Analytical Chemistry Is Science And Art Of Composition Biology Essay


Introduction to Analytical Chemistry

Analytical Chemistry is defined as the "science and the art of determining the composition of materials in terms of the elements or compounds contained". This branch of chemistry, which is both theoretical, and a practical science, is practiced in a large number of laboratories in many diverse ways while analytical method, is a specific application of a technique to solve an analytical problem. Methods of analysis are routinely developed, improved, validated, collaboratively studied and applied. In analytical chemistry it is of prime importance to gain information about the qualitative and quantitative composition of substances and chemical species, that is, to find out what a substance is composed and exactly how much. In quantitative analysis the question is how much is present? The research work in this thesis is based on this criterion.

Instrumental methods of Chemical analysis

Instrumental method is an exciting and fascinating part of chemical analysis that interacts with all areas of chemistry and with many other areas of pure and applied sciences. Analytical instrumentation plays an important role in the production and evaluation of new products and in the protection of consumers and environment. This instrumentation provides lower detection limits required to assure safe foods, drugs, water and air. Instrumental methods are widely used by Analytical chemists to save time, to avoid chemical separation and to obtain increased accuracy.

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Most instrumental techniques fit into one of the four-principal areas mentioned below.

Spectrophotometric techniques

UV and Visible Spectrophotometry

Fluorescence and Phosphorescence Spectrophotometry

Atomic Spectrophotometry (emission &absorption)

Infrared Spectrophotometry

Raman Spectrophotometry

X-Ray Spectrophotometry

Nuclear Magnetic Resonance Spectroscopy

Mass Spectroscopy

Electron Spin Resonance Spectroscopy

Electrochemical Techniques






Chromatographic Techniques

High Performance Liquid Chromatography

Gas chromatography

High Performance Thin Layer Chromatography

Thin Layer Chromatography

GC- MS (Gas chromatography - Mass Spectroscopy

LC-MS (Liquid Chromatography - Mass Spectroscopy)

Flow chart for Classification of chromatographic techniques

Fig 1: Flow Chat for Classification of Chromatographic Techniques


Liquid chromatography is a separation method in which the components of a sample partition between two phases - one of these phases is a stationary bed with a large surface area and the other is a liquid which percolates through the stationary bed. The sample is carried by the mobile liquid phase through the column. Samples partition (equilibrate) into the stationary liquid phase, based on their solubility in the phases and/ or molecular size solubilities. The compounds of the sample separate from one another based on their affinities for the stationary bed. This type of chromatography process is called elution.

High Performance Liquid Chromatography (HPLC) is the fastest growing analytical technique for the analysis of drugs. Chromatographic separation in HPLC is the result of specific interaction between sample molecules with both the stationary and liquid mobile phases. HPLC has been rapidly developed with the introduction of new pumping methods, more reliable columns and wide range of detectors. HPLC is also being automated which involve automated sampling, separation, detection, recording, calculation and printing of results. Due to the high selectivity and sensitivity achieved by HPLC methods, it became the most selective method for the analysis in wide range of drugs. HPLC is one of the most versatile instruments used in the field of pharmaceutical analysis today with following advantages.

High resolving power.

Faster separation.

Continuous monitoring of the column effluent.

Accurate quantitative measurement.

Repetitive and reproducible analysis using same column.

HPLC offers a wide choice of chromatographic separation methodologies from normal to reverse phase and whole range of mobile phases using isocratic or gradient elution techniques. Various detectors available for HPLC are electrochemical detectors, refractive index detectors, fluorescence detectors, radiochemical detectors, mass-sensitive detectors and Ultra-violet (UV) detectors.

To develop a new HPLC method for any drug, knowledge of its molecular weight, polarity, ionic character, pKa values, wavelength of absorption, purity of compound and the solubility should be known. Method development involves considerable effort and time. The most commonly applied method is reversed phase and reverse coupled with ion-pairing. These two techniques probably account for more than 85% of the applications for a typical pharmaceutical compound. The typical pharmaceutical compounds are considered to be an active pharmaceutical ingredient of molecular weight of less than 1,000 Daltons. Depending on the number of active compounds to be resolved or separated, the more complex is the separation, the more gradient elution will be advantageous over isocratic mode. Optimization can be started only after reasonable chromatogram has been obtained.

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Fig 2: Pictorial Representation of HPLC Instrument

Fig 3: Schematic Diagram of HPLC Instrument

Important Components in HPLC

The Mobile Phase Delivery System

In modern HPLC The mobile phase delivery system plays an important role. After preparation of Mobile phase it should be filtered through vacuum and then subjected to degassing by performing sonication. Degassing should be performed in order to remove solubilised gases. If any gases are present they will form bubbles and they may cause damage to the Detector and results in detector noise. Hence in order to prevent these abstructions sinication is must.

There are two types of Mobille phase delivery systems based on number of reservoir systems present .They are 1)Binary 2) Quaternary

1) Binary:

It consists of two reservoir systems named as A and B.

These systems are useful for simple isocratic and gradient elutions.

The disadvantage of the method is complex mixtures are difficult to separate through this binary systems.

2) Quaternary

It consists of four reservoir systems. named as A,B,C, and D.

Now a days these are the most commonly using pumping systems.

The mobile phase composition can be varied randomly in this type of systems,and is very useful for the separation of complex mixtures.

Mixing of solvents

In HPLC most of the separations are done by using combination of different solvents based on the mode of separation i.e isocratic or gradient.

There are two types of mixing of solvents based on isocratic and gradient separation.

Low pressure mixing:

In isocratic elution the mobile phase composition remains constant throughout the experiment. In this elution the the solvent reservoirs are connected to the mixing manifold,the mixed solvents enters into the pump This type of mixing requires low pressure and is called low pressure mixing .

In this process the solvents are allowed to enter into mixing manifold through valves in which mixing takes place according to the amount of solvent entering into the mixing manifold From this the mobile phase enters into pumps the sample valve and then into the column.

Solvent Reservoir  valves  Mixing manifold  pump  Sample valve

High pressure mixing:

In gradient elution the mobile phase composition varies with time. As the composition changing, the pressure required for the mixing increases hence this is also named as high pressure mixing.In this type of mixing the solvents passes through individual pumps and get mixed in the mixing manifold.

Solvent Reservoir  pumps  Mixing manifold  Sample valve


Pump is required to deliver a constant flow of mobile phase at pressures ranging from 1 - 550 bar pumps capable of pressure up to 8000 psi provide a wide range of flow rates of mobile phase, typically from 0.01-10ml min-1. Low flow rates (10-100l min-1) are used with micro bore columns, intermediate flow rates (0.5-2ml min-1) are used with conventional analytical HPLC columns, and fast flow rates are used for preparative or semi preparative columns and for slurry packing techniques

There are a number of different types of pumps that can provide the necessary pressures and flow-rates required by the modern liquid chromatography.

In the early years of the LC, there were two types of pumps in common use; They are the pneumatic pump, where the necessary high pressures were achieved by pneumatic amplification, and the syringe pump, which was simply a large, strongly constructed syringe with a plunger that was driven by a motor.

The Pneumatic Pump

The pneumatic pump has a much larger flow capacity than the piston type pumps but, nowadays, is largely used for column packing and not for general analysis. The pneumatic pump can provide extremely high pressures and is relatively inexpensive, but the high pressure models are a little cumbersome and, at high flow rates, can consume considerable quantities of compressed air.

The Syringe Pump

The syringe pump is a large, electrically operated simulation of a hypodermic syringe. Although used in the early days of LC,it is rarely used today as, due to its design, it can provide only a limited pressure and the volume of mobile phase available for use is restricted to the pump volume. Unless the separation is stopped while the pump is refilled and the development subsequently continued, the pump can only elute solutes that have retention volumes equal or less than the pump capacity.

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Besides these two pumps the other pumps in usage includes The Single Piston Reciprocating Pump, The Rapid Refill Pump, The Twin-Headed Pump and The Diaphragm Pump etc.

There are two modes of elution processes: Isocratic elution and Gradient elution.

Isocratic elution: -

In an isocratic elution, a sample is injected onto a given column and the mobile phase remains unchanged through the time required for the sample components to elute from the column. No single isocratic elution can separate a complex mixture with adequate resolution in a reasonable time and with good detectiblility. The isocratic separation of samples with widely varying k' (partition ratio) values typically exhibits poor resolution of early-eluting bands, difficult detection of late-eluting bands, and unnecessarily long elution times. To adequately handle samples that have both weakly retained and strongly retained substances, the rates of individual band migrations must be changed during a chromatographic run by solvent programming.

Gradient Elution:

Gradient elution also called Solvent Programming, which involves changing the mobile-phase composition either stepwise or continuously as elution proceeds during the chromatographic run. The main purpose of gradient elution is to move strongly retained components of the mixture faster, but having the least retained component well resolved. Usually all the sample components are initially retained at the top of the column. Starting with the low content of the organic component in the eluent, the least retained components get separated. Strongly retained components will sit on the adsorbent surface on the top of the column, or will move very slowly.

When the amount of organic component in the eluent is increased, the strongly retained components will move faster and faster, because of the steady increase of the competition for the adsorption sites.

Gradient elution also increases the quasi-efficiency of the column. In the isocratic elution, the longer a component is retained, the wider its peak. In gradient elution, especially with the smooth gradient shape without a flat region, the tail of the peak is always under the influence of the stronger mobile phase when compared to the peak front. Thus, molecules on the tail of the chromatographic zone (peak) will move faster. This will tend to compress zone and narrow the resultant peak.

A potential twofold or greater increase in sensitivity is achievable for gradient elution versus isocratic elution, even for samples that do not require gradient elution.

Injection System

Injection ports are of two basic types, (A) those in which the sample with injected directly into the column and (B) those in which the sample is deposited before the column inlet and then swept by a valving action into the column by the mobile phase

Modern injectors are based on injection valves, which allow the sample at atmospheric pressure to be transported to the high-pressure mobile phase immediately before the column inlet. In LC however, there is a third type of valve which is similar to the external loop valve but contains an extra loading port and behaves like an internal loop valve The basic difference between this type of valve and the normal external loop sample valve is the introduction of an extra port at the front of the valve. This port allows the injection of a sample by a syringe directly into the front of the sample loop.

Position (A) shows the inject position. Injection in the front port causes the sample to flow into the sample loop. The tip of the needle passes through the rotor seal and, on injection, is in direct contact with the ceramic stator face. Note the needle is chosen so that it's diameter is too great to enter the hole.

After injection, the valve is rotated to position (B) and the mobile phase flushes the sample directly onto the column. The sample is actually forced out of the beginning of the loop so it does not have to flow through the entire length of the loop. This type of injection system is ideally suited for quantitative LC, and is probably by far the most popular injection system in use.


Column is the heart of HPLC instrument Columns are constructed of heavy-wall, glass-lined metal tubing or stainless steel polish internally to a mirror finish They withstand high pressures (up to 680 atm) and the chemical action of the mobile phase

Silica is the most common material used for the packing of HPLC Columns. They are the one which can withstand to high pressures and often used for low molecular analytes in the modern HPLC systems.They can withstand to pH range from 2 to 7.5.

The particle size plays an important role in the separation of compounds.The columns with smaller particles are more efficient when compared with the columns with large particle size columns. particle size effects the back pressure in the column i.e it will decrease the back pressure and increases the efficiency of the column.

Particle size affects the back-pressure of the column and the separation efficiency. Column back-pressure and column efficiency are inversely proportional to the square of the particle diameter. This means that as the particle size decreases, the column back-pressure and efficiency increase.

Different particle sizes involves 3 µm,5 µm,and 7 µm.

The efficiency of separation depends on the particle sizes i.e as the particle size decreases the resolution of separation increases

The particle sizes of 10 µm packing can be used for semi preparative purposes

The particle sizes of (15-20) μm packing can be used for preparative purposes.

Different sizes of columns involves 100 mm, 150 mm, and 250 mm.


The detector of an HPLC is the component that gives a response due to the eluting sample compound and subsequently signals a peak on the chromatogram. It is positioned immediately after the column in order to detect the compounds as the substance elute from the column.. There are many types of detectors that can be used with HPLC.

Ideal Characteristics of a Detector

It should be equally sensitive to all eluted peaks

The ideal detector give the response (area) proportional to the amount

injected, irrespective of the size of sample.


It should be Cheap, reliable and easy to use.

Should not be affected by change in temperature or mobile phase composition.

It should be able to monitor small amounts of compound.

Some of the more common detectors include: Refractive Index (RI), Ultra-Violet (UV), Fluorescent, Radiochemical, Electrochemical, Near-Infra Red (Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR), and Light Scattering (LS).

Photometric detectors:

These normally operate in the UV region of the spectrum and are the most extensively used detectors in pharmaceutical analysis. They comprise essentially a light source, a dispersing device to select an appropriate wavelength per measurement, a flow cell in which they absorbency of the eluent is measured, and a photo multiplier tube are diode to ensure the intensity of transmitted light.

Photometric detectors are of five principle types

Single wavelength detectors

Multi-wave length detectors

Variable wavelength detectors

Programmable Detectors

Diode array detectors

Ultra-Violet (UV) detectors

Based on electronic transitions within molecules.

Most common type of detector for LC

Fixed wavelength, Hg lamp 254 nm

Tunable wavelength, selectable for specific wavelengths,

monochromators or filters. Still limited to single wavelegths.

1 pg LOD

Variable Wavelength measures at one wavelength at a time, but can detect over a wide range of wavelengths.

Diode Array measures a spectrum of wavelengths simultaneously

Refractive Index (RI) detectors

Passes visible light through 2 compartments, sample &