1.3.2 High performance liquid chromatography

The technical advances in column and detector technology that have increased GC's ability to routinely monitor trace level pollutants, have parallels in high pressure liquid chromatography (HPLC). Beginning its development in the mid-sixties, the technique basically suffered from a lack of reliable and easy to use column packings. Since the advent of bonded phase however, packed columns are providing perfectly reproducible results in routine analysis and instrument development, particularly of the detectors, has been impressive. The search for increased laboratory productivity has led to the development of fully automated systems, making HPLC an ideal instrument for the analysis of an extensive range of thermolabile compounds not suited to GC analysis. A large variety of modern agro-chemicals including methyl carbamates and glyphosate are relatively polar, non-volatile compounds far better suited to separation by liquid chromato-graphy. The advent of larger non-volatile molecules in the domain of environ­mental pollutants, together with liquid chromatography's unrivaled prospects for on-line sample preparation and automation will undoubtedly move this technique to a much higher echelon in the near future.

1.3.2.1 Hplc columns

The most commonly used analytical HPLC columns in environmental analysis are 25cm long x 4.6mm i.d., packed with 5 to 10(im spherical ODS particles. In recent years, columns of reduced length and i.d., packed with smaller particles have led to reductions in solvent usage, increases in detectability, speed of analysis, efficiency and ease of connection to spectroscopic detectors. For these reasons, columns with i.ds of 2.1mm are protected with a guard column cartridge to ensure long column life, enhanced reproducibility and repeatability.

1.3.2.2 Hplc detectors

UV detector/diode-array detector

In spite of being the most universal detector in HPLC, the refractive index detector's low sensitivity and incompatibility with gradients make it less popular than UV detectors. The first UV detectors for HPLC were adaptations of UV instruments originally designed for standard, large volume (2 ml) sample cells, giving relatively poor sensitivity. Detector technology has improved dramatically since those early days, some single wavelength detectors now offering 2 to 3 orders of magnitude more sensitivity than their antecedents. High sensitivity UV spectra became a reality with the introduction of the diode-array UV-Vis detector, in which an 'array' of photodiodes (over 200) simultaneously monitor the UV-Vis spectrum, thereby providing UV-Vis spectra to be collected with scanning rates that allow highly sensitive and undistorted spectra to be taken continuously from fast eluting peaks. The data from this simultaneous-wavelength UV detector are processed and evaluated with software that features spectral library search, specified wavelength signal extraction for enhanced selectivity, baseline correction and much more. The software can be set up to automatically match UV spectra against known standards, identifying peaks and testing peak purity by comparing the spectra collected at the start and end of each peak.

This automatic evaluation is an ideal screening technique that allows analysts to investigate only the suspect cases. Specifically designed for small volume HPLC columns, UV-Vis diode-array detectors use flow cells selected for their specific chromatographic qualities to provide full UV-Vis spectra at sensitivities which, not so long ago, were difficult to achieve even with fixed wavelength detectors. This considerable improvement in detection limits has directly influenced the quality of results by permitting analysts to question whether the compound being quantified really is the compound of interest. Compared to single wavelength detection, which provides no information about peak purity, the diode-array's full spectra comparison capabilities provide results with a far greater confidence level. In conjunction with solid phase extraction, relatively high sample enrichment factors can be achieved, improving sensitivity to levels where pesticides for example, can be detected at less than 0.05 ug/1 in drinking water.

Fluorescence detector

The fluorescence detector (FLD) is based on the measurement of emitted rather than absorbed light. Certain molecules re-emit part of the absorbed light (usually UV), in the form of lower energy, higher wavelength light, which can in turn be measured and used to determine the compound's concentration. Despite the fluorescence detector's relatively small application range, its great popu­larity is due to its very high selectivity and sensitivity and to the fact that many environmental pollutants such as polycyclic aromatic hydrocarbons are fluorescent. The detector's excitation and emission wavelengths may be selected independently and the fluorescence signal itself can be measured very sensitively compared to UV-Vis, where the signal consists of the difference between full light intensity and the slightly attenuated intensity caused when an analyte peak passes through the cell. In FLD, apart from some limited scatter and stray light, no signal is generated until a fluorescent compound reaches the detector. By automatically adjusting excitation and emission wavelengths as a function of time and the compound to be detected, optimal conditions can be set for each individual compound during an HPLC separation. The on-line combination DAD/FLD is extremely powerful for the elucidation and sensitive detection of PAHs in water samples. The extreme sensitivity of fluorescence detection has encouraged the development of reliable and automatic pre- and post-column reactors to convert non-fluorescent compounds into fluorescent derivatives, allowing their detection by FLD.

Electrochemical detector

Technological advances have also occurred in the field of electrochemical detection (ECD). The contamination problem originally associated with electrochemical detectors has been largely solved by the introduction of automatic, self-cleaning electrodes, making the computer-controlled ECD a reliable component of HPLC systems. Compounds which are readily oxidized or reduced, for example phenols, mercaptans, amines, aromatic nitro and halogenated compounds, aldehydes, ketones and especially benzidines lend themselves to electrochemical detection. By carefully choosing the potential applied to the electrodes, both the selectivity and the detection limits may be enhanced.

Liquid chromatography/mass spectroscopy coupling

Some years ago, mass spectrometry revolutionized analytical chemistry, becoming one of the most essential and powerful tools of the environmental laboratory. While a mass spectrometer can be coupled directly to a gas chromatograph, the coupling to a liquid chromatograph requires an LC/MS interface. The best known interfaces for environmental analysis are the particle beam interface and the thermospray interface. With particle beam, either pure electron impact or chemical ionization spectra may be generated to identify unknown pollutants. The thermospray interface, while not recommended for structure elucidation, is the preferred technique for the quantitative analysis of target compounds in the ion monitoring mode.