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READING
Skoog D. A., Holler F. J., and Crouch S. R., Principles of Instrumental Analysis, 6th
edition, Harcourt Brace College Publishers, 2007. Chapters 26 and 28.
A. INTRODUCTION
B B
H = A+ + Cu = A + + (C S + C M )u
u u
the C coefficient which relates the linear velocity of the mobile phase to mass transfer
between phases, can be expressed as a sum of two coefficients C S and C M , related to
the stationary and mobile phase respectively. The C M coefficient is directly
proportional to the square of the diameter of the particles, leading to the conclusion that a
decrease in the size of the particles of the stationary phase supporting material will result
in the decrease of the theoretical plate height ( H ). However, use of smaller size
particles (3-10 µm) requires high pumping pressures (several thousands psi) for achieving
separation within reasonable time periods.
Whereas in gas chromatography the mobile phase does not interact with the
analytes and serves only to transport analytes through the column, in liquid
chromatography, the mobile phase interacts with the analyte, thus plays a very important
role in affecting separation parameters such as retention times and resolution. The role of
the mobile phase on separation adds versatility, flexibility and the range of forces that can
be exploited to achieve separation of various complex mixtures by liquid
chromatography.
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Ki =
[i]s
[i]M
ni , s Ws
k i' = = Ki
ni , M VM
where
[i ]s = concentration of i in s, mol/g
[i ]M = concentration of i in M, mol/L
'
Experimentally, k i is determined from the retention time for component i, t R,i ,
and from the void time, t 0 . The void time is usually obtained from the time required for
either the solvent peak, or an unretained component of the mixture, to elute from the
column.
t R,i − t 0
k i' =
t0
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Ideally, in a separation, the capacity factors (k’) for all components should lie
between 2 and 5 to effect good baseline resolution of the peaks in a limited period of
time. For a given stationary phase the k' of a particular component can be controlled by
changing the polarity of the mobile phase. Tables of solvent polarity or elution strength
have been prepared [2, 3]. However, the exact order of elution strength will depend on
the stationary phase and the components examined. Fine control of elution strength of
the mobile phase is obtained by using binary and ternary mixtures of solvents. In reverse
phase HPLC the most common solvent mixtures are H2O and methanol (CH3OH) or H2O
and acetonitrile (CH3CN).
Unfortunately, in complex mixtures k' can vary from zero to, more than twenty
for a single solvent strength. This leads to the "general elution problem" where no one
set of conditions is effective in removing all components from a column in a reasonable
time period, while still attaining resolution of each component. Since solvent polarity has
a strong effect on k' in reverse-phase chromatography, it is convenient to use a binary
solvent mixture (e.g. H2O and CH3OH) consisting of two solvents of differing polarity,
and change the percentage of each in the mixture during the elution of the sample. This
is known as gradient elution chromatography. In gradient elution, the solvent
composition changes over time as shown in Figure 1.
Conc.
of A
in A&B
Time
Figure 1. Solvent gradient of solvent A with solvent B.
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Thus, the mobile phase polarity, or elution strength, varies during the chromatogram.
Note that various gradient shapes can be applied. Gradient elution will not be used this
semester, however an example of its use is discussed below.
Gradient elution is customarily used to separate components of a homologous
series. A typical experiment in Instrumental Analysis laboratory involves separation of a
homologous series of para-hydroxybenzoate esters. A mixtures containing the methyl-,
ethyl-, propyl-, butyl…nonyl hydroxybenzoates is separable within reasonable time by
applying gradient elution. Use of gradient elution allows good resolution of the lower
molecular weight components, while eluting the octyl and nonyl esters in a reasonable
period of time.
The solubilization of a solute in a solvent involves intermolecular interactions of
the solute (i) and solvent (j) with energy Eij [4, 5]. For an organic molecule consisting of
several different functional groups, e.g. CH3-(CH)n-Y, it is possible to break the overall
interaction energy up into the sum of individual interaction energies,
E ij = E CH , j + nE CH , j + E y , j
3 2
Martin first showed [6] that the log of the distribution coefficient, K i , for a solute
between two phases j and l is approximately proportional to the sum of the differences of
the individual interaction energies as illustrated below for CH 3 − (CH 2 ) n − Y .
( ) ( ) (
log K iα E CH , j − E CH ,l + n E CH , j − E CH ,l + EY , j − EY ,l
3 3 2 2
)
'
The Martin equation thus predicts that if E CH 2, j > E CH 2,l , log K i (and log k i )
will increase as the number, n, of a certain functional group in a homologous series
increases. In reverse phase chromatography, the E CH 2, S > E CH 2, M since the mobile
phase is polar and the octadecyl phase is not. Thus the hydroxybenzoate methyl ester will
be less retained than the hydroxy-benzoate nonyl ester.
B. GOALS OF EXPERIMENT
In this experiment, high performance reversed phase liquid chromatography is used
to:
1) Separate the various substances contained in four common beverages from caffeine.
2) Determine the concentration of caffeine in these beverages.
Separation will be conducted under ISOCRATIC conditions (i.e. elution at constant
solvent composition). This experiment illustrates the power of HPLC in analyzing
components in complex mixtures. Traditional method for the determination of caffeine is
via extraction followed by spectrophotometric quantitation. HPLC allows for rapid
separation and quantitation of caffeine from the many other substances found in these
beverages including tannic acid, caffeic acid and sucrose.
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Column
The column is a commercially packed 5-µm octadecyl (C18) bonded phase column
with a length of 25 cm.
Detector
The UV-50 Variable Wavelength detector is a double beam manual
spectrophotometer, equipped with a deuterium and a tungsten lamp, and a
monochromator, to provide wavelengths from 200nm to 720 nm. The optical path length
is 1 cm and the sample flow cell volume is 8-µL. The wavelength of measurement will
be 254 nm, the optimum wavelength for caffeine determination.
Injector
The chromatograph is fitted with an automatic VALCO sample injector
comprised of a sampling valve, an external 10-µL sample loop and a fill-port fitting
assembly. The injector is controlled by the microprocessor. A programmed event 4
injects the content of the sample loop onto the column, and a programmed event 0 returns
the valve to the load position.
4. Always use the instrument gradient program in order to gradually change to the
desired composition.
5. The instrument should be on for at least 30 minutes to equilibrate.
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D. EXPERIMENTAL PROCEDURES
2. Standards
Pipette 5-, 10-, 15- and 20-mL portions of the stock 10-ppm caffeine solution
provided into 25-mL volumetric flasks, and dilute to volume with the solvent mixture.
Pipette 1-mL of the 100 ppm solution of acetaminophen provided into 25-mL
volumetric flask, and dilute to volume with the 10-ppm solution of caffeine. The
stock solutions are already filtered so the standards do not need filtration.
3. Instant Coffee
Put ~150-mL of hot tap water into a 250-mL Erlenmeyer and set the flask on a hot
plate to boil (heat the indicated amount of water for the other sample at the same
time). Spoon out the amount of instant coffee appropriate for one cup, about one
rounded teaspoon (1.5 to 2.5 g), onto a tarred weighing paper and record the mass of
coffee to the nearest 10 mg. Do not use more than 2.5 g. Once the water has boiled,
stir in the coffee and set aside to cool. When the coffee has cooled, transfer it
quantitatively to a 200-mL volumetric flask and make up to the mark with water.
4. Tea
Bring to boil about 400-mL of hot tap water in a 500-mL flask. Meanwhile, weigh
one tea bag and one emptied tea bag. Once the water has boiled, prepare the tea as
you normally would, recording the time you steep the tea to the nearest 0.5-minute.
Once brewed, remove the tea bag. When the tea has cooled, transfer quantitatively to
a 500-mL volumetric flask and dilute to the mark with tap water.
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2. While pumping, use the COARSE ZERO Knob on the detector to move the pen to the
zero position on the recorder, with the attenuator set at 8. This sets the zero
absorbance reading of the detector using our mobile phase as the blank.
4. Flush and fill the sample loop with one of your standard solutions.
5. To practice filling the sample loop and injecting use the following program
TIME 0.0 EVNT 0
TIME 1.0 EVNT 4
TIME 1.2 EVNT 0
6. In order to optimize reproducibility; use one continuous fill of the sample loop.
Therefore, use the 1000-µL syringe so as to have sufficient sample in the syringe for
refilling the sample loop between injections; and use the following program to make
multiple injections at set intervals without stopping the pump.
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E. QUESTIONS
1. Plot the peak areas and peak heights calibration curves for caffeine using units of ppm
for concentration. Use the triangulation method to obtain peak areas (peak height
times peak width at one-half peak height).
2. Use linear regression analysis to obtain the best straight line through the experimental
points [1,7].
4. From the calibration curves report the concentration of caffeine in the injected
beverage samples, and in the original samples in ppm. Evaluate the overall standard
deviation for each of your results.
6. Calculate the capacity factor of caffeine using the retention time of acetaminophen as
t0 .
7. Explain the rationale for using a reverse-phase C18 column for the determination of
caffeine.
8. Derive the relationship between retention time and capacity factor from the following
relations:
v = L / t 0 = average linear velocity of mobile phase
vi = L / t R,i = φ i,M × v = average linear velocity of i in mobile phase
ni , M
φ i,M =
ni , s + ni , M
F. REFERENCES
1. Skoog D. A., Holler F. J., and Crouch S. R., Principles of Instrumental Analysis, 6th
Ed., Harcourt Brace & Company, Orlando, Florida, 2007, Chap. 26 and Chap.28
2. Snyder L. R., Kirkland J. J., Introduction to Modern Liquid Chromatography, John
Wiley, Toronto, 1979, Pp. 218-225.
3. Karger B. L., Snyder L. R., Horvath C., An Introduction to Separation Science, John
Wiley, Toronto, 1973, Pp. 271-274.
4. Ibid. Pp. 55-57, 275-276.
5. Giddings J. C., Unified Separations Science, John Wiley & Sons Inc., New York,
1991, Pp. 24-30.
6. A.J.P. Martin A. J. P., Biochem. Soc. Symp. 3, 4 (1949).
7. Skoog D. A., West D. M., Holler, Analytical Chemistry: An Introduction, 5th Ed.,
Saunders College, Philadelphia, 1990, Chapter 4, Sec. 4B-3 to 22-6.
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