Post-Column Derivatisation - Fluorescence Detectors

Modern column LC has been developed to a very high level by the introduction of selective stationary phases of small particle sizes, resulting in efficient columns with large plate numbers per metre. The development of HPLC equipment has been built upon the achievements in column technology, but the weakest part is still the detection system. UV/vis and fluorescence detectors offer tremendous possibilities, but because of their specificity it is possible to detect components only at very low concentrations when using a specific chromophore or fluorophore. 

With on-line post-column detection the complexity of the chromatographic equipment increases. An additional pump is required for the pulseless and constant delivery of the reagent. 

Type of detector Supplier Detection Part No. HPLC Instrument Part No. 

UV - visible (continued...) 190-600 nm Hewlett Packard wavelength detector CE 1200 Series 1000 

---

Often it is required to detect compounds with no or only very weak chromophores such as sugars and amino acids. Refractive index detectors and mass sensitive detectors can be used but they are relatively insensitive in the context of biological sample concentrations. Indirect detection using a UV or fluorescent eluent can also be employed. However, the most common approach is the use of derivatisation. Derivatisation of some chemically reactive moiety on the analyte can be performed in two modes. In post-column derivatisation the sample is separated first and then reacted with a flowing stream of derivatising reagent being pumped into... 

Several different types of detectors have been used for the quantitation of the biogenic amines however, currently the most popular techniques are electrochemical detection and fluorescent detection, usually coupled with pre- or post-column derivatisation techniques. The specific details of these detection techniques are discussed elsewhere (Chapter 3) and in the present section only the basic principles as applied to the biogenic amines will be reviewed together with a comparison between the two techniques in terms of their respective advantages and disadvantages. 

Through the choice of stationary phase and eluent composition, the selectivity can be modulated, but the eluent must meet the requirements of the detection system. Although the conductivity detector is still the most popular, other types of detection can be applied for different analytes. These include electrochemical (amperometric, pulsed and integrated amperometric, potentiometric), photometric (UV-Vis, indirect photometric following post column derivatisation, chemiluminescence, refractive index), and fluorescence. 

Details concerning the types of detectors used in HPLC are given in Table 3.2. The most commonly used detectors are those based on spectrophotometry in the region 185-400 nm, visible UV spectroscopy in the region 185-900 nm, post-column derivatisation with fluorescence detection (see Section 3.2.1), conductivity and those based on multiple wavelength UV detectors using a diode array system detector (see Section 3.2.2). Other types of detectors available are those based on electrochemical principles, refractive index, differential viscosity and mass detection. 

As mentioned above, the most commonly used method for the analysis of cationic surfactants has been HPLC coupled with conductometric, UV, or fluorescence detectors, the latter typically utilizing post-column ion-pair formation for enhanced sensitivity. Analysis by GC is only possible for cationic compounds after a derivatisation step [33] because of the ionic character of this compound. However, structural information might be lost. 

---