measured. Background is corrected by subtracting the deuterium lamp signal from the hollow-cathode lamp signal. This method is limited in the spectral range on account of the spectra emitted by a deuterium lamp from 190 to 400 nm. Background can also be measured by taking readings at a non-absorbing line near the resonance line and then subtracting the results from the measurement at the resonance line. Another method for the correction of background absorption is the Zeeman effect (based on the Zeeman splitting of the absorption line in a magnetic field). This is particularly useful when the background absorption shows fine structure. It permits an efficient background correction in the range of 185 to 900 nm.

Choice of the operating conditions

           After selecting the suitable wavelength and slit width for the specific element, the need for the following has to be ascertained:
           — correction for non-specific background absorption,
           — chemical modifiers or ionization buffers to be added to the sample as well as to blank and standard solutions,
           — dilution of the sample to minimize, for example, physical interferences,
           — details of the temperature programme, preheating, drying, pyrolysis, post-atomization with ramp and hold times,
           — inert gas flow,
           — matrix modifiers for electrothermal atomization (furnace),
           — chemical reducing reagents for measurements of mercury or other hydride-forming elements along with cold vapour cell or heating cell temperature,
           — specification of furnace design.

Method

           (Note Evaluate and select the type of material of construction, pretreatment, and cleaning of analytical labware used in the Atomic Absorption Spectrophotometric analyses. The material must be inert and, depending on the specific application, resistant to caustics, acids, and/or organic solvents, i.e., plastic.)
           The preparation of the sample may require a dissolution, a digestion (mostly microwave-assisted), an ignition step or a combination thereof in order to clear up the sample matrix and/or to remove carbon-containing material. If operating in an open system, the ignition temperature should not exceed 600°, due to the volatility of some metals, unless otherwise stated in the monograph.
           Operate an atomic absorption spectrometer in accordance with the manufacturer’s instructions at the prescribed wavelength. Introduce a blank solution into the atomic generator and adjust the instrument reading so that it indicates maximum transmission. The blank value may be determined by using solvent to zero the apparatus. Introduce the most concentrated standard preparation and adjust the sensitivity to obtain a maximum absorbance reading. Rinse in order to avoid contamination and memory effects. After completing the analysis, rinse with water or acidified water. 
           If a solid sampling technique is applied, full details of the procedure are provided in the monograph. 
           Ensure that the concentrations to be determined fall preferably within the linear part of the calibration curve. If this is not possible, the calibration plots may also be curved and are then to be applied with appropriate calibration software.
           Determinations are made by comparison with standard preparations with known concentrations of the element to be determined either by the method of direct calibration (Method I) or the method of standard additions (Method II).

METHOD I: DIRECT CALIBRATION

           For routine measurements three standard preparations and a blank solution are prepared and examined.
           Prepare the solution of the substance to be examined (test preparation) as prescribed in the monograph. Prepare not fewer than three standard preparations of the element to be determined, the concentrations of which span the expected value in the test preparation. For assay purposes, optimal calibration levels are between 0.7 and 1.3 times the expected content of the element to be determined or the limit prescribed in the monograph. For purity determination, calibration levels are the limit of detection and 1.2 times the limit specified for the element to be determined. Any reagents used in the preparation of the test preparation are added to the standard and blank solutions at the same concentration. Introduce each of the solutions into the instrument using the same number of replicates for each of the solutions to obtain a steady reading.

            Calculation Prepare a calibration curve from the mean of the readings obtained with the standard preparations by plotting the means as a function of concentration. Determine the concentration of the element in the test preparation from the curve obtained.

METHOD II: STANDARD ADDITIONS

            Add to at least three similar volumetric flasks equal volumes of the solution of the substance to be examined (test preparation) prepared as prescribed.  Add to all but one of the flasks progressively larger volumes of a standard preparation containing a known concentration of the element to be determined to produce a series of solutions containing steadily increasing concentrations of that element known to give responses in the linear part of the curve, if possible. Dilute the contents of each flask to volume with solvent.
Introduce each of the solutions into the instrument, using the same number of replicates for each of the solutions, to obtain a steady reading.

            Calculation Calculate the linear equation of the graph using a least-squares fit and derive from it the concentration of the element to be determined in the test preparation.

            Validation of the method  
            
Satisfactory performance of methods prescribed in monographs is verified at suitable time intervals.

LINEARITY

           Prepare and analyze not fewer than four standard preparations over the calibration range and a blank solution. Perform not fewer than five replicates.
           The calibration curve is calculated by least-square regression from all measured data. The regression curve, the means, the measured data and the confidence interval of the calibration curve are plotted. The operating method is valid when:
           — the correlation coefficient is at least 0.99,
           — the residuals of each calibration level are randomly distributed around the calibration curve. Calculate the mean and relative standard deviation for the lowest and highest calibration level.
           When the ratio of the estimated standard deviation of the lowest and the highest calibration level is less than 0.5 or greater than 2.0, a more precise estimation of the calibration curve may be obtained using weighted linear regression. Both linear and quadratic weighting functions are applied to the data to find the most appropriate weighting function to be employed. If the means compared to the calibration curve show a deviation from linearity, two-dimensional linear regression is used.

ACCURACY

           Verify the accuracy preferably by using a certified reference material. Where this is not possible, perform a test for recovery.

RECOVERY

           For assay determinations a recovery of 90 per cent to 110 per cent is to be obtained. For other determinations, for example, for trace element determination the test is not valid if recovery is outside of the range 80 per cent to 120 per cent at the theoretical value. Recovery may be determined on a suitable standard preparation (matrix solution) which is spiked with a known quantity of analyte (middle concentration of the calibration range).

REPEATABILITY

           The repeatability is not greater than 3 per cent for an assay and not greater than 5 per cent for an impurity test.

LIMIT OF QUANTIFICATION

           The limit of quantification can be estimated by calculating the standard deviation of not less than six replicate measurements of a blank solution, divided by the slope of a standard curve, and multiplying by 10. If validating a procedure using the method of standard additions, the slope of standards applied to a solution of the substance being examined (test preparation) is used. Other suitable approaches can be used.

APPENDICES • 2.3 ATOMIC SPECTROMETRY: EMISSION AND ABSORPTION
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หมายเหตุ / Note : TP II 2011 PAGE 377-378