EXPERIMENT VIII
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
SEPARATION AND ANALYSIS OF MIXTURES
These files are in Adobe Acrobat format, if you are using Netscape Navigator or Internet
Explorer and have Adobe Acrobat Reader installed (If you do not; Acrobat Reader can be
downloaded for free from Adobe) these files should open directly in your browser.
INTRODUCTION
Increasingly, the determination of low concentrations of active ingredients (either desired
or undesired) in complex mixtures, sold for human consumption, has become more necessary.
Federal regulations have imposed strict limits on the type and concentrations of a host of
substances sold as foods or drugs. Such requirements demand analytical techniques that are fast
and reliable and combine the separation (to alleviate interferences) and analysis steps in a single
operation.
Chromatography is the most widely used technique for the analysis of non-inorganic
mixtures. Gas chromatography (where the sample must be volatilized) and liquid
chromatography (where the sample can be determined in the liquid state) are the most common
methods in general use. High Performance Liquid Chromatography (HPLC) is the method of
choice whenever the sample cannot easily be converted to the gas phase.
In this laboratory, you will use the technique of HPLC to determine either the
concentration of caffeine in a soft drink, coffee or tea, or caffeine in one of a variety of analgesic
(pain relief) formulations.
THEORY
Figure I depicts the main components of a modern HPLC system and their
interrelationships. In HPLC, a solution containing the compound(s) of interest is injected into a
loop which has been calibrated to contain a specified volume (a 20 L loop injector is a
commonly used size). The valve switch is then rotated, allowing a sample stream of mobile
phase (the eluent) to sweep the sample from the loop onto a column, packed with a suitable
stationary phase, where the separation occurs. The eluent is delivered from a pump at a constant
rate, (on the order of 1 mL/min) at a pressure sufficiently high to overcome the backpressure of
the column. Pressures of 1000-2000 psi are commonly necessary. An upper limit of 4000 psi is
normally set on the instrument. Recall that the separation efficiency is inversely proportional to
the particle size of the column packing material. High pressures are required to force a liquid
through a tightly-packed column filled with small particle material, and the availability of high
pressure solvent delivery systems is directly responsible for the "high performance".
Assuming that a suitable column has been chosen for the separation of interest, all
components should pass through the column and "elute" at different times (differential
migration). This time differential is due to the differences in the distribution (partitioning) of the
various components between the mobile phase (eluent) and stationary phase (column packing),
which arise from the physical/chemical differences among the components of the mixture. Thus,
each component will pass through the detector and be identified separately.
The time for a substance to pass through the column, termed the retention time, is
therefore, related to the identity of the compound and is the basis for qualitative analysis.
Quantitative information is obtained from the area or height of the peak produced by the
detector.
Figure I : Diagram of a Typical HPLC
Several different approaches to HPLC detection exist. Perhaps most common is a
detector based on the absorbance of ultraviolet or visible light, a UV/Vis absorbance detector.
These detectors are, in reality, miniature UV/Vis spectrophotometers, equipped with a flowthrough cell, allowing continuous monitoring of the eluent. The wavelength selected corresponds
to the region of the electromagnetic spectrum where the compound(s) of interest and/or their
associated chromophores absorb light. In the linear dynamic range of the calibration curve the
absorbance is proportional to the concentration of the compound of interest. The data is recorded
and presented using a chromatographic integrator. The absorbance measured by the detector
produces a peak with a characteristic retention time; each peak has an area which is listed as
well. Measurement of a series of standards, along with the unknown, allows the use of the
"standard series" method, which produce the calibration curve to determine the concentration of
the unknown compound.
PART I
DETERMINATION OF CAFFEINE IN AN ANALGESIC TABLET
PROCEDURE
Preparation of Eluent
If the eluent solution is not already prepared, make approximately 250 mL of an
aqueous 1% (v/v) acetic acid solution using HPLC-grade water. Add approximately 160
mL of HPLC-grade methanol to this solution; the resultant 60/40 (v/v) solution can be
used as the eluent and transferred to the appropriate solvent reservoir. Sparge the mobile
phase liquid with helium prior to use.
Preparation of Standard Solutions
Weigh approximately, but accurately 250 mg of Caffeine, transfer quantitatively
to a 250-mL volumetric flask and dilute to the mark with HPLC grade methanol to make
an approximate but accurately known 1000 ppm. solution. Dilute 50 mL of this solution
to 500 mL. The concentration of this solution should be approximately 100 ppm and
should be known to three significant figures. This is the stock solution. From the stock
solution prepare by successive dilution six solutions, each in a 100-mL volumetric flask,
of concentrations 10, 20, 40, 50, 60, and 80 ppm respectively, using HPLC grade
methanol as the dilutant. Your concentrations do not have to be exactly these values but
they must be accurately known.
Preparation of Sample
Accurately weigh (+0.1 mg) one tablet of your over-the-counter analgesic.
Dissolve this tablet in HPLC-grade methanol in a 100-mL volumetric flask and dilute to
the mark with methanol. This is the unknown solution.
Preparation of the Calibration Curve
Following the instructors' guidelines, fill the sample loop with the most
concentrated standard solution, inject and record the chromatogram, taking special note
of the retention time and peak area of the caffeine peak. Repeat this procedure with each
of the other standard solutions as well as the blank (pure methanol).
Determination of the Caffeine in the Analgesic Preparation
Obtain a chromatogram of the unknown solution using the same procedure as was
used for the preparation of the calibration curve.
CALCULATION OF RESULTS
You will submit the results of this experiment in the form of a laboratory report
(see p.p. 1-3 of these laboratory instructions). The data will fit a graph of standardized
peak area vs. concentration. If appropriate, you should perform a linear regression
analysis to determine the sensitivity of the method (slope) and the concentration. Be
certain to correct for dilution of the original sample and report the results as mg
caffeine/tablet.
PART II
DETERMINATION OF CAFFEINE IN A SOFT DRINK, COFFEE OR TEA
PROCEDURE
The eluent, standard solutions instrument settings and approach to preparing a
calibration curve are identical to that given in PART I of this experiment.
The unknown will consist of two different brands or types of soft drink, coffee or
tea. Ideally, one would be "regular" and the other "caffeine-free". The soft drink coffee
and tea may be analyzed directly (no dilution). It is, however, recommended that for the
soft drink it be allowed to stand in an open container for at least ½ hour to lose its
carbonation (CO2).
CALCULATION OF RESULTS
You will submit the results of this experiment in the form of a laboratory report
(see p.p. 1-3 of these laboratory instructions). The data will fit a graph of peak area vs.
concentration. If appropriate, you should perform a linear regression analysis to
determine the sensitivity of the method (slope) and the concentration. Report the results
of the analysis as mg caffeine/12-oz. serving of [Brand Name].