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# Bioanalytical Systems

Analytical chemistry is the branch of chemistry. In the analytical chemistry, the chemical composition of substances are identified and separated by using different methods. Bio analytical chemistry is the branch of analytical chemistry.

This includes the study of the biological compounds like proteins, peptides and carbohydrates etc. The study of bio molecules that are most commonly analyzed in bio analytical chemistry: amino acids, proteins and nucleic acids.

The structure of these bio molecules and their physical and chemical characteristics and some of the functions of these bio molecules and how they interact with each other in the cell.

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## Mass Spectrometry

1. Mass Spectrometry is a technique for detecting unknown compounds and knowing about molecular structure of those compounds. This is a method for weighing molecules.
2. This is based on the migration of ion (charged particle) in an electric or magnetic field.
3. The motion of ion is affected by the mass to charge ratio (m/z).
4. This ratio is used to measure the mass of an ion as the charge of an electron is known.
5. The Mass Spectrometer-In mass spectrometry technique, mass spectrometer is used to measure the character of the molecules.
6. It changes the molecule into ions and makes them able to move in external electric and magnetic fields.
7. The formation and manipulation of ions are done in a vacuum because they are very reactive and short-lived.
8. The pressure should also be maintained (10-5 to 10-8 torr). In general procedure of mass spectrometry, after the ionization process of ions, they become focused into a beam.
This beam is bent in the magnetic field. Thus these ions are electronically measured and all this information is gathered with the help of computer. The three main components of a mass spectrometer are
• The ion source
• The Mass Analyzer
• The Detector
Ion source is also called heart of spectrometer. The sample is ionized generally in cation by loss of electrons. The electrons from a heated filament are bombarded on sample. This is known as an EI source (electron-impact). Gases and volatile liquid samples are leak into the ion source from a reservoir while non-volatile solids and liquids can be introduced directly. The Cations and anions formed by the electron bombardment (red dots in figure) are attracted towards other electrodes and charged repeller plate respectively.

The cation's electrode has slits for passing the ions as a beam. A perpendicular magnetic field bent the ion beam in an arc. The radius of arc is inversely proportional to the mass of ion. Heavier ions are less deflected than Lighter ions. Thus the ions of different mass can be focused on the detector with varying the strength of the magnetic field. The Residual energy of the collision between high energy electrons and a molecule may be reason for converting the molecular ion to fragment into neutral pieces and smaller fragment ions.

The molecular ion is a radical cation but the fragment ions can be either radical cations or carbocations.
It depends on the nature of the neutral fragment.

M: + e-  $\rightarrow$ 2e- + M+

M+ $\rightarrow$ M.+ (radical cation) + F (fragment neutral species)
(Or)
M+ $\rightarrow$M+ (carbocation) + F (fragment neutral species)

The Nature of Mass Spectra

The mass spectrum are presented as a vertical bar graph and each bar represents an ion having a particular mass-to-charge ratio (m/z). The length of the bar shows the relative abundance of the ion.
The most intense ion has an abundance of 100 which is called as the base peak.

If the sample is assume as single pure compound, and then the highest-mass ion in a spectrum is usually for the molecular ion while lower-mass ions are for fragments from the molecular ion. Atomic mass is defined in the terms of the unified atomic mass unit or Dalton.

For example, the mass spectra of gaseous compounds carbon dioxide CO2, propane C3H8 and cyclopropane C3H6 are similar in size. The nominal mass of CO2 and C3H8 is 44 Da and C3H6 is 42 Da.
The molecular ion of CO2 and C3H6 is the strongest ion in their spectra and it is moderately strong in propane.

The mass spectrometer can easily differentiate between different isotopes of a given element. The molecules of bromine have only two atoms, the five peaks in its spectrum show that natural bromine consist equal mixture of isotopes having atomic masses of 79 and 81 Da respectively.

So the bromine molecule may be formed with two 79Br atoms (mass 158 Da) and two 81Br atoms (mass 162 Da) and the combination of 79Br-81Br (mass 160 Da). The fragmentation of Br2 molecule to a cation gives equal sized ion peaks at 79 and 81 Da.

So the isotopic composition of bromine is 50.50% 79Br and 49.50% 81Br. The nature of the fragments also provides information about the molecular structure but it's difficult to observe mass spectrum with short life time of molecular ion. The most stable molecular ions of organic compounds are those from aromatic rings, conjugated pi-electron systems and cycloalkanes etc. But alcohols, ethers and branched alkanes usually show the tendency toward fragmentation. For example mass spectrum of dodecane.

Since this molecule don't have heteroatom and non-bonding valence shell electrons. But the radical cation of the molecular ion (m/z = 170) get delocalized over all the covalent bonds. Fragmentation of C-C bonds takes place due to the weakness of C-C bonds than C-H bonds.

Thus the mixture of alkyl radicals and alkyl carbocations are obtained. The positive charge are on the smaller fragment, so there is a homologous series of hexyl (m/z = 85), pentyl (m/z = 71), butyl (m/z = 57), propyl (m/z = 43), ethyl (m/z = 29) and methyl (m/z = 15) cations with a set of alkenyl carbocations (e.g. m/z = 55, 41 &27).

The presence of a functional group especially heteroatom with non-bonding valence electrons (N, O, S, halogen etc.) can change the fragmentation pattern of a compound due to the localization of the radical cation of the molecular ion on the hetero-atom.

## Bio Analytical Systems

These are the system which is based on the detection, identification and the separation of components especially the biological compounds. Chromatography technique is used for separation and identification of compounds in the bio analytical system.

## Chromatography

Chromatography technique is used to isolate the various substances of a mixture. Various types of chromatography are gas, liquid, paper, thin layer and gel chromatography etc. All are very useful in
various fields like applied sciences, forensics labs, and athletics etc.

Basic operation of chromatography

• The process is based on fact that the different molecule behave in different ways when they dissolved in solvent and moved in absorbent medium.
• In chromatography two phases are present, the one is mobile phase and another is stationary phase.
• The liquid is in the mobile phase which pumps through bed of particles while the particles are in the stationary phase.
• A mixture of the sample molecules is introduced into the liquid.
• The mixture of molecules is migrated by the mobile phase through the stationary phase.
• A chromatography column is used in separation process.
• The chromatography column can be made of glass or metal and a packed bed or in open tubular column.
• The particles (stationary phase) are present in packed bed column.
• The Solvent (the mobile phase) moves through the column containing the sample mixture.
• The solvent can be either a liquid or a gas, depends upon the type chromatography.
• The partitioning of solutes between the phases is the main cause of separations.

1. Feed Injection-The sample is injected into the mobile phase. The mobile phase flows by the pump.
2. Separation in the Column-As the sample flows through the column, the different components of sample will adsorb to the stationary phase with different degrees. The components with strong attraction to the support move slowly than the components with weak attraction.
3. Elution from the Column-The components with the less attraction for the stationary phase (less adsorbed) will elute first while those with the greatest attraction for the stationary phase (strong adsorbed) will be in last.
4. Detection-Then all the different components are collected. A detector is used to analyze them by measuring their property related to concentration and characteristic of composition.

Example-This figure shows a simple separation. A solvent carries a solution of solutes A and B.
(a) it show the initiation of process as the solvent flows with solutes. (b) It shows that solute B is moving faster than A. (c) and (d) the solute B first separate while solute A still flows. (e)The solute A has a strong affinity for the stationary phase than solute B and A get separated.

The output of the column can be altered by alter the pH of the solvent or temperature. This affect the emerge timing of individual species.

## Spectrometry

This is a technique of observing and measuring the wave length of radiations in a particular range. For example, mass spectrometry, absorption and emission spectrometry, optical spectrometry etc.

## Electrophoresis

Electrophoresis is a most powerful technique for molecular separation. This method is based on the migration of ions in an electric field. The oppositely charged ions migrate towards their respective electrodes under the influence of electric field. The one electrode is always at ground for safety reasons. The migration rate of ions depends on their charge, size, and structure and thus can be easily separated. This technique is especially used for biological samples like protein mixtures or DNA fragments. Electrophoresis can be one dimensional (one plane of separation) or two dimensional.
Some specific techniques of electrophoresis are disc, capillary and gel electrophoresis.

Instrumentation
• An electrode apparatus is used in electrophoresis.
• This consists of a high-voltage supply; electrodes and buffer while filter paper, cellulose acetate strips, gel of polyacrylamide, and capillary
• tube are used for supporting the buffer.
• The support is formed into gel with in tube.
• After completing the separation the support is stained to analyze the separated components.

The isoelectric focusing is used for improving resolution in which the support gel maintains a pH gradient.
As a protein moves down the gel, it reaches a pH which is equal to its isoelectric point.
The protein becomes neutral at this pH and not able to migrate.
It gives a sharp band on the gel. Some of the techniques of electrophoresis are described below-

Discontinuous electrophoresis (Disc)

In this technique, two gels is used that are buffered at different pH. When proteins move from one gel to the other they give sharp bands with high resolution.

Capillary Electrophoresis

Capillaries tubes with small diameter are used with high electric field which reduce the separation time and give perfect band for separations. Gel Electrophoresis -SDA (sodium dodecyl sulfate) and PAGE (polyacrylamide gel electrophoresis). This is also called SDA-PAGE. This is useful for analyzing the molecular weight of protein. They SDA form complexes with proteins and the size of complex determine the migration rate of protein in gel.

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