Thin Layer Chromatography Identification Test for Atenolol and Chlorthalidone in Tablets following European Pharmacopeia Monograph on Silica Gel G 254 Plates


Atenolol is a selective β1 receptor antagonist, a drug belonging to the group of beta blockers, a class of drugs used primarily in cardiovascular diseases.

Chlorthalidone is a diuretic drug used to treat hypertension, originally marketed as Hygroton in the USA. It is described as a thiazide diuretic. It is often used in the management of hypertension and edema.

With this application note we illustrate how silica gel G 254 plates are suitable for analysis of Atenolol and Chlorthalidone in a combination drug following the experimental conditions per the European pharmacopeia identification TLC test for Atenolol (version 9.0).







Experimental Conditions
TLC Plate Silica Gel 60G F254  20 x 20 cm  
Injection volume 5 µL of each solution
Detection UV @ 254 nm
Mobile Phase 18 M Ammonia and n-butanol  30:150  (v/v)
Plate development Line the walls of chromatographic tank with filter paper. Pour into the chromatographic tank sufficient quantity of mobile phase Saturate the chromatographic tank replace the lid and allow to stand for 20-25˚C for 1 hr. Apply the prescribed volume of the solutions in sufficiently small portions to obtain bands at an appropriate distance from the lower edge.

When the solvent has evaporated from the applied spots place the plate in as vertical as possible close the tank and allow the plate to run for 15 cm .Remove the plate and dry the plate and observe under UV light at 254 nm
Diluent    Methanol
Development 15 cm
Drying In air
Standard 1.0% (w/v) of atenolol in methanol and 0.25 % (w/v) of Chlorthalidone in methanol
Sample Remove any film coating on the tablet powder and shake the quantity of powdered tablet containing 0.1 g of atenolol with 10 ml of methanol for 15 minutes and filter.

TLC plate development
TLC Plate Development





spectrum for altenolol

Spectrum for Altenolol


spectrum for chlorthalidone

Spectrum for Chlorthalidone


Chromatographic Data (Standard Solution )

  Compound Rf Area
1 Atenolol 0.24 25166
2 Chlorthalidone 0.68 38676


Altenolol standard


Chlorthalidone standard



Chromatographic Data (Sample Solution )

  Compound Rf Area
1 Atenolol 0.24 22533
2 Chlorthalidone 0.69 31061

Sample solution

Standard Repeatability - Altenolol
Standard Repeatability - Altenolol

STD 1 25045
STD 2 25166
STD 3 25453
STD 4 25092
STD 5 25346
Mean 25220.4
Standard Deviation 154.97
RSD (%) 0.6

Standard Repeatability - Chlorthalodine
Standard Repeatability - Chlorthalodine

STD 1 38676
STD 2 37752
STD 3 37102
STD 4 37598
STD 5 37899
Mean 37805
Standard Deviation 511.3
RSD (%) 1.4


  1. The principal spot in the chromatogram obtained with the test solution and the two principal spots correspond in position, size and intensity to those of the principal spots in the chromatograms obtained with standard solutions.
  2. The repeatability for Atenolol and Chlorthalidone standards, was 0.6 and 1.4 %, respectively.


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