Experiment 4:

1
Experiment 4

• TLC and HPLC of ANALGESICS

2
Objectives

• To learn the separation techniques of Thin Layer
  Chromatography and HPLC chromatography.
• To use these techniques to separate and identify
  active ingredients in an unknown analgesic
  sample.
• To identify the unknown analgesic based on active
  ingredients present.

3
ANALGESICS
4
THIN LAYER CHROMATOGRAPHY

• Sample and standards are applied on origin line
  of POLAR TLC plate.
• TLC Plate is placed in a developing chamber
  containing a nonpolar, organic solvent.
• The substance dissolves in the solvent, and is
  carried up the plate. The polarity of the
  substance determines how far up the plate the
  substance travels.
• Once developed, the spots are visualized using a
  UV lamp.
• The spots are marked with a pencil, and the
  distances traveled by the spots are measured.
• The distance the unknowns spot has traveled is
  called the Rf value, and compared to Rf values of
  standard solutions, can be used to identify
  compounds.

5
THIN LAYER CHROMATOGRAPHYCalculation of Rf values
The Rf value is defined as the distance the
center of the spot moved divided by the distance
the solvent front moved (both measured from the
origin)
6
THIN LAYER CHROMATOGRAPHY Rf values

• Rf values can be used to aid in the
  identification of a substance by comparison to
  standards.
• The Rf value is not a physical constant, and
  comparison should be made only between spots on
  the same sheet, run at the same time.
• Two substances that have the same Rf value may
  be identical those with different Rf values are
  not identical.

7
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(TLC vs.
Normal Phase HPLC)
Note A high TLC Rf value a low HPLC retention
time!
8
HPLC CHROMATOGRAMS OF ANALGESICS
9
HPLC OF ANALGESICS
Unknown X
By comparison of sample retention times to
standard retention times, the active ingredients
can be identified.
10
COMPOSITION OF ANALGESIC TABLETS
11
FOR MORE INFORMATION...

• Please refer to Appendices E and F in the back of
  your laboratory manual for further explanation of
  theory behind chromatography.

12
SAFETY CONCERNS

• All solvents used in todays experiment are
  flammable, eye, and skin irritants. Be sure to
  wash your hands before leaving the laboratory.
• Safety goggles are required!

13
WASTE MANAGEMENT

• Place waste solvent from TLC sample preparation
  and TLC developing chambers into container
  labeled, Organic Waste (TLC).
• Place all used TLC capillary tubes in the
  broken glass container.
• TLC chambers should be left with the lids removed
  in the lab drawer. Do not clean with soap,
  water, OR acetone!