Atomic absorption spectroscopi
Modern atomic absorption spectrometers
Atomic absorption spectroscopy (AAS) is a spectroanalytical
procedure for the quantitative determination of chemical elements using
the absorption of optical radiation (light) by free atoms in the gaseous
state.
In analytical chemistry the technique is used for determining the
concentration of a particular element (the analyte) in a sample to be
analyzed. AAS can be used to determine over 70 different elements in
solution or directly in solid samples used in pharmacology, biophysics and toxicology research.
Atomic absorption spectroscopy was first used as an analytical
technique, and the underlying principles were established in the second
half of the 19th century by Robert Wilhelm Bunsen and Gustav Robert Kirchhoff, both professors at the University of Heidelberg, Germany.
Atomic absorption spectrometry has many uses in different areas of chemistry such as:
Clinical analysis: Analyzing metals in biological fluids and tissues
such as whole blood, plasma, urine, saliva, brain tissue, liver, muscle
tissue, semen
Pharmaceuticals: In some pharmaceutical manufacturing processes,
minute quantities of a catalyst that remain in the final drug product
Water analysis: Analyzing water for its metal content.
Principles
The technique makes use of absorption spectrometry to assess the
concentration of an analyte in a sample. It requires standards with
known analyte content to establish the relation between the measured
absorbance and the analyte concentration and relies therefore on the Beer-Lambert Law.
In short, the electrons of the atoms in the atomizer can be promoted
to higher orbitals (excited state) for a short period of time
(nanoseconds) by absorbing a defined quantity of energy (radiation of a
given wavelength).
This amount of energy, i.e., wavelength, is specific to a particular
electron transition in a particular element. In general, each wavelength
corresponds to only one element, and the width of an absorption line is
only of the order of a few picometers (pm), which gives the technique
its elemental selectivity. The radiation flux
without a sample and with a sample in the atomizer is measured using a
detector, and the ratio between the two values (the absorbance) is
converted to analyte concentration or mass using the Beer-Lambert Law.
Instrumentation
Atomic absorption spectrometer block diagram
In order to analyze a sample for its atomic constituents, it has to
be atomized. The atomizers most commonly used nowadays are flames and
electrothermal (graphite
tube) atomizers. The atoms should then be irradiated by optical
radiation, and the radiation source could be an element-specific line
radiation source or a continuum radiation source. The radiation then
passes through a monochromator
in order to separate the element-specific radiation from any other
radiation emitted by the radiation source, which is finally measured by a
detector.
Atomizers
The atomizers most commonly used nowadays are (spectroscopic) flames
and electrothermal (graphite tube) atomizers. Other atomizers, such as
glow-discharge atomization, hydride atomization, or cold-vapor
atomization might be used for special purposes.
Flame atomizers
The oldest and most commonly used atomizers in AAS are flames,
principally the air-acetylene flame with a temperature of about 2300 °C
and the nitrous oxide system (N2O)-acetylene flame with a
temperature of about 2700 °C. The latter flame, in addition, offers a
more reducing environment, being ideally suited for analytes with high
affinity to oxygen.
A laboratory flame photometer that uses a propane operated flame atomizer
Liquid or dissolved samples are typically used with flame atomizers. The sample solution is aspirated by a pneumatic analytical nebulizer, transformed into an aerosol,
which is introduced into a spray chamber, where it is mixed with the
flame gases and conditioned in a way that only the finest aerosol
droplets (< 10 μm) enter the flame. This conditioning process is
responsible that only about 5% of the aspirated sample solution reaches
the flame, but it also guarantees a relatively high freedom from
interference.
On top of the spray chamber is a burner head that produces a flame
that is laterally long (usually 5–10 cm) and only a few mm deep. The
radiation beam passes through this flame at its longest axis, and the
flame gas flow-rates may be adjusted to produce the highest
concentration of free atoms. The burner height may also be adjusted, so
that the radiation beam passes through the zone of highest atom cloud
density in the flame, resulting in the highest sensitivity.
The processes in a flame include the following stages:
Desolvation (drying) – the solvent is evaporated and the dry sample nano-particles remain;
Vaporization (transfer to the gaseous phase) – the solid particles are converted into gaseous molecules;
Atomization – the molecules are dissociated into free atoms;
Ionization
– depending on the ionization potential of the analyte atoms and the
energy available in a particular flame, atoms might be in part converted
to gaseous ions.
Each of these stages includes the risk of interference in case the
degree of phase transfer is different for the analyte in the calibration
standard and in the sample. Ionization is generally undesirable, as it
reduces the number of atoms that are available for measurement, i.e.,
the sensitivity.
In flame AAS a steady-state signal is generated during the time
period when the sample is aspirated. This technique is typically used
for determinations in the mg L-1 range, and may be extended down to a few μg L-1 for some elements.
Electrothermal atomizers
Electrothermal AAS (ET AAS) using graphite tube atomizers was pioneered by Boris V. L’vov at the Saint Petersburg Polytechnical Institute,
Russia, since the late 1950s, and further investigated by Hans Massmann
at the Institute of Spectrochemistry and Applied Spectroscopy (ISAS) in
Dortmund, Germany.
Although a wide variety of graphite tube designs have been used over
the years, the dimensions nowadays are typically 20–25 mm in length and
5–6 mm inner diameter. With this technique liquid/dissolved, solid and
gaseous samples may be analyzed directly. A measured volume (typically
10–50 μL) or a weighed mass (typically around 1 mg) of a solid sample
are introduced into the graphite tube and subject to a temperature
program. This typically consists of stages, such as:
Drying – the solvent is evaporated
Pyrolysis – the majority of the matrix constituents is removed
Atomization – the analyte element is released to the gaseous phase
Cleaning – eventual residues in the graphite tube are removed at high temperature.
The graphite tubes are heated via their ohmic resistance using a
low-voltage high-current power supply; the temperature in the individual
stages can be controlled very closely, and temperature ramps between
the individual stages facilitate separation of sample components. Tubes
may be heated transversely or longitudinally, where the former ones have
the advantage of a more homogeneous temperature distribution over their
length. The so-called Stabilized Temperature Platform Furnace
(STPF) concept, proposed by Walter Slavin, based on research of Boris
L’vov, makes ET AAS essentially free from interference. The major
components of this concept are:
Atomization of the sample from a graphite platform inserted into the
graphite tube (L’vov platform) instead of from the tube wall in order
to delay atomization until the gas phase in the atomizer has reached a
stable temperature;
Use of a chemical modifier in order to stabilize the analyte to a
pyrolysis temperature that is sufficient to remove the majority of the
matrix components;
Integration of the absorbance over the time of the transient
absorption signal instead of using peak height absorbance for
quantification.
In ET AAS a transient signal is generated, the area of which is
directly proportional to the mass of analyte (not its concentration)
introduced into the graphite tube. This technique has the advantage that
any kind of sample, solid, liquid or gaseous, can be analyzed directly.
Its sensitivity is 2–3 orders of magnitude higher than that of flame
AAS, so that determinations in the low μg L-1 range (for a typical sample volume of 20 µL) and ng g-1
range (for a typical sample mass of 1 mg) can be carried out. It shows a
very high degree of freedom from interferences, so that ET AAS might be
considered the most robust technique available nowadays for the
determination of trace elements in complex matrices.
Specialized atomization techniques
While flame and electrothermal vaporizers are the most common
atomization techniques, several other atomization methods are utilized
for specialized use.
Glow-discharge atomization
A glow-discharge (GD) device serves as a versatile source, as it can simultaneously introduce and atomize the sample. The glow discharge occurs in a low-pressure argon gas atmosphere between 1 and 10 torr. In this atmosphere lies a pair of electrodes applying a DC
voltage of 250 to 1000 V to break down the argon gas into positively
charged ions and electrons. These ions, under the influence of the
electric field, are accelerated into the cathode surface containing the
sample, bombarding the sample and causing neutral sample atom ejection
through the process known as sputtering.
The atomic vapor produced by this discharge is composed of ions, ground
state atoms, and fraction of excited atoms. When the excited atoms
relax back into their ground state, a low-intensity glow is emitted,
giving the technique its name.
The requirement for samples of glow discharge atomizers is that they
are electrical conductors. Consequently, atomizers are most commonly
used in the analysis of metals and other conducting samples. However,
with proper modifications, it can be utilized to analyze liquid samples
as well as nonconducting materials by mixing them with a conductor (e.g.
graphite).
Hydride atomization
Hydride generation techniques are specialized in solutions of
specific elements. The technique provides a means of introducing samples
containing arsenic, antimony, tin, selenium, bismuth, and lead into an
atomizer in the gas phase. With these elements, hydride atomization
enhances detection limits by a factor of 10 to 100 compared to
alternative methods. Hydride generation occurs by adding an acidified
aqueous solution of the sample to a 1% aqueous solution of sodium
borohydride, all of which is contained in a glass vessel. The volatile
hydride generated by the reaction that occurs is swept into the
atomization chamber by an inert gas, where it undergoes decomposition.
This process forms an atomized form of the analyte, which can then be
measured by absorption or emission spectrometry.
Cold-vapor atomization
The cold-vapor technique an atomization method limited to only the
determination of mercury, due to it being the only metallic element to
have a large enough vapor pressure at ambient temperature. Because of
this, it has an important use in determining organic mercury compounds
in samples and their distribution in the environment. The method
initiates by converting mercury into Hg2+ by oxidation from nitric and sulfuric acids, followed by a reduction of Hg2+ with tin(II) chloride.
The mercury, is then swept into a long-pass absorption tube by bubbling
a stream of inert gas through the reaction mixture. The concentration
is determined by measuring the absorbance of this gas at 253.7 nm.
Detection limits for this technique are in the parts-per-billion range
making it an excellent mercury detection atomization method.
Radiation sources
We have to distinguish between line source AAS (LS AAS) and continuum
source AAS (CS AAS). In classical LS AAS, as it has been proposed by
Alan Walsh, the high spectral resolution required for AAS measurements
is provided by the radiation source itself that emits the spectrum of
the analyte in the form of lines that are narrower than the absorption
lines. Continuum sources, such as deuterium lamps, are only used for
background correction purposes. The advantage of this technique is that
only a medium-resolution monochromator is necessary for measuring AAS;
however, it has the disadvantage that usually a separate lamp is
required for each element that has to be determined. In CS AAS, in
contrast, a single lamp, emitting a continuum spectrum over the entire
spectral range of interest is used for all elements. Obviously, a
high-resolution monochromator is required for this technique, as will be
discussed later.
Hollow cathode lamp (HCL)
Hollow cathode lamps
Hollow cathode lamps
(HCL) are the most common radiation source in LS AAS. Inside the sealed
lamp, filled with argon or neon gas at low pressure, is a cylindrical
metal cathode containing the element of interest and an anode. A high
voltage is applied across the anode and cathode, resulting in an
ionization of the fill gas. The gas ions are accelerated towards the
cathode and, upon impact on the cathode, sputter cathode material that
is excited in the glow discharge to emit the radiation of the sputtered
material, i.e., the element of interest. Most lamps will handle a
handful of elements, i.e. 5-8. A typical machine will have two lamps,
one will take care of five elements and the other will handle four
elements for a total of nine elements analyzed.
Electrodeless discharge lamps
Electrodeless discharge lamps
(EDL) contain a small quantity of the analyte as a metal or a salt in a
quartz bulb together with an inert gas, typically argon, at low
pressure. The bulb is inserted into a coil that is generating an
electromagnetic radio frequency field, resulting in a low-pressure
inductively coupled discharge in the lamp. The emission from an EDL is
higher than that from an HCL, and the line width is generally narrower,
but EDLs need a separate power supply and might need a longer time to
stabilize.
Deuterium lamps
Deuterium HCL
or even hydrogen HCL and deuterium discharge lamps are used in LS AAS
for background correction purposes. The radiation intensity emitted by
these lamps is decreasing significantly with increasing wavelength, so
that they can be only used in the wavelength range between 190 and about
320 nm.
Xenon lamp as a continuous radiation source
Continuum sources
When a continuum radiation source is used for AAS, it is necessary to
use a high-resolution monochromator, as will be discussed later. In
addition, it is necessary that the lamp emits radiation of intensity at
least an order of magnitude above that of a typical HCL over the entire
wavelength range from 190 nm to 900 nm. A special high-pressure xenon short arc lamp, operating in a hot-spot mode has been developed to fulfill these requirements.
Spectrometer
As already pointed out above, there is a difference between
medium-resolution spectrometers that are used for LS AAS and
high-resolution spectrometers that are designed for CS AAS. The
spectrometer includes the spectral sorting device (monochromator) and
the detector.
Spectrometers for LS AAS
In LS AAS the high resolution that is required for the measurement of
atomic absorption is provided by the narrow line emission of the
radiation source, and the monochromator simply has to resolve the
analytical line from other radiation emitted by the lamp. This can
usually be accomplished with a band pass between 0.2 and 2 nm, i.e., a
medium-resolution monochromator. Another feature to make LS AAS
element-specific is modulation of the primary radiation and the use of a
selective amplifier that is tuned to the same modulation frequency, as
already postulated by Alan Walsh. This way any (unmodulated) radiation
emitted for example by the atomizer can be excluded, which is imperative
for LS AAS. Simple monochromators of the Littrow or (better) the
Czerny-Turner design are typically used for LS AAS. Photomultiplier
tubes are the most frequently used detectors in LS AAS, although solid
state detectors might be preferred because of their better signal-to-noise ratio.
Spectrometers for CS AAS
When a continuum radiation source is used for AAS measurement it is
indispensable to work with a high-resolution monochromator. The
resolution has to be equal to or better than the half width of an atomic
absorption line (about 2 pm) in order to avoid losses of sensitivity
and linearity of the calibration graph. The research with
high-resolution (HR) CS AAS was pioneered by the groups of O’Haver and
Harnly in the USA, who also developed the (up until now) only
simultaneous multi-element spectrometer for this technique. The
break-through, however, came when the group of Becker-Ross in Berlin,
Germany, built a spectrometer entirely designed for HR-CS AAS. The first
commercial equipment for HR-CS AAS was introduced by Analytik Jena
(Jena, Germany) at the beginning of the 21st century, based on the
design proposed by Becker-Ross and Florek. These spectrometers use a
compact double monochromator with a prism pre-monochromator and an
echelle grating monochromator for high resolution. A linear charge
coupled device (CCD) array with 200 pixels is used as the detector. The
second monochromator does not have an exit slit; hence the spectral
environment at both sides of the analytical line becomes visible at high
resolution. As typically only 3–5 pixels are used to measure the atomic
absorption, the other pixels are available for correction purposes. One
of these corrections is that for lamp flicker noise, which is
independent of wavelength, resulting in measurements with very low noise
level; other corrections are those for background absorption, as will
be discussed later.
Background absorption and background correction
The relatively small number of atomic absorption lines (compared to
atomic emission lines) and their narrow width (a few pm) make spectral
overlap rare; there are only very few examples known that an absorption
line from one element will overlap with another. Molecular absorption,
in contrast, is much broader, so that it is more likely that some
molecular absorption band will overlap with an atomic line. This kind of
absorption might be caused by un-dissociated molecules of concomitant
elements of the sample or by flame gases. We have to distinguish between
the spectra of di-atomic molecules, which exhibit a pronounced fine
structure, and those of larger (usually tri-atomic) molecules that don’t
show such fine structure. Another source of background absorption,
particularly in ET AAS, is scattering of the primary radiation at
particles that are generated in the atomization stage, when the matrix
could not be removed sufficiently in the pyrolysis stage.
All these phenomena, molecular absorption and radiation scattering,
can result in artificially high absorption and an improperly high
(erroneous) calculation for the concentration or mass of the analyte in
the sample. There are several techniques available to correct for
background absorption, and they are significantly different for LS AAS
and HR-CS AAS.
Background correction techniques in LS AAS
In LS AAS background absorption can only be corrected using
instrumental techniques, and all of them are based on two sequential
measurements, firstly, total absorption (atomic plus background),
secondly, background absorption only, and the difference of the two
measurements gives the net atomic absorption. Because of this, and
because of the use of additional devices in the spectrometer, the
signal-to-noise ratio of background-corrected signals is always
significantly inferior compared to uncorrected signals. It should also
be pointed out that in LS AAS there is no way to correct for (the rare
case of) a direct overlap of two atomic lines. In essence there are
three techniques used for background correction in LS AAS:
Deuterium background correction
This is the oldest and still most commonly used technique,
particularly for flame AAS. In this case, a separate source (a deuterium
lamp) with broad emission is used to measure the background absorption
over the entire width of the exit slit of the spectrometer. The use of a
separate lamp makes this technique the least accurate one, as it cannot
correct for any structured background. It also cannot be used at
wavelengths above about 320 nm, as the emission intensity of the
deuterium lamp becomes very weak. The use of deuterium HCL is preferable
compared to an arc lamp due to the better fit of the image of the
former lamp with that of the analyte HCL.
Smith-Hieftje background correction
This technique (named after their inventors) is based on the
line-broadening and self-reversal of emission lines from HCL when high
current is applied. Total absorption is measured with normal lamp
current, i.e., with a narrow emission line, and background absorption
after application of a high-current pulse with the profile of the
self-reversed line, which has little emission at the original
wavelength, but strong emission on both sides of the analytical line.
The advantage of this technique is that only one radiation source is
used; among the disadvantages are that the high-current pulses reduce
lamp lifetime, and that the technique can only be used for relatively
volatile elements, as only those exhibit sufficient self-reversal to
avoid dramatic loss of sensitivity. Another problem is that background
is not measured at the same wavelength as total absorption, making the
technique unsuitable for correcting structured background.
Zeeman-effect background correction
An alternating magnetic field is applied at the atomizer (graphite
furnace) to split the absorption line into three components, the π
component, which remains at the same position as the original absorption
line, and two σ components, which are moved to higher and lower
wavelengths, respectively (see Zeeman Effect).
Total absorption is measured without magnetic field and background
absorption with the magnetic field on. The π component has to be removed
in this case, e.g. using a polarizer, and the σ components do not
overlap with the emission profile of the lamp, so that only the
background absorption is measured. The advantages of this technique are
that total and background absorption are measured with the same
emission profile of the same lamp, so that any kind of background,
including background with fine structure can be corrected accurately,
unless the molecule responsible for the background is also affected by
the magnetic field
using a chopper as a polariser reduces the signal to noise ratio.
While the disadvantages are the increased complexity of the spectrometer
and power supply needed for running the powerful magnet needed to split
the absorption line.
Background correction techniques in HR-CS AAS
In HR-CS AAS background correction is carried out mathematically in
the software using information from detector pixels that are not used
for measuring atomic absorption; hence, in contrast to LS AAS, no
additional components are required for background correction.
Background correction using correction pixels
It has already been mentioned that in HR-CS AAS lamp flicker noise is
eliminated using correction pixels. In fact, any increase or decrease
in radiation intensity that is observed to the same extent at all pixels
chosen for correction is eliminated by the correction algorithm. This
obviously also includes a reduction of the measured intensity due to
radiation scattering or molecular absorption, which is corrected in the
same way. As measurement of total and background absorption, and
correction for the latter, are strictly simultaneous (in contrast to LS
AAS), even the fastest changes of background absorption, as they may be
observed in ET AAS, do not cause any problem. In addition, as the same
algorithm is used for background correction and elimination of lamp
noise, the background corrected signals show a much better
signal-to-noise ratio compared to the uncorrected signals, which is also
in contrast to LS AAS.
Background correction using a least-squares algorithm
The above technique can obviously not correct for a background with
fine structure, as in this case the absorbance will be different at each
of the correction pixels. In this case HR-CS AAS is offering the
possibility to measure correction spectra of the molecule(s) that is
(are) responsible for the background and store them in the computer.
These spectra are then multiplied with a factor to match the intensity
of the sample spectrum and subtracted pixel by pixel and spectrum by
spectrum from the sample spectrum using a least-squares algorithm. This
might sound complex, but first of all the number of di-atomic molecules
that can exist at the temperatures of the atomizers used in AAS is
relatively small, and second, the correction is performed by the
computer within a few seconds. The same algorithm can actually also be
used to correct for direct line overlap of two atomic absorption lines,
making HR-CS AAS the only AAS technique that can correct for this kind
of spectral interference.
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