Evolution of Paper Chromatography

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Paper Chromatography

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Dr. Saurabh Arora

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Director : Auriga Research Ltd., Arbro Pharmaceuticals Ltd. (Analytical Division)

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Dr. Deepak Bhanot

Vice President : Training & Development

E-mail : [email protected]

Auriga Research Ltd.

And

Arbro Pharmaceuticals Ltd.

Analytical Division,

4/9 Kirti Nagar Industrial Area, New Delhi - 110015 (INDIA)

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Authors Profile

Dr. Saurabh Arora

Holds a PhD in Pharmaceutics and has invented a patented nano technology based delivery system for curcumin the active constituent of haldi .

Has a number of national and international research publications and patents to his credit.

Heading the testing laboratory and research business at Arbro and Auriga for close to 10 years.

Has designed and setup 4 state of the art testing laboratories in New Delhi, Baddi and Bangalore.

Leading a team of over 250 scientists and professionals in 4 laboratories which serve more than 10,000 customers each year from the food, retail, hospitality, nutraceutical, pharmaceutical, cosmetics, agri, medical device, research, academics and real-estate industries.

Firm believer that sharing knowledge and experience is the only way to move the society further.

Established www.lab-training.com in June 2011 to share his team’s expertise in the field of testing with scientists and students around the world, the site today has over 10,000 subscribers and offers online training courses on various techniques used in testing laboratories across industries.

He also established www.foodsafetyhelpline.com in January 2013 as a one stop solution for the people in the food industry to stay up-to-date, understand and implement the requirements of the Food Safety and Standards Act and the Food Safety and Standards Authority of India (FSSAI). The site has a simple objective to help food businesses understand and comply with the requirements of this new and rapidly evolving food law which has been put in place to provide safe and hygienic food to all the citizens of India.

He also regularly speaks and presents at industry meets, conferences, workshops and webinars on a variety of topics related to the testing and quality of products.

He has completed a training on “Lean Labs: Lab Performance Optimisation” and has been implementing the principles of Lean Six Sigma for labs which has helped the business grow at over 30% each year.

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Awards –

He received the Award for “10 best training and development practices in private sector” on behalf of Arbro Pharmaceuticals Ltd. At the Excellence in Training and Development Awards 2013.

Arbro was also adjudged among the top 100 SMEs of India at the BOI India SME 100 Awards in 2012

Dr Deepak Bhanot

A seasoned professional having nearly 30 years expertise in sales and product support of sophisticated analytical instruments manufactured by reputed global suppliers. After completing his graduation and post graduation from Delhi University and IIT Delhi he went on to Loughborough University of Technology, UK for doctorate research in analytical chemistry He is a key contributor to the technical content on the on line site..

He is the Centre Incharge for IGNOU sponsored post Graduate Diploma in Food safety and Quality Management for last several years and coordinates laboratory training and project guidance guidance to enrolled students.In addition he also coordinates the training activities of trainees sponsored to the laboratory by Biotech Consortium India Ltd under the Biotech Industrial Training Programme.

His mission is to develop training programs on analytical techniques and share his experiences with broad spectrum of users ranging from professionals engaged in analytical development and research as well as young enthusiasts fresh from academics who wish to embark upon a career in analytical industry.

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Content

Chapter 1 : Evolution of Paper Chromatography

Chapter 2 : Theory of Paper Chromatographic Separations

Chapter 3 : Types of Paper Chromatography

Chapter 4 : Types of papers and their applications in Paper Chromatography

Chapter 5 : Choice of solvents for Paper Chromatography

Chapter 6 : Sample Preparation and Application

Chapter 7 : Visualization of spots in Paper Chromatography

Chapter 8 : Applications of Paper Chromatography

Chapter 9 : What makes Paper Chromatography a Popular Technique even today?

Chapter 10 : 10 Common Interview Questions on Paper Chromatography

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Evolution of Paper Chromatography

Developed Chromatographic Sheet

Chromatography today is a top ranking technique for separation and quantification of components of organic liquid or gas phase mixtures. Several innovations of the technique have contributed to speed of analysis and lowering of detection limits but it is remarkably surprising that all the variants ranging from simplest paper chromatography, column chromatography, thin- layer chromatography to most advanced techniques such as LC – MS - MS are widely accepted and are in common use in college/university. industrial and sophisticated research and development laboratories across the world. Even to this day no claim can be made that any one technique has made the popularity of another chromatographic technique outdated or obsolete.

The evolution of the earliest liquid chromatography technique – Paper chromatography which has provided an initial approach to determine the components of liquid mixtures and has paved the future for more sophisticated instrumental techniques.

Historical

Chromatography, true to its literal Greek translation, means colour writing. It was successfully applied by Dr Michael Tswett in 1906 for separation of plant pigments by allowing vegetable extracts to percolate through a column packed with calcium carbonate powder. The extract comprising of different coloured substances appeared as distinct coloured bands in

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the packed column. The coloured bands were extracted sequentially by washing down the column with the liquid carrier phase.

Paper chromatography is a variant of liquid chromatography where the components of the mixture are separated by the unidirectional flow of the liquid mobile solvent over the filter paper to which the spot of the mixture is applied.

Earliest work on paper chromatography dates back to 1855 when Runge separated inorganic mixtures using impregnated filter paper.In 1865, Schoenbein made the observation of upward movement of aqueous salt solution through capillary action when a paper strip was dipped into the solution.

Martin and Synge in 1941 made significant contribution to liquid - liquid partition chromatography through analysis of amino acids in protein hydrolysates using partition chromatography which cut down the separation times drastically. In 1952 Martin and Synge were honoured with the Noble prize for their path breaking discovery.

Today paper chromatography is commonly employed for separation of mixtures of amino acids, plant pigments, sugars, inorganic ionic species and phenolic constituents of wood, etc.

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Theory of Paper Chromatographic Separations

Rf Calculation in Paper Chromatography

Principle of Separation

Partition or distribution of components of the sample between a pair of liquid phases is the underlying principle of chromatographic separations. In paper chromatography separations the component is distributed between the water held between the pores of filter paper and the liquid phase which travels along the filter paper. Separation results from the differences in affinities towards water and mobile phase when travelling under capillary action between the pores of the filter paper.

Separation of the sample components results from a combination of different factors:

Solubility

Partitioning of the solute molecules between the stationary liquid phase and the developing liquid is governed by differences in solubility of solutes in the two phases which in turn is dependent on differences in polarities between the molecules.

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Propelling Force

Propelling force tends to pull the substance in the direction of flow of developing solvent. This force depends upon the flow rate and solubility of the substance in the developing solvent.

Retarding Force

Retarding force tends to prevent the substance from moving ahead with the mobile phase solvent. This depends on the partitioning of the solute between the mobile and stationary phases

Quantitatively the separation is expressed in terms of retardation factor (Rf) where

Rf = the distance travelled by the solute/ distance travelled by the solvent front

Rf is a unit less quantity. A value of 0 implies that the solute has not moved at all from the application spot and on the other hand if Rf is 1 it means that the solute has no affinity for the stationary phase and has moved the entire distance along with the solvent front.

Rf values provide a fair confirmation on identity of two solutes that have moved the same distance and are of same colour. It cannot be deduced that such compounds are always same as it is quite possible for two compounds to have same Rf value and colour.

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Types of Paper Chromatography

Over the years different types of paper chromatography have evolved which have been classified by the direction of flow of the mobile liquid phase

Ascending Paper Chromatography

As the name suggests the developing solvent moves in an upward direction. Sufficient quantity of mobile phase is poured into the development chamber. Sample and reference are spotted on the line drawn a few centimeters from the bottom edge of the paper suspended from a hook or clip at the top. Alternately, the paper can be rolled in the shape of a cylinder and kept inside the development chamber. Solvent moves up through capillary action. The rate of rise of solvent front decreases with time and some chromatograms can be left overnight for the components to effectively separate. It is important to cover the container with a plate so that the atmosphere inside the development chamber is saturated with the solvent vapour. Separation of the atmosphere with the solvent vapour prevents evaporation of the solvent as it rises up the paper.

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Descending Paper Chromatography

In this technique the solvent front travels down the length of paper suspended from the top inside the developing chamber. The mobile phase is kept in a trough in the upper chamber. The paper with spotting on the line drawn a few centimeters from the top is clamped to the top. Prior to elution the jar is covered and equilibrated with the mobile phase vapour.

The solvent moves downwards through the combined capillary action and gravitational pull. The advantage of this technique is that development can be continued indefinitely even though the solvent runs off at the bottom end of the paper

Ascending – Descending Chromatography

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In the hybrid version the solvent first travels upwards on the paper which is folded over a rod and it continues with its travel downwards after crossing the rod. This arrangement permits longer development period for better resolution of complex mixtures

Two-Dimensional Chromatography

Two dimensional chromatography helps resolve substances having similar Rf values.

A single spot of the mixture is applied close to one end of the baseline on the square filter paper. The paper is allowed to develop by either ascending or descending mode and on completion allowed to dry. The filter paper is now rotated 90° so that the edge having the series of separated spots is at the bottom and allowed to develop in the same or a different solvent phase

The arrangement helps to resolve the spots in two-dimensions by spreading the spots on the filter paper. Horizontal or Circular Paper Chromatography

Horizontal or Circular Paper Chromatography permits separation of sample components in the form of concentric circular zones through radial movement of liquid phase. A circular shaped filter paper is employed and different arrangements are used.

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Arrangement – 1

Arrangement – 1 : Circular Paper Chromatography

A narrow slit about 2mm wide is cut from the circumference to the centre and the wick formed is bent so as to dip in the solvent contained in a petri dish. Sample spotting is done along the circumference of the smaller inner circle. The petri dish is covered after securing the filter paper to the top and allowed to develop. The mobile phase reaches the filter paper through capillary action and spreads radially outwards thereby resolving the sample components radially.

Arrangement – 2

Arrangement – 1 : Circular Paper Chromatography

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In this arrangement there is no need to cut a wick. A small capillary is inserted through an adjustable collar placed inside the solvent inside the petri dish. After applying the sample spot to the centre of a circular filter paper it is covered with a petri dish cover and allowed to develop. As before the solvent is drawn up by capillary action of the filter paper and spreads radially outwards thereby resolving the sample constituents radially.

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Types of papers and their applications in Paper Chromatography

Papers for Chromatographic Separations

Paper chromatography makes use of paper which acts as a stationary phase. Paper essentially consists of cellulose fibers which are polymers having – OH functional groups sticking out of the polymer chains. These groups lead to retention and separation of surface absorbed molecules. In practice the separating molecules equilibrate between the layer of adsorbed water and the mobile phase solvent.

Different types of paper have been tried but Whatman No1 and 3 filter papers are found more suitable for analytical work. Chromatography paper is commonly available as 18”X22” rectangular sheets. Apart from Whatman other popular manufacturers of chromatographic paper are Schleicher and Schull, Macherey Nagel, and Eaton-Dikeman but Whatman appears to be a popular choice.

Choice of Paper

Coarseness of the fibers and packing density of papers decides between speed and resolution. Fast papers are useful for major applications and when high resolutions are necessary slow papers are preferred

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Examples of Whatman filter paper

Fast Medium Slow

No 4 No 7 No 2

No 17 No 3 and 3 MM

Modified papers

In addition to pure cellulose several modified versions are also available to meet the specific separation requirements which include acetylated papers, silicone oil impregnated papers, silica and alumina impregnated papers and papers impregnated with ion exchange materials. Some typical applications of modified papers are

Type of paper Typical applications

Ion exchange papers Charged molecule or ionic separations

Silica or Alumina low polarity molecules such as amines steroids, fatty acids, impregnated papers vitamins and pesticides

insecticides, pigments and metal Acetylated papers cations

cationic separations of proteinated Carboxylated papers amines and amino acids

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Choice of solvents for Paper Chromatography

Chromatographic Solvents

A solvent plays an important role in resolution of sample constituents on the filter paper. For the purpose use can be made of a single pure solvent, undiluted solvent with water or a mixture of miscible solvents. The greater the distance between the resolved spots (measured by Rf values) better is the solvent mixture combination.

Sample Solubility

Solubility plays a critical role. Ideally the constituents should have small but definite solubility in the solvent combination. If the solubility is very high the constituent will move with the solvent upto the solvent front and on the other hand low solubility will result in hardly any movement from the point of application.

Sample Polarity

Each sample constituent has a polarity different from other constituents. Solvent polarity can be modified by changing the proportion of solvent mixtures. Increase in solvent polarity will improve the spatial movement along the filter paper.

Criteria for selection of a working solvent system

 Solvents should not be toxic or carcinogenic. In such cases adequate handling care should be exercised.

 The solvent mixture composition should not change with time

 Solvent constituents should not chemically react with any of the sample constituents

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 Solvents should not interfere with the detection of spots on the developed paper

 Differences in Rf values of any two components should be at least 0.05 for differentiation between two closely spaced spots.

Commonly used solvent combinations

In general solvents can be made by saturating an organic solvent such as n – butanol with water. Several solvent combinations have lesser amounts of water so that polar compounds move slowly. Modifications can be further made by adding an acid, base or a complexing agent. Some suitable solvent combinations for different categories of polar compounds are suggested:

Strongly polar compounds – ethyl acetate: butanol: acetic acid: water in 80:10:5:5 proportion

Polar compounds – 10% methanol or less in dichloromethane

Strongly basic compounds – 10% ammonium hydroxide in methanol and make 1 – 10% mixture of this in dichloromethane

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Sample Preparation and Application

Spotting of Paper Chromatographic Sheet

Developing the Chromatogram involves three stages:

 Sample preparation

 Sample application

 Developing of the chromatogram

Sample Preparation

Samples can come either as liquids or solids. Liquids can be spotted without any pretreatment. However, solid samples need to be dissolved in a suitable solvent as only liquids can be applied to the chromatography paper.

Spotting and Developing

Spotting of the sample is done with the help of a capillary tube or automated applicator.. The sample is applied as a neat spot on a horizontal line drawn with a pencil close to one edge. Allow the spot to dry and then immerse the paper in the developing chamber as per the selected technique with the marked spot above the solvent level.

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The solvent begins to move and draws the sample components differentially along with it. At the end of the development take out the paper and mark the solvent front with another line .Allow the paper to dry in drying cabinets with the provision of electrical heating before visualisation

Commonly Encountered Problems

 Overlapping and oversized spots- Such spots can be differentiated by using other techniques such as two-dimensional chromatography

 Uneven advance of solvent front – It is a common problem which can lead to inaccuracies in calculation of Rf values due to uneven advance of spots. The reasons for this could be unevenness in cutting of paper sheet or not sufficient solvent in chamber to travel up the plate

 Streaking – high concentrations often lead to streaking instead of single well resolved spots. The problem can be overcome by dilution of the sample

 Washing away of applied spot leads to errors. Sample spot gets submerged in the developing solvent if care is not exercised at time of immersion of paper into the developing chamber.

 Spotting above or below the marked pencil line can lead to errors in calculation of Rf values

Spot visualization is a pre-requirement for interpretation of the chromatogram.

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Visualization of Spots in Paper Chromatography

UV Viewing Cabinet

Coloured spots are easily observed on developed chromatograms. However, different approaches need to be adopted when colourless components are to be observed. It is convenient to classify such methods as specific or non-specific.

Non-specific methods

Iodine chamber – the developed plate is suspended in a closed jar containing a few crystals of iodine for about a minute. In presence of iodine vapour most organic compounds appear as brown spots.

UV viewing cabinet – majority of colourless compounds can be viewed under illumination with UV light in a UV viewing cabinet. Commonly the cabinets are equipped with a long wavelength (366 nm) and short wavelength ( 254 nm) light sources. Under exposure of selected source coloured spots appear on a dark background and under exposure to light from short wavelength source dark spots are seen against a bright background. In either case the spots should be marked with a pencil while observing in the cabinet for Rf calculations.

Specific Methods

Amino acids ,primary and secondary amines –A 0.2% ninhydrin solution in water saturated with butanol is sprayed on briefly heated paper chromatogram when deep blue or purple spots begin to appear.

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Alkaloids – Dragendroff’s agent gives orange or orange – red precipitates in presence of alkaloids

Phenols – Ferric chloride solution spray results in appearance of different coloured spots – read, blue, green or purple when phenols are present.

Aldehydes and Ketones – 2,4 – dinitro phenyl hydrazine in a mixture of methanol and sulphuric acid (Brady’s reagent) spray results in bright orange or yellow colour spots in presence of aldehydes and ketones.

Paper chromatography is a versatile analysis technique and has wide range of applications which will be taken up in a subsequent article

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Applications of Paper Chromatography

Paper chromatography has evolved over the years and has found widespread applications in separation of molecules of different polarities. Innumerable applications have been reported in analysis of different classes of compounds such as:

 Amino acids and organic acids

 Alkaloids

 Polysaccharides

 Proteins and peptides

 Natural and artificial pigments

 Inorganic cations

 Plant extracts

Typical applications in key areas are briefly outlined here

Reaction monitoring

In a chemical reaction over a period of time the concentration of reactants decreases whereas the concentration of products increases. Developing the chromatogram over different time intervals by spotting the reactants can give a fair idea on the progress of reaction. Traditionally the technique was used for qualitative monitoring but availability of densitometers made quantitative estimations possible. However, rapid methods using spectroscopic techniques are limiting the application of paper chromatography as a reaction monitoring option.

Isolation & Purification

Paper chromatography has been put to use as a purification and isolation technique for components of mixtures. The separated components on the paper are cut, dissolved in suitable solvents and their absorption characterised at specific wavelengths using spectrophotometric methods. This approach is being superseded by techniques such as Preparative HPLC.

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Foods

Foods & Beverages

Paper chromatography has been primaly used for analysis of food colours in synthetic drinks and beverages, ice creams, jams &jellies, sweets,etc.Colours both natural and synthetic are added to foods to improve their acceptability and to make them more popular. Only edible colours are permitted for use so identification and quantification becomes all the more important to ensure that no non- permitted colouring agents are added to the foods.

Forensics

Forensic

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A forensic scientist is often faced with a severe limitation on the quantity of sample available for analysis Paper chromatography offers a viable analysis option for samples available in milligrams or microlitre quantities.

The major applications are for identification and comparison against reference standards for drugs and their metabolites in viscera, explosive residues from blast sites, inks used in forgery of documents and paint pigments investigations in hit and run road accident cases.

Pharmaceuticals

Pharmaceutical Products

Paper chromatography provides useful information on development of new drug molecules, reaction completion and progress of manufacturing processes. It also offers a cost-effective alternative to monitoring the active ingredients in drug forms. It is also used for identifying colors added in various pharmaceutical formulations.

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What makes Paper Chromatography a Popular Technique even today?

Paper Chromatogram

Paper chromatography techniques have retained their ground and are extensively used in separation of different classes of compounds ranging from foods, pharmaceuticals, forensic investigations, environmental monitoring, etc. Chromatography in general has seen phenomenal growth in terms of separation and quantitative estimations, softwares, applications and increased automation for matching the requirements of high throughput laboratories.

Over the years several versions of chromatographic technique have evolved such as TLC, HPTLC, HPLC, UHPLC, GC, Supercritical fluid chromatography. Advanced detection techniques such as fluorescence, refractive index, mass selective detectors coupled with automated sample introduction options have contributed significantly to separation science. Today scientists are fast approaching the ultimate barriers in terms of resolution and limits of detection of constituents in diverse sample matrices.

Benefits of Paper Chromatography

 Chromatography has seen outstanding growth but as mentioned earlier no basic separation technique has been obsoleted with the advent of advanced techniques. Paper chromatography is no exception and it maintains a noticeable presence in most laboratories even to this day. In this article several factors are highlighted which contribute to acceptance and popularity of Paper Chromatography technique.

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 The technique enables a visual presentation of separated sample components. Some compounds are not visible in visible light but these can be observed under exposure to UV light. This feature serves as a good demonstration exercise for school and undergraduate students

 Visible spots tend to fade out after some time. It is possible to take pictures and retain the chromatogram for future reference and confirmations. The results are semi-quantitative in nature but with accessories such as densitometers the scope for quantitative estimations increases

 Simple calculation of Rf of separated spots not involving any software.

 Minimum amount of sample pretreatment is necessary

 Low cost of operation requiring minimal training. Except for chromatography paper, applicators and solvents there is no major requirement of expensive consumables

 No requirement for maintenance as there are no operational breakdowns

 Solvent requirement is small and solvent mixtures can be prepared fresh every time.

 Choice of solvents and filter papers and chromatography techniques affords improvement in separation and identification of spots.

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10 Common Interview Questions on Paper Chromatography

Jon Interview

Question 1 – What is the basic principle of Paper Chromatography?

Answer – 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.

Question 2 – Why the developing solvent mixture is prepared fresh before use?

Answer – 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.

Question 3-. Why is it necessary to cover the developing chamber during the paper development?

Answer – 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

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Question 4-What are the common techniques used for detecting colourless spots?

Answer - 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.

Question 5– Why should the samples have reasonable solubility which is neither too high or too low in the developing solvent mixture

Answer - 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.

Question 6-What information you get from the Retardation factor value?

Answer - 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.

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Question 7 – Can you remember the various paper chromatography techniques?

Answer – 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.

Two - dimensional paper chromatography – After developing the chromatogram in either ascending or descending mode it is taken out, dried and developed again after turning by 90° in either the same liquid or another liquid mobile phase.The spots spread across the sheet and closely overlapping spots get resolved.

Circular paper chromatography – the mobile phase moves radially outwards from the centre of a circular piece of paper. In this mode the mixture components get resolved radially.

Question 8 – What are essential criteria for selection of suitable solvents for paper chromatography?

Answer – 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.

Question 9-Why paper chromatography has retained its applicability in the face of a emergence of advanced instrumental techniques?

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Answer- 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.

Question 10-What are the limitations of paper chromatography technique?

Answer - 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.

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Conclusion

We believe that you enjoyed the free E-course on Paper Chromatography. The course provided you an insight into the constituent parts of paper Chromatographic system and their contributions towards the overall accuracy and precision of your results. Apart from a general introduction the course was designed keeping in mind the requirements of the separation scientist.

The last chapter of the course provides answers to 10 common questions that you may be faced with as you move up your career ladder. However, learning is a lifelong process and there will be several unanswered questions and queries which will be coming up in your mind from time to time. Our suggestion to you would be to post such queries or comments on the site and we shall try to offer clarifications to the best of our ability based on our expertise and experience.

In case the e- course has awakened your desire to go deeper into the subject of separations you are welcome to join the Certificate Course on HPLC which is available round the year. For more details on this advanced treatment of the subject go to the http://lab-training.com/product/join-our-certificate-course-on-hplc/

Once again we take the opportunity to thank you for your interest. Please feel free to participate actively by contributing articles in areas off your interest and offer your valuable comments and suggestions.

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