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Chromatography Techniques: Principles and Applications, Assignments of Pharmacognosy

Maulana Abul Kalam Azad University of Technology Pharmacognosy

An overview of various chromatography techniques, including gas chromatography, liquid chromatography, thin layer chromatography, and paper chromatography. It covers the principles, advantages, and practical requirements of these techniques, as well as their applications in the separation and identification of chemical and biological mixtures. The role of the mobile phase, stationary phase, and other instrumental components in the chromatographic process. It also highlights the differences between adsorption chromatography and partition chromatography, and the factors that influence the separation and elution of compounds. The information presented in this document can be useful for students and researchers in the fields of analytical chemistry, pharmaceutical sciences, and related disciplines.

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2023/2024

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INTRODUCTION

Chromatography is a technique used to separate and identify the components of a

mixture.

• Works by allowing the molecules present in the mixture to distribute themselves

between a stationary and a mobile medium.

• Chroma -"color" and graphein - "to write”

PURPOSE OF CHROMATOGRAPHY

Analytical

Determine Chemical composition of a sample

* Preparative

Used to purify sufficient quantities of a substance

CHROMATOGRAPHY TERMS

Chromatograph : equipment that enables a sophisticated separation

EX. Gas chromatography or Liquid chromatography

* Eluent: Fluid entering column/ solvent that carries the analyte.

* Eluate: Mobile phase leaving the column.

* Stationary phase - Immobilized phase

Immobilized on the support particles or on the inner wall of the column

tubing.

Examples : Silica layer - Thin Layer Chromatography

Mobile phase - Moves in a definite direction. Liquid (LC), Gas (GC). The mobile

phase moves through the chromatography column (the stationary phase) where the

sample interacts with the stationary phase and is separated.

• Retention time: Time takes for a particular analyte to pass through the system (from

the column inlet to the detector) under set conditions.

• Sample (Analyte) :Substance analyzed in chromatography.

• Solvent: Any substance capable of solubilizing another substance.

• Chromatogram:Visual output of the chromatograph.

Separation - Different peaks or patterns on the chromatogram correspond to different

components of the separated mixture.

Chromatogram:Visual output of the chromatograph.

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INTRODUCTION

Chromatography is a technique used to separate and identify the components of a mixture.

Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium.
Chroma - "color" and graphein - "to write” PURPOSE OF CHROMATOGRAPHY Analytical Determine Chemical composition of a sample

Preparative Used to purify sufficient quantities of a substance CHROMATOGRAPHY TERMS Chromatograph : equipment that enables a sophisticated separation EX. Gas chromatography or Liquid chromatography
Eluent: Fluid entering column/ solvent that carries the analyte.
Eluate: Mobile phase leaving the column.
Stationary phase - Immobilized phase Immobilized on the support particles or on the inner wall of the column tubing. Examples : Silica layer - Thin Layer Chromatography Mobile phase - Moves in a definite direction. Liquid (LC), Gas (GC). The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.

Retention time: Time takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.
Sample (Analyte) :Substance analyzed in chromatography.
Solvent: Any substance capable of solubilizing another substance.
Chromatogram:Visual output of the chromatograph. Separation - Different peaks or patterns on the chromatogram correspond to different components of the separated mixture. Chromatogram:Visual output of the chromatograph.

Separation - Different peaks or patterns on the chromatogram correspond to different components of the separated mixture Adsorption Chromatography:

When a mixture of compounds dissolved in the mobile phase moves through a stationary phase
The compound which has more affinity towards stationary phase travels slower and the compound which has lesser affinity towards stationary phase travels faster
Hence compounds are separated
No two compounds have the same affinity for a combination of a stationary phase, mobile phase and other conditions.
Examples: GSC, TLC, Column Chromatography, HPLC Partition Chromatography
When two immiscible liquids are present, a mixture of solutes will be distributed according to their partition co-efficient.
When a mixture of a compounds are dissolved in the mobile phase and passed through the liquid stationary phase travels slower.
The component which is more soluble in mobile phase travels faster.
Hence compounds are separated.
No two compounds have the same affinity for a combination of a stationary phase, mobile phase and other conditions.
Examples: GLC, Paper Partition Chromatography etc. Based on the modes of Chromatography: i. Normal Phase Chromatography ii. Reserve Phase Chromatography There are two modes viz- Normal Phase and Reserve Phase. These modes are based on the polarity of stationary and mobile phase. i. Normal Phase Chromatography: Stationary Phase is Polar and Mobile Phase is Non-polar ii. Reserve Phase Chromatography: Stationary Phase is Non- polar and Mobile Phase is Polar

Normal phase is not widely used in Pharmacy, Reserve Phase is widely used in analytical Pharmacy. Polar – Polar interaction= Affinity is more Nonpolar- Nonpolar interaction= affinity is more

Principle

The principle of separation is ADSOPTION.
No two compounds have the same affinity for a combination of a stationary phase, mobile phase and other conditions.
The compound which has more affinity towards stationary phase travels slower and the compound which has lesser affinity towards stationary phase travels faster
The components are separated on a thin layer chromatographic plate based on the affinity of the components towards the stationary phase. Advantages
Simple method and cost of the equipment is low.
Rapid technique and not time consuming like column chromatography
Separation of μg of the substances can be achieved.
Any type of compound can be analysed.
Detection is easy and not tedious.
Capacity of the thin layer can be altered. Hence analytical and preparative separation can be made
Corrosive spray reagents can be used without damaging the plates.
Need less solvent, stationary phase and time for every separation when compared to column chromatography.
Efficiency of separation: very small particle size can be used which increase the efficiency of separation. Practical Requirements i. Stationary Phase ii. Glass Plate iii. Preparation and Activation of TLC plates iv. Application of Sample v. Development tank vi. Mobile Phase vii. Development Technique viii.Detecting and visualising agents Stationary Phase:

There are several adsorption which can be used as Stationary Phases.
Some times they have to be mixed with water or other solvents to form a slurry for preparing thin layer chromatographic plates. Name Composition Adsorbent : Water ratio Silica gel G Silica gel + CaSO4 1 : 2 Silica gel GF Silica gel + Binder + Fluorescent indicator 1 : 2 Al2O3G Al2O3+ Binder 1 : 2 Cellulose Powder Cellulose with binder 1 : 6 Preparation and Activation of TLC plates
The slurry, which is a mixture of stationary phase and solvent in a specific ratio.
After preparing slurry, TLC plates can be prepared by using following methods. Pouring, Dipping, Spraying, Spreading Application of Sample:
The spot of extract should be kept atleast 2 cm above the base of the plate and the spotting area should not be immersed in the mobile phase in the development tank. Development tank:
The purpose of development tank to hold the TLC plate.
To avoid the wastage of solvent, new type of development tank have hump in the middle.
The development tank should be lined inside with filter paper moistened with the mobile phase to saturate the atmosphere. If this kind of saturation of the atmosphere is not done, ‘ EDGE EFFECT ’ occurs where the solvent front in the middle of TLC plate moves faster than that of the edge Mobile Phase: The solvent or the mobile phase used depends upon various factor: Nature of the substances to be separated Nature of the stationary phase used Mode of chromatography Separation to be achieved- Analytical or Preparative Examples: Petroleum ether, Carbon tetrachloride, Cyclohexane, Ehter, Acetone, Benzene, Tolune etc.

Distance travelled by solute Distance travelled by solvent front

The Rf value ranges from 0 to 1. ideal values are from 0.3 to 0.
When the Rf value of the sample and reference compound is same, the compound is identified by its standard.
When the Rf value differs, the compound may be different from its reference standard. Rx Value: Rx value is nothing but the ratio of distance travelled by the sample and the distance travelled by the standard. Rx value is always closer to 1. Rx Value = Distance travelled by the sample Distance travelled by the standard Application
Separation of mixture of drugs of chemical or biological origin, plant extract
Separation of carbohydrates, vitamins, antibiotics, proteins, alkaloids, glycosides etc.
Identification of drug (* next slide).
Identification of related compounds in drugs (* next slide).
To detect the presence of foreign substances in drugs (* next slide).
To detect the composition products in drugs (* next slide). COLUMN CHROMATOGRAPHY INTRODUCTION Column chromatography is a technique which is used to separate a single chemical compound from a mixture.
It separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates
This technique can be used on a small scale as well as large scale to purify materials that can be used in future experiments
This method is a type of ADSORPTION chromatography technique. Column Chromatography Principle The mobile phase along with the mixture that needs to be separated is introduced from the top of the column
The movement of the individual components of the mixture is at different rates.

The components with lower adsorption and affinity to stationary phase travel faster when compared to the greater adsorption and affinity with the stationary phase.
The components that move fast are removed first whereas the components that move slowly are eluted out last.
The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of the components is expressed as: Rf= the distance travelled by solute/ the distance travelled by the solvent Column Chromatography Procedure Mobile phase – This phase is made up of solvents and it performs the following functions:

It acts as a solvent – sample mixture can be introduced in the column.
It acts as a developing agent – helps in the separation of components in the sample to form bands.
It acts as an eluting agent – the components that are separated during the experiment are removed from the column
Some examples of solvents used as mobile phase based on their polarity are – ethanol, acetone, water, acetic acid, pyridine, etc. Stationary phase – It is a solid material which should have good adsorption property and meet the conditions given below:
Shape and size of particle: Particles should have uniform shape and size in the range of 60 – 200μ in diameter.
Stability and inertness of particles: high mechanical stability and chemically inert. Also, no reaction with acids or bases or any other solvents used during the experiment.
It should be colourless, inexpensive and readily available.
Should allow free flow of mobile phase
It should be suitable for the separation of mixtures of various compounds. Column Chromatography Applications
Column Chromatography is used to isolate active ingredients.
It is very helpful in Separating compound mixtures.
It is used to determine drug estimation from drug formulations
It is used to remove impurities.
Used to isolation metabolites from biological fluids.

In general the paper contains 98-99% of α-cellulose, 0.3 – 1% β - cellulose. Other modified papers •Acid or base washed filter paper •Glass fiber type paper. •Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc. •Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used for reverse phase chromatography. •Impregnation of silica, alumna, or ion exchange resins can also be made. Paper chromatography mobile phase •Pure solvents, buffer solutions or mixture of solvents can be used. Examples- Hydrophilic mobile phase •Isopropanol: ammonia: water 9:1: •Methanol : water 4: •N-butanol : glacial acetic acid : water 4:1: Hydrophobic mobile phase •dimethyl ether: cyclohexane kerosene : 70% isopropanol •The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated. •If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied. Chromatographic chamber •The chromatographic chambers are made up of many materials like glass, plastic or stainless steel. Glass tanks are preferred most. •They are available in various dimensional size depending upon paper length and development type. •The chamber atmosphere should be saturated with solvent vapour. Steps in Paper Chromatography

Selection of Solid Support
Selection of Mobile Phase
Saturation of Tank
Sample preparation and Loading

Development of Chromatogram
Drying of Chromatogram
Detection 1.Selection of Solid Support Fine quality cellulose paper with defined porosity, high resolution, negligible diffusion of sample and favouring good rate of movement of solvent. 2.Selection of Mobile Phase Different combinations of organic and inorganic solvents may be used depending on the analyte. Example.Butanol: Acetic acid: Water (12:3:5) is suitable solvent for separating amino- acids. 3.Saturation of Tank The inner wall of the tank is wrapped with the filter paper before solvent is placed in the tank to achieve better resolution. 4.Sample Preparation and Loading If solid sample is used, it is dissolved in a suitable solvent. Sample (2-20ul) is added on the base line as a spot using a micropipette and air dried to prevent the diffusion. 5.Development of the Chromatogram Sample loaded filter paper is dipped carefully into the solvent not more than a height of 1 cm and waited until the solvent front reaches near the edge of the paper. 6.Drying of Chromatogram After the development, the solvent front is marked and the left to dry in a dry cabinet or oven. 7.Detection Colourless analytes detected by staining with reagents such as iodine vapour, ninhydrin etc. Radiolabeled and fluorescently labeled analytes detected by measuring radioactivity and florescence respectively. Rf value Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base line. The distance travelled relative to the solvent is a constant

The sample is either a gas or a liquid that is vaporized in the injection port. The mobile phase for gas chromatography is a carrier gas, typically helium because of its low molecular weight and being chemically inert.
The pressure is applied and the mobile phase moves the analyte through the column. The separation is accomplished using a column coated with a stationary phase Principle of Gas chromatography
The equilibrium for gas chromatography is partitioning, and the components of the sample will partition (i.e. distribute) between the two phases: the stationary phase and the mobile phase.
Compounds that have a greater affinity for the stationary phase spend more time in the column and thus elute later and have a longer retention time (Rt) than samples that have a higher affinity for the mobile phase.
Affinity for the stationary phase is driven mainly by intermolecular interactions and the polarity of the stationary phase. Parts of Gas Chromatography 1.Carrier gas 2.Sample injection system 3.The separation column 4.Liquid phases 5.Detector 6.Recorder Carrier Gas Helium, N , H, Argon are used as carrier gases.
Helium is preferred for thermal conductivity detectors because of its high thermal conductivity relative to that of most organic vapours.
N is preferable when a large consumption of carrier gas is employed.
Carrier gas from the tank passes through a toggle valve, a flow meter, (1-1000 ml/min), capillary restrictors, and a pressure gauge (1-4 atm).
Flow rate is adjusted by means of a needle valve mounted on the base of the flow meter

and controlled by capillary restrictors.

The operating efficiency of the gas chromatograph is directly dependant on the maintenance of constant gas flow. Sample injection system
Liquid samples are injected by a microsyringe with a needle inserted through a self- scaling, silicon-rubber septum into a heated metal block by a resistance heater.
Gaseous samples are injected by a gas-tight syringe or through a by-pass loop and valves.
Typical sample volumes range from 0.1 to 0.2 ml. The separation column
The main part of the gas chromatography is the column which is made of metals bent in U shape or coiled into an open spiral shape.
Copper is useful up to 2500
Sweage lock fittings make column insertion easy.
Several sizes of columns are used depending upon the requirements. Liquid phases
An infinite variety of liquid phases are available limited only by their volatility, thermal stability and ability to wet the support.
No single phase will serve for all separation problems at all temperatures. Detector
Detectors sense the arrival of the separated components and provide a signal.
These are either concentration-dependent or mass dependant.
The detector should be close to the column exit and the correct temperature to prevent decomposition. The procedure of Gas Chromatography

Sample Injection and Vapourization
Separation in the Column
Detecting and Recording Results Sample Injection and Vapourization a) A small amount of liquid sample to be analyzed is drawn up into a syringe. b) The syringe needle is positioned in the hot injection port of the gas chromatograph and

Applications :

GC analysis is used to calculate the content of a chemical product, for example in assuring the quality of products in the chemical industry; or measuring toxic substances in soil, air or water.
Gas chromatography is used in the analysis of: (a) air-borne pollutants (b) performance-enhancing drugs in athlete’s urine samples (c) oil spills (d) essential oils in perfume preparation
GC is very accurate if used properly and can measure picomoles of a substance in a 1 ml liquid sample, or parts-per-billion concentrations in gaseous samples.
Gas Chromatography is used extensively in forensic science. Disciplines as diverse as solid drug dose (pre-consumption form) identification and quantification, arson investigation, paint chip analysis, and toxicology cases, employ GC to identify and quantify various biological specimens and crime-scene evidence. Advantages :
The use of longer columns and higher velocity of carrier gas permits the fast separation in a matter of a few minutes.
Higher working temperatures up to 5000C and the possibility of converting any material into a volatile component make gas chromatography one of the most versatile techniques.
GC is popular for environmental monitoring and industrial applications because it is very reliable and can be run nearly continuously.
GC is typically used in applications where small, volatile molecules are detected and with non-aqueous solutions.
GC is favoured for non-polar molecules. Limitations Compound to be analyzed should be stable under GC operation conditions.
They should have a vapor pressure significantly greater than zero.
Typically, the compounds analyzed are less than 1,000 Da, because it is difficult to vaporize larger compounds.

The samples are also required to be salt-free; they should not contain ions.
Very minute amounts of a substance can be measured, but it is often required that the sample must be measured in comparison to a sample containing the pure, suspected substance known as a reference standard. Introduction:
The technique of High Performance liquid Chromatography is so called because of its improved performance when compared to classical column and Thin layer Chromatography.
The development of HPLC from classical column chromatography can be attributed to the development of smaller particle sizes.
Smaller particle size is so important since they offer more surface area over the conventional larger particle sizes.
A porous particle of 5μ offers a surface area of 100 - 860 sq. meters/ gram with an average of 400 sq. meters/ gram. These offers very high plate counts upto 100000 sq. meters/ gram Principle of Separation: Mobile Phase
The principle of separation is adsorption.
Separation of compounds takes place based on the difference in the affinity of the compounds towards stationary phase as in the normal and reverse phase.
The lesser the affinity of the sample particles towards the stationary phase the faster the time of elution of the sample.
Due to different interaction between stationary phase and different sample, the molecules move at different rate, therefore separation can be done Solvent storage bottle
Gradient controller and mixing unit
De-gassing of solvents
Pump
Pressure gauge
Pre-column
Sample introduction system

That is, smaller the particle size of the stationary phase the greater is the resistance to the flow of solvents.
Hence high pressure is recommended. Requirements of Pumps
Generation of pressure of about 5000 psi.
Pulse free output & all materials in the pump should be chemically resistant to solvents.
Flow rates ranging from 0.1 to 10 mL/min
Pumps should be capable of taking the solvent from a single reservoir or more than one reservoir containing different solvents simultaneously. Guard column:
Guard columns are placed anterior to the separating column.
This protects and prolongs the life & usefulness of the separating column.
They are dependable columns designed to filter or remove:- a) particles that clog the separating column, b) compounds and ions that could ultimately cause ‘baseline drift’, decreased resolution, decreased sensitivity and create false peaks. c) Compounds that may cause precipitation upon contact with the stationary or mobile phase. Analytical column:
This is the most important part of HPLC which decides the efficiency of separation
Length- 5 to 25 cm ,Internal Diameter 3 to 5mm.
Particle size of packing material is 3 to 5μm.
LC columns achieve separation by different intermolecular forces b/w the solute & the stationary phase and those b/w the solute & mobile phase. C18: Octa Decyl Silane (ODS), CN: Nitrile Column C8: Octyl Column, NH2: Amino Column C4: Butyl Column Detectors:
Absorbance (UV/Vis or PDA)
Refractive index (detects the change in turbidity)

Fluorescence (if the analyte is fluorescent)
Electrochemical (measures current flowing through a pair of electrodes, on which a potential difference is
imposed, due to oxidation or reduction of solute)
Conductivity (for ions)
Light scattering
Mass spectrometry (HPLC-MS) Application:
Qualitative analysis
Checking the purity of a compound
Presence of impurity
Quantitative analysis
Multicomponent analysis or determination of mixture of drugs
Isolation and identification of drugs
Purification

Chromatography Techniques: Principles and Applications, Assignments of Pharmacognosy

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INTRODUCTION