PHS 3303 – BIOMEDICAL LABORATORY III

SPRING 2019 LAB GUIDE

LAB2
PARTITION COEFFICIENT

 Felicia Carvalho, B. Pharm, M.Sc. Ph.D.

Pharmaceutical Sciences Department
St Johns University, NY

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PHS 3303 – BIOMEDICAL III LAB 2 - Partition Coefficient
Lipophilicity ( as known as hydrophobicity) of a drug greatly affects its distribution in biological tissues; The cell
membrane consists two lipid layers intercepted with some hydrophilic pores. Only small water soluble molecules can permeate
through these hydrophilic pores. The cell membrane is lipophilic (hydrophobic) hence drugs have to be sufficiently
hydrophobic to cross the membrane. Most drugs are transferred across the membrane by passive diffusion. The rate of
diffusion is dependent on several factors; molecular size, solubility in surrounding aqueous phase, concentration of the drug,
permeability and tissue blood flow. The greater the concentration of the drug at the absorption site, the greater is it’s
absorption. Hence the drug must have sufficient aqueous solubility. Drug permeability on the other hand increases with it’s
hydrophobicity as the drug must diffuse through the lipophilic cell membrane. For gastrointestinal and topical preparations,
the drug must overcome the biological membrane barrier to reach blood circulation. Hence a good drug candidate must have
both hydrophilic and hydrophobic properties for absorption and distribution in the biological tissue.
The relative solubility of a substance an organic solvent (water-immiscible)and in water is a measure of its lipophilicity.
The term “partition coefficient” is used to express the ability of a drug to distribute itself between two immiscible (lipid and
aqueous) solvents. The partition coefficient (abbreviated as P or Kpc) is given as the ratio of concentration of the drug in the
non-polar lipid phase vs the polar aqueous phase.
Kpc = concentration of drug in lipid phase
concentration of drug in aqueous phase
The partition coefficient relates to the neutral (non-ionized or zwitterion)species of the drug. Similar to other
equilibrium constants, partition coefficients are constant at a constant temperature and measured at dilute concentrations.
When determining the drug partition coefficient, the drug must be in the same chemical form in both solvents and not react,
ionize or form dimers and complexes with each other.
The solvent system most suitable to test the in-vivo partitioning ability is n-octanol:water. n-Octanol is a non-polar
solvent that better mimics a bilayer lipid membrane because the water content of n-octanol is similar to that of the membrane.
However, the boiling point of n-octanol is high (198°C); therefore, in lab experiments, other non-polar solvents with low
boiling point such as dichloromethane (boiling point from 39°C to 40°C) are used. Dichloromethane is a colorless and volatile
organic solvent that is not miscible in water commonly used in extraction procedures. The density of the solvents must also
considered. The order of density of some solvents is n-octanol < ethyl acetate < water < dichloromethane. It is important to
remember that when 1-Octanol is used as the nonpolar solvent, the water layer will be the bottom layer.
Think: In a system of dichloromethane and water, which layer is the bottom layer?
For the pharmacokinetic processes of absorption, distribution, and excretion, the drug must be able to distribute
itself: 1) between aqueous extracellular fluids (blood, interstitial fluid, urine) and lipid membrane, and 2) between lipid
membrane and aqueous cytosol. In this sense, the drug goes through partitioning between two immiscible aqueous and lipid
solvents. In general, the drug must be sufficiently lipophilic to passively diffuse through the lipid cell membrane, but not so
lipophilic that it cannot diffuse into the cytosol or solubilize in the aqueous cell environment.
Partition coefficient is an important factor for predicting its pharmacological effects, designing, dosage forms, and
selecting excipients and routes of administration. The partition coefficient is useful to predict the behavior and fate of drugs in
the body. Highly polar (hydrophilic drugs) are poorly absorbed in the GI tract and skin and may need to be administered via
the IV route. Lipid soluble drugs may be better absorbed in GI tract when they are sufficiently soluble in the aqueous
environment. Permeabilty enhancers may be needed in topical dosage forms to improve absorption. During drug
development, the drug molecular structure is modified to include hydrophobic groups that improve lipophilicity. Very
hydrophobic drugs have high partition coefficient values and tend to partition (cross) favorably across the lipid blood brain
barrier to produce CNS effects.
Materials:
Dichloromethane Micropipettes 1000 l
Distilled water Micropipette tips for 1000 l
Conical vials fitted with a screw cap Macropipette aid
Semi-Micro UV cuvettes Kim Wipes
Large test tubes Spectrophotometer
Goggles 2- 5mL pipette
Cuvette racks Test tube rack
Ephedrine 5 mg/mL ( in water) Acetaminophen 200 g/ mL (in water)

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PERSONNEL SAFETY AND PROTECTIVE EQUIPMENT
Safety first! Must have proper attire
 Fully covered shoes, proper full length labcoat, no dangling hairstyles at all times
 Wear safety goggles, Gloves (some materials may not be compatible with organic solvents)
o Nitrile gloves are excellent general use & limited contact. Good for most solvents, oils, greases, and some
acids and bases
o Latex gloves are not suitable for organic solvents
 No eating, drinking, chewing gum or smoking.
Warning:
 Dichloromethane : The hoods at the table must be turned on during the lab activity
Good practices :
 Evaluate the safety of the chemicals and drugs used and ensure that you use the correct protective equipment
 Wash your hands thoroughly with soap or detergent before leaving the laboratory.
LAB ACTIVITY
Drug concentration and Extraction volumes
Drug Acetaminophen Ephedrine
Concentration 50 µg/mL 5 mg/mL
Drug solution 800uL 800 uL
Dichloromethane 800uL 800 uL

Method outline

1. Compare the molecular structure of acetaminophen and ephedrine base

o Predict their lipid/aqueous partition properties
2. Perform volume calculations for the standard curve concentrations
3. Create a record table for each standard dilution in each test tube and for absorbance observations
4. Write the method. May be updated/corrected once the activity is complete
5. Extract drug.
o Dichloromethane 0.8 mL is provided to you in a wheaton conical vials
o Add 0.8 mL of respective drug solution (see image above)
o Shake every 4 minutes for a total of 4 times (~16 mins)
o Allow to equilibrate (15mins)
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 6. Analyze concentration of aqueous phase after dilution ( 0.4 mL aqueous extract with 1.6 mL water = 2 mL)
7. Calculate concentration of drug in aqueous phase, dicloromethane and the partition coefficient
8. Compare the KPC of the two drugs. Conclude wether your predictions are valid.

PARTITION COEFFICIENT (Kpc) CALCULATION: (write units)

Concentration of ephedrine in the organic layer =
Concentration of ephedrine in the aqueous layer =
Concentration of acetaminophen in the organic layer =
Concentration of acetaminophen in the organic layer =

COMPARE PARTITION COEFFICIENT (Kpc) RESULTS

Group # Drug System Kpc DCM/W
Ephedrine base Dichloromethane /Water
Acetaminophen Dichloromethane /Water

IN-LAB ASSIGNMENT
1. What is density? What are its units? How does density play a role in a mixture of two (or more) liquids?
2. Define the two terms Polar and Nonpolar.
3. What makes a drug? i) polar ii) non polar
4. Will enantiomers and diastereoisomers differ in their partition coefficient. Justify your answer.
5. Can the polarity of a drug be modified. How?
6. Fill in the table below. From the list below, select the terms (A-J) associated with polar & non-polar and write the
term(s) in the corresponding boxes.
Polar Nonpolar

What is it?

What belongs here?

A B C D E
Hydrophobicity Hydrophilicity Lipophobicity Lipophilicity Acetanilide

F G H I J
Ephedrine Aqueous Solvent Organic Solvent Water Dichloromethane

POST-LAB ASSIGNMENT
 Submit Lab report. Guidelines are provided by your instructor
o Must use observations of both drugs for your analysis and discussion
 Read Lab 3 guide
 Come prepared for Lab 3 Quiz