A High pressure liquid chromatography will take a liquid or dissolved sample of a

mixture of substances, put it through a long pipe (column), separate the substances,
and spit them out one after another. The separation happens because of two phases
made of certain chemicals for the substances to "get stuck" to, a moving (mobile)
phase and an unmoving (stationary) phase. The moving phase is a liquid, that will carry
substances through the pipe. The pipe contains the stationary phase.
When HPLC analysis are start, the mixture of substances, gets pushed right into the
pipe by mobile phase. There, different substances keep going in and out of the
stationary phase. Some substances like to do this more and longer than others, so they
will remain longer in the pipe, while others don't want to get stuck to the stationary
phase, and prefer flowing with the mobile phase. In the end the mixture of substances
leave the pipe at different times. At the end of the pipe usually have a detector, that
can tell you that a substance just came out of the system, and depending on how big
the signal the detector gives is, how much of it came out.

The stationary phase is non polar or hydrophobic and the mobile phase is polar and usually
consists of a mix of solvents, such as water and acetonitrile. The mobile phase is pumped at
high pressure through the system. The non polar part of the compound injected will interact with
the stationary phase which slows it down as it moves through the system. So, if a mixture of
compounds is injected, each one will interact differently with the stationary phase and the mobile
phase, thus facilitating separation. After the sample passes through the column, it is detected by
ultraviolet absorption. For many compounds this is usually at 254nm. The time taken for a
sample to pass through the system is recorded as its retention time and is one of the
characteristics used to identify a compound. Retention times can vary from a few minutes to an
hour. The area under a peak is used for calculating the concentration of a sample.

Figure 1 shows what happens to a sample containing a mixture of compounds after injection
into the column. The compounds bind to the column and are flushed out at different times,
depending on whether they are more likely to stick to the column or the mobile phase as it is
pumped through. The time that each compound elutes (or flushes out) from the column is known
as that compounds retention time (Rf).

Figure 1: Compounds of differing polarities (indicated as darkening shades of blue) are injected
into the HPLC column (entire cylinder). The mobile phase is pumped through the column, and
the addition of solvent along a concentration gradient (shown as a black dotted line)
continuously decreases the overall polarity of the mobile phase (Y-axis). Compounds are able to
stick to either the column or the mobile phase, depending on how polar they are. Compounds
will eventually stick to the mobile phase when their polarity matches that of the mobile phase.
They will then dissociate from the column and will be eluted at a particular time (X-axis) during
the run. This time is known as the Rf for that compound.

Used to separate solids or liquids dissolved in liquids which cannot be studied by gas
chromatography. HPLC requires a pump, an injector, a column filled with some active substance
that effects the separation and a detector that tells when a substance exits the column. One
method of separation would involve a column packing that is polar so that a solution of solutes
of differing polarity would have the solutes held on the column packing for differing periods of
time, depending on the polarity of the solute. Thus less polar solutes would be eluted first and
very polar solutes would be eluted last.

HPLC is a separation technique that involves: the injection of a small volume of liquid sample
into a tube packed with tiny particles (3 to 5 micron (m) in diameter called the stationary phase)
where individual components of the sample are moved down the packed tube (column) with a
liquid (mobile phase) forced through the column by high pressure delivered by a pump. These
components are separated from one another by the column packing. These separated components
are detected at the exit of this tube (column) by a flow-through device (detector) that measures
their amount. An output from this detector is called a liquid chromatogram.

The HPLC system gives out two types of data images. One is called a chromatogram, and the
other is called a spectrum. The chromatogram is a graph that monitors the signal in the detector
over time. As chemicals are detected by the instrument, the signal increases, and the
chromatogram displays a "peak." Each peak in the chromatogram indicates the presence of a
chemical in the sample. The chromatogram from last week is shown in Figure 1 below. Each
peak is labeled with retention time. Retention time indicates how long it takes for a compound to

come out of the HPLC column. Notice there are 2 peaks. The peak labeled 9.994 minutes is
actually methylnaphthalene and the peak at 13.468 minutes is phenanthrene.

Mobile phases

Mobile phase is solvent which carries the analyte of liquid or solid. For chromatography to work
effectively, we need the components of the mobile phase to separate out as much as possible as
they move past the stationary phase. Mobile phase is an important parameter in reversed-phase
HPLC. Type of mobile phase used may have a big effect on the retention. It can promote an
ionization of the analyte molecules. The mobile phase in reversed-phase chromatography has to
be polar and it also has to provide a reasonable competition for the adsorption sites for the
analyte molecules.If the preparation method is not the same as the method described in the
material for the analysis method, differences can occur in the mobile phase that affect the
chromatograms and analysis results.

One reference/standard solution were prepared and injected into the HPLC. One vial were
injected sixx times to get average peak area value which wil be used to determine the assay
value in the sample.
. Shake the five caffeine solutions to insure adequate mixing.
Pipette 5 mL of coffee into a CLEAN and DRY 50 mL volumetric flask and then dilute to the
mark with HPLC grade water. Be sure to filter the sample into a clean vial. Rinse the filter before

collecting the sample by filtering the first 1-2 mLs to waste. Record three chromatograms for
each coffee sample.