About Post-Column HPLC
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 tosee 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 increasesensitivity 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.
The chemical requirements for post-column derivatization are generic.
- 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.
- 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.
- 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.
- 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.
- 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.
Refractive Index Sensitivity
- RI sensitivity applies only to UV-vis detectors. There are two sources of RI noise in post-column applications:
- RI discontinuities due to imperfect mixing.
RI discontinuities due to temperature gradients in the eluant/reagent stream as it leaves a heated reactor.
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.
- suppress boiling in the reactor
- prevent outgassing in the detector flowcell.
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.