Chromatography Part 3, Thin Layer Chromatography TLC

Clinical Pharmacy, Instrumental Analysis section

A study in Instrumental Analysis dedicated for professionals & students

Done By Ismail Mortada

B.Sc.Pharmacy & Health Sciences, Clinical Pharmacy Section

 

In past articles I spoke about general instrumental analysis methods including chromatography in general, paper chromatography & now it’s the time to speak about Thin Layer Chromatography called TLC

TLC relies upon the production of a uniform layer of stationary phase held on a glass plate or other supporting medium, The most popular media used are microcrystalline cellulose & Kieselguhr, the last had been defined before in another article but microcrystalline cellulose in brief is a natural polymer which is widely used in pharmaceutical manufacturing processes for different reasons, It’s a purified, partially depolymerized cellulose prepared by treating alpha-cellulose, obtained as a pulp from fibrous plant material, with mineral acids. The degree of polymerization is typically less than 400. Not more than 10% of the material has a particle size of less than 5 m m like the pictures below in general

The past mentioned stationary phases holders are normally used in partition chromatography where a liquid is help as a stationary phase as mentioned before, in case the chromatography method was wanted to be adsorption method so Alumina is used normally with silica gel, the last had been used for both partition & adsorption methods

The solid layers are prepared by applying a slurry of the chosen medium in a suitable solvent (water is the most commonly used solvent) on a clean glass plates, if the material adhere badly, a binding agent such as CaSO4 is often incorporated in small quantities, the slurry is spread on the plate by mean of commercial spreading appartus, this appartus is used to help producing a uniform spreaded layer & to control the thickness of the layer depending on the experiment or process itself, the thickness of the layers might varie between 0.25 - 0.5 up to 1 mm

TYPES OF STATIONARY PHASES

There is a wide range of adsorbents commercially available such as the following

Silica gel , silicic acid; a partition method basically but can be used for both types

Alumina, aluminium oxide; an adsorption method

Kieselguhr or diatomaceous earth; a partition method

Microcrystalline cellulose; a partition method

Other commercial adsorbents

A) SILICA GEL OR SILICIC ACID

Is the most commonly used type & is slightly acidic in nature, & used in both partition & adsorption methods, a binding agent is normally incorporated to improve it’s binding properties to the glass plate, an agent such as Calcium Sulphate Hemihydrate, this can creat problems in TLC of inorganic compounds, but it had been found that adsorbents will adhere quite satisfactory in the absence of the binder if they are ground to a smaller particle sizes (I relate it to the magnetic power or exactly charges achieved or something similar to that normally), 2 UV indicators had also been incorporated, either in a single form or together, these are Sodium fluorescein & Sodium salt of hydroxy pyrene sulphonic acids (they fluoresce at 254 nm & 365 nm, respectively) thus providing a contrasting background for compounds which adsorb at this frequency, in the separation of organic bases (like alkaloids) which are basic in nature usually a small amount of ammonia or diethylamine is added to the solvent system to enhance the dissolving properties because the CaSO4 is acidic in nature also

B) ALUMINA OR ALUMINIUM OXIDE

It is a slightly basic adsorbent (used basically in adsorption methods) with less resolution compared to silica, it is also more chemically reactive adsorbent so that it is possible for sensitive compounds to be degraded while the plate is being loaded that’s why you need to be careful while using it with sensitive compounds, sometimes in the separation of acids a small amount of glacial acetic acid is added to the solvent system to enhance the resolution properties, it is available with or without plaster of CaSO4 & fluorescin indicator

C) Kieselguhr or diatomaceous earth

It is a neutral adsorbent (used basically in partition methods) having a lower resolving power, it can be mixed with silica gel usually to give a less active mixed adsorbent & to increase it’s adsorption capacity , but it is mainly used as a support for the stationary phase during partition TLC , it is available normally with or without a binder

D) Microcrystaalline cellulose

It is used only as a support for the stationary liquid phase in partition chromatography TLC, the advantages of cellulose TLC over PC (Paper Chromaatography) is that running times are greatly reduced compared with those for the same solvent system on paper, & sensitivity is greatly increased, this is explained partially by the reduced fibre length in cellulose for TLC & partialy by the higher specific surface area, both of which tends to make the spots more compact & because of it’s fibrous nature, cellulose gives satisfactory layers without a binder needed in the method

E) Other commercial adsorbents

Those especially used in TLC include the following

Polyamides, used basically in TLC for flavonoids & other organic compounds

Magnesium silicate

PLATE COATING

 

Plates should be cleaned with a detergent solution first, then rinsed with hot water or with an organic solvent to free the surface from grease, this oil or grease will prevent a professional adherance of the future layers which are supposed to be spreaded on the glass plate & finally dried before using it. The appropriate adsorbent is a slurry made with distilled water; the proportion of adsorbent to water varies with the type of the adsorbent (for example for silica gel it is 1:2), after that to mix the slurry throughly it is preferable to stir it mechanically, for cellulose plates high speed stirring with a blender is advisable. The usual thickness of layers of TLC is 0.25 nm (for analytical purposes), & thicker layers (0.5 to 1 mm) are used for preparative purposes. Normal chromatoplates are prepared in standard sizes 5 X 20, 10 X 20 & 20 X 20 cm, as shown in the pictures using a glass plate with the stationary phase slurry covering it by manual ways in the lab

Activation & storage of the plates

Plates for adsorption chromatography are normally left for about 30 minutes after spreading, then placed in a metal racks & kept in an oven for at 105 degree C. for 30-60 minutes to drive off the water content but using higher temperatures will lead to the destruction of the paper itself or the plate, Then the activated plates should be stored in a desiccator until being used, The activity of Alumina plates is governed to a greater extent by the temperature to which are heated & the storage after activation is important because any subjection to humidity will lead to adsorb & absorb water in the plate which will lead to the in-activation of the plate again

LAYER IMPREGNATION

It is used for partition chromatography basically, water or an aqueous solution can be used as a stationary phase & in this case the coated plates (with silica gel, cellulose or Keiselghur) are allowed to dry at room temperature and heated for 10 minutes, In some cases hydrophylic or hydrophobic liquids (such as in reverse methods discussed in past articles) are used as a stationary phase, impregnation is performed as in PC in that case & the layers are allowed to dry without heating

Precoated plates are available commercially with a wide range of coating materials to make it easier for the chromatographer

SAMPLE PREPARATION & APPLICATION

It is the same as in PC (Paper Chromatography) but unlike PC in sample application, the drawing of the base line should be avoided since it scraps the coated layer, so several plastic templates are available to protect the coated layer surface during the loading process, For preparatice TLC mechanical applicators are used, so as shwon in the picture down this is a WRONG WAY OF DRAWING THE LINE unlike PC method

PLATES DEVELOPMENT

Development of TLC is normally done by the Ascending methods (explained in PC before), It should be carried in a saturated tank, After development, the solvent front is marked & the plates are dried rapidly in a stream of warm air

Variation in TLC techniques

The basic techniques in adsorption TLC consist of a single development, if this proved in adequate results another techiniques are used, the following techinques are used such as

Multiple development

Tow-Dimensional development

Stepwise development

Other techiniques

MULTIPLE DEVELOPMENT

If a single development showed partial separation, the plate can be dried and re-runned a number of times in the same solvent system

STEPWISE DEVELOPMENT

When a mixture contains groups of components differing considerably in polarity it may be possible to obtain a separation on a single plate by running it progressively with different solvents, The first solvent used should be polar (like methanol) in order to separate the polar components & it is runned only part way up the plate, if a 2′nd non-polar solvent (such as cyclohexane) is then run the whole lenght of the plate it may separate in the upper portion those non-polar compounds that ran together at the first solvent front

Regarding the question of why that we use the polar solvent first, this is because it will dissolve the polar compound AND WILL CARRY the non-polar component, then after that and from that half way trip we’ll start the second development using the non-polar solvent

TWO DIMENSIONAL DEVELOPMENT

It is exactly the same in PC, Paper Chromatography

OTHER TECHNIQUES

They include a lot of type such as the following

Multi-brand TLC: In this techinique different adsorbents are diposited as brands on a single 20 X 20 plate, in pc we were cutting it to strands but in TLC we can just scratch different lines by using a normal spatula for example

Gradient TLC: This technique is based on the continous production of varying mixture of adsorbents, A commercial appartus is available for gradient TLC

Partition TLC & Reversed phase partition TLC; Is similar to PC, this can be done by saturation with solvent vapour or by impregnation

VISUALIZATION\LOCATION OF SUBSTANCES

As in PC, physical, chemical & biological methods can visualize the developed chromatoplates, on using chemical locating reagents, heating is often necessary, corrosive reagents can also be used (like acids) which was not suitable to be used in PC

FACTORS AFFECTING Rf VALUES OR REPRODUCIBILITY OF THE VALUE

In TLC, in order to obtain a reproducible Rf values, the following basic 10 factors should be considered

Quality of adsorbent In which particle size differences exist between adsorbents from different manufacturers may affect the time of development but not the Rf value itself

Thickness of the layer, There is no evidence that the increase of the layer thickness in itself affects the Rf values, it is likely that distortion of spots & variations in the running position will occur when layer thickness varies across a single plate so for analytical purposes, thick layers are preffered normally

Activity of the layer, It depends on the time & temperature of heating (if any occured), on the storage conditions (as mentioned before) after activation & the length of time a plate is held in storage

Relative Humidity, Which will affect the activity of the layer (as mentioned before) during both storage & sample application unless precautions are taken to protect the plate, by increasing humidity the plate will be de-activated or in-activated

Amount of sample applied, The amount of sample required for most compounds is ranging from 10 to 20 micro-gram with larger amounts Rf values tend to increase or decrease, impurities can also affect the Rf values in adsorption TLC + overloading the sample will overcome the adsorption capacity & will deviate the method totally

Solvent Quality, This can be controlled by using analytical grade solvents, when solvent mixtures are required they should be renewed at frequent intervals

Temperature, Increase in temperature raises Rf values slightly, therefore temperature should be controlled during TLC, however chromatography is less affected by temperature changes

Equilibrium between liquid & vapour in tanks, The saturation of the atmosphere in the tank by lining the sides and enclosing the tank is necessary, this shortens the running time; & decreased Rf values are obtained with an unsaturated atmosphere is demonstrated by the so-called "edge effect", The Rf values at the edge of the plate which had been run in an un-lined tank are higher than those at the center, because of the greater rate of solvent evaporation at the plate edges while the solvent front itself runs level

Technique: The angle at which a plate lies in the tank has no effect on Rf values, but the use of ascending or descending techniques may well do so

Pre-adsorption of solvent vapour by adsorbent: It is necessary only for some forms of partition TLC, Rf values are then lowered because less solvent runs up the plate during development since some had already been adsorbed or runned while vapours are available

PREPARATIVE THIN LAYER CHROMATOGRAPHY

The optimum layer thickness is normal 1 mm,  But layers upto 5 mm thick are also used sometimes, All thick layers should be dried slowly, since there is a danger of cracking

A sample for examination is dissolved in the minimum of solvent, & the solution is applied as a band along the origin, This can be done by applying the sample as series of spots that merge into one another (by using a small capillary or syringe), mechanical applicators are available, development of the plate can be done after that normally

Location of zone presents a problem except for coloured compounds, various non-destructive methods can be applyed to the whole plate for example UV light & iodine vapour

However; if chemical methods (destructive agents) are to be used; this requires careful application of the reagent on a strip at one edge (this is done by covering the developing plates with a non-coated plate of the same size or a larger one, & leaving the uncovered strip at one edge), one disadvantage of locating portions of the zones is that brands do not always run evenly on thick layers

The separated zones can be removed by scrapping off adsorbent with a spatula or razor blade, the pure components can be freed from the aadsorbent by the addition of a suitable organic solvent followed by filtration

Chromatography Part 3, Thin Layer Chromatography TLC

Clinical Pharmacy, Instrumental Analysis section

A study in Instrumental Analysis dedicated for professionals & students

Done By Ismail Mortada

B.Sc.Pharmacy & Health Sciences, Clinical Pharmacy Section

 

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التصنيفات : Chromatography studies, Instrumental Analysis studies | السمات:Chromatography, Instrumental Analysis, mobile phase, stationary phase, Thin Layer Chromatography, tlc
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