Sample introduction

The chromatographic process begins with the injection of the sample at the injector end of the column before the chromatography system is equilibrated with the mobile phase. Separation of components takes place as the analytes and mobile phase are pumped through the column. As the mobile phase can be adjusted to the changing polarity of the mixture in gradient elution mode, a much larger number of compounds can be sep- arated in a mixture. Additionally, in gradient elution mode, the chroma- tographic peaks become sharper in a much shorter time. An isocratic flow system is also chosen to minimize baseline drift. Also, isocratic elution is more simple, precise, accurate and stable than a gradient system, and thus is more suitable for quality control and routine analysis (Sun and Su, 2000). Components eluting from the column appear as peaks on the data station.

5.1.5 Detector

The choice of an appropriate detector is crucial because of the diversity in the physico-chemical properties of natural products. The refractive index detector is the most simple and least expensive detector. This de- tector has been useful for detecting compounds such as carbohydrates and polymers, but it lacks sensitivity and is very susceptible to changes in ambient temperature, pressure and flow rate. Furthermore, it cannot

be used for gradient elution. Because of these limitations, the refractive index detector has been replaced by other detectors.

The UV detector is the most simple and most widely used among all HPLC detectors. The relationship between the intensity of light trans- mitted through the detector cell and solute concentration is given by Beer’s law. The magnitude of the extinction coefficient of an analyte at a given wavelength controls the sensitivity of the detection. This detector cannot be used for natural products lacking chromophores. Also, mobile- phase constituents having high UV cut-off wavelenths should be avoided because they might prevent the detection of analytes with weak chromo- phores (Ozkan, 2007). The photodiode array detector is a multiple wave- length UV detector and is also more versatile than the fixed wavelength detector. The UV spectra of the constituent can be monitored even during the separation. For those compounds possessing a weak chromophore, an unspecific wavelength close to 200 nm is used for detection (Ganzera

et al., 2004).

Fluorescence detectors afford greater sensitivity. Here, the molecular ab- sorption of photons triggers the emission of another photon with a longer wavelength. This difference in wavelengths, that is, absorption versus emis- sion, provides more selectivity. Fluorescent light is measured against a very low light background. Compared with UV, fluorescence detectors have scarcely been used for the detection of natural products. Most of the applications are related to very sensitive detection of aflatoxins in food because this class of natural product contains natural fluorescence (Jaimez et al., 2000). The de- tection of natural products that do not fluoresce naturally has been achieved successfully after the addition of fluorescent tags (Kristl et al., 2005). Similar to fluorescence, chemiluminescence can be defined as the emission of light from a molecule or atom in an electronically excited state produced by a chemical reaction at an ordinary temperature without any associated gen- eration of heat. Chemiluminescent nitrogen detection is a relatively new technology for the detection of nitrogen-containing molecules, including a broad range of pharmaceuticals with very high sensitivity up to the femto- gram range (Li et al., 2003). As many compounds are not chemiluminescent, they can only be detected by chemiluminescence after being derivatized (Ohba et al., 2002). The analysis of flavonoids (limit of detection 3 ng ml–1₎

in phytopharmaceuticals containing Hippophae rhamnoides has been re- ported. The procedure was based on the chemiluminescent enhancement of the flavonols by a cerium (IV) rhodamine 6G system in a sulfuric acid medium (Zhang and Cui, 2005). Natural products with electrochemical ac- tivity are common. Natural products with electroactive groups are readily measurable and detectable by liquid chromatography with electrochemical detection. This technique can be applied to a large number of analytes in either the oxidation or reduction mode. Functional groups such as phenols, aromatics, amines, thiols, quinolones, etc., are sensitive to oxidation, while functionals groups such as olefins, esters, ketones, aldehydes, ethers, quinones, etc., are compatible with reduction. Cells of an electrochemical detector consist of three electrodes, the working, counter and reference

electrodes. Although two designs exist for these electrodes, they can be aligned in several different geometries. The eluent is directed through the electrode in coulometric systems, while the eluent passes over the electrode in amperometric systems. Electrochemical detection is usually performed with maintaining the potential of the working electrode at a fixed value rela- tive to the potential of the working electrode at a fixed value, which is meas- ured by the reference electrode. The electrochemical reaction is driven by the fixed potential difference applied between the working and the reference electrodes. The current produced is measured as a function of the elution time, allowing detection at the picomolar level. The electrochemical detector differs from other detectors because it alters the sample; however, it is less se- lective than the fluorescent detector. This method of detection is inexpensive, sensitive and widely accepted. The accessible potential range, the number of compounds that are active in this range and half-width of the individual sig- nals determine the selectivity of the electrochemical detector (Ozkan, 2007). Multi-step potential time wave forms involving pulse techniques are also available for electrochemical detection. Here, uniform and reproducible elec- trode activity is maintained, realizing amperometric or coulometric detection (LaCourse and Modi, 2005). Proper use of electrochemical detectors requires knowledge of redox reactions and their dependence on mobile-phase com- position. Study of the electrochemical reactions mechanism of natural prod- ucts may supplement the establishment of their interaction with living cells (Guo et al., 1997).

The evaporative light scattering detector (ELSD) was introduced in 1966 (Ford and Kennard, 1966). Any analyte that is less volatile than the mobile phase, regardless of the optical, electrochemical or other analyte properties, can be detected using ELSD. The eluent is nebulized using a flow of nitrogen, and the resulting aerosol is transported through a heated drift tube. Volatile components and solvents are evaporated in the drift tube. The solid fraction remaining is subsequently introduced into a de- tection cell. A light beam is directed on to the particles, which causes scat- tering of the incident light detected by a photodiode or photomultiplier. Nebulizer gas flow and drift tube temperatures are the most important parameters affecting the ELSD signal response. Gas flow rate influences the droplet size of the column effluent before evaporation occurs. Higher flow rate results in the formation of smaller aerosol droplets and less scat- tering of light, causing lower sensitivity but allowing a stable baseline. Drift tube temperature facilitates the evaporation of the nebulized aerosol so that the light scattering response of the non-volatile solute can be de- termined exclusively. In comparison to the semi-volatile mobile phase, a higher temperature is needed for non-volatile analytes, as well as a mobile phase with high aqueous content. The new generation ELSD detector is able to vaporize eluent at a low temperature, thereby facilitating the detection of semi-volatile analytes (Megoulas and Koupparis, 2005; Guillarme et al., 2008). In contrast to the UV detector, which is concentration dependent, the ELSD is mass dependent. Therfore, theoretically, ELSD generates a similar response for equal amounts of mass present, and thus a universal

response factor. The ELSD response also depends on the volatility of the compound and the mobile-phase composition in the case of gradient elution, which makes such quantification procedures experimentally inaccessible. Thus, the interplay of several factors leads to a non-linear re- sponse. This disadvantage renders linear regression for calibration curves inaccurate (Vervoort et al., 2008). ELSD has been widely used as an alter- native to the UV detector for analytes lacking chromophores or with weak chromophores (Wolfender, 2009). ELSD techniques are frequently used in combination with UV detection.

Dixon and Peterson (2002) introduced a charged aerosol detector as a new technology for the universal HPLC detector. The first step of the charged aerosol detector is similar to ELSD, and the dried particle stream is charged with a corona discharge needle. The current generated is meas- ured by an electrometer (Vervoort et al., 2008).

The flame ionization detector (FID), which is a general detector of gas chromatography, has been applied to HPLC (Guillarme et al., 2008). HPLC-FID coupling implies working with 100% water as the mobile phase; however, water is a very weak mobile phase in reverse-phase li- quid chromatography at room temperature. For selected compounds with appropriate stationary phases, separations have been achieved with pure water at ambient temperature (Foster and Synovec, 1996).