The Pharmaceutics and Compounding Laboratory
Extraction of Drugs

Laboratory Procedure

I. STANDARD CURVE

A 1.50 × 10-2 M stock solution of salicylic acid is supplied. From the stock solution, prepare the following standards by serial dilution with water. Put the solutions in liquid scintillation vials and label the vials.

Standard (M) Serial Dilution Scheme Absorbance
7.50 x 10-4    
3.75 x 10-4    
7.50 x 10-5    
3.75 x 10-5    
7.50 x 10-6    
3.75 x 10-6    

Determine the UV absorbance of the standards at 300 nm using UV grade cuvettes. Use water as a blank. Construct a Beer's Law plot of absorbance vs. concentration. Determine the slope, intercept, and R2 value. Remember, [0,0] is a valid point.

Video View a video demonstration on how to use a spectrophotometer


II. PARTITION COEFFICIENT DETERMINATION

Make three series of scintillation vials that each contain 0.20 ml of the 1.50 × 10-2 M salicylic acid stock solution and 5.0 ml of the different buffer solutions (approximate pHs will be 2.6, 3.0, 3.6, 4.0, 4.6). Write a series code and the pH of the buffer solution on the vial for identification; e.g., B - 2.6, B - 3.0, ... DM - 4.6.

To the first series, do nothing; these are the starting concentrations (see below). To the second series, add 5.2 ml of methyl-tert-butyl ether, and to the third series, add 5.2 ml of dichloromethane. Cap the vials immediately after adding the solvent.

CAUTION:
When adding methyl-tert-butyl ether and dichloromethane, wear gloves and work in the fume hoods. Also, at the end of the experiment, dispose of these two solvents in the waste containers in the fume hoods.
DO NOT POUR THEM DOWN THE SINK!

Gently shake the vials for 10 minutes; then let the vials stand for an additional 10 minutes. Transfer part of the aqueous phase to UV grade cuvettes using transfer pipets. The aqueous phase in the methyl-tert-butyl ether series will be the bottom layer, but it will be the top layer in the dichloromethane series. PLEASE WATCH THIS DIFFERENCE!

NOTE: Have air expelled from pipet bulb before putting transfer pipet in the vial.

Read the absorbance of the samples on the spectrophotometer at 300 nm. When you analyze the aqueous phase in each vial, you are quantitating [HA]aq + [A-]aq since both species of salicylic acid absorb at 300 nm. Use water for the blank. Record the absorbance values in the table below.

Series Aq. phase Absorbance Starting Conc. [HA]aq + [A-]aq [HA]org PC1 1/PC1 [H3O1] 1/[H3O+]
Buffer - 2.6
Buffer - 3.0
Buffer - 3.6
Buffer - 4.0
Buffer - 4.6
    xxxxx
xxxxx
xxxxx
xxxxx
xxxxx
xxxxx
xxxxx
xxxxx
xxxxx
xxxxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
xxx
BE - 2.6
BE - 3.0
BE - 3.6
BE - 4.0
BE - 4.6
  xxxxx
xxxxx
xxxxx
xxxxx
xxxxx
           
DM - 2.6
DM - 3.0
DM - 3.6
DM - 4.0
DM - 4.6
  xxxxx
xxxxx
xxxxx
xxxxx
xxxxx
           

NOTES:

1. The "Starting Concentration" is calculated using Beer's Law and the buffer series data.

2. The total [HA]aq + [A-]aq concentration is calculated using Beer's Law and the absorbances from the methyl-tert-butyl ether and dichloromethane series' aqueous phases.

3. [HA]org is the difference between the buffer series' Starting Concentration and the total [HA]aq + [A-]aq concentration for each corresponding pH value.

4. PC' is then calculated as the quotient of [HA]org and [HA]aq + [A-]aq.

Prepare a plot of PC' versus pH for each extracting solvent. Explain why PC' is related to pH in the manner shown in your plots.


III. pKa DETERMINATION

The data you have collected can be used to determine the pKa of salicylic acid. The mathematical derivation for the method has been given in the laboratory introduction section. Prepare a plot of 1/PC' versus 1/[H3O+] for each extracting solvent. The intercept mathematically is 1/PC, and the intercept value is used to determine the Ka from the slope which is mathematically Ka/PC. Determine the intercept, slope, and R2 value for each extracting solvent. Calculate the resulting Ka for salicylic acid.

Video View a video demonstration on how to use a pH meter