CHEM 231 Lab

Technique Primer

7. High-performance Liquid Chromatography (HPLC)

While there are many analytical instruments found
in the typical chemical laboratory, perhaps the most
common and most flexible is the HPLC. Based on the
principle of chromatographic separation, HPLCs are
used to determine the composition of complex
mixtures (such as plant extracts), to establish the
provenance of artifacts (such as evidence at a crime
scene), to gauge the extent of a reaction, or to verify
The sample is introduced into the HPLC system by
way of an injector. When the injector is turned to
the “load” position (counterclockwise), a syringe is
used to fill up (or load) a sample loop of a precise
volume. When the injector is then turned to the
“inject” position, the loop is placed in line with the
column (Figure 3).
20 uL loop

the purity of a product.

The typical HPLC consists of four key components
(Figure 1). Solvent is pumped at high pressure pump

through a column packed with derivatized silica gel. solvent

Upstream of the column is an injector to allow for

column
introduction of a sample. Downstream of the
column is a detector (typically one that measures
UV-Vis absorbance).
injection
port

injector column
solvent pump
detector

Figure 1. Basic components of the HPLC system waste

20 uL loop

As individual components exit the column, they

create an absorbance signal in the detector,
resulting in a peak on the HPLC chromatogram pump

(Figure 2). The y-axis of the chromatogram is a solvent

measure of the intensity of absorbance (in units of

column
mAU, or milli-Absorbance Units). The x-axis is in
units of time (typically minutes), and is used to
determine the retention time (tR) for each peak. For
example, compound 2 has a peak absorbance of
about 45 mAU and a retention time of about 3.9 injection
port

min, whereas compound 5 has a peak absorbance of

about 12 mAU and a retention time of about 7.6
min.
waste

Figure 3. HPLC Injector plumbing in the “load” (top) and

“inject” (bottom) positions

When making up a sample for the HPLC, a good

target concentration is about 1 mg/mL. Usually,
acetonitrile is a good solvent to use for making up
samples. If your HPLC trace shows more intense
absorbances than 1500 mAU, then the sample is too
concentrated & should be diluted. Keep in mind that
HPLC is performed in “reverse phase” mode, so that
the stationary phase retains non-polar compounds
Figure 2. An HPLC chromatogram (or “trace”) of a multi-
component mixture
more strongly than polar ones. Thus, non-polar
compounds tend to have longer retention times.