Simultaneous Determination of Caffeine and Phenolic Compounds in Tea and Coffee - Caffeine: Chemistry, Analysis, Function and Effects - page 143

using different conditions. Without doubt, caffeine is the main focus, but other
alkaloids and different phenolic classes can be simultaneously analyzed.
In general, flow-rates used in the methods are in the 1.0-1.2 mL min 21
range, but flow rates as high as 2.2 mL min 21 and as low as 0.2 mL min 21 have
been reported. Flow-rate is limited by the system backpressure, which is
influenced by the mobile and stationary phases. For example, due to its higher
viscosity, methanol produces a higher column backpressure than acetonitrile,
which limits flow-rates (Figure 8.2). As can be seen, the correct choice of
mobile and stationary phases and temperature can expand the range of
workable flow-rate, which can be explored to reduce analysis time.
Conventional RP-C18 columns of 5 mm particles are the most used
stationary phases for the separation of alkaloids and phenolics. Usually,
relatively long columns are necessary to separate a greater number of
compounds. Higher performance can be achieved with columns of smaller
particles (1.7-3.5 mm), but systems that can withstand higher pressures are
required (ultra-high performance liquid chromatography - UHPLC). It was
recently reported a UHPLC method for the determination of different
phenolics and caffeine in tea and tea extracts (Nov´ kov´ et al 2010). The
separation of 30 compounds was achieved in less than 19 min. In a similar way,
the separation of a large number of phenolics and alkaloids in teas was
reported using a similar column (Zhao et al 2011). Chromatograms of both
methods are shown in Figures 8.3 and 8.4. However, in both cases very low
flow-rates were used due to the high backpressure generated by the small
particle column.

d n 0 t 2 n g | 0

Figure 8.2

System pressure using different columns (PC: conventional particle
column, FC: fused-core column, MC: monolithic column) at different
temperatures, flow rates, and mobile phase compositions. Illustrates the
effect of the column used and how, depending on its characteristics,
higher flow-rates can be used to reduce analysis time. Reproduced from
Manchon et al 2011 with permission from Springer.