Thermo Scientific
| Reagents, Solvents and Accessories
29
Thermo Scientific
Derivatization and Visualization Reagents for HPLC
Thermo Scientific Derivatization
Reagents for HPLC
Designed to provide selectivity and improve sensitivity.
The lack of a universal HPLC detector that provides high
sensitivity (as well as some degree of selectivity)
established the need for suitable derivatization procedures.
Derivatization is the chemical modification of an existing
compound, producing a new compound that has properties
more suitable for a specific analytical procedure. It is an
analytical tool that can be used to provide both selectivity
and improved sensitivity.
There are several requirements for a derivatization protocol:
1. At least one acidic, polar functional group must be
available for reaction on the parent compound.
2. A single derivative should be formed per parent
compound.
3. The reaction should be reproducible under the given time
and reaction conditions.
4. The reaction should proceed quickly and easily under
mild conditions.
5. The reaction byproducts (if any) should not interfere with
the chromatography, or with detection of the sample.
Pre- and post-chromatographic techniques are both used in
HPLC derivatization. In addition, off-line and on-line
reactions have been employed with both techniques.
Pre-chromatographic (or pre-column techniques) offer
more than greater selectivity and sensitivity in detection.
Pre-column techniques can be used to enhance stability,
improve resolution, improve peak symmetry and increase
or decrease retention of solutes. FDAA (Marfey’s Reagent)
allows separation and quantification of optical isomers of
amino acids (Figure 2). Post-chromatographic
(or post-column) techniques are used primarily to provide
selectivity and improve sensitivity.
We offer a variety of HPLC detection reagents for
pre- and post-chromatographic techniques. All compounds
and formulations are purified for chromatography,
minimizing artifact formation.
5
10
15 20
Minutes
L-DOPA
D-DOPA
Marfey-OH
Figure 1. Separation of D- and L-DOPA on 100 mm x 4.6 mm C18.
Conditions: A) 0.05 M triethylamine phosphate, pH 3.0;
B) acetonitrile. Linear gradient: 10 to 40% B in 45 minutes, 2.0 ml/minute,
25˚C, 340 nm.
