Chemistry Reference
In-Depth Information
Thin-layer chromatography (TLC)
In this technique, the
R
f
value of the drug is related mathematically to the
partition coefficient. A thin-layer plate, or a paper sheet, is pre-coated with
organic phase (usually paraffin or octanol) and allowed to dry. Sample is
applied to the origin and the plate is allowed to develop. The mobile phase
used is either water or a mixture of water and a miscible organic solvent
(such as acetone) to improve the solubility of the drug.
Once the plate has developed, the spots are visualised (using an ultra-
violet lamp if the drug possesses a chromophore, or iodine vapour if it does
not) and the
R
f
for each spot is determined. The
R
f
is the distance moved by
the spot divided by the distance moved by the solvent front, and is expressed
as a decimal. The
R
f
can be related to the partition coefficient by equations
of the type
k
P
——----
(1/
R
f
)
1
(2.5)
where
k
is a constant for the given system, which is determined by running a
number of standard compounds of known
P
in the system and calculating
k
.
The TLC method of determining
P
works best for compounds of
similar structure and physical properties. The advantages of using this tech-
nique to determine
P
are that many compounds can be run simultaneously
on one plate, and very little sample is required. On the other hand, finding
suitable standards can be difficult, and mobile phases containing a large
amount of aqueous solvent may take many hours to run up a large TLC
plate.
High-performance liquid chromatography (HPLC)
This method of analysis relies on the same chemical principles as the deter-
mination by TLC, except that the efficiency (and the cost) of the technique
has increased greatly. Instead of the
R
f
value, the retention time of the drug
is measured and related to
P
by equations similar to equation (2.5) for TLC.
The retention time, as its name suggests, is the time taken for the sample to
elute from the HPLC column. The major drawback with using this tech-
nique to determine
P
is detecting the drug if it does not possess a chro-
mophore, when a UV detector cannot be used. In cases like this, use must
be made of an HPLC system connected to a refractive index (RI) detector
or an electrochemical detector (ECD).
