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What and Why of Post-Column Reaction
Chromatography is a science of separations. High Performance Liquid
Chromatography (HPLC) like other forms of chromatography, is used
to separate complex mixtures into their components. There are many
flavors of HPLC, but what they have in common is that the separation
takes place in solution. Having separated a mixture, you need to
see the components. The most popular detectors use either
UV/VIS light absorption, or fluorescence. Unfortunately, many substances
are difficult to detect. Moreover, you want to see the components
of interest without distraction from the background.
Post-column derivatization, also known as post-column reaction,
renders visible certain compounds that are normally invisible. This
trick is accomplished after the separation by performing a chemical
reaction on the substances that gives them an easily-detectable
physical property. Typically you use a reaction that produces a
strong color or makes a fluorescent product. You can increase the
sensitivity of detection by several orders of magnitude in favorable
cases. Most reagents are selective for a particular class of substances,
so analytes of that class are more easily seen against a complex
background. So, post-column derivatization is used to increase sensitivity
and selectivity in HPLC analysis.
The post-column reaction system mixes the stream of eluant from
the HPLC column with a stream of reagent solution. The mixture usually
flows through a reactor to allow enough time for the chemical reactions
to complete. If the reaction is slow, the reactor may be heated
to speed things up. Some reactions need two or more reagents added
in sequence. Finally the mixed streams pass into the detector, typically
UV/VIS absorbance or fluorescence. Of course a practical system
requires metering pumps, pulse-dampeners, thermostats, and safety
systems to give reliable results.
Examples of the chemistry and hardware are given in the catalog
and user's manuals.
Chemical Requirements
The chemical requirements for post-column derivatization are generic.
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Stability of Reagent: The minimum reagent stability sufficient
for routine work is one day. This means that the yield and signal-to-noise
ratio for a given sample must remain constant for at least 8 hours.
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Completeness of Reaction: The analytical separation is complete
when the reagent is mixed with the column effluent. Therefore,
in order to minimize band spreading, it is important to keep the
volume small between the mixing tee and the detector. If the reaction
is slow (in excess of one minute), an elevated temperature can
be used to decrease the reaction time.
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Reproducibility: Unless the system consistently produces the
same signal for the same sample, quantitation is impossible. Because
the reaction is occurring ³on the fly² as the combined
column and reagent stream flows toward the detector, the reproducibility
is linked to the flow-rate precision of the pumps and to the temperature.
Accordingly, even an incomplete reaction will be as repeatable
as the retention time for any given species. The completeness
of the reaction, then, is not strictly necessary for reproducibility,
but it is important for maximum sensitivity.
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Minimal Detector Response of Reagents: The color or background
fluorescence of the reagent (or its by-products) represents a
continuous noise source. Because the reagent is present in excess
relative to the analyte, the analyte's signal could be obliterated
by the reagent's strong background signal. Pickering's Chromatographic
Grade® eluants and reagents are guaranteed to produce the
absolute minimum possible detector background signal in post-column
applications.
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Solubility: All species must remain in solution, including the
combined components of the eluants and the reagent(s), as well
as the newly formed derivative(s). Precipitates can block capillary
tubes, burst reactors and foul detector flowcells.
Ninhydrin chemistry provides a good example of multiple solubility
considerations. Ninhydrin reagents contain a lithium acetate buffer,
ninhydrin, hydrindantin, and a water-miscible organic solvent. The
organic solvent is necessary to maintain both the hydrindantin and
the new purple chromophore (derivative) in solution. Also the presence
of lithium ion in the formula precludes the use of eluants containing
phosphate, because lithium phosphate is insoluble and would precipitate
at the point of mixing.
Detector Considerations
Refractive Index Sensitivity
RI sensitivity applies only to UV-vis detectors. There are two
sources of RI noise in post-column applications:
In either case, when such inhomogeneities enter the flowcell, they
bend light into the wall or off the photomultiplier tube, causing
detector noise. The noise usually correlates with the piston cycles
of the pumps, thus limiting the detector to low-sensitivity applications.
Most flowcells in modern UV-vis detectors are designed to minimize
the effects of RI.
In order to minimize the temperature-related RI effects mentioned
above, some manufacturers have a capillary heat exchanger at the
flowcell entrance. In some instances this heat exchanger has an
internal diameter of 0.12 mm (0.005 inches), which can result in
post-column pressures in excess of 42 bar (600 psi). Since this
can exceed the pressure rating of a heated reactor made with fluorocarbon
tubing, this small-diameter heat-exchanger tube may need to be replaced
with a 0.25 mm (0.010 inch) i.d. tube.
Detector Pressure Ratings
When the eluant-reagent stream from the heated reactor reaches
the detector, it can release dissolved gas as it cools. The Pickering
Laboratories derivatization instruments place a back-pressure of
7 bar (100 psi) on the detector flowcell in order to prevent the
formation of bubbles.
The back-pressure regulator can be factory-adjusted to accommodate
flowcells with a lower back-pressure rating, depending upon the
reactor temperature. but a setting lower than 3.2 5 bar (70 psi)
is not recommended for reactor temperatures over 100° C.
Operating an HPLC system with a post-column derivatization system
can be as routine as regular LC. The benefits from this LC/post-column
combination include minimal sample pre-treatment, greatly improved
sensitivity, and enhanced selectivity for compounds that would normally
be much more difficult to detect.
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