Seminar Report On Chromatography

Chromatography is a powerful separation method that finds applications in all branch of science. Chromatography was invented and named by the Russian botanist Mikhail Tswett shortly after the turn of the last century. He employed this technique to separate various plant pigments such as chlorophylls and xanthophylls by passing solutions of these compounds through a glass column packed with finely divided calcium carbonate. The separated species appeared as colored bands on the column, which accounts for the name he chose for the method (Greek chroma meaning color and graphein meaning writing).The application of chromatography have grown explosively in the last half century, due not only to the development of several new types of chromatographic techniques but also to the growing need by scientists for better methods for characterizing complex mixtures. The tremendous impact of these methods on sciences is attested by the 1952 Nobel Prize in chemistry that was awarded to A.J.P.Martin and R.L.M.Synge for their discoveries in the field.

Chromatography involves a sample or sample extract being dissolved in a mobile phase, which may be a gas, a liquid or a supercritical fluid. The mobile phase is then forced through an immobile, immiscible stationary phase, which is fixed in place in a column or on a solid surface. The two phases are chosen so that the components of the sample distribute themselves between the mobile and stationary phases to varying degrees. Those components strongly retained by the stationary phase move only slowly with the flow of mobile phase. In contrast, components that are weakly held by the stationary phase travel rapidly. As a consequence of these differences in migration rates, sample components separate into discrete bands, or zones, that can be analyzed qualitatively and quantitatively.¹

1.1 Methods in chromatography²

Chromatographic techniques can be classified according to the nature of the stationary and mobile phases as follows:

Adsorption chromatography

• Gas solid chromatography
• Liquid column chromatography
• High performance liquid chromatography
• Thin layer chromatography

Partition chromatography

• Gas solid chromatography
• Super critical fluid chromatography
• Liquid-liquid chromatography
• Paper chromatography
• High performance liquid chromatography

Pore exchange chromatography

• Ion exchange chromatography
• High performance liquid chromatography

Permeation chromatography

• Size exclusion chromatography

Affinity chromatography

• DNA affinity chromatography

1.2 HPLC

Liquid chromatography is the most widely used of all of the analytical separation techniques. The reasons for the popularity of the method are its sensitivity, its ready adaptability to accurate quantitative determinations, its easy to automation, its suitability for separating non-volatile species or thermally fragile ones, and above all.¹The first instrumental liquid chromatography was constructed by Csaba Horvath at Yale University in 1964, and was described as a high-pressure liquid chromatography (HPLC).³

High-pressure liquid chromatography (HPLC), sometimes called high-performance liquid chromatography, is a separation technique based on a solid stationary phase and a liquid mobile phase. Separations are achieved by partition, adsorption, or ion-exchange processes, depending upon the type of stationary phase used. HPLC has distinct advantages over gas chromatography for the analysis of organic compounds. Compounds to be analyzed are dissolved in a suitable solvent, and most separations take place at room temperature. Thus, most drugs, being non-volatile or thermally unstable compounds, can be chromatographed without decomposition or the necessity of making volatile derivatives.⁴

1.2.1 Classification of HPLC⁵

This classification primary based on internal diameter (ID) of the column employed for HPLC.

System                        Flow rate                    Column

Range                           ml/min                        Dimension

Micro                            0.001-0.1                    0.3-1 mm i.d

Semi-micro                   0.1-0.4                        1-3 mm i.d

Analytical                    0.4-2                           4-8 mm i.d

Semi preparative          2-10                            10-20 mm i.d

Preparative                   10-150                        20-50mm i.d

1.2.2 Bonded phase of HPLC

Two bonded phases are defined depending on relative polarity of two phases.

·         Normal phase chromatography

·         Reversed phase chromatography

1.2.2.1 Normal phase chromatography

Normal phase chromatography is a chromatographic technique that uses organic solvents for the mobile phase and a polar stationary phase. Here, the less polar components elute faster than the more polar components.⁶ Normal-phase liquid-liquid chromatography uses a polar stationary phase (often hydrophobic) and a less polar mobile phase. To select an optimum mobile phase, it is best to start with a pure hydrocarbon mobile phase such as heptanes. If the sample is strongly retained, the polarity of the mobile phase should be increased, perhaps by adding small amounts of methanol and dioxane.⁷

Mechanism: Retention by interaction of the stationary phase polar surfaces with polar part of the sample molecules

Stationary phase: SiO_2, Al₂O₃, -NH₂,-CN, -Diol, -NO₂.

Mobile phase: Heptanes, hexane, Cyclohexane, chloroform, dioxane etc.⁵

1.2.2.2 Reversed phase chromatography

Reversed-phase high performance liquid chromatography (RP-HPLC) is a well-known technique exceptionally for the simultaneous determination of pharmaceutical dosage forms.⁸ Reverse-phase chromatography uses a hydrophobic bonded packing, usually with an octadecyl, octyl functional group and a polar mobile phase.⁷

Mechanism: Retention by interaction of the stationary phase non-polar hydrocarbon chain with non-polar part of the sample molecules

Stationary phase: N-octadecyl (C18), n-octayl (C8), Phenyl, (CH₂)_n-CN, (CH₂)_n-diol.

Mobile phase: Methanol, acetonitrile/water and buffer are used sometimes with additives of THF or dioxane.⁵

1.2.3 Mode of operation⁹

·         Isocratic elution

·         Gradient elution

1.2.3.1 Isocratic elution

A separation in which the mobile phase composition is remains constant throughout the analysis.

1.2.3.2 Gradient elution

A separation in which the mobile phase composition is altered gradually during the separation process is described as a gradient elution.

1.2.4 Simultaneous HPLC analysis

Simultaneous HPLC analysis is carried out in order to estimate or analyze two or more drugs at a single run.

1.2.4.1 Application of simultaneous HPLC analysis

• Analysis of two or more drugs possible in a single run.
• Reduced cost of analysis.
• Reduced time of analysis.
• There is a possibility of studying the intermediate of compounds.

1.3 Identification and Quantification process of active ingredients in dosage form

Separation of mixture by chromatographic process is a central part of analytical and preparative chemistry.¹¹

Chromatographic techniques are

• Adsorption chromatography
• Partition  chromatography
• Size exclusion chromatography

Spectroscopic techniques are

• Ultra Violet and Visible spectroscopy
• Infrared Spectroscopy
• Nuclear Magnetic Resonance
• Mass Spectroscopy
• Atomic Absorption Spectroscopy
• Atomic Emission spectroscopy

The direct conjugation of these chromatographic techniques with spectroscopic examination of the separated fractions constitutes several powerful analytical partnerships.¹⁰

1.4 HPLC Instrumentation

• Reservoir
• Degasser
• Pump
• Injector
• Column
• Detector
• Integrator
• Software
• Miscellaneous

1.4.1 Solvent reservoir²

The solvent reservoir are glass or stainless steel containers capable of holding up to 1-2 liter of mobile phase, which may be consist of pure organic solvents or aqueous solution of salt or buffers.

1.4.2 Degasser¹¹

Degasser is a device, which removes the dissolved gases in the mobile phase.  There are two types of degasser, are online and offline. Helium and vacuum degasser are available as online degasser. Offline degasser means an external device like sonicator, vacuum aspirator, etc.

DGU-10A, DGU-12A, DGU-14A, DGU-3A are some of the models with two, three or four channel.        

1.4.3 Pump

The pumping systems deliver metered amounts of the mobile phase from the solvent reservoirs to the column through high –pressure tubing and fittings. Modern  systems consists one or more computer controlled metering pumps that can be programmed to vary the ratio of mobile phase components, as is required for gradient elution chromatography, or to make an isocratic mobile phase (mobile phase having a fixed ratio of solvents).¹²

Basically the solvents in the reservoir succeed through a suction filter, which is made of 5mm mesh.

Various configurations of HPLC pumps are

A) Isocratic system                 - consists of only one pump.

B) Binary gradient system      - consists of two pumps and additionally a mixer

                                                  is required before the injector stage.

C) Ternary gradient system     - consists of three pumps, additionally a mixer is                                                                     required before the injector stage.

D) Low Pressure Gradient     -  can be used to take four solvents using

      Elution system                    a flow channel selection valve, additionally a mixer is         .                                                  required before the injector stage.

Maintenance and verification

Set the flow rate and solvent was collected in a measuring cylinder. The set was confirmed and flow rate was measured for 10min.  Also pay attention for leakage at every joint.¹³

1.4.4 Injector¹²

After dissolution in th mobile phase or other suitable solvent, sample that are to be chromatographed are injected, either manually by syringe or by fixed loop injectors, or automatically by autosamplers. An autosampler consists of a carousel or rack to hold sample vials with tops that have a pierceable septum or stopper and an injection device to transfer samples from the vials to loop from which it is loaded into the chromatograph.

1.4.5 Guard column¹

Guard column is introduced before the analytical column to increase the life of analytical column by removing not only particulate matter and contaminants from the solvents but also sample components that bind irreversibly to the stationary phase. The composition of the guard column packing should be similar to that of the analytical column.

1.4.6 Column¹⁴

This is the heart of the HPLC system. The separation of the components in the sample takes place. Most separations are based upon partition mechanisms utilising chemically modified silica as the stationary phase and polar solvents as the mobile phase. The surface of the support, e.g. the silanol groups of silica, is react with various silane reagents to produce covalently bound silyl derivatives covering a varying number of active sites on the surface of the support. The nature of the bonded phase is an important parameter for determining the separation properties of the chromatographic system.

Commonly used bonded phases are shown below:

Octyl                                 Si-[CH₂]₇-CH₃                        C₈                       

Octadecyl                          Si-[CH₂]₁₇-CH₃                      C₁₈             

Phenyl                               Si-[CH₂] _n-C₆H₅                     C₆H₅          

Cyanopropyl                      Si-[CH₂]₃-CN                        CN            

Aminopropyl                     Si-[CH₂]₃-NH₂                       NH₂           

Diol                                   Si-[CH₂]₃-O-CH (OH)-CH₂-OH

Maintenance and verification

Connect the column in HPLC system was showed with the direction, run a test sample as per the column performance certificate.  Data from the manufacturer was compared with the results.  System suitability test of the column will measured accurate performance of the column.

1.4.7 Detector¹²

A detector consists of a flow through cell mounted at the end of column and capable of detecting various types of components in the eluents. The recommended volume of the detector flow cell is 3 µl to 20 µl. Ultraviolet/visible spectrophotometers, including diode array detectors, are the most commonly employed detectors. A beam of U.V radiation passes through the flow cell and into the detector. As compound elute from the column, they passes through the cell and absorb the radiation, resulting in measurable energy level changes.

Fluorescence spectrophotometers, differential refractometers, electrochemical detectors, mass spectrometers, light scattering detectors, radioactivity detectors or other special detectors may also be used. Fluorimetric detectors are sensitive to compounds that are fluorescent or that can be converted to fluorescent derivatives. Differential refractometers detectors measure the difference between the refractive index of the mobile phase alone and that of the mobile phase containing chromatographed compound as it emerges from the column. Electrochemical detectors are suitable for measuring nanogram quantities of easily oxidisable compounds like phenols and catechols.

1.4.8 Integrator¹⁵

Integrator is a recording device, which plots the output of the detector.  The graph is called chromatogram. The chromatogram consists of the retention time and area calculation. Retention time is used to find qualitative result and area is used to quantitative calculation.

1.4.9 Software¹⁵

Modern chromatographers are using the HPLC with personal computer. Improved technology and advanced processors make the further advantage like controlling the complete system, Integration and storage of mass data, faster communication via networking, etc.

Various software packages we marketed and continuing packages are:

            01) SPD-M6A                                     06) EZCHROM

            02) CLASS LC10                               07) CLASS VP SINGLE

            03) CLASS CR10                               08) CLASS VP MULTI

            04) CLASS M10A                              09) CLASS VP PDA

1.4.10 Fraction Collector

Fraction Collector is an optional part of the HPLC system, gets connected after the detector.  Fraction collector is designed to collect the sample, which has been separated by the column to the desired vial with the given information on time base, level of sensitivity. Models available are FRC-10A, FCB-100.  FRC-10A is controlled through a controller and FCB-100 is controlled through Integrator with program.

1.4.11 Rinsing Pump

Rinsing pump is attached to the pump for continuous washing of the pump head, leading the long life to the plunger seals, plunger, etc.  This is recommended to those who use buffer as mobile phase, where possible of deposition of salt.  Rinsing pump uses only water. 

1.5 HPLC Method Development

Best column, best mobile phase, best detection wavelength, efforts in their selection can make a world of difference while developing HPLC method for routine analysis. Determining the ideal combination of these factors assures faster delivery of desire result-a validated method for separation.

• On sample properties as much knowledge as possible should be collected.
• Chromatography method is selected according to the sample properties.
• The sample is chromatographed with HPLC conditions where all compounds elute in a reasonable time.
• The HPLC method is optimized with regard to analysis time, resolution, selectivity and sensitivity.