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Pharmaceutical analysis is the branch of science which deals with identification of substances and determination of amount present in particular sample. Pharmaceutical analysis also deals with bulk materials, dosage forms and more recently, biological samples in support of biopharmaceutical and pharmacokinetic studies1. Analysis can be divided into areas called qualitative and quantitative analysis.
STEPS IN ANALYSIS OF AN ANALYTE ARE2
SEPARATION OF ANALYTE FROM INTERFERING SUBSTANCE IN SAMPLE
SELECTION OF A METHOD (BASED ON THE NATURE OF THE SUBSTANCE & DEGREE OF ACCURACY)
PROPER INTERPRETATION OF DATA
Qualitative Analysis: deals with identification of the substance.
Qualitative Analysis: deals with the determination of how much of the constituent is present.
Pharmaceutical analysis deals not only with the medicaments (drugs and formulations), but also with their precursor that is with the raw materials whose degree of purity, which in turn decides the quality of medicaments.
The analytical chemist is often confronted with the difficulty of selecting the most suitable method for the required determination. Not only he must be familiar with the practical details of the various techniques and of the theoretical principles upon which they are based, he must also be conversant with the conditions under which each method is reliable and fast. It is therefore important to emphasize that the analytical method should be as accurate as required and not as accurate as possible, otherwise, validation of method will be difficult..
The instrumental technique can be categorized into five techniques
The spectral methods are based on the nature of absorption or emission of electromagnetic radiation by the system being analyzed.
UV - Visible spectroscopy
Fluorescence and Phosphorescence spectroscopy,
X ray radiation technique
Nuclear magnetic resonance
Electron spin resonance
Turbidimetry, Nephloturbidimetrymetry, etc.,
The Electro chemical method based on interdependence of Electro chemical properties and composition of system can be classified as-
High performance thin layer chromatography
Super critical chromatography
High performance liquid chromatography
Modern pharmaceutical formulations are complex mixtures containing one or more therapeutically active ingredients, to a number of inert materials like diluents, disintegrants, colors and flavors. In order to ensure quality and stability of the final product, the pharmaceutical analyst must be able to separate the mixtures into individual components prior to quantitative analysis.
Amongst the most powerful techniques available to the analyst for the separation of these mixtures, a group of highly efficient methods which are collectively called as chromatography.
It is a group of technique for the separation of compounds of mixture that depends on the affinities of the solutes between two immiscible phases. One of the phases is affixed bed of large surface area, while the other is a fluid which moves through the surface of the fixed phase. The fixed phase is called stationary phase, and the other is termed as the mobile phase3, 4.
Depending on the type of chromatography employed, the mobile phase may be a pure liquid or a mixture of solutions (Eg Buffer) or it may be gas (pure or homogenous mixture).
Chromatographic methods can be classified according to the nature of the stationary and mobile phases.
The different types of chromatography are
Ion exchange chromatography
Size exclusion or gel permeation chromatography.
The modern instrumental techniques of GLC and HPLC provide excellent separation and allow accurate assay of very low concentrations of wide variety of substance in complex mixtures.
COMMON TYPES OF PHASE FOR SEPARATION TECHNIQUE
Gas (helium, nitrogen or hydrogen)
Viscous liquid such as squalane, poly ethylene glycol or polymethyl siloxane. Adsorbent solid such as silica, molecular sieves, alumina and porous polymers. Phase is housed in a column of glass or metal
Liquid water or organic solvents such as methanol, acetonitrile, propanol or hexane
Solid silica or a polymer such as polysaccharide or polystyrene housed in a column made of
Electrolyte or buffer solution
Buffer-filled capillary column , subject to applied voltage , creating migration of charges species
Super critical fluid chromatography
Carbon dioxide in super critical state; may have modifier such as methanol. Other choices include pentane, hexane, sulphur hexa fluoride and isopropanol
Modified silicas or polymer used in packed columns and cross-linked polymethyl siloxanes in capillary columns
Aqueous solutions of acids, bases or salts. these solutions are sometimes modified with water-miscible organic solvents such as acetonitrile or methanol
Ion exchange resins, alkyl bonded porous silica resins, and styrene-divinyl benzene polymers
Planar chromatography (thin layer chromatography)
Solvent mixtures such as hexane-acetone, chloroform, ethyl acetate, ethyl acetate- methanol, butanol, acetic acid, water, or methanol or acetonitrile
Pre coated layers silica gel, aluminium oxide, cellulose, polyamide or ion exchange material supported by glass, plastic sheets, or aluminum foil.
Adsorption chromatography5, 6
In adsorption chromatography, the mobile phase containing the dissolved solutes passes over the surface of the stationary phase. Retention of the component and their consequent separation depends on the ability of the atoms on the surface to remove the solutes from the mobile phase and adsorb them temporarily by means of electrostatic forces. Usually silica or alumina is utilized as the adsorbent with relatively non polar solvents such as hexane as the mobile phase in normal phase adsorption whereas in reversed phase adsorption non polymer beds with relative polar solvents such as water, acetonitrile methanol as mobile phase .
In partition chromatography an inert solid material such as silica gel or diatomaceous earth serves to support a thin layer of liquid which is the effective stationary phase. As the mobile phase containing the solutes passes in close proximity to this liquid phase, retention and separation occur due to the solubility of the analytes in the two fluids as determined by their partition coefficients. The method suffers from disadvantage due to some solubility of stationary phase in the mobile phase. Hence precautions must be taken to limit dissolution of stationary phase.
Ion exchange chromatography
In ion exchange chromatography the stationary phase consists of a polymeric resin matrix on the surface of which ionic functional groups, eg. Carboxylic acids or quaternary amines have been bonded chemically. As the mobile phase passes over this surface, ionic solutes are retained by forming electrostatic chemical bonds with the functional groups. The mobile phase used in this type is always liquid.
Size exclusion chromatography8, 9
In size exclusion chromatography the stationary phase is a polymeric substance containing numerous pores of molecular dimensions. Solutes whose molecular size is sufficiently small will leave the mobile phase to diffuse into the pores. Large molecules which will not fit into the pores remain in the mobile phase and are not retained. This method is mostly suitable for the separation of mixtures in which the solutes vary considerably in molecular size. The mobile phase in this type may be either liquid or gaseous.
High performance liquid chromatography10
HPLC is one of the most useful tools available for quantitative analysis. Reversed phase chromatography refers to the use of a polar mobile phase with a non polar stationary phase in contrast to normal phase is employed with a non polar mobile phase.
Liquid chromatography is based upon the phenomenon that, under the same conditions, each component in a mixture interacts with its environment differently from all other components. Since HPLC is basically a separating technique, it is always used in conjunction with another analytical tool for quantitative and qualitative analysis.
Advances in column technology, high pressure pumping systems and sensitive detectors has transformed liquid column chromatography into a high speed, high efficiency method of separation. This advanced technology is based upon the use of small bore (2.5 mm-internal diameter) columns and small particle size (3-50 µm) that allow fast equilibrium between stationary and mobile phases. This small particle column technology requires high pressure pumping system capable of delivering the mobile phase at high pressure, as much as 300 atmospheres, to achieve flow rates of several ml per minute. Since it is often necessary to use small amounts of analyte (usually less than 20 µg) with the column packing, sensitive detectors are needed.
With this technology, liquid chromatography can give high speed separations comparable in many cases to those achieved by gas chromatography, with the advantage that nonvolatile or thermally unstable samples can be chromatographed without decomposition or the necessity for making volatile derivatives.
Types of HPLC Techniques11
Based on modes of chromatography
Normal phase chromatography
Reverse phase chromatography
Based on principle of separation
Ion exchange chromatography
Ion pair chromatography
Size exclusion chromatography
Chiral phase chromatography
Based on elution technique
Based on the scale of operation
RP- HPLC is widely in use due to the factors
Many compounds such as biologically active substances, have limited solubility in non polar solvents that are employed in normal phase chromatography.
Ionic or highly polar compounds have high heats of adsorption on straight silica or alumina columns and therefore can elute as a tailing peaks.
Column deactivation from polar modifiers is a problem in liquid-solid chromatography which frequently can lead to irreducibility in chromatography systems.
Ionic compounds can be chromatographed via ion exchange chromatography. This mode of chromatography is tedious because precise control of variables such as pH and ionic strength is required for reproducible chromatography.
Mobile phase characteristics
Following points are considered for the selection of a mobile phase.
The viscosity generally increases with the number of carbons in the solvent. Straight chain alcohols show a very pronounced relationship of this nature. For example, to achieve 1ml/min flow rate in a 4.6X250 mm column packed with 5 µm octasilane material, a pressure of 1500 psi is required with methanol. Solvents of low viscosity are needed to be compatible with the limitations of the pump. Also as viscosity increases, the efficiency of the system, as measured by the number of theoretical plates decreases. The sensitivity of the detection is related to the difference between the respective refractive indices, i.e. the greater the difference, greater is the sensitivity. The UV cutoff is defined as the wave length below which the solvent will absorb more than 1.0 absorbance units.
The polarity of the solvent is a measure of the dielectric constant or the ability to elute a particular type of compound. The vapour pressure of a solvent plays an important role in mobile phase selection. Solvent reservoir could easily change in composition due to the evaporation of one of the more volatile constituents. The flammability of the mobile phase is a safety consideration. Careful attention should be paid to adequate ventilation and waste solvent disposition.
Mobile phases used in Reverse Phase HPLC12
The mobile phase in HPLC, however, has a great influence on the retention of the solutes and the separation of component mixtures.
The primary constituent of reversed phase-mobile phase is water. Water miscible solvents such as methanol, ethanol, acetonitrile, dioxan, tetrahydrofuran and DMF are added to adjust the polarity of the mobile phase. The water should be of high quality, either distilled or demineralised. The most widely used organic modifiers are methanol, acetonitrile and THF. Methanol and acetonitrile have comparable polarities but the later is an aprotic solvent. This factor may be important if hydrogen bonding plays a significant role in the separation. When inorganic salts and ionic surfactants are used, the mobile phase should be filtered before use since these additives frequently contain a significant amount of water insoluble contaminants that may damage the column. Reverse phase mobile phases are generally noninflammable due to high water content. Degassing is quite important with reverse phase mobile phases.
Solvent delivery system:
An ideal pump should generate pressures up to 6000 psi with a pulse-free output giving a flow rate from 10mL/min to less than 1mL/min. Solvent delivery system is to ensure the delivery of a precise, reproducible, constant and pulse-free flow of mobile phase. High pressure pumps are required to force the solvents through packed stationary phase beds.
Injectors should provide the possibility of injecting the liquid sample within range of 0.1 to 100 ml of volume with high reproducibility and under high pressure (up to the 4000 psi). They should also produce minimum band broadening minimize possible flow disturbances. In liquid chromatography, the liquid samples may be injected directly and solid samples need only be dissolved in a appropriate solvent.
Reverse Phase HPLC Detectors11, 13
Detectors for HPLC fall into two general categories. Differential detectors or bulk property detectors provide a differential measurement of a bulk property that is possessed by both the solute (analyte) and the mobile phase. These detectors are generally nonspecific and respond to a wide range of compounds. Eg. Refractive index detectors. The solute property or selective detectors measures a property of the sample (analyte) which is not possessed by the mobile phase, Eg. Ultraviolet and fluorescence detectors.
The ideal HPLC detectors would possess the following properties:
Produce reproducible and predictable responses.
It should give a response to any analyte.
Its response should be linear over a wide range of sample concentration.
It should be rugged and not sensitive to change in temperature or mobile phase composition.
Pumps must be constructed from the materials that are inert to all mobile phases. Materials commonly used are glass, stainless steel, Teflon and sapphire. The pump must be capable of generating pressure up to 5000 psi at flow rates of up to 3ml/min for analytical operations. The solvent flow from the pump should be pulseless or should be dampened in order to remove pulses. Since the presence of pulses in the solvent flow may cause superior results with some detectors. HPLC pumps can be classified in to two groups according to the manner in which they operate constant flow rate pumps and constant pressure pumps
Modern HPLC is a technique for making precision, separation of complex mixture and offers high resolution separating capability to solve problems faster and better.
Reverse Phase Ion-Pair Chromatography10
The solvents generally employed in Reverse phase- chromatography consist of water and an organic modifier for neutral compounds. For basic substances and acidic substances an aqueous buffer system plus an organic modifier is employed. The typical buffer consists of acetate and phosphate salts at a pH value that favors the formation of a unionized form of a solute.
Characteristics of Column Packing7
Most column packing is based on a silica matrix. These packing are rigid and can withstand pressure in excess of 10,000 psi. Silica particles are also available in a wide range of porosity. Since the surface of silica contains silanol groups, organic moieties may be chemically bonded to the surface for bonded phase chromatography. Other rigid solids which can be used as support materials. Eg. Particles of polystyrene cross linked with divinyl benzene.
The polymers are more resistant to chemical attack than silica particles and may find more uses with mobile phase of high and low pH values. The size of the packing material has a major effect on the resolving power of the system. As the particle size decreases, the height equivalent to theoretical plate also decreases. However, as the diameter of the particles decreases, the resistance of solvent flow increases. Particle diameter of 5-10µm are used in analytical applications while particles of 37-50 milli microns can be used for preparative scale HPLC where high solvent flow rates are required.