The UV Absorption Detector

Differentiating equation (1) with respect to solute concentration  

AA is commonly employed to define the detector sensitivity where the value of AA is the change in absorbance that provides a signal-to-noise ratio of 2. 

Where Ac is the detector concentration sensitivity or the Minimum Detectable Concentration which is the parameter of importance to chromatographers. 

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The basic theory, principles, sensitivity, and application of fluorescence spectrometry (fluorometry) were discussed in Chapter 8. Like the UV absorption detector described above, the HPLC fluorescence detector is based on the design and application of its parent instrument, in this case the fluorometer. You should review Section 8.5 for more information about the fundamentals of the fluorescence technique. 

As with the UV absorption detector, the sample compartment consists of a special cell for measuring a flowing, rather than static, solution. The fluorescence detector thus individually measures the fluorescence intensities of the mixture components as they elute from the column (see Figure 13.10). The electronic signal generated at the phototube is recorded on the chromatogram. 

If appropriate cleanup is accomplished and if the analyte or analytes have sufficient absorption, UV-absorption detection is a highly reliable method of quantification. However, the UV-absorption detector... 

Concentration sensitive detectors provide an output that is directly related to the concentration of solute in the mobile phase passing through it. In GC the katherometer would be an example of this type of detector whereas in LC the UV absorption detector would be typical of a concentration sensitive detector. 

The cell volume was about 12 pi, which is too large for use with modern high efficiency LC microbore columns. Nevertheless, the detecting system is an interesting association as the combined performance of the UV absorption detector and the refractive index detector approaches that of the universal detector. 

Detectors. The requirements for detectors for preparative work are different from those for analytical operation, For analytical work a very high sensitivity and a small volume with a very small distribution are essential- In preparative work, the concentrations of samples are important and overloading of the detection signal often occurs and may cause problems. The same principles of detection are used in both preparative and analytical work. The two detectors that have the widest range of applications and are the most often used in preparative chromatography are the UV absorption detector, with adjustable wavelength (suppression of too intense signals), and the refractive index (RI) detector. 

More recently (1984), Baba and Housako (7) described another bifunctional detector but this time based on the UV absorption detector combined with the electrical conductivity detector. A diagram of their detector is shown in Figure 2. The UV absorption system is very similar to that of the DuPont bifunctional detector. UV light is collimated through the cell and focussed by a second quartz lens onto a photo diode, the output from which, is processed by suitable electronic circuitry in the usual manner. 

Electrochemical detection in HPLC has become established for some, more specialized, applications such as catecholamine analysis though it can be exploited for a far wider range of compounds. The work in this paper has attempted to investigate some of the basic properties of an electrochemical detection system and some more difficult applications. The detector has a linear dynamic range and precision that are comparable with those of other detectors for HPLC. It is, however, more dependent on temperature than, for example, the UV absorption detector and must be operated in a temperature controlled environment to obtain the lowest detection limits. For many electroactive compounds with moderate oxidation potentials, the electrochemical detector can yield sub-nanogram detection limits. 

This is because the UV absorption detectors are set in the aromatic region of the spectrum where the phenolic compounds being determined have high extinction coefficients, and this decreases the possibility of interference. The cis and trans isomers of the phenolic acids can be separated by both HPLC... 

With uv absorbance detectors, we have to consider the uv absorption of the mobile phase, which always increases as the wavelength decreases. The uv cut-off of solvents indicates the useful wavelength range of the solvent and means the wavelength below which the solvent has an absorbance of 1 or more when measured in a 1 cm cell. Aliphatic hydrocarbons cut off at about 210 nm the best polar solvents for low wavelength work are methanol and acetonitrile, which cut off at 205 and 190 nm, respectively, provided they are pure. Acetonitrile is difficult to purify, and is consequently expensive. 

While a UV absorption detector is fairly sensitive, it is not universally applicable. The mixture components being measured must absorb light in the UV region in order for a peak to appear on the recorder. Also, the mobile phase must not absorb an appreciable amount at the selected wavelength. 

A UV detector was not used to determine continuously the HEC since the UV absorption maximum changed with the HEC concentration. The following linear relationship was found between the maximal wavelength (in nm) and the concentration (between 0 and 0.003 g/mL) ... 

To be effective, the detector must be capable of responding to concentration changes in all of the compounds of interest, with sensitivity sufficient to measure the component present in the smallest concentration. There are a variety of HPLC detectors. Not all detectors will see every component separated by the column. The most commonly used detector is the variable ultraviolet (UV) absorption detector, which seems to have the best combination of compound detectability and sensitivity. Generally, the more sensitive the detector, the more specific it is and the more compounds it will miss. Detectors can be used in series to gain more information while maintaining sensitivity for detection of minor components. 

HPLC separations were carried out with a reverse-phase column (pBondapak C-18) using methanol-water (45 55) at 1.8 mL/min for 13.5 min followed by methanol-water (60 40) for 20 min. A Waters 6000A pump, Model 440 UV absorption detector and a fraction collector (LKB Multirac) were used. Fractions eluting from the column were collected and assayed by LSC. Retention times of fenitrothion, AF, and MNP under these conditions were 27.0, 11.0, and 7.5 minutes, respectively. 

Fewer compounds absorb in the UV region than in the IR region, and the UV absorption pattern of a compound is not as distinctive (not as narrow) as is its IR "fingerprint." On the other hand, UV analyzers provide better selectivity if the sample contains air and humidity because these materials do not absorb in the UV region. UV analyzers are also more sensitive than IR detectors. On an equal-path-length basis, the UV absorbance of liquids is stronger than that of vapors in proportion to their densities. 

Unfortunately, the LC-MS combination is less successful. In part, this may be due to technological interfacing problems, but even if these are solved, LC-MS is unlikely to provide the same degree of universality (large molecules will remain a problem), spectral information and reproducibility as the GC-MS combination. For the moment, the combination of LC with a multichannel UV absorption detector is a more realistic proposition. 

This chapter describes the final configuration of the chromatographic system (column and instrument) after the optimization of the phase system (the combination of the stationary and the mobile phase) has been completed. The entire optimization process is illustrated in figure 7.1. This figure shows the different stages in the process from the moment at which it has been decided (either on the basis of literature information or on the basis of figure 2.1) which chromatographic method should be used. For example, it may have been decided that RPLC is the method of choice. It should also be decided what kind of detector will be used. For instance, we may choose to use a UV absorption detector. 

Many detection principles require a finite volume of eluent. For example, a UV absorption detector yields a signal that is directly proportional to the optical pathlength (Beer s law, see eqn.5.21). The volume of the detector flow cell is usually well-defined and its contribution to aejc, and hence its effects on the observed dispersion ctg, can be discussed in quantitative terms (see section 7.4.2). 

Pederson described a specific HPLC method for the determination of dipyridamole in serum [74]. The HPLC system used was a Waters model 600 liquid chromatograph equipped with a U6K injector, a pBondapak Ci8 column (30 cm x 39 mm) (10 pm), and a model 440 dual channel filter absorbance detector in conjunction with a Tarkan W + W 600 recorder. The mobile phase was a 75 25 mixture of methanol and a 0.02 M solution of sodium acetate (adjusted to pH 4 with acetic acid). The solvent flow rate of 2 mL/min was produced by an applied pressure of approximately 2000 p.s.i. Detection of the analyte was made at the UV absorption maximum of 280 nm. 

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