An analysis is broadly divided into two types:
- Qualitative Analysis: It gives information about the identity of atomic and molecular species or the functional groups in samples. It is used only to determine the presence and absence of the compound.
- Quantitative Analysis: It establishes the relative amount of one or more of the species (analyte) in numerical terms. It measures the concentration or amount of each substance in a sample.
Analytical Method
Qualitative and/or quantitative analyses are carried out for one or more analytes using a specified technique/analytical method.
Classification of Analytical Methods
Various methods of analysis can be broadly classified into two categories; Classical methods and Instrumental methods.
Classical Methods:
These are based on the traditional method of analysis and may be divided as:
- Qualitative: Use for identification by colour, odours, boiling points, melting point, limit test, etc.
- Quantitative: Use for determination of mass or volume (e.g. volumetric and gravimetric analysis).
Volumetric Analysis is used to determine the exact volume of the solution of known concentration required to react completely with the solution of the substance to be analysed.
(i) Neutralization acid-base titration: It involves neutralization acid-base reaction in presence of water as solvent.
(ii) Non-aqueous acid-base titration: It involves a reaction between acid and base in presence of a non-aqueous solvent i.e. organic solvents.
(iii) Complexometric titration: It is a complex formation reaction. In this titration, the reacting substance reacts with a standard solution to form a soluble but very slightly dissociated complex.
(iv) Precipitation titration: It is a precipitation formation reaction. In this titration, the reacting substance reacts with the standard solution to form a precipitate or slightly soluble salt.
(v) Oxidation-reduction titration: In this titration simultaneousoxidation–reduction reaction occurs. It includes all the methods where one reacting substance is oxidized while the other gets reduced.
Gravimetric Analysis is the quantitative determination of an analyte based on the mass of a solid by the process of isolating a definite compound of an element in pure form. In this technique, substance underdetermination is converted into an insoluble precipitate which is collected and weighed.
Instrumental Methods:
These methods are based upon the measurement of some physical properties such as conductivity, electrode potential, light absorption or emission, mass-to-charge ratio, and fluorescence of substance. When a non-instrumental method is not possible, an instrumental method is the only answer to the problem.
The instrumental methods have the advantage of being less time-consuming than classical methods. Various factors such as cost of instruments, sensitivity, accuracy, precision, and reproducibility have to be taken into consideration before selecting an analysis method.
Qualitative: Chromatography, electrophoresis, and identification by measuring physical properties (e.g. spectroscopy, electrode potential).
Quantitative: Use for measuring property and determining its relationship to concentration (e.g. spectrophotometry, mass spectrometry) often, the same instrumental method is used for qualitative and quantitative analysis.
Advantages of Instrumental Methods:
- A small amount of a sample is required for analysis.
- Determination is comparatively fast.
- Various complex mixtures can be analyzed with or without their separation.
- High sensitivity and selectivity.
- Results obtained by the instrumental method are reliable and accurate.
Limitation of Instrumental Methods:
- Instrumental methods are comparatively costly.
- Need for regular maintenance of the instrument
- Skilled personnel required.
- Specialized training is required for handling instruments.
- The sensitivity and accuracy depend upon the type of instrument.
- Selectivity of an instrument is required for specific samples.
- Sometimes cross checking of results with other methods is needed.
Types of Instrumental Methods:
There are many techniques available for the analysis of analytes, which can be broadly classified as:
- Electroanalytical methods (chemical potential electrons)
- Analytical separation methods (chemical equilibrium detectors)
- Analytical spectroscopic methods (chemical energy photons)
- Miscellaneous methods
Electro Analytical Methods
Table of Contents
Electrochemical methods are powerful and versatile analytical techniques that offer high sensitivity, accuracy, and precision with relatively low-cost instrumentation.
1. Potentiometry is useful means of characterizing an acid of which the potential is measured across the analyte without using an indicator. It gives information about the composition of the sample through the potential appearing between two electrodes.
2. Voltammetry is the study of current as a function of applied potential. It is used to designate the current-voltage measurement obtained at a given electrode.
3. Coulometry use for the determination of the amount of matter transformed during an electrolysis reaction by measuring the amount of electricity (in Coulombs C’) consumed or produced
4. Polarography is a special case of voltammetry referring to the current-voltage measurement acquired using a dropping mercury electrode with a constant flow of mercury drop.
5. Amperometry is used for the determination of any analyte concentration by measurement of the current generated in a suitable chemical reaction.
6. Conductometry is a measurement of the electrical conductivity of ionic solutions by applying an electric field between two electrodes.
7. Electrogravimetry is based on an analysis that is carried out by passing an electric current for a sufficient length of time to ensure complete oxidation or reduction of the analyte to a single product of known composition. It is a method used to separate and quantify ions of a substance, usually a metal. Examples: Cd, Cu, Ni, Ag, Sn, Zn.
Analytical Separation Methods
Chromatographic is a method of separating a mixture of the compound into the individual compound through equilibrium distribution between two phases viz, a stationary phase and a mobile phase. Chromatography is based on the difference in the rate at which the compound of a mixture moves through a porous medium under the influence of some solvent or gas.
Chromatographic methods are broadly classified based on separation that takes place on a planar surface or in a column. In column chromatography, the stationary phase is placed in a narrow column through which the mobile phase moves under the influence of gravity or pressure. In planar chromatography, the stationary phase coats a flat glass, metal, or plastic plate and is placed in a developing chamber containing the mobile phase.
Depending upon the stationary phase used, chromatographic methods are broadly divided into two types namely Adsorption chromatography and Partition chromatography. Adsorption chromatographic separation is based on the principle of adsorption at solid-liquid interphase, whereas separation by partition chromatography is based upon the differences in partition characteristics of the individual components of a mixture between a liquid stationary phase and liquid or gaseous mobile phase.
(a) Column Chromatography: Column chromatography is characterized by the use of relatively wide-diameter glass columns packed with a finely divided stationary phase with the mobile phase percolating through the column under gravity. There are five types of column chromatography:
1. Adsorption Chromatography: Sample components from the mixture are selectively adsorbed on the surface of the packing material used as the stationary phase. The liquid solvent is used as the mobile phase and the solid as the stationary phase. The adsorbent is an active solid such as alumina, charcoal, or silica gel, which may be packed in a column or spread on a plate. In Normal-Phase Chromatography (NPC), the stationary phase is polar and the mobile phase is non-polar. In NPC non-polar compounds prefer the mobile phase and elute first whereas polar compounds prefer the stationary phase and elute later. In Reverse-Phase Chromatography (RPC), the stationary phase is non-polar and the mobile phase is polar. Elution of compounds in RPC has commonly reversed to that of NPC i.e. polar compounds eluting first and non-polar ones later.
2. Partition Chromatography: The sample component from the mixture is partitioned between the liquid stationary phase (liquid held stationary on inert solid support e.g. silica gel, kieselguhr, etc.) and the liquid mobile phase. The distribution of compounds between two immiscible liquid phases takes place according to their relative solubilities.
3. Bonded-Phase Chromatography (BPC): To overcome the problems related to conventional liquid-liquid chromatography (partition chromatography), such as loss of stationary phase from the support material, the stationary phase may be chemically bonded to the support material. In this both monomeric and polymeric phases have been bonded to a wide range of supporting materials.
4. Ion Exchange Chromatography: In this constituent of the sample is selectively retained by ion exchange resin packing material. This is a reversible reaction, in which free mobile ions of a solid (ion exchange) are exchanged for different ions of similar charge present in the solution. The technique is widely used for the separation of ionisable compounds, mostly for aprotic ions, e.g. quaternary ammonium compounds, and for compounds such as amino acids which are difficult to extract in the uncharged form.
5. Gel Permeation Chromatography or Gel Exclusion Chromatography: In this column is packed with a porous material (permeable gel) and liquid as a mobile phase. The constituents penetrate the pores and exclude from the pores according to their molecular size and shape, which leads to a difference in the rates at which molecules pass down the column.
(b) Paper Chromatography: It is partition chromatography in which the substances are distributed between two liquids i.e. one is stationary liquid held on the fibres of the paper (stationary phase) and the other is moving liquid as a mobile phase. The mixture components to be separated travel at different rates and appear as spots on the paper.
(c) Thin Layer Chromatography (TLC): Thin Layer Chromatography is similar to paper chromatography except for that thin layer of sorbent (e.g. silica gel or cellulose powder) coated on an inert and rigid supporting material i.e. a glass plate or plastic foil so as the separation process occurs flat on a two-dimensional surface. Mostly it is used for qualitative analysis as it does not provide quantitative information of high precision and accuracy.
(d) High-Performance Thin-Layer Chromatography (HPTLC): HPTLC has a firm place as a reliable method for quantitation at micro and nanogram levels for the drug present single or in the multicomponent formulation. HPTLC has evolved through conventional TLC through improvements in the quality of the sorbent layers, methods of sample applications, new development techniques and availability of scanning densitometers for in-situ quantitative analysis. HPTLC plates are prepared using specially purified silica gel with an average particle diameter of 5-15 µm with narrow particle size distribution.
HPTLC gives a better choice of analysis as it can handle several samples of divergent nature and composition by several analysts at the same time.
(e) Gas Chromatography (GC): Gas chromatography is a process of separation of a mixture in its constituents by a moving gas phase passing over a stationary sorbent. The technique is similar to liquid-liquid chromatography except that the mobile liquid phase is replaced by a moving gas phase. In Gas-Liquid Chromatography (GLC), the constituents are separated by distributing themselves between the mobile gas phase and a thin layer of non-volatile liquid coated on an inert support as a stationary liquid phase as per their partition coefficient. In Gas-Solid Chromatography (GSC), the constituents are separated by distributing themselves between the mobile gas phase and the adsorbent. Separation in GSC is carried out as per their adsorptive behaviour.
(f) High Performance (Pressure) Liquid Chromatography (HPLC): It is a separation technique in which a sample is passed through a column packed with solid particles. In this system, pressure is applied to the column and forces the mobile phase through at a much higher rate. The resolving power of the chromatographic column increases with column length and the number of theoretical plates per unit length. High-Performance Liquid Chromatography is in some respects more adaptable than Gas Chromatography as the choice of mobile and stationary phases is wider and it is not limited to volatile and thermally stable samples only.
(g) Supercritical Fluid Chromatography (SFC): SFC is pressurized flow path chromatography that utilizes carbon dioxide as the mobile phase. It is a form of normal phase chromatography having a principle similar to High-Performance Liquid Chromatography (HPLC). As the supercritical phase represents a state in which liquid and gas properties converge (critical temperature and critical pressure), supercritical fluid chromatography is sometimes called “Convergence Chromatography.”
(h) Electrophoresis and Capillary Electrophoresis (CE): Electrophoresis is the process of migration of charged species (ions) through solutions by attraction or repulsion under the influence of an electric field. Capillary Electrophoresis (CE) is electrophoresis performed in a capillary tube. It is the most efficient separation technique available for the analysis of both large and small molecules.
Capillary Electrophoresis is used for the separation of many compounds such as amino acids, carbohydrates, peptides and proteins, vitamins, dyes, surfactants, pesticides, inorganic ions, organic acids and chiral drugs.
Analytical Spectroscopy
Analytical methods employing electromagnetic radiation or light energy for the qualitative or quantitative determination of compounds are known as the Spectroscopic method. They are broadly divided into two types namely Absorption Spectroscopy and Emission Spectroscopy. In Absorption Spectroscopy, the light energy is absorbed by the sample whereas in emission spectroscopy, the light energy is first absorbed and immediately it is emitted by the samples. Some of the spectroscopic methods are,
1. Ultra Violet-Visible Spectroscopy: UV-visible spectroscopy is a type of absorption spectroscopy in which light of the UV-visible region (200-750 nm) is absorbed by the molecule. Absorption of the radiations leads to the excitation of the electrons from the ground state to a higher energy state. The energy of the ultraviolet radiation can be calculated as it is equal to the energy difference between the ground state and higher energy states.
UV-visible spectroscopy obeys the Beer-Lambert law, which states that: “when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation is directly proportional with the thickness of the absorbing solution as well as the concentration of the solution”.
2. Atomic Absorption Spectroscopy (AAS): Atomic absorption spectrometry (AAS) is an analytical technique that measures the concentrations of elements.
Atomic Absorption Spectrometry (AAS) is a technique for measuring quantities of chemical elements present in environmental samples by measuring the absorbed radiation by the chemical element of interest. This is done by reading the spectra produced when the sample is excited by radiation. The atoms absorb ultraviolet or visible light and make transitions to higher energy levels. Atomic absorption methods measure the amount of energy in the form of photons of light that are absorbed by the sample.
3. Infrared Spectroscopy (IR): Infrared spectroscopy is an absorption method in the wavelength region of 700 nm – 1 mm. Infrared spectroscopy is a technique based on the vibrations of the atoms of a molecule.
An infrared spectrum is commonly obtained by passing infrared radiation through a sample and determining what fraction of the incident radiation is absorbed in a particular energy. The energy at which any peak in an absorption spectrum appears corresponds to the frequency of vibration of a part of a sample molecule.
Infrared spectroscopy provides mostly information about the presence or absence of certain functional groups.
4. Mass Spectroscopy (MS): A mass spectrometer is an instrument that measures the mass-to-charge ratio (m/z) values and their relative abundances of ions. In Mass Spectroscopy, a bombardment of the sample with electrons is carried out and the detection of resulting molecular fragments takes place. It provides information about the molecular mass and atom connectivity.
5. Raman Spectroscopy: Raman spectroscopy measures the vibrational motions of a molecule like infrared spectroscopy. The physical method of observing the vibrations is, however, different from infrared spectroscopy. In Raman spectroscopy one measures the light scattering while infrared spectroscopy is based on the absorption of photons. In Raman spectroscopy, a sample is illuminated with a monochromatic laser beam which interacts with the molecules of the sample and originates a scattered light.
6. Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR is the absorption of radio frequencies by atomic nuclei within a sample that is placed in a magnetic field. In NMR, excitation of the nucleus of atoms takes place through radiofrequency irradiation. It provides extensive information about molecular structure and atom connectivity.
7. Polarimetry: Polarimetry is a sensitive non-destructive technique for measuring the optical activity exhibited by inorganic and organic compounds. A compound is considered to be optically active if linearly polarized light is rotated clockwise (+) or counterclockwise (−) when passing through it. The amount of optical rotation is determined by the molecular structure and concentration of chiral molecules in the substance.
8. Fluorimetry and Phosphorimetry are a determination of fluorescence and phosphorescence.
(a) Fluorescence is the re-emission of longer wavelength (lower frequency) photons (energy) by a molecule that has absorbed photons of shorter wavelengths (higher frequency). Absorption and emission of energy are characteristics of a molecule during the fluorescence process.
Light is absorbed by molecules for a very short period which causes electrons to become excited to a higher electronic state. All of the excess energy is not lost by collisions with other molecules and the electron returns to the ground state. Energy is emitted during the electron’s return to its ground state. The emission of light is an instantaneous process and starts immediately after the absorption of light and stops as soon as the incident light cut off.
(b) Phosphorescence is the re-emission of a longer wavelength. When a beam of light is incident on certain substances, they emit light continuously even after the incident light is cut off. The emitted light is always a longer wavelength than absorbed light due to limited energy loss by the molecule before emission.
9. Nephelometry and Turbidimetry: Turbidity is an expression of an optical property of a medium a cuvette containing suspended particles, which causes light to be scattered and absorbed rather than transmitted in straight lines through the medium.
(a) Nephelometry is involved with the measurement of scattered light. The components of a nephelometry are the same as a light spectrophotometer except that the detector is placed at a specific angle from the incident light. Detectors may be placed at 90°, 70° or 37° depending on the angle at which most scattered light is found.
(b) Turbidimetry is a concern with measuring the amount of transmitted light (and calculating the absorbed light) by particles in suspension to determine the concentration of the substance.
The amount of scattered light depends on the size and number of particles in suspension. Since the amount of scattered light is far greater than the transmitted light in a turbid suspension, nephelometry offers higher sensitivity than turbidimetry.
10. Flame Photometry: Flame photometry is a process wherein the emission of radiation by the neutral atom is measured. The neutral atoms are obtained by the introduction of the sample into flame. Hence, the name flame photometry. Since radiation is emitted it is also called flame emission spectroscopy.
Miscellaneous Methods
1. Thermal Gravimetric Analysis: Thermal Gravimetric Analysis (TGA) uses stoichiometry and heat ratios to determine the percentage of the mass of a solute. Test and analysis are carried out by gradually increasing the temperature of a sample, whilst plotting weight against temperature so effectively monitoring material transformation where compounds degrade or form at different temperatures.
2. Differential Scanning Calorimetry (DSC): Differential Scanning Calorimetry (DSC) is a thermo analytical technique that is used to demonstrate the energy phenomena produced during the heating or cooling of a substance and to determine the changes in enthalpy, specific heat and the temperatures at which these occur.
The DSC instrument measures the heat flow in and out of both a sample and reference crucible during a controlled temperature programme. The sample crucible usually contains the substance (or a mixture of substances) under study and the reference crucible is either left empty or is loaded with an inert reference material relevant to the sample under investigation.
3. Refractometry: Refractometry is the technique of measuring a substance’s refractive index to access its composition and purity. The speed of light in a vacuum is always the same, but when light moves through any other medium it travels more slowly since, it is constantly being absorbed and re-emitted by the atoms in the material. The refractive index of a medium is a measure of how much the speed of light is reduced inside the medium related to the speed of light in a vacuum. A refractometer is an instrument used to measure the refractive index of materials.
4. Radioactivity: Radioactivity methods involve the measurement of the intensity of radiation from naturally radioactive material and are used
- In the determination of trace elements.
- In examining geological specimens.
- In quality control in semiconductor manufacturing.
Radioactivity is the process in which unstable atomic nuclei lose energy by emitting radiation in the form of particles or electromagnetic waves. These radiations are capable to ionize the atoms and molecules along their track. As these radiations can cause cancer and death, they concern health and safety.
5. Kinetic Methods: Kinetic methods involve the study of an increase in the speed of a reaction by adding a small amount of catalyst to the reaction mixture. These methods are found to be useful:
- In determining sub microgram amount of appropriate organic substances.
- In determining the amount of a substance in a solution.
- In clinical chemistry.
Hyphenated Techniques:
The hyphenated technique is developed from the coupling of two different analytical techniques with the help of a proper interface. Mainly a separation technique and an on-line spectroscopic detection technology are combined which is useful for the analysis of bioactive compounds from synthetic as well as natural origins. Chromatography produces pure or nearly pure fractions of chemical components in a mixture whereas spectroscopy produces selective information for identification using standards or library spectra.
Some of the advance hyphenated techniques are,
- Gas Chromatography-Mass Spectroscopy (GC-MS)
- Liquid Chromatography-Mass Spectroscopy (LC-MS)
- Capillary Electrophoresis–Mass Spectroscopy (CE-MS)
- Gas Chromatography-Fourier Transfer Infra-Red Spectroscopy (GC-FTIR)
- Liquid Chromatography-Fourier Transfer Infra-Red Spectroscopy (LC-FTIR)
- Liquid Chromatography-Nuclear Magnetic Resonance Spectroscopy (LC-NMR)
- Liquid Chromatography-Nuclear Magnetic Resonance Spectroscopy-Mass Spectroscopy (LC-NMR–MS)
Selection of an Analytical Method:
To select an analytical method, it is essential to understand clearly the nature of the analytical problem. In general, some of the following points should be considered for selecting any analytical method for any qualitative or quantitative measurement.
- Available sample amount.
- Physical and chemical properties of the sample.
- Interference in a sample.
- Speed, ease, skill and cost of analysis.
- Several samples are to be analyzed.
- Accuracy and precision are required.
- The concentration range of the analyte.
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