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1. It should be VOLATILE. 
2. It should IMPART COLOUR to the different spots. 
3. It should not REACT with VARIOUS compounds which are being separated. 

The application of the mixture as a SPOT on the original line on the FILTER paper strip or ADDITION of mixture to the COLUMN, is called loading (or SPOTTING).

The application of the mixture as a spot on the original line on the filter paper strip or addition of mixture to the column, is called loading (or spotting).

1. It can be USED for a MIXTURE CONTAINING any NUMBER of COMPONENTS. 
2. Very small quantities of the substances can be effectively detected and separated from a mixture. 

RUSSIAN BOTANIST M. Tswett (1906). 

Russian botanist M. Tswett (1906). 

It helps in the separation of amino acids, PROTEINS, PEPTIDES, nucleic acids, etc.

It helps in the separation of amino acids, proteins, peptides, nucleic acids, etc.

1. NATURE of the COMPOUND. 
2. Nature of the SOLVENT. 
3. TEMPERATURE. 

Rf (retention factor) of a SUBSTANCE is defined as the ratio of the DISTANCE moved up by the SOLUTE from the point of its APPLICATION to the distance moved up by the SOLVENT from the same point.

Rf (retention factor) of a substance is defined as the ratio of the distance moved up by the solute from the point of its application to the distance moved up by the solvent from the same point.

By MEANS of CAPILLARY ACTION.

By means of capillary action.

During CHROMATOGRAPHY, if the components to be separated are colourless, then these separated components on chromatogram are not visible. Their presence is detected by development, which INVOLVES spraying a suitable reagent (called DEVELOPING reagent) on the chromatogram, or placing the chromatogram in iodine CHAMBER when various components BECOME visible. This process is called developing of chromatogram.

During chromatography, if the components to be separated are colourless, then these separated components on chromatogram are not visible. Their presence is detected by development, which involves spraying a suitable reagent (called developing reagent) on the chromatogram, or placing the chromatogram in iodine chamber when various components become visible. This process is called developing of chromatogram.

WATER ABSORBED on CELLULOSE constituting the paper serves as the stationary phase and ORGANIC solvent as MOVING phase.

Water absorbed on cellulose constituting the paper serves as the stationary phase and organic solvent as moving phase.

Paper chromatography, COLUMN chromatography, THIN LAYER chromatography, GAS chromatography. 

Paper chromatography, column chromatography, thin layer chromatography, gas chromatography. 

Generally solvents having low viscosities are EMPLOYED in CHROMATOGRAPHY. This is due to the fact that the RATE of flow of a solvent VARIES inversely as its viscosity.

Generally solvents having low viscosities are employed in chromatography. This is due to the fact that the rate of flow of a solvent varies inversely as its viscosity.

It is based on the differential migration of the INDIVIDUAL COMPONENTS of a MIXTURE through a — stationary phase under the INFLUENCE of a MOVING phase.

It is based on the differential migration of the individual components of a mixture through a — stationary phase under the influence of a moving phase.

It is technique for rapid and efficient SEPARATION of components of a mixture and purification of COMPOUNDS. It is BASED on differential migration of the VARIOUS components of a mixture through a stationary phase under the influence of a moving phase.

It is technique for rapid and efficient separation of components of a mixture and purification of compounds. It is based on differential migration of the various components of a mixture through a stationary phase under the influence of a moving phase.

Fast or UHPLC technique makes use of small particles below 2 μ size Use of such particle sizes result in high resolution and as small columns can be used it results in completion of analysis in much less time thereby reducing CONSUMPTION of EXPENSIVE SOLVENTS.

We hope you had a great LEARNING experience through the introductory free e-learning HPLC course. I SHALL remain in contact with you for our offerings on advance versions of the HPLC e-learning courses and subsequent introduction covering other analytical techniques.

Fast or UHPLC technique makes use of small particles below 2 μ size Use of such particle sizes result in high resolution and as small columns can be used it results in completion of analysis in much less time thereby reducing consumption of expensive solvents.

We hope you had a great learning experience through the introductory free e-learning HPLC course. I shall remain in contact with you for our offerings on advance versions of the HPLC e-learning courses and subsequent introduction covering other analytical techniques.

Plate theory concept was introduced to explain efficiency of columns. The concept ASSUMES that a state of instantaneous equilibrium EXISTS between the CONCENTRATION of solute in stationary phase and the mobile phase and further the column is imagined to be divided into a number of theoretical PLATES. Any analyte spends a finite time in each plate and this is the equilibrium time. Smaller the plate height the greater is the number of plates in a given LENGTH (HETP) and better is the column resolution.

Plate theory concept was introduced to explain efficiency of columns. The concept assumes that a state of instantaneous equilibrium exists between the concentration of solute in stationary phase and the mobile phase and further the column is imagined to be divided into a number of theoretical plates. Any analyte spends a finite time in each plate and this is the equilibrium time. Smaller the plate height the greater is the number of plates in a given length (HETP) and better is the column resolution.

The desirable features of a detector are

• Sensitivity towards solute over MOBILE phase.
• Low dead volume to ELIMINATE memory EFFECTS
• Low noise
• Low DETECTION limits
• LARGE dynamic linear range

The desirable features of a detector are

When the composition of the mobile PHASE is not changed through the CHROMATOGRAPHIC run the OPERATION is TERMED as isocratic. It can involve a single solvent or a mixture of two or more SOLVENTS mixed in a fixed proportion. In gradient operation the composition at start of run is programmed to change at a predetermined rate and the composition at the end of run is different from the composition at the start.

When the composition of the mobile phase is not changed through the chromatographic run the operation is termed as isocratic. It can involve a single solvent or a mixture of two or more solvents mixed in a fixed proportion. In gradient operation the composition at start of run is programmed to change at a predetermined rate and the composition at the end of run is different from the composition at the start.

A BULK property detector responds to some property of mobile PHASE and sample COMBINATION passing through it at any point of time such a Refractive index or Electrochemical detector whereas a specific property detector is RESPONSIVE only to the characteristic property of the eluting molecule and is INDEPENDENT of changes in mobile phase composition such as UV-Vis and Fluorescence detectors.

A bulk property detector responds to some property of mobile phase and sample combination passing through it at any point of time such a Refractive index or Electrochemical detector whereas a specific property detector is responsive only to the characteristic property of the eluting molecule and is independent of changes in mobile phase composition such as UV-Vis and Fluorescence detectors.

The most commonly used DETECTOR in HPLC is the UV-VIS detector. The reason for its predominant use is that it GIVES specific RESPONSE to a particular compound or class of compounds. Most of the organic compounds ABSORB at specific wavelengths covered in the available WAVELENGTH range of the detector.

The most commonly used detector in HPLC is the UV-VIS detector. The reason for its predominant use is that it gives specific response to a particular compound or class of compounds. Most of the organic compounds absorb at specific wavelengths covered in the available wavelength range of the detector.

Mobile PHASES entrap air from the ATMOSPHERE and this trapped air gets released as small bubbles under high pressures encountered during the HPLC analysis. Such bubbles can lead to noise in DETECTOR response or hinder flow of mobile PHASE through columns. In order to overcome such problems degassing of mobile phase becomes ESSENTIAL.

Mobile phases entrap air from the atmosphere and this trapped air gets released as small bubbles under high pressures encountered during the HPLC analysis. Such bubbles can lead to noise in detector response or hinder flow of mobile phase through columns. In order to overcome such problems degassing of mobile phase becomes essential.

In size exclusion chromatography the SEPARATION does not involve chemical interactions between eluting molecules and stationary phase. The separation takes place on the basis of molecular size with larger molecules eluting first and small molecules in the END. Small molecules are retained LONGER in the PORES of the stationary phase therefore they get eluted LAST.

In size exclusion chromatography the separation does not involve chemical interactions between eluting molecules and stationary phase. The separation takes place on the basis of molecular size with larger molecules eluting first and small molecules in the end. Small molecules are retained longer in the pores of the stationary phase therefore they get eluted last.

Reverse phase Chromatography has the widest range of applications. The stationary phase COMPRISES non polar organic chains bound to inert silica surface and MOBILE phase comprises of aqueous or aqueous-organic MIXTURES comprising of polar solvents of varying degrees of polarity. The elution sequence is polar followed by LESS polar and least polar or non polar COMPOUNDS eluting last through the column.

Reverse phase Chromatography has the widest range of applications. The stationary phase comprises non polar organic chains bound to inert silica surface and mobile phase comprises of aqueous or aqueous-organic mixtures comprising of polar solvents of varying degrees of polarity. The elution sequence is polar followed by less polar and least polar or non polar compounds eluting last through the column.

Paper chromatography has some limitations such as:

• Semi-quantitative in nature.
• Overlapping of spots of components having close Rf values.
• Higher concentration of components often leads to streaking instead of well-defined spots.
• Errors in Rf calculations can result from uneven FLOW of solvent front. This can be caused by running out of solvent at the bottom of the CHAMBER, uneven cutting of the filter paper or unevenness of the bottom of the DEVELOPMENT chamber.
• Improper sample spotting, spotting below the marked LINE resulting in dipping into the solvent or ACCIDENTAL dipping of spot into solvent while inserting the paper into the solvent chamber.

Paper chromatography has some limitations such as:

Chromatographic technique of ANALYSIS has seen an impressive growth over time. Such advances have increased laboratory throughputs lowered limits of detection and has made FORAYS into new areas of applications. Paper chromatography has RETAINED its GROUND till date and is popular in laboratories across the world. Some of the reasons for this are:

• Low cost of analysis and freedom from maintenance.
• Separated spots are visible for coloured compounds and colourless compounds can be viewed by using alternate techniques.
• Minimum operation and training requirements.Solvent consumption is MUCH less as compared to more sophisticated techniques.
• Paper chromatography serves as a good demonstration of basic concepts of separation for school and undergraduate students.

Chromatographic technique of analysis has seen an impressive growth over time. Such advances have increased laboratory throughputs lowered limits of detection and has made forays into new areas of applications. Paper chromatography has retained its ground till date and is popular in laboratories across the world. Some of the reasons for this are:

Solvents are SELECTED on the BASIS of solubility of the sample components. In general it is advisable to keep in MIND:

• Solvents are not toxic or carcinogenic.
• SOLVENT constituents of mixture should not react with any of the sample constituents.
• Solvents selected should not interfere in detection of separated spots.
• Solvents should not be highly volatile as loss of components can RESULT in change of mixture composition.

Solvents are selected on the basis of solubility of the sample components. In general it is advisable to keep in mind:

PAPER CHROMATOGRAPHY separations are classified in accordance with the direction of flow of mobile phase along the filter paper.

• Ascending paper chromatography – the carrier liquid moves from bottom upwards.
• Descending paper chromatography – the carrier liquid trough is on TOP and mobile phase moves downward on the filter paper.
• Ascending – descending paper chromatography – The paper is rolled downward over the rod at the top. On reaching the top in ascending mode it starts downward movement in the NEXT phase.

Paper chromatography separations are classified in accordance with the direction of flow of mobile phase along the filter paper.

Retardation factor Rf is a measure of the separation of a particular COMPONENT. It is expressed as

Rf = distance MOVED by the component spot/ distance moved by solvent front

Rf is a unit LESS quantity and lies between 0and 1.A value of 0 indicates no separation has taken place and 1 represents that the component has moved ENTIRE length alongwith the solvent front. In CASE two spots have same value of Rf it indicates that they are not resolved. At least a difference of 0.05 is necessary to discern the separation between two spots.

Retardation factor Rf is a measure of the separation of a particular component. It is expressed as

Rf = distance moved by the component spot/ distance moved by solvent front

Rf is a unit less quantity and lies between 0and 1.A value of 0 indicates no separation has taken place and 1 represents that the component has moved entire length alongwith the solvent front. In case two spots have same value of Rf it indicates that they are not resolved. At least a difference of 0.05 is necessary to discern the separation between two spots.

The samples should have a medium solubility in the developing solvent MIXTURE.Too high a solubility will lead to transfer of the component alongwith the solvent FRONT and on the other HAND if the solubility is too low the component will not be carried by the solvent mixture and will remain close to the INITIAL applied spot. In either case the resolution of the mixture components will be low. THUS reasonably good resolution can be obtained for medium solubility of compounds in the solvent mixture.

The samples should have a medium solubility in the developing solvent mixture.Too high a solubility will lead to transfer of the component alongwith the solvent front and on the other hand if the solubility is too low the component will not be carried by the solvent mixture and will remain close to the initial applied spot. In either case the resolution of the mixture components will be low. Thus reasonably good resolution can be obtained for medium solubility of compounds in the solvent mixture.

It is easy to distinguish coloured spots visually but for colourless COMPOUNDS alternate techniques need to be adopted which can be specific or non-specific.

• A common non-specific method is suspension of developed chromatogram in iodine vapour. Most organic compounds show up as BROWN spots.
• The sheet is viewed in a UV VIEWING cabinet under 366 nm and 254 nm wavelength lamp ILLUMINATION. On observation the spots need to be carefully marked with a pencil for Rf calculations.
• Under specific methods amines and amino acids are observed by spraying heated paper on development with 0.2% hydrazine. Deep blue or purple spots begin to appear.
• Alkaloids – Dragendroff’s reagent spray results in orange or orange yellow spots.
• Aldehydes&Ketones – 2,4-DNPH spray in methanol and sulphuric acid results in orange or yellow spots.

It is easy to distinguish coloured spots visually but for colourless compounds alternate techniques need to be adopted which can be specific or non-specific.

During the chromatogram development chamber is covered.This is essential as the environment inside the chamber should remain SATURATED with the solvent vapour. Development times can VARY from about an HOUR to several HOURS and a saturated environment prevents losses due to evaporation.

During the chromatogram development chamber is covered.This is essential as the environment inside the chamber should remain saturated with the solvent vapour. Development times can vary from about an hour to several hours and a saturated environment prevents losses due to evaporation.

The DEVELOPING liquid PHASE comprises of a pure solvent but more often it is a mixture of two or more SOLVENTS in specified proportions. In case solvents are mixed and stored for long periods there could be loss of volatile COMPONENT which will alter the mixing proportions.

The developing liquid phase comprises of a pure solvent but more often it is a mixture of two or more solvents in specified proportions. In case solvents are mixed and stored for long periods there could be loss of volatile component which will alter the mixing proportions.

Paper chromatography is a form of liquid chromatography where the components of a mixture of organic COMPOUNDS get SEPARATED as unique spots by unidirectional flow of the developing liquid mobile PHASE solvent mixture over the filter paper to which a spot of the sample is applied. The DISTANCE travelled by each COMPONENT is specific under the given set of operational conditions.

Paper chromatography is a form of liquid chromatography where the components of a mixture of organic compounds get separated as unique spots by unidirectional flow of the developing liquid mobile phase solvent mixture over the filter paper to which a spot of the sample is applied. The distance travelled by each component is specific under the given set of operational conditions.

• Condition a column before first use or after long time STORAGE 
• TAKE care not to exceed UPPER temperature limit specified by the manufacturer 
• Avoid injection of solutions which are strongly acidic or basic in nature 
• Rinse columns by injection with blank solvents such as methanol, methylene chloride or HEXANE to remove contamination of column after excessive usage

Isothermal operation is useful when high resolution is required for separating COMPOUNDS having narrow BOILING range. Temperature is set to around mid range of boiling points of constituents. This results in good resolution of low boiling components but band BROADENING of higher boiling components can result DUE to their longer retention in the column.

Isothermal operation is useful when high resolution is required for separating compounds having narrow boiling range. Temperature is set to around mid range of boiling points of constituents. This results in good resolution of low boiling components but band broadening of higher boiling components can result due to their longer retention in the column.

Temperature programming MEANS change of temperature of the column at a rate predetermined rate during the analytical run. This has the same influence on elution time of separated components as gradient programming in HPLC ANALYSIS. Temperature programming helps REDUCE analysis time by permitting EARLY elution of less volatile components.

Temperature programming means change of temperature of the column at a rate predetermined rate during the analytical run. This has the same influence on elution time of separated components as gradient programming in HPLC analysis. Temperature programming helps reduce analysis time by permitting early elution of less volatile components.

On continuous use a COLUMN gradually loses its original resolution power. Column efficiency is expressed on the basis of plate theory CONCEPT. Each component under SEPARATION SPENDS a finite time in each theoretical plate. Smaller the plate height the larger the number of plates (N) and better is the column efficiency.

On continuous use a column gradually loses its original resolution power. Column efficiency is expressed on the basis of plate theory concept. Each component under separation spends a finite time in each theoretical plate. Smaller the plate height the larger the number of plates (N) and better is the column efficiency.

Capillary columns are GENERALLY 10 – 100M long TUBES having an internal diameter ranging from 0.1 – 0.5mm made of flexible material such as fused silica. Common types of capillary columns are

• Wall COATED open tubular (WCOT) – Internal wall is coated with a very fine film of adsorbing liquid 
• Surface coated open tubular (SCOT) – Inner wall is lined with a layer of SOLID support on to which the liquid phase is absorbed. 

The columns are flexible and wound into several turn coils supported on a SS cage inside the column oven

Capillary columns are generally 10 – 100m long tubes having an internal diameter ranging from 0.1 – 0.5mm made of flexible material such as fused silica. Common types of capillary columns are

The columns are flexible and wound into several turn coils supported on a SS cage inside the column oven

Detectors falls into three categories depending upon response to the eluting COMPOUNDS:

• Non-selective – Respond to all component in the gas stream except for the CARRIER gas
• Selective – Respond to a particular class of compounds with common physical or chemical PROPERTIES 
• Specific – Respond to a SINGLE specific compound only in the carrier gas stream

Detectors falls into three categories depending upon response to the eluting compounds:

The detector chosen for particular ANALYSIS should :

• Give reproducible response to changes in concentration of eluting compounds in the carrier GAS stream.
• Should provide a large linear dynamic range 
• Should have high sensitivity 
• Should have small internal volume to give narrow peaks and also facilitate flushing of PREVIOUS sample traces 
• Should PREFERABLY be non-destructive

The detector chosen for particular analysis should :

Inert GASES commonly used in analysis when using FID detector are Nitrogen and Helium. Nitrogen is more commonly used as it is LESS EXPENSIVE than Helium. PURITY of carrier gas should be more than 99.995% and on-line traps should be used to prevent residual moisture or other impurities from entering the system.

Inert gases commonly used in analysis when using FID detector are Nitrogen and Helium. Nitrogen is more commonly used as it is less expensive than Helium. Purity of carrier gas should be more than 99.995% and on-line traps should be used to prevent residual moisture or other impurities from entering the system.

The most commonly used detector is the FLAME ionize detector. The sample is COMBUSTED with the help of fuel gas and oxidant in the detector body. Combustible sample components burn and PRODUCE ions and electrons which can conduct electricity through the flame. A large potential DIFFERENCE is applied at the burner tip and the collector electrode located above the flame and the current between the electrodes is measured. The detector is mass sensitive and response is not affected by carrier gas flow rate changes. However, the detector is not responsive to inorganic gases such as CO, O2, NH3, N2, CS2, CO2, etc.

The most commonly used detector is the flame ionize detector. The sample is combusted with the help of fuel gas and oxidant in the detector body. Combustible sample components burn and produce ions and electrons which can conduct electricity through the flame. A large potential difference is applied at the burner tip and the collector electrode located above the flame and the current between the electrodes is measured. The detector is mass sensitive and response is not affected by carrier gas flow rate changes. However, the detector is not responsive to inorganic gases such as CO, O2, NH3, N2, CS2, CO2, etc.

• In HPLC the mobile phase is a liquid whereas in Gas Chromatography the mobile phase or carrier is a gas.
• HPLC is useful for analysis of SAMPLES which are liable to decompose at higher temperatures. GC INVOLVES high temperatures so compounds are stable at such temperatures.
• Gas Chromatography is applied for analysis of volatile compounds whereas non volatile compounds can be easily analyzed on HPLC
• Gas Chromatography cannot be used for analysis of high molecular weight molecules whereas HPLC has applications for separation and identification of very high molecular weight compounds
• HPLC REQUIRES higher OPERATING pressures than GC because liquids require higher pressures than gases for transport through the SYSTEM
• HPLC columns are short and wide in comparison to GC columns