A New Simple Method for Determination of Partition Coefficient by Normal Phase Thin Layer Chromatography (TLC).

 

Akshada N. Kakade, Shrikant B. Deshmukh, Pankaj P. Chothe, Vishal V. Khot, Dr. Shrinivas K. Mohite, Dr. Chandrakant S. Magdum.

Department of Pharmaceutical Chemistry. Rajarambapu College of Pharmacy, Kasegaon, 415404, Tal: Walwa Dist: Sangli, MS.

*Corresponding Author E-mail: akshadakakade@yahoo.com, dshriraj@gmail.com

 

ABSTRACT:

Owing to the importance of partition coefficient in field of pharmaceutical education  and  research  in  the  present work, We  have  made  and  attempt  to determine  benzene/water  partition coefficient (logP) values  for  drugs  by simple normal  phase TLC  technique. The partition coefficient being  the  relative concentration of  drug  in benzene/water, remains  as  an  important  physiochemical parameter which determine  the absorption  of  drug  in  the body and thus influence it therapeutical response.

 

According to literature the determination of partition coefficient is done by shake-flask method, GC, HPLC and by  potentiometric  method. These methods are accurate but expensive.  Here there is need for simple and efficient method to determine logP value for desire drug compound or pharmaceutical research. This reasserted to come out with  simple  experiment technique like normal phase TLC to  determine  to calculate logP value. And which can be the part of under graduate laboratory practical.  The differential values like Rfb/w and logRfb/logRfw were calculated and found to be closer to reported log P values (i.e from chemical abstract ACD lab), Paracetamol-0.461, Naproxen-0.769, Atenolol-0.22, Hydrochlorthiazide-0.331, Telmesertan-5.11, Pthalic acid-0.85, Sulfathiazoline-0.1, Nimesulide-0.9, Diclofenac-0.2, Benzoic acid-0.9.  Considering the results we have concluded that simple and convenient TLC method was developed to determine logP values, which were matching with reported logP values of drugs bank.

 

KEYWORDS: Partition coefficient, Thin layer chromatography, logP, Relative concentration

 

 

INTRODUCTION:

The partition coefficient being the relative concentration of the drug in benzene and water, remains as an important physico-chemical parameter which determines the absorption of the drug in the body and thus influences its therapeutic response1. The hydrophobicity of a compound plays a major role in determining where drug are distributed within the body after their absorption and as a consequence, how rapidly they are metabolized and excreted2-4. Partition coefficient is also one of the important parameters in deriving quantitative structure activity relationships (QSAR). According to the literature the determination of partition coefficient is done by shake-flask method5, GCmethod6, revers phase RP HPTLC/HPLCmethod7 and by potentiometric method8. Among these existing methods, HPLC method is proven to be an most accurate method.

 

Today, all the Pharmaceutical industries and research institutions are following this method to determine the log P values for the drugs. Academic institutions are following shake-flask method as part of the undergraduate and postgraduate laboratory practical to determine the logP values for the drugs. For the shake-flask method, availability of appropriate simple procedures for the quantitative analysis of the separated benzene or water layers is a major disadvantage. Hence, there is a need for simple and efficient method to determine the logP values for the desired compounds or drugs in pharmaceutical researchas wellas in pharmaceutical education. This re-asserted us to come out with simple experimental techniques like normal phase TLC to calculate the logP value for the organic Drug.

 

 

Material AND Methods:

The benzene used in the experiment was analytical grade solvent and where as water was Distilled water. All the drug samples were procured from the store house of drug testing laboratory and some of the drugs from store of Rajarambapu College of Pharmacy, Kasegaon. Precoated aluminum plates (silica gel GF254, Merck) were used in the present analysis.

 

1] THIN LAYER CHROMATOGRAPHY:

A] Activation of plates:

Glass plates were cut into dimension of 10cm×1.5cm. These plates were kept for activation in hot air oven for about 2 hr at 60°c prior to the experiment.

 

 

B] Preparation of drug for spotting:

About 10mg of the drug was dissolved in an organic solvent in which the drug is soluble in a fusion tube.

 

C] Saturation of chamber:

The two TLC chamber of a dimension 15cm×7cm were used for the study and labeled One of the chamber water and other one as benzene. The chamber were lined with filter paper wetted with the developing solvent so as to maintain an atmosphere saturated with the solvent vapours, which prevents the edging effect. 10ml of water and benzene were transferred to the respective chamber with the petriplates. 

 

D] Spotting of drug:

2 to 5µl of the drug sample was spotted with the aid of capillary maintaining the spot sizeof about 2-3 mm. the spot was allowed to dry completely before taking for the development of chromatography

 

E] Development of chromatography:

The spotted the TLC plates were transferred to the respective saturated chambers. The solvent was allowed to rise for plate length of 8cm and then removed from the chromatographic chamber and allowed to dry. The development of chromatogram was done at a room temperature.

 

F] Detection:

Examination of the TLC plate under ultraviolet light or in iodine chamber did the detection of the compounds. The sensitivity of detection was thus related to the absorptivity of the drug under UV which in turn depends upon its structure.

 

G] Determination of logP:

The distance traveled by the solute and solvent was measured from which retention factor in water and benzene was calculated.(Table no 1) Different values like Rf b, Rf w, Rfb/w and logRfb/w were calculated and tabulated to find out which is the most appropriate one to consider in the determination of partition coefficient for the drugs (Table 1). Rfb/w values were found to be closer to the reported logP values when compared to the logRfb/ w values10.

 

H] Calculation of logP values by using computational tools:

Different algorithms can be used in the calculation of logP values for the drugs by the different softwares. The logP values for the same drugs were calculated by using the computational software from ACD Lab (advanced chemistry development)9

 

Result and discussion:  

Owing to the importance of determination of degree of hydrophobicity or partition coefficient for the drug in the Field of pharmaceutical education and research. In this present word attempt was made to develop a novel. Simple and convenient TLC method to determine the logP values. Since, the principle of thin layer chromatography is Based on two important parameters such as adsorption coefficient, solubility and partition coefficient hence we want to take advantage of the partition coefficient which also has direct influence over the relative migration of solute with respect  to nature of the solvent. Graph of reported logP values vs Rfb/w values obtained from developed TLC method is drawn above(Fig no 1). Most of the reported lop P values of the taken drug matches their result with Rf b/w values of performed TLC methods (Table 1). Considering the above results, we have an opinion that this simple TLC method can be further improved to get still better results. The reverse phase TLC can be tried to mimic the reverse phase HPLC/HPTLC technique for determining the logP values.

 

Table 1: Determination of various parameters like, Rfb/w, logRfb/w, reported logP  values of various drugs.

Drug no.

Drug

Benzene

Water

Rf b/w

logRf b/w

Reported logP11

d-solute

(cm)

d-solvent

(cm)

d-solute

(cm)

d-solvent

(cm)

1

Atenolol

6.3

8

5.4

8

1.164

0.065

1.256

2

Diclofenac

5.4

8

1.8

8

3.35

0.525

4.218

3

Hydrochlorthiazide

2.9

8

6.8

8

0.41

0.3872

0.1

4

Nimusulide

5.5

8

1.8

8

3.12

0.494

3.38

5

Paracetamol

2.5

8

6.2

8

0.4

0.3979

0.46

6

Telmiserton

6.1

8

1.2

8

5.06

0.704

7.8

7

Sulphathiazoline

5.8

8

6.9

8

0.83

0.08

0.9

8

Benzoic acid

6.3

8

4.1

8

1.529

0.184

1.9

9

Naproxen

4.5

8

1.7

8

2.66

0.424

3.31

10

Pthalic acid

5.1

8

6.3

8

0.82

0.0861

0.85

d- Distance traveled by the  solute  or solvent , Rf = Retention factor (d-Solute/d-Solvent), Rf b/w- Retention factor in  benzene water system,  logRf b/w-benzene water partition coefficient. LogP – partition coefficient.

 

Fig no 1 Reported logP values vs Rfb/w values obtained from developed TLC method

 

 

CONCLUSION:

In conclusion, we have developed an easy and convenient experimental method to determine the logP values for the drugs. This method can be used as a valuable alternative to students for determining logP values of drugs.

 

ACKNOWLEDGEMENT:

The authors are very much grateful to Hon. Principal Dr. Chandrakant S. Magdum. and Hon. Vice-Principal Dr. Shrinivas K. Mohite, of Rajarambapu College of Pharmacy, Kasegaon, for their valuable and continued moral support to bring out this research work.

 

REFERENCES:

1.        Dennis AS, Han Van DW, Don KW. Pharmacokinetics and metabolism in drug design. 1st ed. Wiley-VCH, Verlag:2001.

2.        Beckett AH, Stenlake JB. Text book of practical pharmaceutical chemistry. 4th ed. Part-II, CBS, New Delhi: 2005:115-127.

3.        Maria FM, Maria VR, Ariel FM, Horacio RC. Octanol-water partition coefficient of glucose, sucrose, and trehalose.  Carbohydrate Res 340(6); 2005:1207-1211.

4.        Hammers WE, Meurs GJ, De Ligny CL. Correlation between Liquid Chromatographic Capacity Ratio. J Chromatogr 247;1982:1-13.

5.        Krisztina Takacs-Novak, Alex Avdeel. Interlaboratory study of log P determination by shake-flask and potentiometric methods. J Pharm Biomed Anal 14;1996:1405-1413.

6.        Reynaldo Cesar Castells. Determination of gas– liquid partition coefficients by gas chromatography. J Chromatog A 2004;1037:223.

7.        Roman Kaliszan, Piotr Haber, Tomasz B Czek, Danuta Siluk, Klara Valko. Lipophilicity and pKa estimates from gradient high-performance liquid chromatography. J Chromatog A 965; 2002:117.

8.        Claire Barzanti, Rebecca Evans, Jeremy Fouquet, Leonard Gouzin, Nicola M. Howarth. Potentiometric determination of   octanol–water and liposome–water partition coefficients (log P) of ionizable organic compounds. Tetrahedron Lett 48(19); 2007:3337-3341.

9.        www.acdlabs.com (Advanced chemistry labs.)

10.     Alfred Martin “Physical Pharmacy” 4th Edition ,lipponcot williams and willkines , comboden street Baltimo Maryland       21201-2436 USA : 237-242

11.     Reported  log P values available on www.drug bank .com

 

 

 

Received on 02.05.2012        Modified on 02.06.2012

Accepted on 09.06.2012        © AJRC All right reserved

Asian J. Research Chem. 5(7): July, 2012; Page 922-924