To determine partition coefficient of Benzyl penicillin using reverse-phase thin-layer chromatography

 Experiment

Object:

To determine partition coefficient of Benzyl penicillin using reverse-phase thin-layer chromatography.

Requirements:

Chemicals required:

1. Microcrystalline cellulose - 100 g

2. n-Octanol - 20 ml

3. Acetone - 180 ml

4. Benzylpenicillin - 2 mg

5. Ammonia solution

6. Starch iodine solution

Apparatus required:

1. Chromatographic chamber

2. Glass plate 20 cm x 20 cm

3. Distillation unit

Theory: 

The classical shake-flask method offers many disadvantages, for instance, it is time

consuming and requires relatively large amounts of solutes and solvents, and applicable to

the limited compounds having log P range - 3 to 3. Hence, this method is not suitable for

very hydrophilic or hydrophobic compounds.

The reverse-phase TLC technique is simple and less laborious. In addition, it requires

less amount of the sample without having high purity. These features make RP-TLC as a

valuable method for estimation of the partition coefficient log P. The most popular

lipophilicity descriptor estimated by this method is Rm, which is derived from the retention

factor (Rt) according to Bate-Smith and Westal equation:

Rm = log (6 -1) = 10g (1 – R.) – log R

where Rp is calculated on the basis of migration distances of a compound and the

solvent front.

In 1965, Boyce and Milborrow proved that retention factor Rm is useful for determining

the lipophilicity of substances. Later on, Bird and Marshall established the linear relationship

for Penicillins between their Rm and log P value (1.13- 2.83) at pH 4 as follows:

Rm = 1.036 log P-2.628

Procedure:

1. The experiment is performed on 20 cm x 20 cm glass plates coated with a 0.5 mm

layer of microcrystalline cellulose (0.205 g/ml) with water. Plates were dried at room

temperature overnight, heated in an oven at 110°C for 1.5 hr, cooled on the bench

for 10 min.

2. Octanol: Acetone (1:9) mixture is used as mobile phase. Plates are then impregnated

with octanol for 1 hr in the chromatographic chamber.

3. Acetone is allowed to evaporate by keeping plates on the bench for 15 minutes.

4. A spot of Benzyl penicillin (2 mg/ml) is applied and subsequently, the plate is placed

in the buffer pH 4 to reach the same mark.

5. The wet plate is placed in a tank saturated with ammonia vapour for 15 minutes,

dried, sprayed with 10% acetic acid in acetone and then with starch iodine solution.

Benzyl penicillin appears as a white spot on a blue background.

6. Rf value is calculated to determine log P value for penicillin.

Calculations:

Bate-Smith and Westal equation for calculation of Rm value:

Rf value of Benzylpenicillin = 0.8644 => Rm value = log (1 - Rt) - log Rp = - 0.8045

Using Bird and Marshall equation:

log P =(2.628 + Rm)/1.036 = = 1.7601 1.036

Result:

Log P value of Benzylpenicillin using reverse-phase thin-layer chromatography is 1.7601.

Reference:

1. Bird AE, Marshall AC. Reversed-phase thin-layer chromatography and partition

coefficients of penicillins. Journal of Chromatography A 1971, 63, 313-319.

2.Dr. Abhishek Tiwari, Dr. Rajeev Kumar " A practical book of medicinal chemistry" Nirali Prakashan Pune 2019 pp 80-81