|
Introduction
Carbamates is a class of organic pesticides derived from and named
after Carbamic Acid (HOCONH2). This HPLC method utilizes
a complex post column reaction, the chemistry of which is discussed here.
Separation
Upon dilution in pH 3 ultrapure water, the eight unmodified carbamate
analytes are separated using a typical reverse phase HPLC system
including a Waters brand C18 column designed specifically for
carbamate separation as a non-polar stationary phase and
a water-methanol-acetonitrile gradient mobile phase. The mobile
phase gradient begins with an 88:12 water/methanol mixture
at 1.5 mL/min and steps to a 50:50 mixture of methanol and
acetonitrile. This allows acceptable resolution with
near-baseline separation.
Fluorescence
Detection, however, is slightly more challenging. While some
of the carbamate analytes have the necessary functional groups
to absorb in the UV range, several do not. Because of this, more
inclusive methods for carbamate detection have been devised
using a scanning fluorescence detector, the most common of
which is discussed here.1
Fluorescence is a compound's absorption at one wavelength of
light and subsequent radiation at another wavelength. Depending on
functionality, organic compounds can absorb and fluoresce at
wavelengths anywhere from the visible to the far UV regions. Unmodified
carbamates do not fluoresce, but can be made to fluoresce via a
post column derivatization reaction in which each carbamate is
reduced to its molar equivalent of methyl amine and reacted to
form a highly conjugated, and fluorescent, ring structure.
The Reaction
After separation and before detection the analytes
run through a post column derivatization as
pictured below. A column heater brought to 30 C, just
above the ambient room temperature, allows for consistent
retention time while a heater around the post column reaction
coil brought to a significantly warmer 80 C facilitates
the reactions.
Figure 1. The analytes separate in the column, mix and react with the post column reagent in the reaction coil and then move on to the detector.
Two reagents are used. The first, 0.05 N sodium hydroxide in water, hydrolyzes the carbamate pesticide into alcohol and a molar equivalent of methylamine.
Figure 2. Reaction scheme of reduction of the carbamate to methylamine.2
The second, 200 mg/L o-phthalaldahyde (OPA) and 50 ug/L mercaptoethanol in an aqueous 0.05 M sodium borate
buffer solution, react with the methylamine product to form a fluorescent derivative. This fluorescent
derivative, pictured below, is the final product in the post column reaction.
Figure 3. Reaction scheme of addition of OPA and mercaptoethanol to methylamine to form fluorescent derivative. 2
The post column reagents are delivered via reagent managers designed specifically for this and similar reactions. Because of the excess molar amounts of hydroxide, OPA and mercaptoethanol, each carbamate analyte is easily quantified according to detector response.
Additional attention should be paid to the reagent managers. They are often stand alone pumps and are subject to the same potential problems as the HPLC pumps, such as loss of prime. Variation in the post column reagent flow can cause significant variation between runs and between analytes. If one or both pumps losses prime entirely then the post column reaction cannot take place and the detector will show a clean and empty baseline.
Detection
Detection is achieved using a scanning fluorescence detector with a deuterium lamp. This allows for excitation and emission wavelengths to be set according to the specifications of a particular analyte. The derivative pictured above absorbs best at a wavelength of 339 nm and emits at a wavelength of 445 nm. Because each carbamate analyte is converted to this same product, each can be detected at those wavelengths. As a result, the final chromatogram shows excellent selectivity with equivalent response from each analyte.
Resources
1. Aaron, J.J., A. Coly. Luminescence methods in pesticide analysis. Analysis, 2000, 28, 699-709.
2. Waters. Lab Highlights: Post-column derivatization of carbamates I: Chromatography and Detection.
|
