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THIN LAYER CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY

Introduction

Thin layer chromatography (TLC) is a technique in which a solute undergoes distribution between two phases, a stationary phase, acting through adsorption and a mobile phase in the form of liquid.

The adsorbent is relatively thin, uniform layer of dry, finely powdered material applied to glass, plastic or metal sheet. Glass plates are most commonly used. Separation may also be achieved on the basis of partition or a combination of partition and adsorption, depending on a particular type of support, its preparation and its use with different solvent.

Identification can be effected by observation of spots of identical Rf value and about equal magnitude obtained respectively, with an unknown and reference sample chromatograph on the same plate.

Apparatus required

Flat  glass plates of appropriate dimensions.

b)  An aligning tray or plate surface on which the plates can be aligned and                              rested  when coating substance is applied .

c) An adsorbant or coating substance consisting of finely divided adsorbant                             material, normally 5 µm to 40 µm in diameter. A variety of coating                            materials are available, but Silica gel is most frequently used. The adsorbent may contain fluorescing matter to help in visualizing spots that absorb                             ultraviolet light.

A spreader, which when moved over the glass plate, will apply a uniform layer of adsorbent, of a uniform thickness, usually between 150 to 250 µm.

A storage rack to support the plates during drying and transportation.

                 

The apparatus described above are essentially required for the preparation of TLC plates. Ready to use TLC plates are commercially available, which may be used.

f) A developing chamber that can accommodate one or more plates and can be properly closed .

g) Graduated micro pipettes capable of delivering quantities.

h) A reagent sprayer that will emit a fine spray and will not itself be attacked by the reagent.

i) A viewing cabinet, fitted with ultra-violet light, suitable for observation at short (254 nm) and long (366 nm) ultra-violet wavelengths.

Precautions 

The spot must be applied by holding the micro pipette as erect as possible, which avoids undue spreading of the spot and ensures a compact spot, usually 2 to 3 mm in diameter.

The syringes must be cleaned thoroughly, prior to spotting .

Use dedicated syringes, wherever feasible; especially for spotting impurities.

For developing solvents chromatographic grade solvents must be used, which avoids unwanted impurities being introduced on the plate.

All solutions for TLC including the mobile phase must be freshly prepared. TLC solvents may be kept separately to avoid accidental contamination.

If the mobile phase consists of more than two solvents, the solvents must be mixed in the order mentioned, keeping the volume recommended, as accurate as possible.

Unless unsaturated conditions are prescribed the developing chamber must be saturated with the developing solvent, prior to placing the TLC plates in the chamber.

h)  The developing chamber is lined with sheet of filter paper which dips into the       solvent in the base of the chamber which ensures complete saturation of the chamber with solvent vapour.

i) The developing chamber may be covered by a black cloth or Aluminum foil in case of spotting of light sensitive materials. The developing chamber  must be placed on   a firm surface, away from turbulence and use of acids; since these factors tend to spoil the plates. The developing chamber must be of good quality, having a flat bottom to ensure uniform flow of mobile phase.

Before and after spotting, the TLC plate must be inspected for any unwanted spots. The edges of the plate may be cut and rounded up for uniform movement of the mobile phase. The TLC plates must always be handled by holding at the edge, to avoid finger prints on the surface. The plates must be placed in an erect fashion in the developing chamber; which must always be covered.

Cutting of the pre-coated full plates into half plates or quarter plates, must be avoided as far as possible. It is a good practice to always use a full plate.

I)  A narrow strip of coating substance, about 5 mm wide is usually removed from the vertical side of the TLC plate, to prevent accidental loss of the spot near the edge of the plate. In the case of ready to use plates, the narrow strip of coating substance is already removed.

m) The plates after preparation must be protected from moisture and used within three days of preparation. At the time of usage, the plates may be dried.

n) For drying of applied spots, during spotting a gentle current of air or nitrogen is used. Use of hot air from hair dryers must be avoided as the degradation of the spot, may be inadvertently introduced. The spotting must be carried out at least two cm from the bottom of the plate, to avoid direct contact with the mobile phase.

0) The spraying with reagent for development of the spot must be carried out uniformly over the plate. The spray must never be directed for a long time on a portion of the plate, as it results in localised darkening of the TLC plate.

p) The plates after spotting may be wrapped in Aluminium foil and than placed in            a polybag for future reference. It is a good practice to calculate and record the Rf value of experimental spot and standard spot, during the identification test.

Quantitative evaluation -The identification of the raw material is deemed to be satisfactory, if the Rf value of the experimental spot and the standard spot is identical.

In case of degradation products which are denoted by secondary spots; these are compared with main spots of diluted samples (0.5, 1.0 or 2.0 %). These secondary spots are required to be not more intense than the main spot obtained from lower dilution of the parent compound. If more than one secondary spot is observed, the individual intensities of  the secondary spots can be compared with the main spots of lower dilutions and can be added to get a rough idea of degradation products or related substances. 

Advantages -

The technique is simple and less expensive.

It is one of the most important techniques used in stability indicating methods and gives ready information regarding degradation products.

It is useful technique for identification of the raw material, when compared to           an authentic standard.

Disadvantages 

The method is not quantitative.

In absence of availability of impurities for spotting, degradation products cannot be identified.

c) The precision and accuracy depends on the technique employed by an individual and hence can vary substantially from person to person.

Note :

Recent changes in the practice of TLC have, however resulted in improved performance both in terms of separation and quantitative measurements. These developments are referred to as High Performance Thin Layer Chromatography (HPTLC), which has combined HPLC techniques with TLC, to give quantitative measurements of high precision.

Polarimeter - introduction, Defination, apparatus and precautions for use in details

POLARIMETRY

Introduction
The electric fields associated with the beam of monochromatic light, vibrate in all directions perpendicular to the directions of propagation of light. Certain crystalline materials have different refractive indices for light, whose field vibrates parallel or perpendicular to the principal plane of the crystal. As a result, a Nicol prism constructed of this material transmits only light whose electric field oscillates in one plane. Optical activity concerns with the interaction of such plane polarised light with certain materials, particularly solutions of some organic compounds.

When a plane polarised light passes through a medium, it is retarded to an extent indicated by the refractive index of the medium. When the later is optically inactive, both circularly polarised components are retarded to the same extent and the beam emerges from the medium, polarised in the same plane as the incident beam. If the medium is optically active, the components are retarded to different extents and the beam emerges from the medium still plane polarised; but with the plane of polarisation inclined at an angle to the plane of polarisation of the incident beam. If the plane of polarisation is rotated clockwise, the substance is termed 'dextrorotatory', while if the plane of polarisation is rotated anticlockwise, the substance is termed 'laevorotatory'. Dextrorotation is designated (+) and laevorotation is designated (-).

Definitions

a) Optical Rotation : Optical rotation, unless otherwise specified, is measured at the wavelength of the 'D' line of Sodium (wavelength = 589.3 nm), at 25°C, on a layer 1 dm thick. It is expressed in degrees.

b) The specific optical rotation : The specific optical rotation of a liquid substance is the angle of rotation of the plane of polarisation at the wavelength of the 'D' line of Sodium, measured at 25°C unless otherwise specified, calculated with reference to a 1 dm thick layer of liquid and divided by the specific gravity of the liquid at 25°C.

The specific optical rotation of a solid substance is the angle of rotation of the plane of polarisation at the wavelength of the 'D' line of Sodium, measured at 25°C unless otherwise specified, calculated with reference to a 1 dm thick layer of a solution containing one gm of the substance per ml. The specific optical rotation of a solid is always expressed with reference to a given solvent and concentration.

Apparatus
The apparatus for measurement of rotation of a compound is known as the, Polarimeter.

It Consist of following parts

a) Light source -The source of light is usually Sodium vapour lamp, which emits monochromatic light.

b) Polariser - The polariser consists of a fixed Nicol prism at one end to convert ordinary light into plane polarised light.

c) Sample compartment -In this compartment the sample is placed in 1 dm or 2 dm tube. In the case of solids, a suitable solution is made, through which the plane polarised light is passed.

d) Analyser - The analyser consists of a movable Nicol prism, with a scale marked off in degrees. This is placed at the other end of the polarimeter.



e) Eye piece -The eye piece is placed at the analyser end.

Calibration -The polarimeter is calibrated using a solution of previously dried Sucrose and measuring the optical rotation in a 2 dm tube at 25°C for concentrations ranging from 10% w/v to 50% w/v.

The angle of rotation is as given in following table :

Concentration.   Angle of rotation

 g /100ml.            at 25°C

10.0       -             13.33°

20.0        -            26.61°

30.0.       -            39.86°

40.0        -            53.06°

50.0.       -            66.23°


Alternatively a quartz control plate with known optical activity can be used for calibration.

Precautions
The accuracy and precision of optical rotation measurements can be increased, if following precautions are taken

a) The instrument must be in a good condition. The optical elements must be very clean and in exact alignment.

b) Specific attention should be paid to the temperature control of the solution and the polarimeter.

c) Five consecutive readings are taken and the mean of these five readings is used for calculations to improve precision.

d) Polarimeter tube must be filled in such a way as to avoid air bubbles.

e) For tube with removable end plates fitted with gaskets and caps, the end plates must be tightened to ensure a leak proof seal between the end plate and the body of the tube.

f) For substances with low rotatory power, the end plates should be loosened and tightened again after each reading.

g) Spillage of the sample must be avoided.

h) The tube having the sample must be thoroughly cleaned. Liquid and solution of solids must be clear. 

INTRODUCTION AND BASICS OF UV SPECTROSCOPY

INTRODUCTION AND BASICS OF UV SPECTROSCOPY

UV spectroscopy is type of absorption spectroscopy in which light of ultra-violet region (200-400 nm.) is absorbed by the molecule. Absorption of the ultra-violet radiations results in the excitation of the electrons from the ground state to higher energy state. The energy of the ultra-violet radiation that are absorbed is equal to the energy difference between the ground state and higher energy states (deltaE = hf).

Generally, the most favoured transition is from the highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO). For most of the molecules, the lowest energy occupied molecular orbital’s are s orbital, which correspond to sigma bonds. The p orbitals are at somewhat higher energy levels, the orbital’s (nonbonding orbitals) with unshared paired of electrons lie at higher energy levels. The unoccupied or anti-bonding orbitals (pie* and sigma*) are the highest energy occupied orbitals.

In all the compounds (other than alkanes), the electrons undergo various transitions. Some of the important transitions with increasing energies are: nonbonding to pie*, nonbonding to sigma*, pie to pie*, sigma to pie* and sigma to sigma*.

PRINCIPLE OF UV SPECTROSCOPY

UV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution.

The expression of Beer-Lambert law is-

A = log (I0/I) = Ecl

Where, A = absorbance

I0 = intensity of light incident upon sample cell

I = intensity of light leaving sample cell

C = molar concentration of solute

L = length of sample cell (cm.)

E = molar absorptivity

From the Beer-Lambert law it is clear that greater the number of molecules capable of absorbing light of a given wavelength, the greater the extent of light absorption. This is the basic principle of UV spectroscopy.

INSTRUMENTATION AND WORKING OF UV SPECTROSCOPY

UV spectrometers consist of the following parts-

1.       Light Source- Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm.

2.       Monochromator-  Monochromators generally composed of prisms and slits. The most of the spectrophotometers are double beam spectrophotometers. The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism.

3.       Sample and reference cells- One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV region.

4.       Detector- Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.

5.       Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servometer. Generally current generated in the photocells is of very low intensity, the main purpose of amplifier is to amplify the signals many times so we can get clear and recordable signals.

6.       Recording devices- Most of the time amplifier is coupled to a pen recorder which is connected to the computer. Computer stores all the data generated and produces the spectrum of the desired compound.

CONCEPT OF CHROMOPHORE AND AUXOCHROME IN THE UV SPECTROSCOPY

Chromophore- Chromophore is defined as any isolated covalently bonded group that shows a characteristic absorption in the ultraviolet or visible region (200-800 nm). Chromophores can be divided into two groups-

a) Chromophores which contain p electrons and which undergo pie to pie* transitions. Ethylenes and acetylenes are the example of such chromophores.

b) Chromophores which contain both p and nonbonding electrons. They undergo two types of transitions; pie to pie* and nonbonding to pie*. Carbonyl, nitriles, azo compounds, nitro compounds etc. are the example of such chromophores.

Auxochromes- An auxochrome can be defined as any group which does not itself act as a chromophore but whose presence brings about a shift of the absorption band towards the longer wavelength of the spectrum. –OH,-OR,-NH2,-NHR, -SH etc. are the examples of auxochromic groups.

ABSORPTION AND INTENSITY SHIFTS IN THE UV SPECTROSCOPY

There are four types of shifts observed in the UV spectroscopy-

a) Bathochromic effect- This type of shift is also known as red shift. Bathochromic shift is an effect by virtue of which the absorption maximum is shifted towards the longer wavelength due to the presence of an auxochrome or change in solvents.

The nonbonding to pie* transition of carbonyl compounds observes bathochromic or red shift.

b) Hypsochromic shift- This effect is also known as blue shift. Hypsochromic shift is an effect by virtue of which absorption maximum is shifted towards the shorter wavelength. Generally it is caused due to the removal of conjugation or by changing the polarity of the solvents.

c) Hyperchromic effect- Hyperchromic shift is an effect by virtue of which absorption maximum increases. The introduction of an auxochrome in the compound generally results in the hyperchromic effect.

d) Hypochromic effect- Hyperchromic effect is defined as the effect by virtue of intensity of absorption maximum decreases. Hyperchromic effect occurs due to the distortion of the geometry of the molecule with an introduction of new group.

APPLICATIONS OF UV SPECTROSCOPY

1. Detection of functional groups- UV spectroscopy is used to detect the presence or absence of chromophore in the compound. This is technique is not useful for the detection of chromophore in complex compounds. The absence of a band at a particular band can be seen as an evidence for the absence of a particular group. If the spectrum of a compound comes out to be transparent above 200 nm than it confirms the absence of –

a) Conjugation b) A carbonyl group c) Benzene or aromatic compound d) Bromo or iodo atoms.

2. Detection of extent of conjugation- The extent of conjugation in the polyenes can be detected with the help of UV spectroscopy. With the increase in double bonds the absorption shifts towards the longer wavelength. If the double bond is increased by 8 in the polyenes then that polyene appears visible to the human eye as the absorption comes in the visible region.

3. Identification of an unknown compound- An unknown compound can be identified with the help of UV spectroscopy. The spectrum of unknown compound is compared with the spectrum of a reference compound and if both the spectrums coincide then it confirms the identification of the unknown substance.

4. Determination of configurations of geometrical isomers- It is observed that cis-alkenes absorb at different wavelength than the trans-alkenes. The two isomers can be distinguished with each other when one of the isomers has non-coplanar structure due to steric hindrances. The cis-isomer suffers distortion and absorbs at lower wavelength as compared to trans-isomer.

5. Determination of the purity of a substance- Purity of a substance can also be determined with the help of UV spectroscopy. The absorption of the sample solution is compared with the absorption of the reference solution. The intensity of the absorption can be used for the relative calculation of the purity of the sample substance.

WHAT IS TITRATION AND TYPES OF TITRATION

A titration (titrimetry) is a technique where a solution of known concentration is used to determine the concentration of an unknown solution. Typically, the titrant (the know solution) is added from a buret  to a known quantity of the analyte (the unknown solution) until the reaction is complete. Knowing the volume of titrant added allows the determination of the concentration of the unknown. Often, an indicator is used to usually signal the end of the reaction, the endpoint.

Titrations can be classified by the type of reaction. Different types of titration reaction include:

• Acid-base titrations are based on the neutralization reaction between the analyte and an acidic or basic titrant. These most commonly use a pH indicator, a pH meter, or a conductance meter to determine the endpoint.

• Redox titrations are based on an oxidation-reduction reaction between the analyte and titrant. These most commonly use a potentiometer or a redox indicator to determine the endpoint. Frequently either the reactants or the titrant have a colour intense enough that an additional indicator is not needed.

• Complexometric titrations are based on the formation of a complex between the analyte and the titrant. The chelating agent EDTA is very commonly used to titrate metal ions in solution. These titrations generally require specialized indicators that form weaker complexes with the analyte. A common example is Eriochrome Black T for the titration of calcium and magnesium ions.

• A zeta potential titration characterizes heterogeneous systems, such as colloids. Zeta potential plays role of indicator. One of the purposes is determination of iso-electric point when surface charge becomes 0. This can be achieved by changing pH or adding surfactant. Another purpose is determination of the optimum dose of the chemical for flocculation or stabilization

• ·         Gas phase titration

          Gas phase titrations are titrations done in the gas phase, specifically as methods for determining         reactive species by reaction with an excess of some other gas, acting as the titrant. In one common gas phase titration, gaseous ozone is titrated with nitrogen oxide according to the reaction

O₃ + NO → O₂ + NO₂.

After the reaction is complete, the remaining titrant and product are quantified (e.g., by FTIR);  this is used to determine the amount of analyte in the original sample.

Gas phase titration has several advantages over simple spectrophotometry. First, the measurement does not depend on path length, because the same path length is used for the measurement of both the excess titrant and the product. Second, the measurement does not depend on a linear change in absorbance as a function of analyte concentration as defined by the Beer Lambard Law, Third, it is useful for samples containing species which interfere at wavelengths typically used for the analyte.