Calibration and General Test of AAS (Atomic Absorption Spectrophotometry) Instrument

Atomic Absorption Spectrophotometry is designed to determine the amount (concentration) of an object element in a sample, utilizing the phenomenon that the atoms in the ground state absorb the light of characteristic wavelength passing through an atomic vapor layer of the element.
Apparatus Usually the apparatus consists of a light source, a sample-atomizer, a spectroscope, and a photometer, and a recording system. Some are equipped with a background compensation system. For the light source, a hollow cathode lamp and a discharge lamp are mainly used. To the sample-atomizer, the flame type, electrothermal type, and the cold-vapor type are applied.
The cold-vapor flameless type is categorized as the two methods: reduction vaporizing method and heat vaporizing method. The flame type is composed of a burner and a gas-flow regulator, the electrothermal type is composed of an electric furnace and a power source, and the cold-vapor type is composed of a mercury
generator by chemical reduction-vaporization and thermal reduction-vaporization and an absorption cell. For the spectroscope, a grating for light diffraction or an interference filter prism is used. The photometer mainly consists of a detector and a signal treatment system. A recording system is composed of a display and a recording device. A background compensation system is employed for the correction of matrix effects on the measuring system. Several principles can be utilized for background compensation, using the continuous spectrum sources, the Zeeman split spectrum, the nonresonance spectrum, or the self-inversion phenomena.
Procedure Unless otherwise specified, proceed by either of the following methods:

1. Flame Type Fit the specific light source lamp to the lamp housing, and switch on the instrument. Light the source lamp, adjust the wavelength dial of the spectroscope to the wavelength of the analytical line specified, and set at an appropriate current value and slit-width. Using the supporting gas and combustible gas specified, ignite the mixture of these gases, adjust the gas flow rate and pressure, and make the zero adjustment after nebulizing the solvent into the flame. Nebulize the test solution or the standard solution or control solution prepared by the method prescribed elsewhere, and measure the absorbance.
2. Electrothermal type Fit the specific light source to the lamp housing and switch on the instrument. After lighting the lamp and selecting the analytical wavelength specified in the individual monograph, set an appropriate electric current and slit-width. A suitable amount of sample solution, standard solution, or control solution, prepared as specified in the individual monograph, is injected to the furnace and an appropriate stream of inert gas is made to flow through the furnace. The specimen is dried and ashed, and the element included is atomized, on heating at appropriate temperature for an appropriate time in appropriate mode. The atomic absorption specified is observed and the intensity of absorption is measured.
3. Cold-vapor Type Fit the light source lamp specified on the photometer. Light the source lamp, adjust the wavelength dial of the spectroscope to the wavelength of the analytical line specified, and set at an appropriate current value and a slit-width.

Then, in the reduction vaporizing method, transfer the test solution or the standard solution or control solution to the closed vessel, reduce to the element by addition of a proper reducing agent, and vaporize. In the heat vaporizing method, vaporize the sample by heating. Measure the absorbance of the atomic vapor generated by these methods.
Usually, the determination can be done by an appropriate one of the methods given below. In the determination, the interference and background should be considered.

• Calibration Curve Method Prepare standard solutions of at least three different concentrations, measure the absorbances of these standard solutions, and prepare a calibration curve from the obtained values. Then measure the absorbance for the test solution adjusted in concentration to a measurable range, and determine the amount (concentration) of the object element from the calibration curve.
• Standard Addition Method To equal volumes of more than 2 of different test solutions, add the standard solution so that the stepwise increasing amounts of the object element are contained in the solutions, and add the solvent to make a definite volume. Measure the absorbance for each solution, and plot the amounts (concentration) of added standard object element on the abscissa and the absorbances on the ordinate on graph paper. Extend the calibration curve obtained by linking the plots, and determine the amount (concentration) of object element from the distance between the origin and the intersecting point of the calibration curve on the abscissa. This method is applicable only in the case that the calibration curve drawn as directed in (1) above passes through the origin.
• Internal Standard Method Prepare several solutions containing a constant amount of the prescribed internal standard element, and known, graded amounts of the standard object element. Using these solutions, measure the absorbances of the standard object element and the absorabance of internal standard element at the analytical wavelength of each element under the same measuring condition, and obtain the ratios of each absorbance of standard object element to the absorbance of the internal standard element. Prepare a calibration curve by plotting the amounts (concentrations) of standard object element on the abscissa and the ratios of absorbance on the ordinate. Then prepare the test solutions, adding the same amount of internal standard element as in the standard solution. Proceed under the same conditions as for preparing the calibration curve, obtain the ratio of the absorbance of standard object element to that of internal standard element, and determine the amount (concentration) of the object element from the calibration curve.

Note: For this test, avoid the use of reagents and test solutions which can interfere with the determination.

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Technology of Atomic Absorption Spectrophotometry (AAS)

When using atomic absorption spectrophotometry (AAS) as an analytical technique the absorption of light of free atoms is measured. Therefore it is one of the branches of atomic spectroscopy, together with flame photometry (see Standardbase techniques: “Flame Photometry” that measures the intensity of light emitted by free atoms when their electrons return to ground state after the excitation by light). However - unlike flame photometry - AAS is based on the “first half” of the excitation process, while atoms absorb light getting their
electrons from the ground state to a higher energy level.

Figure 1: Photograph of an atomic absorption spectrophotometer

Although the atomic absorption spectrophotometer (fig. 1.) is quite expensive, the technique is very wide-spread, thank to the fact that by AAS it is possible to determine about 70 elements (mainly metals) at very low concentrations. The sample is atomised at a very high temperature (2500-3000 ºC) and the free
atoms have line spectrum. It means that they can only absorb the energy of light at discrete energy levels according to the excitations of electrons. Excitation energies in this case are determined by the difference between the energy level of the ground state and one of the excitation states of their electrons. Only a
light with a concrete wavelength belongs to each of these excitation energies and when this light is absorbed it is missing from the continuous spectra of the electromagnetic radiation: a black line appears in the absorption spectrum of the atom. There are no vibration or rotation energy levels that would widen the lines to brands in the spectrum (like it happens in the case of UV-Vis spectrophotometry, when molecules and ions are measured, see Standardbase technique: “UV-Vis Spectrophotometry”). Using AAS free atoms are “lit” by
monochromatic light (called “resonance radiation” that has got a special wavelength) that belongs to one line of their spectrum and therefore it has the suitable excitation energy mentioned above. Only the examined atoms can absorb it. As a result of absorption, the intensity of light decreases, which is proportional to the number of the examined atoms being present. That makes very sensitive quantitative measurements possible.

 

 

Fig. 2: A schematic diagram of atomic absorption spectrometer

To produce the proper monochromatic light necessary for the AAS, so called “hollow cathode lamps” are used. The cathode of this sort of lamp is made of the metal under investigation (or its alloy). It means that different lamps are used for the determination of each element. It is named after the cylindrical shape of
the cathode that gives direction to emerging beam, and helps re-deposit sputtered atoms back on cathode. The anode is made of tungsten and the electrodes are surrounded by noble gases. At high voltage the cathode produces electrons that speeding up in the electric filed cause the ionisation of noble gas atoms. These high-speed noble gas ions bombard the cathode and therefore sputtering occurs, dislodging atoms from the surface of cathode. These free atoms are excited by the high-speed electrons and then emit the line spectrum
characteristic of the particular element that is the cathode made of.
AAS called a “destructive technique”, because only solutions containing the investigated element can be used. Solid samples should be accurately weighed and then dissolved, often using strong acids (e.g. in cases when soil samples contaminated with heavy metal ions are measured). However only a very small amount of sample is enough, because of the high sensitivity of the technique.
The solvent of the solution is evaporated and all materials present in the sample are vaporised and dissociated to atoms at the very high temperature. (The process in the reality is a bit more complicated, since ions and oxides are also produced, decomposition and association reactions take place too.) The following
atomisation methods are known:

• Flame atomisation
• Graphite furnace atomisation
• Mercury hydride atomisation (this is only mentioned here, but not used while doing Standardbase experiments).

The source of atoms is usually flame (“flame atomisation”). Metals could be measured at ppm concentration (part per million, that is mg kg-1 or mg dm-3 in case of dilute solutions). The sensitivity could be increased when the light travels for longer in the flame. Therefore most of the burners are about 5-10 cm long.
The accuracy is very good, about 1-2%. The sample solution is sprayed (“nebulized”) continuously into the flame (similarly to the flame photometer).
The graphite furnace AAS (GFAAS), a more recent technique is even more sensitive than the traditional, cheaper AAS using flame. Measuremnts could be done at ppb level (part per billion, ppb = 10-3 ppm, that is μg kg-1 or μg dm-3 in case of dilute solutions!). The accuracy is still about 20% in this latter case. The
drying, combustion, vaporisation and atomisation of sample happen in a heated graphite tube that is placed in the way of light. This “graphite furnace” is protected against oxidation by an inert gas (e.g. argon).
The other parts of the atomic absorption spectrophotometer are similar to the one used in the case of UV-Vis spectrophotometer. The monochromator that selects the proper wavelength of the emitted spectra is the usual prism or optical filter. The detector is a photo-electron multiplier that produces an electric sign proportional to the intensity of emitted light. (It contains diodes /electrodes with increasing potential/ between the photocathode and anode, where multiplied electron emission is caused by the electrons bumping in them.) The electric sign is converted and appears as absorbance on the read-out (fig. 2.)

Reference textbooks:

• D. A. Skoog - D. M. West - F. J. Holler: Fudamentals of Analytical Chemistry (Saunders College Publishing, Fort Worth, US 1992.)
• J. Kenkel: Analytical Chemistry for Technicians (Lewis Publishers, Boca Raton, US 1994.)

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Introduction Background of Atomic Absorption Spectroscopy

Atomic absorption spectroscopy (AAS) determines the presence of metals in liquid samples. Metals include Fe, Cu, Al, Pb, Ca, Zn, Cd and many more. It also measures the concentrations of metals in the samples. Typical concentrations range in the low mg/L range.
In their elemental form, metals will absorb ultraviolet light when they are excited by heat. Each metal has a characteristic wavelength that will be absorbed. The AAS instrument looks for a particular metal by focusing a beam of uv light at a specific wavelength through a flame and into a detector. The sample of interest is aspirated into the flame. If that metal is present in the sample, it will absorb some of the light, thus reducing its intensity. The instrument measures the change in intensity. A computer data system converts the change in intensity into an absorbance.
As concentration goes up, absorbance goes up. The researcher can construct a calibration curve by running standards of various concentrations on the AAS and observing the absorbances. In this lab, the computer data system will draw the curve for you! Then samples can be tested and measured against this curve.
It is important to understand the theory behind any instrument if you are to be successful using that instrument. However, AAS theory can be looked at either on a simplified basis or a complex basis. Try looking at the complex first--you may surprise yourself by understanding it!
Look over the following sections before the lab on Wednesday. If you do not understand the background section, go on to the other sections. We will have a chance to review the background in the lab prior to the experiment. You should understand the instrument's set-up and operation prior to lab.

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Atomic Absorption Spectrophotometer

Introduction

Atomic absorption absorption spectroscopy (AA orAAS) is one of the commonest instrumental

methods for analyzing for metals and some metalloids.

Metalloids like antimony, arsenic, selenium, and tellurium are now routinely analyzed by hydride generation AAS (HGAAS; see www.shsu.edu/~chm_tgc/sounds/sound.html and www.shsu.edu/chemistry/primers for animations and primers on that method). Inductively coupled plasma (ICP) is also a powerful analytical, instrumental method for these elements but at this point its much higher cost limits it widespread use as compared toAAS.

As the animation on AAS here shows, the main parts of the AAS system are a hollow cathode lamp, nebulizer, air/acetylene flame, and optical system. Alternate sample introduction systems such as graphite furnaces are also available but will not be discussed here. The job of each are detailed below:

Job of the hollow cathode lamp

Provide the analytical light line for the element of interest

Provide a constant yet intense beam of that analytical line

Job of the nebulizer

Suck up liquid sample at a controlled rate

Create a fine aerosol for introduction into the flame

Mix the aerosol and fuel and oxidant thoroughly for introduction into the flame

Job of the flame

Destroy any analyte ions and breakdown complexes

Create atoms (the elemental form) of the element of interest

Fe0, Cu0, Zn0, etc.

Job of the monochromator

Isolate analytical lines' photons passing through the flame

Remove scattered light of other wavelengths from the flame

In doing this, only a narrow spectral line impinges on the PMT.

Job of the photomultiplier tube (PMT)

As the detector the PMT determines the intensity of photons of the analytical line

exiting the monochromator.

The Hollow Cathode Lamp

The hollow cathode lamp (HCL) uses a cathode made of the element of interest with a low internal pressure of an inert gas.A low electrical current (~ 10 mA) is imposed in such a way that the metal is excited and emits a few spectral lines characteristic of that element (for instance, Cu 324.7 nm and a couple of other lines; Se 196 nm and other lines, etc.). The light is emitted directionally through the lamp's window, a window made of a glass transparent in the UV and visible wavelengths.

Neublizer, Different Oxidants, and Burner Heads, andWaste

The nebulizer chamber thoroughly mixes acetylene (the fuel) and oxidant (air or nitrous oxide), and by doing so, creates a negative pressure at the end of the small diameter, plastic nebulizer tube (not shown in adjacent figure; see figure below). This negative pressure acts to suck ("uptake") liquid sample up the tube and into the nebulizer chamber, a process called aspiration. A small glass impact bead and/ or a fixed impeller inside the chamber creates a heterogeneous mixture of gases (fuel + oxidant) and suspended aerosol (finely dispersed sample). This mixture flows immediately into the burner head where it burns as a smooth, laminar flame evenly distributed along a narrow slot in the well-machined metal burner head.

Liquid sample not flowing into the flame collects on the bottom of the nebulizer chamber and flows by gravity through a waste tube to a glass waste container (remember, this is still highly acidic). For some elements that form refractory oxides (molecules hard to break down in the flame) nitrous oxide (N2O) needs to be used instead of air (78% N2 + 21% O2) for the oxidant. In that case, a slightly different burner head with a shorter burner slot length is used.

The Monochromator and PMT

Tuned to a specific wavelength and with a specified slit width chosen, the monochromator isolates the hollow cathode lamp's analytical line. Since the basis for the AAS process is atomic ABSORPTION, the monochromator seeks to only allow the light not absorbed by the analyte atoms in the flame to reach the PMT. That is, before an analyte is aspirated, a measured signal is generated by the PMT as light from the HCL passes through the flame. When analyte atoms are present in the flame while the sample is aspirated--some of that light is absorbed by those atoms (remember it is not the ionic but elemental form that absorbs). This causes a decrease in PMT signal that is proportional to the amount of analyte. This last is true inside the linear range for that element using that slit and that analytical line. The signal is therefore a decrease in measure light: atomicabsorption spectroscopy.

Acidic Content and Oxidation State of Samples and Standards

The samples and standards are often prepared with duplicate acid concentrations to replicate the

analyte's chemical matrix as closely as possible.Acid contents of 1% to 10% are common.

In addition, high acid concentrations help keep all dissolved ions in solution.

The oxidation state of the analyte metal or metalloid is important inAAS. For instance,AAS analysis of selenium requires the Se(IV) oxidation state (selenite). Se(VI), the more highly oxidized state of the element (selenate), responds erratically and non reproducibly in the system. Therefore, all selenium in Se calibration standards and Se containing samples must be in the Se(IV) form for analysis. This can be accomplished by oxidizing all Se in the sample to selenate using a strong

Flame

oxidizer such as nitric acid or hydrogen peroxide and then reducing the contained selenate to selenite with boiling HCl.

Double Beam Instruments

The light from the HCL is split into two paths using a rotating mirror: one pathway passes through the flame and another around. Both beams are recombined in space so they both hit the PMT but separated in time. The beams alternate quickly back and forth along the two paths: one instant the PMT beam is split by the rotating mirror and the sample beam passes through the flame and hits the PMT. The next instance, the HCL beam passes through a hole in the mirror and passes directly to the PMT without passing through the flame. The difference between these beams is the amount of light absorbed by atoms in the flame.

The purpose of a double beam instrument is to help compensate for drift of the output of the hollow cathode lamp or PMT. If the HCL output drifts slowly the subtraction process described immediately above will correct for this because both beams will drift equally on the time scale of the analysis.

Likewise if the PMT response changes the double beam arrangement take this into account.

Ignition, Flame conditions, and Shut Down

The process of lighting theAAS flame involves turning on first the fuel then the oxidant and then

lighting the flame with the instrument's auto ignition system (a small flame or red-hot glow plug).

After only a few minutes the flame is stable. Deionized water or a dilute acid solution can be aspirated between samples.An aqueous solution with the correct amount of acid and no analyte is often used as the blank.

Careful control of the fuel/air mixture is important because each element's response depends on that mix in the burning flame. Remember that the flame must breakdown the analyte's matrix and reproducibly create the elemental form of the analyte atom. Optimization is accomplished by aspirating a solution containing the element (with analyte content about that of the middle of the linear response range) and then adjusting the fuel/oxidant mix until the maximum light absorbance is achieved. Also the position of the burned head and nebulizer uptake rate are similarly "tuned." Most computer controlled systems can save variable settings so that methods for different elements can be easily saved and reloaded.

Shut down involves aspirating deionized water for a short period and then closing the fuel off first.

Most modern instruments control the ignition and shutdown procedures automatically.

These notes were written by Dr. Thomas G. Chasteen; Department of Chemistry, Sam Houston State University, Huntsville, Texas 77341. Copyright 2000.

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