Spectrophotometry - Atomic Absorption Spectrophotometry

Atomic absorption spectroscopy (AAS) is similar to flame photometry with the difference that it measures the absorption of a beam of monochromatic light by the atoms in the flame. This technique was first introduced by Alan Walsh in Australia in 1954. We will discuss the principle, instrumentation and applications one by one.

Principle:

The basic principle behind the AAS is that the free atoms normally remain in the ground state which are capable of absorbing the energy of their own specific resonance wavelength. If light of the resonance wavelength is passed through the flame containing the atoms (in sample), then part of the light will be absorbed. The atoms absorb UV or visible light and make the transitions to higher energy levels. The absorption will be directly proportional to the number of atoms in the ground state in the flame.

Instrumentation:
The major difference in the instrumentation of AAS and flame spectrophotometry is the presence of a radiation source (a particular resonance wavelength cannot be isolated from the continuous source using a prism or diffraction gratings). So, for this purpose, a hollow cathode lamp is used.

Light Source: (Hollow Cathode Discharge Lamp): It contains a tungsten anode and cathode (as can be seen in the diagram on the right) is a hollow cylindrical tube which is lined by the element to be determined. These are sealed in the glass tube filled with an inert gas like neon or argon at a low pressure. At the end of the cylinder is a window, made up of quartz or pyrex, transparent to the emitted radiation. Each element in question will thus emit monochromatic radiation characteristic of the emission spectrum of that particular element involved. So, each element has its own unique lamp which must be used for the analysis.

Nebulizer: It creates a fine spray of the sample for the introduction in the flame. The aerosol and the fuel and oxidant are mixed thoroughly for the introduction into the flame.

Atomizer: The elements which needs to be analysed needs to be in the atomic state. Here comes the role of atomizer. It breaks down the molecules into the atoms by exposing the analyte to high temperatures in a flame of graphite furnace (as explained in previous post, here).

Monochromator: A monochromator is used to select the specific wavelength of light which is absorbed by the sample and to exclude other wavelengths. The selection of the specific wavelength allows the determination of the element.

Detector: The light selected by the monochromator is directed onto the detector that typically is a photomultiplier tube that converts the light signal to electrical signal proportional to the light intensity.

Applications of Atomic Absorption Spectrometry

• It is highly sensitive technique and can measure upto parts per billion of a gram (ugdm-3)
• It is used to detect the presence of metals as impurity or in alloys.
• The minute levels of the metals could be detected in biological samples like copper in the brain tissues.
• The quantity of elements can be determined be agricultural and food products.
• It can also be used to determine the impurity in the environmental water sources like in the ocean water, river and stream water, waste water, sludge and suspensions.

Spectrophotometry - Flame Photometry

Flame spectrophotometry is a technique in which the intensity of the radiations emitted by a chemical into the flame is determined.  This basic concept of working of flame spectrometer is that, a flame, through its heat, can raise the atoms from a lower energy state to a higher energy state and when it comes back to its ground state, there is emission which is in the form of radiations. And determination of these radiations is by flame spectrophotometer.

Flame photometry can be applied in two ways as emission flame photometry or simple flame photometry and atomic absorption spectrophotometry. We will discuss the principle, instrumentation and applications of the two one by one.

Lets start with emission flame photometry or simply, flame photometry.

Emission Flame Photometry:

Principle:

Here, the solution containing the metallic salt (to be analyzed) is placed into the flame, whereby the solvent is evaporated, leaving behind only the solid. The solid is then dissociated by vaporization. The volatilization of the molecules in the solid produces free atoms which then, due to heat, excites to a higher energy level.  The emission spectrum is produced when the atoms return back to the ground state (as a result of radiation). This is the basic principle of the emission flame photometry.

Instrumentation:

Below is the basic representation of the components which are involved in flame photometry.

Nebulizers: Before the samples get into the flame, they must be converted to a fine spray, i.e., they must be nebulized. This is necessary as the large drops will not be able to stay in the hottest area of the flame for a long time and hence, will be difficult to volatize and excite.

Atomizers or Flames: It converts the sample or the analyte to free atoms. The atomizers can be flame atomizers or graphite rod atomizers.

Flame atomizers: To create flame, we need to mix an oxidant gas and a fuel gas. Generally, air-acetylene flame or nitrous oxide-acetylene flame is used (in the above diagram, this type is depicted). 

Graphite rod atomizers: These uses graphite rod instead of the flame.  The graphite rod is a small cavity in which the sample can be pipetted. These tubes are heated using a high current power supply such that the temperature can raise as high as 2500 degree Celsius. As a result, the sample is vaporized or atomized.

Monochromators: A monochromator is used to select a specific wavelength of light which can be absorbed by the sample while excluding other wavelengths. Generally, a simple filter is used. However, in sophisticated instruments, the prisms or diffraction gratings are used.

Detectors:  The light selected by a monochromator is directed into a detector, which is generally a photomultiplier. It converts the light signal into the electrical signal which is proportional to the intensity of the light.

Applications of flame photometry:

• It is used to determine even the small quantities of metals like lead, calcium, mercury etc.
• So, it is used in the determination of sodium, potassium, calcium, lithium etc. in the biological samples (like serum, interstitial fluids etc.).
• It is used in the determination of lead in the petrol.
• It is used in determination of calcium and magnesium in the cement.

In the next post, we will discuss about atomic absorption spectrometry.

Spectrophotometry - Luminometry

Till now, we have seen various posts on UV-visible spectrophotometry, IR-spectrophotometry and spectrofluorimetry. Now, we will have a look at the next type of spectrophotometry which is luminometry.

As the name suggests, this type of spectrophotometry is associated with the phenomenon of luminescence. Now, the question is “what is luminescence?” Luminescence can be described as the emission of light by certain materials which do NOT result from heating (that is, the emission of light is when the temperature is below that of incandescence). Luminescence is the basic principle behind the working of luminometers. This phenomenon is usually ascribed to oxidative reactions which take place in solution producing molecules in an excited state. Some of these reactions release energy in the form of heat while others release in the form of photons.
Examples of luminescent compounds are luciferin (light emitting compound found in organisms), luminol (chemical exhibiting luminescence).

There are two major categories of luminescence as chemiluminescence and bioluminescence. It is easy to understand them as the name itself suggests the meaning. So, chemiluminescence is the luminescence produced by some chemical means. For example, luminol when oxidized with hydrogen peroxide (H₂O₂) in the presence of a catalyst produces luminescence which is called the chemiluminescence. On the other hand, luminescence which is produced by the interference of an enzyme is referred to as bioluminescence. 

Advantages of luminometry 

There are various advantages of luminometry over spectrophotometry. Firstly, luminometry is more sensitive as around femtomole quantities can be measured. Next advantage is that of a simple instrumentation (as we will see below). In luminometers, wavelength selectors are not required. This is so because the luminescent light is monochromatic as a result of its emission from a specific reaction.

Instrumentation

The basic components of luminometers are:
a. A light-tight chamber in which the cuvette containing the sample can be kept
b. A facility for the addition of luminescent reagents in light-tight fashion
c. A detector (which is generally a photomultiplier)
d. An amplifier
e. A recorder

The light which is emitted by the reaction taking place in the cuvette is measured either as a peak value (which generally measures the concentration of compound of interest) or the rate of change of intensity (which is generally while measuring enzyme intensities). 

Applications:

We will discuss here three main systems which are of frequent use as firefly, bacterial luminescence and luminol chemiluminescence. The principle and applications of each of these are described below:

a. Firefly luminescence and ATP measurement:

Luciferase enzyme catalyses the following reaction in the presence of magnesium:

Here, for each molecule of ATP reacting, one photon of intensity of 562nm is produced. This system is highly specific for ATP if all the reagents are pure. By linking this reaction with various other reactions, it can be used to assay a number of ATP-specific enzymes and their substrates such as creatine kinase, creatine phosphate etc.

b. Bacterial luminescence and coenzymes measurement:

The coenzymes that can be measured by this method are the NADH and NADPH. This system utilizes a purified oxidoreductase obtained from the bacterium Benecka harveyi. The reaction can be then coupled to bacterial luciferase as follows:

Here, bacterial luciferase catalyzes the oxidation of aldehyde by oxygen in the presence of FMNH2 during which a photon of maximum intensity at 495nm is produced.

 

c. Luminol based chemiluminescent assays:

Luminol is oxidized by hydrogen peroxide at pH 10-11 if chromium, copper or iron compounds are used as catalysts. Photons with maximum intensity at 430nm are produced.