An analyte migrates up or across a layer of stationary phase (most commonly silica gel), under the influence of a mobile phase (usually a mixture of organic solvents), which moves

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Chapter 13

Thin-layer chromatography

Shin-Hun Juang, Ph.D.



An analyte migrates up or across a layer of stationary phase (most commonly silica gel), under the influence of a mobile phase (usually a mixture of organic solvents), which moves

through the stationary phase by capillary action.

The distance moved by the analyte is

determined by its relative affinity for the

stationary vs. the mobile phase.


(i) A few μl of sample solution are slowly spotted onto the plate at the origin. If more than ca I μl is applied at once, the spot will spread too far. The spot has to be allowed to dry between each application of 1 μl. Loadings of sample are typically 20 μg.

(ii) The bottom 0.5 cm of the plate is immersed in the mobile

phase contained in a tank and the liquid mobile phase is allowed to travel up the silica gel plate by capillary action.

(iii) The more polar a compound is the more it adsorbs

(partitions into) the silica gel stationary phase, the less time it spends in the mobile phase as it travels up the plate and thus the shorter the distance it travels up the plate in a given time.

The TLC method


Three Components of TLC Analysis

1. Stationary Phase 2. Mobile Phase

3. Spray Regents



or plastic plate 

The silica gel particle size is in the range 2- 25 μm.


Stationary Phases

Silica gel is the most commonly used adsorbant for TLC. The rate at which compounds migrate up a silica gel plate depends on their polarity.


Stationary Phases


The Strength of a Mobile Phase Depends

on the Particular Solvent Mixture Used.


TLC Chromatogram

compound A is less polar than compound B

Rf value

The distance travelled by the compound from the origin divided by the distance

travelled by the solvent from the origin

for compound A, Rf = a/S

for compound B, Rf = b/S


Solvent is allowed to move 10 cm from the origin up a TLC plate. The time taken to develop the plate for this distance is 15 min. On the basis of this information, complete the table below.

(i) 80, 12, 03 (ii) 60, 09, 06 (iii) 40, 06, 09


The more polar a solvent or solvent mixture, the further it will move a polar compound up a silica gel TLC plate.

When non-polar compounds are being analyzed, there will not be a marked increase in the distance migrated with

increasing polarity of the mobile phase since they migrate towards the solvent front under most conditions.

Water many organic compounds are not very soluble



Mobile phases have been developed which ensure that a particular drug will have a quite different Rf value in one system compared with another.




3 4

5 6


(i) 1. 18, 2.25, 3.24 (ii) 1. 40, 2.25, 3.14 (iii) 1. 33, 2.45, 3.14 (iv) 1. 20, 2.28, 3.10


Modification of TLC Adsorbant

1) Treatment of silica gel with KOH

For analysis of basic compounds, silica gel which has been

sprayed with a solution of KOH in methanol may be used.

Basic compounds chromatograph as their free bases.

The mobile phases used in these type of systems also typically contain a basic component. Examples of the mobile phases:

(i) Methanol / strong ammonia solution (100: 1.5)

(ii) Cyclohexane / toluene / diethylamine (75: 15: 10) (iii) Chloroform / methanol (90: 10)

*The use of selective solvent systems is often combined with use

antihistamines / narcotics sympathomimetic bases


2) Silanized silica gel

The surface of the silica gel can be rendered non-polar by reaction with dichlorodimethylsilane or octadecylsilanes (ODS).

Example: identity tests for penicillins

Mobile phase: a solution of ammonium acetate adjusted to pH 5.0 with acetic acid and acetone (90: 10).

Stained: iodine vapour


3) Keiselguhr as an inert support

Keiselguhr in itself does not have strong absorptive properties but it can be coated with a liquid or waxy stationary phase.

Example: triglycerides and fatty acids in fixed oils

The keiselguhr plate is impregnated with a solution containing liquid paraffin in petroleum ether. This renders the surface hydrophobic.

Mobile phase: acetic acid (very polar)

The triglycerides in the fixed oil are only weakly polar and will partition usefully between the liquid paraffin stationary phase and the acetic acid mobile phase. The longer the chain length of the fatty acids in the

triglyceride, the lower the Rf of the triglyceride.

Stained: iodine + starch

Other agents used to impregnate keiselguhr include: formamide and propan-1,2-diol.

In the case of these impregnating agents, the mobile phases used to


Detection of Compounds on TLC plates

 Ultraviolet light

• In order to observe the absorption of UV light by an analyte, silica gel which has been impregnated with a fluorescent

material is used to prepare the TLC plate.

• Light with a wavelength of 254 nm is used to illuminate the plate and if the analyte absorbs UV light it can be seen as a black spot on a yellow background where it quenches the fluorescence of the background.

• If a compound is naturally fluorescent, longer wavelength light at 365 nm may be used to visualize the plate.

Anthraquinones 365 nm


 Location reagents Iodine vapour

brown spots for many organic compounds

the staining is reversible: the iodine may be allowed to evaporate by exposing the plate to air scraped off marked spot containing the compound of interest .

permanent record of the plate: covered to prevent the iodine evaporating, or sprayed with starch solution

Ex. fixed oils and of cetrimide

~specific for a particular type of analyte put into a tank containing iodine crystals


Ninhydrin solution

Potassium permanganate

detection of sugars and sugar like molecules, and drugs with aliphatic double bonds

antibacterial agents: clindamycin / lincomycin / spectinomycin.

This reagent gives pink spots with primary amines and yellow spots with tertiary amines.

aminoglycoside antibiotics such as gentamycin

limit test for aminobutanol in ethambutol

nitrogen-containing drugs in conjunction with Dragendorff reagent.

Dragendorff reagent will produce orange spots with tertiary amines and may be used to overspray plates which have been sprayed in the first instance with ninhydrin.


Alkaline tetrazolium blue

Ethanol/sulphuric acid 20%

specific for corticosteroids, producing blue spots

influclorolone acetonide.

This reagent is used to produce fluorescent spots from

corticosteroids such as dexamethasone or prednisolone by spraying the plate, heating to 120°C and then observing the plate under UV light at 365 nm.


Applications of TLC Analysis


Qualitative Identity Tests


Limit Tests

Where the structure of the impurity is known

A TLC limit test is based on comparison between a concentrated solution of an analyte and a dilute solution of an impurity. The intensities of the spots due to any impurities in the analyte are compared with the intensity of a spot or spots due to standards spotted separately onto the same plate.

1 % w/v This test is a 1 % limit test since 

0.0111 x 100 = 1 %.


Limit Tests

Where the structure of the impurity is unknown

• This type of test is used, for instance, on compounds of natural origin or partly natural origin, which may contain a range of compounds related in structure to the test substance which are co-extracted with the raw starting material.

• The assumption which is made in the type of test described

following is that the related unknown substances will produce a similar intensity of spot to the test substance itself at equal



•The plate is developed in  ethanol/cyclohexane/13.5 M  ammonia (72:30:6), is dried  and is then sprayed with  iodobismuthate reagent,  which is specific for 

nitrogenous drugs.

(i) There should be no secondary spot in the chromatogram of Solution 1  which is more intense than the spot obtained with Solution 2.

(ii) There should be no more than one secondary spot with a Rf value 

higher than that of codeine which is more intense than the spot obtained  with Solution 3.


Three samples of codeine are analyzed as described earlier.

Indicate whether the TLC limit tests shown below pass or fail the samples. Solutions 1-3 appear in numerical order from left to right.


Known and Unknown Standards are Used


TLC Strengths TLC Limitations Identification of compounds and for

determination of the presence of trace impurities

Low theoretical plates available

A huge array of TLC-based tests is available and pharmacopoeial


Low sensitivity

Its flexibility in being able to detect almost any compound, even some inorganic compounds

Not suitable for volatile compound

The entire chromatogram can be seen Requires skillful operation Robust, cheap and flexible


High-performance TLC (HPTLC)

• HPTLC is conducted on TLC plates which are coated with

purified silica gel with a particle range of 2-10 μm, as opposed to 2-25 μm for standard commercial TLC plates.

• The narrower particle size range, a greater number of theoretical plates

• These type of plates may be run in a standard type of TLC tank but optimal performance is obtained from horizontal

development of the plates.


HPTLC Flow Diagram & Instrumentation


Scanning Densitometer

The scanner is connected to a computer.

The scanner features three light sources ,a deuterium lamp , a tungsten lamp and a high pressure mercury lamp.

The scanning speed is selectable between 1 and 100 mm/s


Differences Between TLC and HPTLC


(i) The mobile phase moves more quickly.

(ii) There is little evaporation of solvent from the surface of the plate, which in the case of vertical development can change the composition of the mobile phase as it moves up the plate.

(iii) In the vertical position, if the plate is not in a

saturated atmosphere, solvent at the edge of the plate tends to evaporate, drawing solvent from the centre of the plate and causing the solvent at the edge of the

plate to migrate more quickly. This does not occur when horizontal development is used.

The Advantages of Horizontal Development


Applications of HPTLC

HPTLC can be used for the quantitative analysis of mixtures which include large and complex molecules such as rifampicin.

It would be possible to run many current pharmaceutical limit tests with a much higher degree of accuracy and precision.



(A)Silica gel G   (B)Cellulose  (C)Agarose  (D)Keiselguhr G

胺醣類抗生素(aminoglycoside antibiotics)如gentamycin的薄層 層析定性分析中,常使用下列何種呈色試劑來進行其鑑別?

(A)Iodine vapor   (B)Ninhydrin solution

(C)Alkaline tetrazolium blue  (D)Ethanol/Sulphuric acid 20%


(A)不適合用於精油(volatile oil)的分析






TLC展開後,下列何種定位試劑(location reagent)可使皮質 類固醇在白色背景上產生藍色的點?

(A)Iodine vapor  (B)Ninhydrin solution

(C)Potassium permanganate  (D)Alkaline tetrazolium blue

進行薄層層析時,展開槽未經展開溶媒事先飽和時,下列何 種情況最易發生?

(A)分析時間增加 (B)層析板吸附劑容易脫落

(C)待測物Rf 值之再現性不變 (D)展開溶媒容易與分析物產生 化學反應


(A)Mercuric nitrate (B)Antimony trichloride (C)Ninhydrin  (D)Aniline phthalate


TLC 可用於抗黴菌劑Clotrimazole 藥物原料中之何種已知雜質 的檢查?

(A)Aniline   (B)Phenylpropanolamine   (C)Shikimic acid  (D)Imidazole


(A)Cellulose   (B)Keiselguhr G   (C)Silica gel G   (D)Silica gel GF254


(A)Mercuric nitrate   (B)Antimony trichloride   (C)Ninhydrin  (D)Aniline phthalate




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