eChapters

Enzymes are organic catalysts produced by living organisms. They make possible biochemical reactions. Hence they are called biocatalysts. They are mostly a specialized class of proteins. However, there are some non-protein enzymes like ribozymes, which are RNAses. Enzymes speed up reactions at ambient temperatures. This is in contrast to chemical reactions like saponification of lipids which take place when lipids are boiled for a few hours with concentrated alkali. Enzymes, namely, lipases, hydrolyze lipids at body temperatures in minutes. They are superior to chemical catalysts by a factor of 10⁷ to 10¹⁴ . An enzyme may be a simple protein or a complex protein. They are produced by cells and are mostly globular proteins. Each cell contains an estimated 3000 different enzymes.

The word protein was first coined in 1838 to emphasize the importance of this class of molecules. The word is derived from the Greek word proteios which means “of the first - rank”. Proteins are polymers/macro molecules made up of several amino acids. When there are more than 50 amino acids in a chain, the polypeptide is normally called a protein. Each protein in the cells of living beings has a unique sequence of amino acids that determines its biological function. The proteins are folded into specific defined structures, which are maintained by large number of relatively weak bonds. The three dimensional structure is well defined and suited to it’s specific function.

Lipids are a diverse group of chemical molecules. They are generally insoluble in water but soluble in organic solvents. Lipids include long-chain hydrocarbons, alcohols, aldehydes, fatty acids, and their derivatives, especially, glycerides, phospholipids, glycolipids and sulfolipids. Other compounds like sterols, fatty acid esters of sterols, vitamins A, D, E, and K, carotenoids, and other poly-isoprenoids also are part of the lipids. Oils and fats are mainly triglycerides and neutral molecules. Those in the liquid state at ambient temperatures are called oils while those in the solid state are called fats. Lipids and lipid components have varied uses in the food and non food sectors. Vegetable and animal matter contain a variety of these constituents in different proportions. It is neccessry to evaluate the composition and quality of these constituents from the point of view of their utility.

Carbohydrates are the most abundant biomolecules on earth. They occur in nature as glucose, starch and cellulose etc. Each year, photosynthesis converts more than 100 billion metric tonnes of carbon dioxide and water into starch, cellulose and other plant products. Carbohydrates are polyhydroxy aldehydes or ketones and their derivatives. They provide 4K calories of energy per gram. They promote utilization of lipids and reduce wastage of proteins. They are readily available in roots, tubers, cereal grains, sugarcane and sugar beet.

Organic material especially, seeds, grains, leaves, fruits and nuts contain prominently three groups of compounds. They are carbohydrates, proteins and fats/oils, mostly in that order. They also contain 3-5% minerals and smaller quantities of vitamins and other minor constituents. Cereal grains contain more of carbohydrates while oilseeds and nuts contain more oil. Tree nuts and even oilseeds may contain 30-50% protein while cereal grains contain 5-8% protein only. The starch and sugar contents of oilseeds may be less than 10% while the oil content of cereal grains ranges around 5%. The actual content and the composition of these groups of compounds really account for their quality as well as commercial value.

DNA and RNA are the carriers of genetic information in all the living organisms. The study and analysis of the genetic code can provide information that can be used in clinical diagnostics, drug discovery, genetic engineering and forensic sciences. Hence there is need to understand the techniques in isolation of DNA and RNA followed by their amplification and sequencing.

Titration, also known as titrimetry, is a common laboratory method of quantitative chemical analysis that is used to determine the unknown concentration of an identified analyte.  Since volume measurements play a key role in titration, it is also known as volumetric analysis. A reagent, called the titrant or titrator is prepared as a standard solution. A known concentration and volume of titrant reacts with a solution of analyte or titrand  to determine concentration. The volume of titrant reacted is called titre. Volumetric analysis originated in late 18^th-century in France.

Gravimetric analysis describes a set of methods in analytical chemistry for the quantitative determination of an analyte based on the mass of a solid. A simple example is the measurement of solids suspended in a water sample. A known volume of water is filtered, and the collected solids are weighed.
 
In gravimetry, the analyte needs to be first converted to a solid by precipitation with an appropriate reagent. The precipitate can then be collected by filtration, washed, dried to remove traces of moisture from the solution, and weighed. The amount of analyte in the original sample can then be calculated from the mass of the precipitate and its chemical composition.

In some cases, it is easier to remove the analyte by evaporation. The analyte might be collected—perhaps in a cryogenic trap or on some adsorbent material such as activated carbon—and measured directly. Or, the sample can be weighed before and after it is dried; the difference between the two masses gives the mass of analyte lost. This is especially useful in determining the water content of complex materials such as foodstuffs.

Immunoassays rely on the ability of an antibody to recognize and bind a specific macromolecule in what might be a complex mixture of macromolecules. In immunology the particular macromolecule bound by an antibody is referred to as an antigen and the area on an antigen to which the antibody binds is called an epitope.

In some cases an immunoassay may use an antigen to detect for the presence of antibodies, which recognize that antigen, in a solution. In other words, in some immunoassays, the analyte may be an antibody rather than an antigen.

When a metal M is immersed in a solution containing its own ion Mⁿ⁺, then  an electrode potential  develops. The value of  the  electrode potential  is given by the Nernst equation.

When a fixed voltage is applied to two electrodes dipping into a solution,a current will flow, depending on the conductivity of the solution. Conductivity measurements do not give information about the nature of the ions in solution. However, they are useful in the quantitative measurement of the ions.

Compounds having one or more asymmetric carbon atoms can rotate the plane of polarized light. Such compounds are called optically active. Measurement   of this property is called polarimetry.
 
The magnitude and direction in which the plane of polarized light is rotated depend on 1.The nature  of the compound, 2. The  concentration of the solution, 3. The path length of light, 4. The solvent, 5. The wave length of light and 
6. The temperature. These variables are related and can be presented in the formula

Mass spectrometry (MS) is a  useful  technique which produces spectra of the masses of the atoms or molecules in a sample.  The spectra are used to determine the elemental or isotopic   details  of a sample. The masses of particles and of molecules, are used to elucidate the chemical structures of molecules, such as peptides and other chemical compounds. Mass spectrometry works by ionizing chemical compounds to generate charged molecules and measuring their mass-to-charge ratios.

A sample, which may be solid, liquid, or gas, is ionized, by bombarding with electrons. This may cause some of the sample’s molecules to break into charged fragments. These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field. Ions of the same mass-to-charge ratio will undergo the same amount of deflection. The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.

In 1896, French physicist Becquerel observed that uranium compounds affected a photographic plate that was wrapped inside a black paper. He found that all uranium compounds gave off penetrating rays. He called the production of radiation by uranium compounds “radioactivity”. Madame and Pierre Curie later identified thorium, polonium, and radium as the other radioactive elements.

Nuclear Magnetic Resonance spectroscopy is a powerful and theoretically complex analytical tool. The nuclei of certain isotopes are continuously spinning with an angular momentum, which can give rise to an associated magnetic field.

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) uses an inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. A plasma is a neutral gas containing a good number of positive and negative ions or free electrons. A plasma can only be created. A combustion flame is one example of a plasma. Advantages of ICP-AES are excellent limit of detection and linear dynamic range, multi-element capability, low chemical interference and a stable and reproducible signal. Disadvantages are spectral interferences (many emission lines), high cost and operating expense and the fact that samples typically must be in solution.

Atomic-absorption (AA) spectroscopy uses the absorption of light to measure the concentration of gas-phase atoms. Since samples are usually liquids or solids, the analyte atoms or ions must be vaporized in a flame or graphite furnace. The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy levels. The analyte concentration is determined from the amount of absorption. Applying the Beer-Lambert law directly in AA spectroscopy is difficult due to variations in the atomization efficiency from the sample matrix, and non-uniformity of concentration and path length of analyte atoms (in graphite furnace of AA). Concentration measurements are usually determined from a working curve after calibrating the instrument with standards of known concentration.

When an element burns in a flame it emits radiation characteristic of its identity. Flame photometry is the determination of an element in solution by measurement of emission of light by atoms of the element in flame. Initial tests for identification of a few elements by flame tests are well known. Na burns with yellow flame, K with lilac flame, Ca, with red flame, Cu, with green flame etc. Emission of characteristic radiation by an element and correlation of emission intensity with the concentration of that element form the basis for flame photometry.

The sample to be analyzed is prepared in solution and sprayed under controlled conditions into a flame. The light from the flame enters a monochromator to isolate the desired region of the spectrum. A photocell and an amplifier measure the intensity of the isolated radiation. After proper calibration of the photometer with solutions of known composition and concentration, it is possible to correlate the intensity of a given spectral line with the unknown concentration of that element. A calibration curve prepared using known concentrations is useful. Internal standards and addition of known concentration of sample to be quantified are more useful.

Infrared light (800-1500nm) interacts with molecules at the level of atomic vibrations. An infrared spectrum gives valuable information about the molecular structure. It is rarely used for quantitative purposes and hence concentration measurements are not possible. Infrared light detects vibrations characteristic of functional groups. For example the C=O (carbonyl) group absorbs at 1700 cm-1 (wave number). The main region of interest for analytical purposes is from 2.5- 25µm or 4000- 400 cm-1. At wavelengths below 25µm the radiation has sufficient energy to cause changes in the vibrational energy levels of the molecule, and these are accompanied by changes in the rotational energy levels. The pure rotational spectra of molecules occur in the far infra red region (50-1000 µm) and are used for determining molecular properties.

Spectrophotometry measures how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through the sample solution. Chemical compounds normally absorb or transmit light over a certain range of wavelength. Hence spectrophotometry is used to measure the amount of a known chemical substance. It is one of the most useful methods of quantitative analysis in the laboratory and is widely used.

A  colorimeter or spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through the sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected. Depending on the range of wavelength of light source, it can be classified into two different types, namely,1) UV-visible spectrophotometer which uses light over the ultraviolet range (185-400nm) and visible range (400-700nm) of electromagnetic radiation spectrum and 2) IR spectrophotometer which  uses light over the infrared range (700-15000nm) of electromagnetic radiation spectrum.