Separation of Carvedilol Enantiomers using HPLC by two Different Methods: Mobile Phase Chiral Additive and Chiral Stationary Phase


Ola Mahmoud Younes1*, Fida Am Ali2, Zaid Al Assaf3

1PhD Student, Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria.

2Professor Assistant, Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria.

3Professor, Department of Analytical and Food Chemistry, Faculty of Pharmacy, Damascus University, Damascus, Syria

*Corresponding Author E-mail:



This paper presents the results of HPLC enantioseparation of carvedilol using HPLC by two different methods the first one is reversed phase HPLC (RP-HPLC), In which carboxy methyl-𝛽- cyclodextrin (CM-𝛽-CD) is used as chiral mobile phase additive the second one, which is common used, depends on using chiral stationary phase (polysaccharide type, Chiralcel OD-R column, 250 mm×4.6 mm). In mobile phase chiral additive method, the highest resolution was achieved using Knauer C18 column (250 mm×4.6 mm) and mobile phase made up of a mixture of aqueous solution [pH=4.4, (2.3g/l CM-𝛽-CD)], methanol and acetonitrile with a volumetric ratio of (56.8/ 30.7/12.5) v/v%, flow rate of 0.7mL/min, and column temperature was set at 40°.In chiral stationary phase method ,the mobile phase consisted of acetonitrile, isopropanol, and diethyl amine with a volumetric ratio of (95/ 5/0.1) v/v%, flow rate of 1 ml/min at room temperature. The effects of different conditions on the enantioseparation of carvedilol were investigated. Both methods showed good resolution of carvedilol enantiomers. But the first new one is very inexpensive comparing with the second one.


KEYWORDS: Carvedilol enantiomers, HPLC, chiral selector, carboxy methyl- β cyclodextrin (CM-𝛽-CD), chiral stationary phase.




Carvedilol is a non-cardioselective β-blocker, which also exhibit α-blocking activity. It has vasodilating properties, attributed mainly to its blocking activity on α receptors. Carvedilol is used not only in the management of hypertension and angina pectoris, but also in the therapy of symptomatic heart failure [1].


Like all other β-blockers that are currently used, carvedilol contains an asymmetric carbon atom in the amino-alkanol side chain resulting in the existence of two enantiomers. The chemical structure of carvedilol (1-(4-carbazolyloxy)-3-(2-(2-methoxy) ethylamino)-2-propanol) is presented in Figure 1 [2]


Figure 1 chemical structure of carvedilol [1]


Carvedilol is administered clinically as a racemic mixture of the R (+)- and S(−)-enantiomers. The carvedilol enantiomers exhibit different pharmacological effects, the blockade of the β adrenergic receptor being primarily attributed to the S (−)-carvedilol (50–500 times higher than R(+)-carvedilol), whereas the two enantiomers are considered to be equipotent with respect to the blockade of the α adrenergic receptor. Additionally, each enantiomer displays a unique pharmacokinetic behaviour, where the R (+)-enantiomer attains higher plasma concentrations, bioavailability and protein binding. [3]


To date, many methods are developed for the enantioselective analysis of Carvedilol which include capillary electrophoresis by adding cyclodextrins to the buffer as chiral selectors [4,5,6], and HPLC direct methods using chiral stationary phase [7,8] or indirect methods which involve derivatization of carvedilol by pure chiral reagents.[9]


Cyclodextrins are chiral cyclic oligosaccharides composed of α-(l,4)-linked d-glucose units, beta cyclodextrin (7) glucose units [10] and its derivatives , have been recognized as the most prevalent chiral mobile phase additives due to the nature of being nontoxic, nonflammable, nonvolatile, stable over a wide range of pH and negligible absorption in the UV ranges broadly used in chromatographic detection. [11].


 To the best of our knowledge, this is the first report to use carboxy methyl β-cyclodextrin (CM-β-CD) as chiral mobile phase additive using reversed phase HPLC to separate carvedilol enantiomers on classical C18 column which is available in most laboratories and relatively inexpensive .this research also introduce enantioseparation method of carvedilol using polysaccharide chiral stationary phase (Chiralcel OD-R, cellulose tris 3,5 dimethylphenyl carbamate), this type of chiral stationary phases proved its effectiveness among all other types of chiral stationary phases [7] .Conditions that effect on enantioseparation of carvedilol were studied in both methods.



2.1. Chemicals and reagents:

Standard of racemic Carvedilol , SC-200157, standard of (S)- Carvedilol SC- 212831 (Santacruz Biotechnology), (CM-β-CD) Carboxy methyl beta cyclodextrin 99.3 ),%Degree of substitution 5.79( Zibo Qianhui Biological Technology CO,LTD. Glacial acetic acid, HPLC methanol, acetonitrile, Isopropanol, diethyl amine (DEA) (Panreac, EU. Quimica. SA). Triethylamine (TEA) (Scharlau Chemie S.A., Barcelona-Spain). Water for HPLC (Chem-lab NV, Belgium).


2.2. Instruments:

HPLC system (Jasco, Japan) which is equipped by PU-980 intelligent pump, Jasco UV -970 intelligent detector, manual injector, column C18(250X4.6) Knauer, Germany. Chiralcel column (OD-R, 250X4.6), France. Spectrophotometer Hitachi U-1800, Quartz cuvettes, Sartorius sensitive analytical balance Ag, d=0.1 mg, Germany. Ultrasonic bath (Transsonic 310, εlma®) Germany. pH meter Orion model 410 A. 0.45µ cellulose nitrate filters (Sartorius Stedim), Germany.

2.3. Chromatographic conditions of chiral mobile phase additive method:

Chromatographic separation was conducted in the reversed phase operating mode, with a mobile phase consisting of aqueous solution (which prepared by adding to one liter of HPLC water 3.3ml triethylamine (TEA) and adjust the pH on 3.1 using glacial acetic acid and contains chiral selector CM-β-CD (2.3g/l) /methanol and acetonitril, with the volumetric ratio of (56.8/ 30.7/12.5) v/v, respectively,). The final pH value of mobile phase was adjusted on 4.4 using TEA. The mobile phase was filtered using 0.45µm disposable filter, and degassed by ultrasonic vibration prior to use. The measurements were carried out in an isocratic elution mode with a flow rate of 0.7mL/min, an injection volume of 20𝜇L, UV detection at 250nm and column temperature was controlled at 40°. The column should be conditioned with mobile phase for an hour prior to injection. Samples were dissolved in methanol.


2.4. Chromatographic conditions in chiral stationary phase method:

Chromatographic separation was conducted in the normal phase operating mode, with a mobile phase consisting of acetonitril, isopropanol and diethylamine (DEA) with the volumetric ratio of (95/5/0.1) v/v/v, respectively. The mobile phase was filtered using 0.45 µm disposable filters, and degassed by ultrasonic vibration prior to use. The measurements were carried out in an isocratic elution mode with a flow rate of 1 mL/min, an injection volume of 20𝜇L, at room temperature, and UV detection at 240nm. Samples were dissolved in methanol.


3.    RESULTS:

3.1. Methods development:

3.1.2. Enantioseparation of Carvedilol by chiral mobile phase additive:

Figure 2 shows the enantioseparation chromatogram of Carvedilol under optimal conditions, with retention time of 21.8 min, 29.9 min respectively. Effect of mobile phase composition on enantioseparation:

The composition of mobile phase is very important due to relatively long retention time (tR) of Carvedilol enantiomers, when using aqueous solution and methanol alone, so it was necessary to add acetonitril to give an acceptable retention time and good resolution of enantiomers as shown in table 1.


Table 1 effect of mobile phase composition on resolution

Rs (resolution)

Composition of mobile phase

(Aqueous solution: methanol: Acetonitrile )

No peak till 200 min


tR=107 min








(58:31.5:10.5) Effect of chiral selector concentration on resolution:

The effect of CM-𝛽-CD concentration on enantioseparation was investigated under the pH of 4.4, and the results are summarized in table 2.


Table 2 Effect of CM -𝛽-CD concentration on resolution

Rs (resolution)

Concentration of CM -𝛽-CD

(g/l )







3 Effect of mobile phase pH on resolution

The pH value of mobile phase is one of the most important factors that affects on the resolution of enantioseparation, as shown in table 3, the pH value was chosen to be 4.4 for the subsequent work


Table 3 effect of pH on resolution

Rs (resolution)

pH of mobile phase

Oval: R1.6





3.5 Effect of column temperature on resolution

The effect of column temperature on the resolution of carvedilol enantioseparation was studied in a range of 30°C to 45°C and the column temperature was set on 40 °C (as shown in table 4) to make balance between column pressure, retention time of enantiomers and resolution.


Table 4 effect of temperature on resolution

Rs (resolution)

Temperature (°C)









3.1.3. Enantioseparation of Carvedilol using chiral stationary phase:

Figure 3 shows the enantioseparation chromatogram of Carvedilol on chiralcel OD-R column under optimal conditions, with retention time of 13.97 min, 15.53 min respectively. Effect of mobile phase composition on enantioseparation:

The results showed that with the reduction of the proportion of isopropanol, the enantiomers retention time was extended, however the resolution was affected slightly as shown in Table 5, and DEA was added to improve peak shape.


Table 5 Effect of mobile phase composition on resolution

Rs (resolution)

Composition of mobile phase

(acetonitrile: isopropanol: DEA)






(90:10:0.1) Effect of flow rate on enantioseparation:

At 1mL/min, baseline separation can be obtained and the shapes were fine, so the rate of 1mL/min was taken (Table 6).


Table 6 Effect of flow rate on resolution

Rs (resolution)

Flow rate(ml/min)






0.9 Effect of column temperature on enantioseparation:

The results showed that the increasing of temperature caused not significantly change in the enantiomeric separation. As well as when temperature is 25°C, the best separation enantiomers was obtained. So the temperature 25°C was taken (Table 7).





Table 7 Effect of temperature on resolution

Rs (resolution)

Temperature (°)









New enantioseparation method of carvedilol was introduced using CM- β-CD as chiral additive to the mobile phase , Chiral resolution results from stability constant differences between the inclusion complexes formed by the chiral selector with each enantiomer [12,13], interactions between CD and analyte, might be caused by the formation of hydrogen bonds, electrostatic. or van der Waals interactions which could be affected by the conditions of mobile phase [14].


The optimal conditions of mobile phase to achieve a good resolution were studied considering the solubility of the chiral selector, the column pressure and the retention time of the enantiomers.


CM-β-CD is soluble mainly in water and in methanol better than acetonitrile [15], which would provide a higher concentration of CMβCD for inclusion complex formation, but it was necessary to add acetonitrile in percentage of 12.5% to reduce the retention time and to increase the resolution. When the mobile phase consisted of aqueous solution and methanol alone, the retention time exceed 100 minutes and the separation did not take place.


The effect of mobile phase pH was explored in the range of 3.5-5, at pH of 4.4 the resolution reached its maximum, the experiments showed that adding buffers like phosphatic buffer at pH 4.4 increased both retention time and column pressure, without any enhancement in results.


Column temperature is also an important parameter for enantioseparation. The effect of temperature on the enantioseparation was investigated in the range of 30-45°, at temperature 40° the best resolution was achieved due to higher solubility of both carvedilol and chiral sector.


On the other hand, a second method was used to separate carvedilol enantiomers by using chiral stationary phase. Among the chiral stationary phases, the polysaccharide ones are the most effective and versatile [7] so chiralcel OD-R column was chosen as chiral stationary phase which contains cellulose tris 3,5 dimethylphenyl carbamate as chiral selector, while the composition of the mobile phase and choosing the suitable proportions of acetonitrile and isopropanol had the biggest effect on retention time but minor effect on resolution. good results were also obtained on in combination with EDA as additive. column temperature had small effect on resolution, and 25°C was selected as the optimum temperature.



New chiral separation of carvedilol enantiomers was successfully developed on an achiral C18 column with CM-β-CD as a mobile phase additive, other method was also developed utilizing polysaccharide column as chiral stationary phase.


The chiral stationary phase additive method is simple as it does not need any derivatization steps and uses very common solvents in RP-HPLC, moreover, it overcomes the necessity of using expensive chiral columns and could be applied in all laboratories, and it showed good resolution and very acceptable results comparing to chiral stationary phase method. Chiral stationary phase method introduced better peaks shape and shorter retention time.



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Received on 10.10.2018         Modified on 18.12.2019

Accepted on 12.01.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(4): 1679-1683.

DOI: 10.5958/0974-360X.2020.00304.2