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 –

a) 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.

d) 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.

e) 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 –

a) 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.

b) The syringes must be cleaned thoroughly, prior to spotting.

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

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

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

b) 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.

c) 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.

j) 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.

k) 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 -

a) The technique is simple and less expensive.

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

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

Disadvantages –

a) The method is not quantitative.

b) 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.

How to prepare and Standardize 0.1 N Sodium hydroxide volumetric solution.

Preparation of 0.1 N Sodium hydroxide volumetric solution.

➤ Glass ware and instruments required.

Beaker, Glass stirrer, Graduated Pipettes, Graduated cylinder, Analytical balance, Sucker, Volumetric flask, Fume hood.

➤ Reagents required.

Sodium hydroxide.
Molecular wt NaOH , 40.0
Potassium hydrogen phthalate primary standard. (Previously dried for 2 hour at 120°C)
Phenolphthalein indicator.

➤ Preparation.

Weigh 4.2 g sodium hydroxide pallets reagent grade in beaker, add 150 ml of carbon dioxide free water. Cool to room temperature. Filter through filter paper and transfer to 1000 ml volumetric flask. Dilute up to the mark with same solvent and transfer to tight polyolefin container.

➤ Standardisation.

Accurately weigh about 0.5 g potassium hydrogen phthalate PS, previously crushed lightly and dried for 2 hour at 120°, in 250 ml conical flask. Dissolve in 100 ml carbon dioxide free water. Add 2 drops of phenolphthalein indicator solution. Titrate with 0.1 N / 0.1 M sodium hydroxide till the solution becomes permanent pink. Note this final end point as burette reading in ml.

➤ Calculations.

g KHC₈H₄O₄ x 0.1

N / M= --------------------------------------------------

0.02042 x Burette reading in ml

How to Prepared and Standardize of 0.05 N Potassium Iodate Volumetric Solution.

Preparation of 0.05 N Potassium Iodate Volumetric solution

➤ Glass ware and instruments required.

Beaker, Glass stirrer, Graduated Pipettes, Graduated cylinder, Analytical balance, Sucker, Volumetric flask, Burette, Conical Flask, Fume hood.

➤ Reagents required.

Potassium Iodate Reagent grade,

Molecular Wt. :- KIO₃, 214.0

Potassium Iodide,

Sulphuric Acid Dilute,

Sodium thiosulphate Volumetric Solution (0.05M),

Starch Solution.

➤ Preparation.

Weigh and transfer about 10.7 g of potassium iodate reagent grade into 1000 ml volumetric flask. Dissolve it in sufficient water and make up to the mark. Filter the solution, if it is not clear.

➤ Standardization.

Pipette out 25 ml of 0.05 M potassium iodate solution into 100 ml volumetric flask, and make up with water. To 20 ml of this solution, add 2 g of potassium iodide, and 20 ml of dilute sulphuric acid. Titrate with 0.1 M sodium thiosulphate VS using 1 ml starch solution, added towards end of titration, as an indicator. End point is blue to colourless.

➤ Calculations.

g KIO₃ x 0.005 x 20 x 25

M= ----------------------------------------------------------------------

0.03566 x Burette Reading in ml x 1000

COPLEXOMETRIC TITRATION

A complexing agent is an electron donating ion or molecule,called a ligand, which by it's ability to form one or more covalent bonds with the metal ion, produces a complex, which has different properties from those of the metal ion.thus the metal may not be precipitated from the complex by the usual metal ion precipitants. The stability of the complex ion varies and greater the stability, more marked will be the differences in properties, from of the original cations.

Bonding in Complexes :
The bonds are either ordinary covalent bonds, in which both the metal and the ligand contribute one electron each or co-ordinate bonds in which both electrons are contributed by the ligand.

Chelating agents :
Complexes involving simple ligands, that is those forming only one bond, are described as 'co-ordinate compounds'. Ligands having more than one electron donating group are called 'chelating agents'.

Many organic compounds will chelate metals if they contain groups with an easily replaceable proton ( - COOH, phenolic and enolic-OH) or neutral groups offering a lone pair of electrons ( NH₂, CO and alcoholic OH) and the structure of the molecules are such as to permit the formation of stable rings. The greater the number of rings which can be formed; the chelate is likely to be more stable. Most rings formed in chelates involve the highest valency state of a metal, since these are more stable than those involving lower valency states.

The solubility of metal chelates in water depends upon the presence of hydrophilic groups such as COOH, SO₃ H,NH₂ and OH. When both acidic and basic groups are present, the complex will be soluble over a wide range of pH. When hydrophilic groups are absent, the solubilities of both the chelating agent and the metal chelate, will be low; but they will be soluble in organic solvents. The term 'sequestering agent', is generally applied to chelating agents, which form water soluble complexes with bi or polyvalent metal ions. Thus, although the metal remains in solution, they fail to give normal ionic reactions. Ethylenediamine tetra-acetic acid (EDTA) is a typical sequestering agent and can be represented as.

HOOC-CH₂ CH₂COOH

N-CH₂-CH₂-N

HOOC-CH₂   CH₂COOH

Types of EDTA titrations -

a) Direct titration -

  The solution containing the metal ion to be determined is buffered to the desired pH (usually pH-I0) and titrated directly with the standard EDTA solution. It may be necessary to prevent precipitation of the hydroxide of the metal ion by the addition of some auxiliary complexing agent such as tartrate, citrate or triethanolamine.

b) Back titraton -

Many metals cannot be titrated directly as they may precipitate from the solution in the necessary pH range or they may form inert complexes or  a suitable metal indicator is not available. In such cases, an excess of standard EDTA is added, the resultant solution is buffered to the desired pH and the excess of EDTA is back titrated with a standard metal ion solution e.g. a solution of Zinc chloride / sulphate or Magnesium chloride / sulphate is often used for this purpose. The end point is detected with the aid of metal indicator, which responds to the Zinc or Magnesium ions.

c) Replacement or substitution titration -
Substitution titration may be used for metal ions, that do not react (or react unsatisfactorily) with a metal indicator. The metal cation to be determined may be treated with the Magnesium complex of EDTA, when Magnesium ion is set free equivalent to the cation present and can be titrated with a standard solution of EDTA and a suitable metal indicator.

Metal ion indicators -

The success of EDTA titration depends upon the precise determination of the end point. The requisites of metal ion indicators for use in visual detection of end point include

a) The colour reaction must be such that before the end point, when nearly all metal ion is complexed with EDTA, the solution is strongly coloured.

b) The colour reaction should be specific or at least selective.

c) The metal indicator complex must possess sufficient stability, otherwise because of dissociation, a sharp colour change is not obtained. The metal indicator complex must however be less stable then the metal-EDTA complex to ensure that at the end point EDTA removes metal ion from the metal indicator complex. The change in equilibrium from the metal indicator complex to the metal-EDTA complex, should be sharp and rapid.

d) The colour contrast between the free indicator and the metal-indicator complex, should be such as to be readily observed.

e) The indicator must be very sensitive to metal ions, so that the colour change occurs as near to the equivalence point as possible.

f) The above requirements must be fulfilled within the pH range at which the titration is performed.

Some examples of metal ion indicators -

a) Murexide -

This is the Ammonium salt of purpuric acid and it was probably the first metal ion indicator, to be employed in the EDTA titration. Murexide solutions are reddish violet upto pH = 9, violet from pH -9 to 11 and blue above pH - 11. These colour changes are due to progressive displacement of protons from imido groups.

Murexide forms complexes with many metal ions, only those with Cu, Ni, Co, and Ca are sufficiently stable to find application in analysis. Their colour in alkaline solution are orange (Cu), yellow (Ni & Co) and red (Ca); the colours vary some what with the pH of the solution.

Aqueous solutions of Murexide are unstable and must be prepared every day. Normally, it is better to prepare a mixture of the indicator with pure Sodium chloride in the ratio (1:500) and employ 0.2 to 0.4 g in each titration.

b) Solochrome black ( eriochrome black T ) - This is the most commonly used metal indicator for EDTA titration and the colour can be observed with the ions of Mg, Mn, Zn, Cd, Hg, Pb, Cu, Al, Fe, Ti, Co, Ni and Pt metals. To maintain the pH constant at the value 10, a buffer mixture is added and most of the above metals must be kept in solution with the aid of a weak complexing agent such as Ammonia or Tartrate. The cations of Cu, Co, Ni, Al, Fe (III), Ti (IV) and Pt, form such stable indicator complexes, that the dyestuff can no longer be liberated by adding EDTA, making direct titration with EDTA impracticable. However, with Cu, Co, Ni, Al, a back titration can be carried out, for the rate of reaction of the EDTA complexes with the indicator is slow and it is possible to titrate the excess of EDTA with standard Zinc or Magnesium ions.

Cu, Ni, Co, Cr, Fe or Al even in traces, must be absent when conducting a direct titration with other metals; if the metal ion to be titrated does not react with cyanide ion or with triethanolamine; these substances can be used as masking reagents. The addition of 0.5 to 1 ml of 0.001 M o-phenanthroline prior to the EDTA titration eliminates the blocking effect of these metals with Solochrome black and also with Xylenol orange.

c) Patton and Reeder's indicator - Its main use is in the direct titration of Calcium, particularly in the presence of Magnesium. A sharp colour change from wine red to pure blue is obtained, when Calcium ions are titrated with EDTA at pH values between 12 and 14.

The dyestuff is thoroughly mixed with 100 times its weight of Sodium sulphate and 1 g of the mixture is used in each titration. The indicator is not very stable in alkaline solution.

d) Solochrome dark blue or Calcon - This is sometimes referred as Eriochrome blue black RC and is used in the titrations of Calcium in the presence of Magnesium, which must be carried out at a pH 12.3, in order to avoid the interference with Magnesium. Magnesium is precipitated as Magnesium hydroxide and the colour change for Calcium is from pink to pure blue.

e) Xylenol orange - Direct EDTA titrations of Bi, Th, Zn, Cd, Pb, Co, etc. are readily carried out and the colour change is sharp. By appropriate pH adjustment certain pairs of metal may be titrated, in a single sample solution. Thus Bismuth may be titrated at pH = 1 to 2 and Zinc or lead after adjustment to pH-5 by addition of Hexamine .

f) Calmagite -An important advantage of this indicator is that its aqueous solution is very stable and the colour change is somewhat clearer and sharper.

g) Fast sulphon black F -This indicator is virtually specific for Cu ions. The presence of Ammonia or pyridine is required for colour complex with Copper to form.

h) Bromopyrogallol red -This indicator possesses acid-base indicator properties and is resistant to oxidation. It forms coloured complex with many cations e.g. Bismuth at pH- 2 to 3 in Nitric acid solution, has a colour change from blue to claret red.

i) Thymolphthalein complexone (Thymolphthalexone) -This indicator contains a stable lactone ring and reacts only in an alkaline medium. The indicator may be used for Calcium, where the colour change is from blue to colourless. Manganese and Nickel may be determined by adding an excess of standard Calcium chloride solution; the colour change is from very pale blue to deep blue.

j) Zincon -This indicator is specific for Zinc at pH 9 to 10. It can also be used as an indicator for the titration of Calcium in the presence of Magnesium.

Some practical considerations -The following points must be borne in mind, while carrying out complexometric titrations.

a) Adjustment of pH - For many EDTA titrations, the pH of the solution is extremely critical; often limits of + 1 unit of pH must be achieved for a successful titration. Whenever a buffer solution is added, it must be ensured that the requisite buffering action is in fact achieved. Hence, it is necessary to make certain that the original solution has first been made almost neutral by cautious addition of Sodium hydroxide or Ammonium hydroxide. When acid solution containing the metallic ion is neutralised by the addition of alkali, care must be taken to ensure that the metal hydroxide is not precipitated.

b) Concentration of metal ion to be titrated - Most titrations are successful with 0.25 millimole of the metal ion in a volume of 50 to 150 ml of solution. If the concentration of metal ion is too high, then the end point may be difficult to judge.

c) Amount of indicator - The addition of too much indicator is a fault which must be guarded against; as end point anticipation, which is of great practical value; may be virtually lost if too much of indicator is added; as the colour is too intense. In general a satisfactory colour is obtained by the use of 30 to 50 mg of a solid mixture.

d) Attainment of the end point - In many EDTA titrations, the colour change in the neighbourhood of the end point is very slow; hence cautious addition of the titrant, coupled with continuous stirring of the solution is recommended.

e) Detection of colour change - The detection of the end point of the titration is dependent upon the recognition of a specific change in colour and for many observers affected by colour blindness, this may be difficult. This difficulty may be overcome by replacing the human element with a colourimeter or a spectrometer.

DETERMINATION OF WATER BY KARL FISCHER REAGENT

PRINCIPLE

For the determination of water; Karl Fischer in the year 1935, proposed a reagent prepared by the action of Sulphur dioxide, upon a solution of Iodine in a mixture of anhydrous Pyridine and anhydrous Methanol.

Water reacts with this reagent, In a two stage process as shown.

SO₂

3C₅H₅N + I₂ + SO₂ + H₂O = 2 C₅H₅NH⁺I^- + C₅H₅N+ O^-

OSO₂OCH₃

C₅H₅N+ ---SO₂ + CH₃OH = C₅H₅N

Thus, each molecule of Iodine is equivalent to one molecule of water. The end point of the reaction is conveniently determined electrometrically, using the dead-stop end point procedure. If a small e.m.f. is applied across two platinum electrodes immersed in the reaction mixture, a current will flow, as long as free Iodine is present, to remove Hydrogen and depolarise the cathode. When the last traces of Iodine have reacted, the current will decrease to zero or very close to zero. Conversely, the technique may be combined with a direct titration of the sample with the Karl Fischer reagent. Here the current in the electrode circuit, suddenly increases at the first appearance of unused Iodine in the solution.

The original Karl Fischer reagent, prepared with an excess of Methanol, was some what unstable and required frequent standardisation. It was found that the stability was improved by replacing Methanol with 2-methoxy ethanol.

Present day Karl Fischer reagents have also replaced Pyridine, with a base. Hence, these are advertised as "pyridine free reagents".

The method is clearly confined to those cases, where the test substance does not react either with the components of the reagent or with the Hydrogen iodide formed during the reaction with water. Hence, this method is unsuitable for

a) Oxidising agents such as Chromates, Dichromates, Copper (II) and Iron (III) salts, higher oxides and peroxides.

MnO₂+ 4C₅H₅NH⁺+ 2I^-= Mn⁺⁺ + 4 C₅H₅N + I₂ +2H₂O

b) Reducing agents such as, Thiosulphates, Tin(Il) salts and sulphides.

c) Compounds which can be regarded as forming water with the components of the Karl Fischer reagent e.g.

1. Basic oxides -

ZnO + 2C₅H₅NH⁺ = Zn⁺⁺ + 2C₅H₅N + H₂O

2. Salts of weak oxy acids -

NaHCO₃ + C₅H₅NH⁺ = Na⁺ + C₅H₅N + CO₂+ H₂O

Instrument

The instrument used for determination of water by K.F. is METROHM 701 KF TITRINO, consisting of a titrant dispenser, with a facility to draw Methanol and K.F. reagent in a closed system provided with guard tubes containing desiccant which prohibits ingress of moisture and key board for programming.

The titration vessel is conditioned by neutralizing the inherent moisture present in the titration vessel; using K.F. reagent.

The water equivalent factor of the K.F. reagent is determined daily, by titrating known quantities of water, in duplicate, with the reagent.

The water content of the sample is found out by titrating known amount of the sample.

The instrument is calibrated once a month by titrating known quantity of Disodium tartrate in duplicate and comparing the mean result obtained, with the specification for water content of Disodium tartrate.

Precautions

a) The desiccants in the guard tubes must be regularly replaced.

b) The stop cocks must be lubricated with Silicone grease and not with petroleum grease, which reacts with the K.F. reagent.

c) The water equivalent factor of the K.F. reagent must be determined daily and must be within 1% R.S.D., to ensure accuracy of the water determination.

d) The water equivalent factor of the K.F. reagent is normally around 5 to 6 mg of water per ml of the reagent. On repeated use, this factor goes down and the reagent having a factor of 4.5 or less, must never be used; as the volume of the reagent required for the titration increases, reducing the sensitivity of the determination of the water content.

e) It is advisable to mix the sample in Methanol for at least one minute, prior to titration with the K.F. reagent.

f) During the determination of water, the sample and the Methanol must be thoroughly mixed.

Pharmaguidea1

THIN LAYER CHROMATOGRAPHY

THIN LAYER CHROMATOGRAPHY

INTRODUCTION :-

Apparatus required –

Advantages -

How to prepare and Standardize 0.1 N Sodium hydroxide volumetric solution.

How to Prepared and Standardize of 0.05 N Potassium Iodate Volumetric Solution.

COPLEXOMETRIC TITRATION

DETERMINATION OF WATER BY KARL FISCHER REAGENT

DETERMINATION OF WATER BY KARL FISCHER REAGENT

PRINCIPLE

Instrument

Precautions

THIN LAYER CHROMATOGRAPHY

Report Abuse

Labels