Development Team

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Environmental Sciences

Environmental Chemistry

Atomic Spectroscopy, X-ray & Electrophoresis Paper No: 16 Environmental Chemistry

Module: 31 Atomic Spectroscopy, X-ray & Electrophoresis

Development Team

Principal Investigator

&

Co- Principal Investigator

Prof. R.K. Kohli

Prof. V.K. Garg & Prof. Ashok Dhawan Central University of Punjab, Bathinda

Paper Coordinator

Prof. K. S. Gupta

University of Rajasthan, Jaipur

Content Writer Dr. Swagat K. Mohapatra University of Rajasthan, Jaipur Content Reviewer Dr. Y. P. Singh

University of Rajasthan, Jaipur

Anchor Institute Central University of Punjab

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Atomic Spectroscopy, X-ray & Electrophoresis Description of Module

Subject Name Environmental Sciences Paper Name Environmental Chemistry Module Name/Title

Atomic Spectroscopy, X-ray & Electrophoresis

Module Id EVS/EC-XVI/31

Pre-requisites A basic knowledge of Spectroscopy.

Objectives

Atomic Spectroscopy:

1. What is if Flame Test, and how its relation to Atomic Spectroscopy.

2. Introduction to Atomic Spectroscopy, and how does it work

3. Types of Atomic Spectroscopy: Atomic Absorption Spectroscopy & Atomic Emission Spectroscopy & their uses in environmental studies

X-ray:

1. Introduction to X-ray

2. Types of X-ray spectroscopy based on the interaction of X-rays with matter 3. Description on X-ray Fluorescence & X-ray diffraction, & their application.

Electrophoresis :

1. What is electrophoresis, and its principle.

2. What are the different forms of electrophoresis?

3. Use of the Electrophoresis technique in various Environmental analyses.

Keywords

Absorption spectroscopy, emission spectroscopy, X-ray fluorescence, X-ray diffraction, Bragg’s law, electrophoresis

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Atomic Spectroscopy, X-ray & Electrophoresis Module 31: Atomic Spectroscopy, X-ray & Electrophoresis

Contents

1. Atomic Spectroscopy: Introduction 2. Atomic Absorption Spectroscopy (AAS) 3. Application of AAS

4. Flame Photometry

5. Application of Flame Photometry

6. Inductive Coupled Plasma (ICP) Spectrophotometry 7. Application of ICP Spectrophotometry

8. X-ray: Introduction

9. X-ray Fluorescence (XRF) 10. X-ray Diffraction (XRD)

11. Single Crystal XRD and Bragg’s Law 12. X-ray Powder Diffraction

13. Application of XRD

14. Electrophoresis: Introduction 15. Different forms of Electrophoresis 16. Application

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Atomic Spectroscopy, X-ray & Electrophoresis 1. Atomic Spectroscopy:

Introduction:

When a sample of an alkali or alkaline earth metal salt is shown in the Bunsen flame, a characteristic color in the flame is found. For example:

Lithium  crimson Sodium  yellow Potassium violet Calcium  orange Strontium  crimson Barium  green

This experiment is known as flame test, has been used for many qualitative analyses for metal ions, and comes under atomic emission spectroscopy, called flame atomic emission spectroscopy. In the Bunsen flame, the metal ions are reduced into their corresponding atoms. The metal absorbs energy from the flame, and rises to an excited energy state in their atomic form. Then the atom comes back to its lower energetic state by emitting the absorbed energy in the form visible light of specific wavelength (figure 1).

Atomic spectroscopy is the analytical technique used for the qualitative as well quantitative analysis of the elements. This was first introduced in 1859, when Bunsen, and Kirchhoff found that the radiation emitted from the Bunsen flame is due to the characteristic element present in the sample, as said above. The potential of atomic spectroscopic techniques were then well established. Atomic spectroscopy is divided into two types: 1) atomic absorption spectrophotometry (AAS), and 2) atomic emission spectrophotometry (AES). Further AES is divided into two categories; i) flame atomic emission spectrophotometry or flame photometry, and ii) inductively coupled plasma-atomic emission spectrophotometry (ICP-AES).

FIGURE 1

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The basic difference between AAS and AES is as follows; i) AAS occurs only when the metal atoms remain in their ground or lower energetic state, and ii) AES occurs when the metal atoms are in their excited or higher energetic state.

1.1. Atomic Absorption Spectrophotometry (AAS):

Alan Walsh first developed the AAS technique, and since then it has become an important analytical tool. AAS is an analytical technique that provides the quantitative information on elements present in a sample. It uses the characteristic wavelength of light absorbed by an element, which corresponds to the energy required to move electron from ground state to excited state, to measure the concentration of that element.

How it works? Atoms present in different elements absorb specific wavelength of light, and this light of particular wavelength is analyzed to find the presence of that particular element. For instance, a lead lamp emits light from excited lead atoms which is the standard wavelength to be absorbed by the lead atoms present in any sample.

In AAS, first the sample is vaporized into the ground state free atoms, called atomization, and then a beam of electromagnetic radiation emitted from excited lead atoms is passed through the vaporized sample. Some of the radiation is absorbed by the lead atoms present in the sample. The absorption will be higher when there is a greater number of lead atoms present in the sample (the amount of light absorbed is directly proportional to the number of lead atoms). A calibration curve is prepared by running several such samples with known lead concentrations, following the same conditions as in the case of unknown samples. The amount of standard absorbs is compared with the calibration curve to calculate the concentration of lead present in the unknown sample.

Application of AAS:

AAS has been a very useful tool in many areas of chemistry, environmental science, pharmaceutical science, Industry, etc.

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i) Environmental analysis: AAS helps to monitor our environment, like finding out the levels of various elements in water, air, and soil.

For example; the presence of elements like calcium, copper, iron, magnesium, potassium, sodium, zinc, and many other in soil can be analyzed using AAS. These metal ions are extracted using an extractable solution like 0.05N HCl in 0.025N H2SO4or ammonium acetate from a known weight of air-dried ground soil. The sample is then filtered and the extract can be directly used for the analysis by AAS to find out the level of the metal ions. This method is useful for evaluate the nutrient quality of soil, and further the data can be used in deciding the fertilizer needed for a soil-cropping situation.

AAS is useful to determine the elements present in natural water, sea and other saline water.It is also used to determination the elements like cadmium, chromium, cobalt, copper, lead, manganese, nickel, and zinc in airborne particles, called metallic air pollutants.

ii) Food analysis: AAS technique is used in the analysis of minerals concerning the food and human health safety. The toxic metals like lead, cadmium, copper, chromium, cobalt, etc can be analyzed using atomic spectrophotometry method. Before analysis, all the organic substances should be destroyed by dry washing or wet-oxidation method. The sample can then be taken for analysis. If the concentration of the element of interest is low, then its concentration can be raised via chelation and extraction. For instance, if the element tin is to be analyzed, then methanol can be used to improve its signal.

Recently, AAS technique is used for the determination of minerals present in Pomegranate. It is found that 100gm of pomegranate contains 0.3-0.36 mg of sodium and 50-95 mg of potassium, and 0.1 μg of copper. No lead and iron were detected.

iii) Clinical Science: AAS technique is widely used in various clinical analyses, such as determination of iron in hemoglobin, determination of calcium, sodium, magnesium, and potassium in urine, etc.

This is also important in the determination of lead in blood, and urine using an extraction method for the concentration of lead,

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iv) Pharmaceuticals: In many pharmaceutical industries, AAS technique is used for various analyses.

For example, this method is used to monitor the amount of Vitamin B12 by measuring the amount of cobalt present in the sample. There is one atom of cobalt present for every molecule of Vitamin B12.This method also able to determine the level of zinc in insulin as low as 5 μg zinc, which helps to check the purity and activity of crystalline and protamine zinc insulins.

v) Industry: Many raw materials are tested using AAS before metal extraction. It is widely used to examine that the major elements are present and any toxic impurities present are lower as per the standard. For example in concrete, where calcium must be a major constituent than lead, which should be low due to its high toxicity.

vi) Mining: AAS also helps to determine the amount of a specific metal present in the rock before any further extraction. It also tells the worthiness in mining the rocks to extract any metal.

1.2. Flame Photometry:

Flame photometry is one the simplest atomic emission spectroscopy technique, which is very similar to the flame test, as described above. It useful for the detection of alkali and alkaline earth metals, which has relatively low excitation energy, such as sodium, potassium, calcium, etc.

How it works? Flame photometry based on the fact that samples of alkali or alkaline earth metals undergo thermal dissociation when shown to a flame, and some of their atoms produced are excited to a higher energetic state. On return of these atoms to their ground state, they emit radiation, which generally lies in the visible region of the electromagnetic spectrum. Each element emits light of a particular wavelength, as given in the table 1. Within a particular concentration the intensity of the emission is directly proportional to the number atoms coming back to their ground state. In other words, this is proportional to the absolute quantity of the species vaporized in the flame, i.e. the amount of light emitted is directly proportional to the concentration of the sample. The emitted light then isolated by an optical filter, and a photo detector measures the intensity of that light. The signal obtained upon further processing gives the detail of the required element.

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Atomic Spectroscopy, X-ray & Electrophoresis TABLE 1

Element Emission

wavelength (nm)

Flame color

Barium 554 Green

Calcium* 622 Orange

Lithium 670 Crimson

Potassium 766 Violet

Sodium 589 Yellow

*Calcium is determined by using calcium hydroxide band emission at 622 nm.

Application of Flame Photometry:

Flame photometry has been used as a primary analytical technique in many environmental studies, analysis of biological fluid, material science, etc. Recently this technique has been employed in determining the sodium content in sea water (used for analysis of Marina beach water sample, Chennai).

Sodium and Potassium are two very essential elements, needed in proper level for normal function of human cell. Increase or decrease in their level affect severely the nervous system and heart. Flame photometry technique has been useful in measuring the sodium and potassium level in the biological fluids, such as in blood,red blood cells, plasma, and urine.Calcium content was also determined in biological materials using this method. Determination of elements like sodium, potassium, calcium and magnesium in plant tissues (e.g. sugar cane) were carried out using this method.

Flame photometer has been used to study its suitability for the determination of Na2O and K2O in Portland cement and cement raw materials like argillaceous limestone, and clays. This is also used in the quantitative analysis of Iodine present in common salt.

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1.3. Inductive Coupled Plsma (Atomic Emission or Mass) Spectrophotometry:

Inductively Coupled Plasma (ICP): Argon gas when flows through a copper foil connected to radiofrequency generator, the slower central argon flow is covered by a stronger outer flow, and this slower moving argon gas become inductively coupled to the radiofrequency region. The moving electrons and nuclei are ripped apart in opposite directions by the magnetic field producing a gas called plasma, containing both electrons and positively charged argon ions. This has a very high temperature of about 5,000 – 10,000 degree Kelvin. This plasma is useful for ionizing almost all elements of high excitation energies.

How it works? When a high temperature ICP source combines with an atomic emission spectrophotometer (AES) creates an ICP-AES, and on combination with a mass spectrophotometer (MS) create ICP-MS.

In ICP-AES, the sample elements are quickly excited to their higher energetic state upon giving plasma energy. When these excited atoms return to their ground lower energetic state, they emit light of a specific wavelength corresponding to that element. The element is detected based on the characteristic wavelength of the light emitted, and the concentration of each element is analyzed using the intensity of that light.

In ICP-MS, the high temperature ICP source excites the atoms of the elements in the sample into their corresponding ions. Then these ions are separated and recorded by a mass spectrophotometer.

Application of ICP Spectrophotometry:

Recently Inductive coupled CP spectrophotometry established as a cost-effective analytical technique used for multi-elemental analysis, with a wide range of concentration (μg/L level) of major and trace element analysis in the field of environmental, agricultural and food, biological and clinical, geological, and archaeological research.

i) Environmental: ICP spectrophotometry technique has a wide application in many areas of environmental analysis, such as soil, sediments, water, air, etc.

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Water analysis include: determination of metals life As, Fe, Cr, Cd, Cu, Mn, Pb, and Zn in sea water;

determination of phosphorus in municipal water, determinations of heavy metals in city dust samples, etc.

This technique is used to determine the metals, and metal compounds in suspended particulate matter in ambient air. For example, this technique has been used to check the suitability of the air in the workplace, and occupational air exposure of workers.

ICP-MS technique has also been used in determining the isotopic compositions of radio nuclides such Uranium and Plutonium in the environment.

ii) Agricultural and Food: This technique is largely used in the analysis of various agricultural and food materials.

For example, concentration of elements like Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn were measured in food items like fruits, vegetables, and meat by ICP spectrophotometry via a nitric-perchloric acid digestion procedure.

iii) Biological and Clinical: ICP spectrophotometry has played an important role in the field biological and clinical uses. Estimation of essential, toxic and therapeutic elements, important for medical as well as forensic laboratories, is carried out using this method. For examples, determination of Cu, Cr, and Ni in urine sample, Al in blood, Cu in brain tissue, Se in liver, B, P, and S in bone are done using ICP method.

iv) Geological: ICP is also being used in the field Geological research, such as elemental compositions in rocks, soil, and sediments. For example, determination of uranium in ore grade material, various metal in river sediments. This method is also proved to be useful for estimating the origin of rock formations, and for marine geochemistry.

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Atomic Spectroscopy, X-ray & Electrophoresis 2. X-ray:

Introduction: In electromagnetic spectrum X-ray region lies between 0.1 and 200 Å (Angstrom unit, 1 Å = 10^-8 cm). However, only a small portion of this total X-ray region (0.1 to 25 Å) is used by the conventional X-ray spectrometer, known as analytical X-ray region. Similar to other electromagnetic waves, X-ray travel in straight lines at a constant speed on vacuum, and they are not affected under the influence of an electric or magnetic field. However, they undergo reflection and refraction, and can be superimposed to set up interference patterns.

Interaction of X-ray radiations with Matter: The interaction of X-rays with matter can proceed in three different ways:

i) Absorbed or transmitted through the sample (medical X-ray, used to see inside the materials).

ii) Diffracted or scattered from a crystalline material (X-ray powder diffraction, and single crystal X- ray diffraction, XRD – used to identify the crystalline material).

iii) Cause the generation of X-rays of different color (X-ray fluorescence, XRF – used to determine elemental composition).

2.1. X-ray Fluorescence (XRF):

Introduction: An X-ray Fluorescence (XRF) spectrometer is mainly involved for regular, and relative non-destructive determination of elemental compositions of a material sample. It is used for analysis of rocks, minerals, sediments and fluids. There are two types of XRF spectrometers: Wavelength dispersive X-ray fluorescence (WDXRF), and energy dispersive X-ray fluorescence (EDXRF).

Principle: XRF spectrometer is based on the fact that atoms of a material sample when excited by the primary X-ray radiations, electrons from the innermost K-shell are released, and the resultant gaps are

5 The Principle

X-ray fluorescence analysis has its basis in the phenom- enon that, when atoms in a material sample are excited by the primary X-radiation, electrons from the innermost shells are released; the resultant vacancies are then filled by electrons from the outer shells.

During these transitions, fluorescent radiation is generated that is characteristic for each element. This is read by the detector and provides information on the composition of the sample.

Applications

Because ED-XRFA is capable of determining the composition of materials and measuring thin coatings and coating systems, there is a wide variety of applications for this technology. Examples include:

In the electronics and semiconductor industries, thin gold, palladium and nickel coatings are ascer - tained on contacts or on traces.

In the watch and jeweller y industries or in precious metal refining, accurate knowledge of the composi - tion of precious metal alloys is required.

For quality and incoming goods inspections, exact compliance with material specifications is essential.

In the photovoltaic industr y, for example, the composition and thickness of a photovoltaic film deter - mines its efficiency, while in contract electroplating, it is necessar y to measure the coatings of masspro - duced parts.

For manufacturers and importers of electronic goods, it is critical to be able to monitor compli - ance with the Restriction of Hazardous Substances (RoHS) Directive.

The toy industr y is also dependent on the reliable detection of har mful substances.

FISCHERSCOPE X-RAY measurement systems are opti - mally suited for all these purposes.

COATING THICKNESS

MATERIAL AN ALYSIS

Advantages of the X-ray fluorescence analysis (XRFA)

Fast and non-destructive measurement of coating thickness (single and multiple layers) Analysis of solids, powders and liquids Trace analysis of harmful substances High precision and trueness Very broad range of applications

Accurate measurement irrespective of magnetic and electric proper ties of base material Very simple sample preparation: little to none Safe method without the use of environmentally

hazardous chemicals

N o consumables required, therefore cost-effective

FIGURE 2

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then filled by the outer shell electrons (figure 2). During these transitions, fluorescent radiation is generated which is characteristic to each element. This is read by the detector, and gives the information on the composition of sample material.

Application: XRF has a wide range of application in many different areas for both quantitative and qualitative analysis, such as environmental analysis, geology, recycling, etc.

 XRF spectrometer is used for quantitative analysis of contaminants in solids, characterization of radioactive elements (e.g. uranium), detect the compositions of rocks and minerals, characterization of material for recycling. It also suitable for the analysis of metals and metalloids present dust samples.

 Given that children extensively use plastic toys, the toy industry is also very dependent on XRF technique to monitor the level of any harmful substance content. Recently XRF has been used to detect the level of hazardous metal content in contemporary toys. Recently G. Z. Miller in his study (Advancement of the Science, 2013) used a handheld XRF spectrometer and showed that the level of hazardous metal content in plastic toys from the year 1970s and 1980s exceed the current US and European limits.

 Electronic product manufacturer also get the help from XRF to monitor the compliance with the restriction of hazardous substance directive.

 It is used in determining the major, precious, trace element analysis, and characterization of rocks, ores and soil.

 In watch and jewelry industries or in refining process of precious metals, XRF is needed to find out the accurate knowledge of the composition of the precious metal alloys to be refined.

 It is used for the identification of materials such alloy and waste further processing or recycling.

2.2. X-ray Diffraction (XRD):

XRD is an analytical method, mainly used for the identification of crystalline materials, and determination of unit cell dimensions. Max von Laue first explored this technique in 1912, and demonstrated that crystalline substances could act as diffraction gratings for X-ray radiations. There

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are two types of XRD techniques: i) Single crystal XRD, and ii) X-ray powder diffraction.

Single crystal XRD is used for the determination of unit cell dimension and the position of the atoms within a crystal lattice. This is particularly useful for the characterization of new materials and to find out the answers regarding the molecular arrangement of the crystalline substance.

X-ray powder diffraction is recognized as one of the important technique in the field of environmental science, geology, material science, and engineering for rapid identification of crystalline substance. In this case, the sample needs to be pure, finely ground and homogenized for finding out the bulk composition. It is further used for the detailed characterization of crystalline substances and detection of unit cell dimension. Identification of fine-grained minerals is also carried out using this tool.

2.2.1. Single crystal XRD and Bragg’s Law:

As a beam of X-ray is made up of several waves. The waves interact with each other, and such interaction is called interference. When all the waves in a bundle remain in a phase i.e. all their crests and troughs happen at the same position, the waves are said to be in constructive interference with one another and their amplitude combine with each other and result a wave of high amplitude (figure 3).

When the waves are not in the same phase, then destructive interference take place, and the amplitude of the waves reduces (figure 3). In the worst case, the waves which are out of phase by an odd multiple of ½ [(2n + 1)/2, n = 1,2,3, etc.] have no amplitude, and thus fully destroyed.

FIGURE 3:INTERFERENCE BETWEEN WAVES

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Atomic Spectroscopy, X-ray & Electrophoresis In XRD, X-rays hit the atoms in crystals in

such way that it produces interference. The interaction is assumed because of the atoms in the crystal cause the reflection of the waves.

However, as in a crystal structure, atoms are arranged in an orderly manner, the reflection happen due to the planes of atoms.

As shown in the figure 4, let’s say two beam of X-rays (Ray 1 and 2) of wavelength λ

enters into a crystal through one of the atomic planes faced at angle of θ to the incoming beam monochromatic X-rays.

The incident Ray 1 reflects back from the upper atomic plane with an angle equal to its angle of incidence θ. Similarly incident Ray 2 also follows the same path with the same angle. However, Ray 2, as shown in the figure 4, travels a distance 2a farther than the Ray 1. When this distance 2a become equal to an integral number of wavelengths (nλ), then diffracted Rays 1 and 2 occur in one phase, and result a constructive interference.

If 2a ≠nλ, then destructive interference will happen between the diffracted Ray 1 and 2, and as a result the diffracted rays will not be stronger compared to the incidental rays.

Thus for a constructive interference

𝑛𝜆 = 2𝑎

From trigonometry, the distance 2a can be expressed in terms of the distance between the atomic planes d, as

𝑎 = 𝑑 𝑠𝑖𝑛𝜃

FIGURE 4:BRAGG’S LAW

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Atomic Spectroscopy, X-ray & Electrophoresis or, 2𝑎 = 2 𝑑 𝑠𝑖𝑛𝜃

or, 𝑛𝜆 = 2𝑑 𝑠𝑖𝑛𝜃 − − − − − 𝐸𝑞(1)

Eq (1) is called Bragg’s Law for X-ray diffraction, formulated by Sir W. H. Bragg and W. L. Bragg, who got the Nobel Prize in physics in 1915 for their analysis of crystal structure using X-rays.

or, 𝑑 = 𝑛𝜆

2 𝑠𝑖𝑛𝜃− − − − − 𝐸𝑞(2)

Thus Eq (2) tells once we know the wavelength λ of the incident X-rays, and the angle θ of diffracted X-rays, then we can find out the spacing d between the atomic planes. We could further re-orient the crystal to measure the spacing d between all the atomic planes in the crystal, ultimately lead us to find the crystal structure of the molecule and the size of the unit cell.

2.2.2. X-ray powder diffraction:

In this technique, the sample needs to be crushed to a fine powder. In the powder form, there are number of grain particles of random orientations. As a result, most of the different atomic planes might lie parallel to the surface in some of the grains. Thus upon scanning through an angle θ of incident X-ray from 0 to 90 °, all the angles can be measured where diffraction occur, and each of these angles would be linked with a different atomic spacing.

2.3. Application of XRD:

XRD has many applications in the field of environmental science, agriculture, geology, material science, chemistry, forensic science, and the pharmaceutical industry, and many more.

 In the environmental studies, XRD techniques have been used to quantitatively analyze the amount of amorphous substances present in particulate matter. XRD is useful to study the areas affected by acid mine drainage, to detect the secondary minerals, and fine-grained precipitates.

 In the field of agriculture, it is used for the qualitative and quantitative analysis of minerals both in top and sub soils. It also helps to classify the soil based on its nutrient rich minerals and to find out

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Atomic Spectroscopy, X-ray & Electrophoresis the soil fertility potential.

 In geology, it helps to study the ore genesis, mineral-resource assessments, etc. It is one the important tool to determine the lateral and vertical variations in mineral matter, and major, minor, and trace elements coal beds.

 XRD is useful in the field of conservation science and archaeometry. XRD also helps to find out the answer behind the stability and deterioration processes of art and museum objects.

3. Electrophoresis

Introduction: Frederic Reuss in 1807 first demonstrated that dispersed clay particles in water migrate under the influence of an electric field. This is due to the presence of charge interface between the clay particles and water, and the rate of migration depends on; i) the strength of the field, ii) net charge, size and shape of molecules, and iii) ionic strength, viscosity and temperature of the medium in which particles are moving. The above process is called as Electrophoresis, the movement of ions in a solution under the influence of a uniform electric field.

Thus Electrophoresis became an analytical tool, used to study the properties of single charged species and as a separation technique for separation of molecules by size, charge, or binding affinity. This technique is mainly used to separate proteins from each other, such as: i) proteins in biological fluids like serum, urine, etc; ii) proteins in erythrocytes: hemoglobin, and iii) nucleic acids; DNA and RNA.

Ultimately, Arne Tiselius bagged the Nobel Prize in chemistry in 1948, for his work on electrophoresis and adsorption analysis, particularly for his discoveries on the complex nature of serum proteins. He examined horse serum, and observed it as a mixture of four components, one albumin and 3 globulin.

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Atomic Spectroscopy, X-ray & Electrophoresis Principle:

When amixture consisting of electrically charged molecules, kept under an influence of electric field of strength E, themolecules will flow towards the electrode of opposite charge (figure 5). However the velocity of each of the molecule will be different, and depend on its own physical characteristics and the experimental conditions. The velocity of movement v, of the charged molecule in an

electric field can be expressed as:

𝑣 =𝐸 × 𝑞 𝑓

where ‘f’ = frictional co-efficient, defined as the frictional resistance to the mobility of the molecule and depends on the mass of the molecule, its degree of compactness, viscosity of

electrolyte (usually a buffer), and the porosity of the matrix in which experiment is carried out. ‘q’ = net charge, determined by the number positive and negative charges present in the molecule.

Different forms of Electrophoresis:

i) Paper Electrophoresis: It is generally used for the analysis and separation of small molecules such as amino acids, and nucleotides, not suitable for proteins. In this case the paper is moistened with buffer, and place between electrodes in electrophoresis chamber.

ii) Capillary Electrophoresis: In this case, the material and the electrophoresis medium are placed in a long, fine-bore capillary tube. A very small amount of sample (in nano liter) is kept at one end of the capillary and subjected to electrophoresis. This method is suitable for separation of DNA molecules of size as small as a single nucleotide.

iii) Gel Electrophoresis: In this method, separation occurs in aqueous buffer supported within a

Electrophoresis

Separation of solutes based on different rates of migration though an electric field (through background electrolyte [running buffer].

Anions move toward the anode.

Note that charge and size influence the movement of charged particles, in opposite ways.

FIGURE 5:MIGRATION OF IONS IN AN ELECTROPHORETIC CHAMBER

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polymeric gel matrix. This technique has many advantages than the above; such as i) larger samples can be accommodated for preparative scale electrophoresis, ii) the nature of the gel matrix can be varied to fit for a particular use. Two major gel matrixes are used in this system, i) agarose (a polygalactose polymer) gel, and ii) polyacrylamide gel.

Application:

i) Environmental Monitoring: As mentioned above, the electrophoresis is an efficient tool mainly used in clinical laboratories to separate proteins. In addition recently it, particularly capillary electrophoresis, has shown to be useful in organic pollutant analysis, such as pesticides, phenols, pharmaceuticals, nitro aromatic compounds, amines, and etc. This technique is also useful for trace metal analysis in many environmental analyses. Recently this method is used for monitoring toxic and hazardous chemicals from hazardous waste sites.

ii) Pharmaceutical Industry: Electrophoresis techniques also finds it usefulness in the characterization of small-molecule pharmaceuticals. It determines the physiochemical properties, identification, purity and stability analysis, and cleaning certification of the drug substance, its precursor, process chemicals, the drug product, etc.

iii) Source tracking contamination in Food, and other materials: When a particular microbial organism contaminant is found in a certain product, electrophoresis can be helpful to track this organism to its source. This is specifically important in food, pharmaceutical, and various manufacturing systems industries.

Suggestions for further Readings:

(Atomic Spectroscopy) : 1. Textbooks:

i) Basic Atomic and Molecular Spectroscopy by J. Michael Hollas, Royal Society of Chemistry, 2002.

ii) Optical Spectroscopy in Chemistry and Life Sciences by W. Schmidt, Wiley.

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iii) Inductively Coupled Plasma Spectrometry and its Applications, Edited by Steve J. Hill, Blackwell Publishing.

iv) Inductively Coupled Plasma – Optical Emission Spectroscopy: A Review, S. Ghosh, V. L.

Prasanna, B. Sowjanya, P. Srivani, M. Alagaraja, D. Banji, Asian J. Pharm. Ana.2013, 3, 24-33.

2. Websites:

Atomic Spectroscopy:

i)http://jpkc1.szpt.edu.cn/2008/spjy/UploadFile/ /introduction%20of%20analysis/atiomic ii) http://faculty.sdmiramar.edu/fgarces/labmatters/instruments/aa/

Atomic Absorption Spectrophotometry:

i) http://www.nuigalway.ie/chemistry/level2/courses/CH205_atomic_absorpti

ii) http://faculty.rmu.edu/~short/chem3550/chem3550-references/RSC-AA-Leaflet.pdf Flame Photometry:

i) http://www.chem.purdue.edu/courses/chm424/PDF ii)

http://www.rktech.hu/dokumentaciok/Sherwood/A%20Guide%20to%20Single%20Channel%20Flame iii) http://www.jenway.com/adminimages/PFP7_Manual(3).pdf

Inductively Coupled Plasma:

i) http://crustal.usgs.gov/laboratories/icpms/What_is_ICPMS.pdf ii) http://ir.uz.ac.zw/jspui/bitstream/10646/816/3/03_Maham

iii)http://ocw.mit.edu/courses/earth-atmospheric-and-planetary-sciences/12-119-analytical-techniques- for-studying-environmental-and-geologic-samples-

(X-ray) : 1. Textbooks:

i) Introduction to Crystallography by Donald E. Sands, Dover Publications Inc. 1994.

ii) X-ray Diffraction: A Convenient Pathway Towards Structure by SoumenGhosh and

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Atomic Spectroscopy, X-ray & Electrophoresis SantuChakraborty, New Academic Publishers, 2014.

2. Websites:

X-ray Fluorescence:

i)http://dugi-doc.udg.edu/bitstream/handle/10256/7563/1EDXRF-project.pdf?sequence=1

ii) http://www.asdlib.org/onlineArticles/ecourseware/Palmer/ASDL%20Intro%20to%20XRF.pdf iii) http://nptel.ac.in/courses/103108100/

X-ray Diffraction:

i) http://www6.cityu.edu.hk/appkchu/AP5301/Lecture-5.ppt, https://www.google.co.in

ii)http://ocw.mit.edu/courses/materials-science-and-engineering/3-091sc-introduction-to-solid-state- chemistry-

iv) http://www.soest.hawaii.edu/HIGP/Faculty/sksharma/GG711/GG711Lec07Xray

(Electrophoresis):

1. Textbooks:

i) Arne W. K. Tiselius, Electrophoresis and adsorption analysis as aids in investigations of large molecular weight substances and their breakdown products, Nobel Lecture, December 13, 1948.

ii) Electrophoresis in Practice: A Guide to Methods and Application of DNA and Protein Separations, by R. Westemeier, Wiley 4^th Edition.

2. Websites:

i) http://cdn.intechopen.com/pdfs-wm/35088.pdf ii) http://www.uvp.com/pdf/ab-1000-02.pdf

iii) http://www.chem.uky.edu/courses/che554/3_Chromatography/