1.1
Section 1
IntroductIon
Pinnacle Operators Manual
Pickering Laboratories Inc.
Section 1
intrOduCtiOn
1.1
1.1
1.2
1.4
1.6
What is Post-column derivatization?
Requirements for a Successful Post-column Method
Design of a HPLC system
Designing a Post-column system
Design of the Pinnacle PCX
What is Post-column Derivatization?
This is a method which renders visible certain compounds that are normally invisible. Since this reaction
occurs after, or post-separation, it is referred to as post-column deriviatization. The analytes of interest are
separated on the column first, and then reacted with a chemical that will render them detectable at a desirable
wavelength, voltage, or any number of various means of detection.
Post column derivatization enhances the sensitivity of HPLC by several means:
1) Most reagents are selective for a particular class of substances, so analytes of that class are more easily seen
against a complex background.
2) Since the separation is performed first, the matrix of the sample is either washed off of the column before
the analytes, or is retained by the column. This leaves a very pure sample of analyte to react. This eliminates
the need for extensive sample clean-up, and provides a very reproducible reaction because there are no matrix
interferences.
The Pinnacle PCX post-column derivatization instrument automatically mixes the stream of effluent flowing
from the HPLC column with a stream of reagent solution. The mixture flows through a reactor to allow enough
time for the chemical reactions to complete. In many cases, the reaction is very slow at room temperature. For
this reason, the reactor can be heated. There are some methods that require two or more reagents added in
sequence. This is done by the addition of a second reagent pump. In many cases, the second reaction occurs
at a much faster rate, and can be efficiently accomplished at room temperature. After the reaction is complete,
the derivatives flow into the detector, where the absorbance or the fluorescence (usually) is measured by the
HPLC system. These two means of detection are the most common, but they are certainly not the only means of
detection.
Requirements for a Successful Post-column Method
There are many things to take into consideration when developing a method and instrument for post-
column derivatization. For example, many pumps have a periodic motion when drawing and dispensing that
To Next Page
Loading...