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CHE425L HPLC
DETERMINATION OF CAFFEINE IN BEVERAGESBY HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY
READING
Skoog D. A., Holler F. J., and Crouch S. R., Principles of Instrumental Analysis, 6
th
 edition, Harcourt Brace College Publishers, 2007. Chapters 26 and 28.
A. INTRODUCTION
Liquid chromatography (LC) refers to chromatography in which the mobile phaseis a liquid. The four basic types of LC are partition chromatography; adsorption orliquid-solid chromatography; ion exchange chromatography and size exclusion/ gelchromatography. They differ in the exact nature of the stationary phase, thus themechanism/ processes insuring differential retention of analytes. Early LC utilized longglass columns with wide diameters (1 to 5 cm), and solid support particles of diameter inthe 150- to 250-
µ
m range to insure reasonable flow rates (still less than 1mL per minute).Under these conditions, separations could take up to several hours.High-performance liquid chromatography (HPLC) was developed to increasespeed and efficiency in liquid chromatography. Decreasing the size of the solid supportmaterial increased efficiency; i.e. decreases the height of theoretical plates. In the vanDeemter equation [1], covered in the GC module,
uC C u B ACuu B A H 
 M S
)(
+++=++=
 the
C 
coefficient which relates the linear velocity of the mobile phase to mass transferbetween phases, can be expressed as a sum of two coefficients
S
C 
and
 M 
C 
, related tothe stationary and mobile phase respectively. The
 M 
C 
coefficient is directlyproportional to the square of the diameter of the particles, leading to the conclusion that adecrease in the size of the particles of the stationary phase supporting material will resultin the decrease of the theoretical plate height (
 H 
). However, use of smaller sizeparticles (3-10
µ
m) requires high pumping pressures (several thousands psi) for achievingseparation within reasonable time periods.Whereas in gas chromatography the mobile phase does not interact with theanalytes and serves only to transport analytes through the column, in liquidchromatography, the mobile phase interacts with the analyte, thus plays a very importantrole 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 canbe exploited to achieve separation of various complex mixtures by liquidchromatography.1
 
CHE425L HPLCIn bonded phase liquid chromatography, the stationary phase is composed of arelatively low molecular weight solvent-like molecule covalently bound to a solid supportparticle that is packed in the column. The solid support in modern high performancecolumns typically consists of 3 to 10 µm-diameter porous silica gel particles (5 µm in thisexperiment). In reverse phase bonded phase (RPBP) chromatography, the bonded phaseis non-polar and the mobile solvent phase is polar. And, in normal phase bonded phase(NPBP) chromatography, the bonded phase is polar, and the mobile solvent phase is non-polar. The column in this experiment is packed with an octadecyl (C
18
) reverse-phasebonded stationary phase.In chromatography, a component injected onto the column will be distributedbetween the mobile, M, and the stationary, S, phases according to its affinity for bothphases. Thus, in reverse phase HPLC, a polar molecule that interacts more strongly withthe M phase will elute quickly from the column. In contrast, a non-polar molecule wouldinteract more strongly with the S phase and so would elute more slowly from the column.The retention time of a component on the column is related to the capacity factor, , fora column, which is in turn related to the distribution coefficient, , of the analyte ibetween the S and M phases [1].'
i
k 
i
K 
 
[ ][ ]
ii
 M si
K 
=
 
 M si M isii
V W K nnk 
==
,,' where
s
W 
= weight of S phase in column (g)
 M 
V 
= volume of M phase in column (L)= concentration of i in s, mol/g= concentration of i in M, mol/L
[ ]
s
i
[ ]
 M 
i
Experimentally,'is determined from the retention time for component
i
, ,and from the void time, . The void time is usually obtained from the time required foreither the solvent peak, or an unretained component of the mixture, to elute from thecolumn.
i
k 
i R
t 
,0
t 
00,'
t t t k 
i Ri
−=
 2
 
CHE425L HPLC
Use of solvent mixtures
Ideally, in a separation, the capacity factors (k’) for all components should liebetween 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 bychanging the polarity of the mobile phase. Tables of solvent polarity or elution strengthhave been prepared [2, 3]. However, the exact order of elution strength will depend onthe 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 reversephase HPLC the most common solvent mixtures are H
2
O and methanol (CH
3
OH) or H
2
Oand acetonitrile (CH
3
CN).
Use of solvent gradient elution
Unfortunately, in complex mixtures k' can vary from zero to, more than twentyfor a single solvent strength. This leads to the "general elution problem" where no oneset of conditions is effective in removing all components from a column in a reasonabletime period, while still attaining resolution of each component. Since solvent polarity hasa strong effect on k' in reverse-phase chromatography, it is convenient to use a binarysolvent mixture (
e.g.
H
2
O and CH
3
OH) consisting of two solvents of differing polarity,and change the percentage of each in the mixture during the elution of the sample. Thisis known as
gradient elution chromatography
. In gradient elution, the solventcomposition changes over time as shown in Figure 1.
Conc.of Ain A&BTimeFigure 1.
Solvent gradient of solvent A with solvent B.3
of 00

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