The ultraviolet detectors

The ultraviolet detector is probably the detector in most common use today. This is available as a fixed wavelength detector and as a variable wavelength spectropho- 

The great and obvious limitation of the ultraviolet detectors is that they are restricted to ultraviolet absorbing compounds. Fortunately a large number of biochemically important substances absorb ultraviolet light and this detector is thus the first choice for general chromatographic work, unless the detector is chosen for a specific application. 

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Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

If unidentified peaks are detected the stability of the protein under the chromatographic conditions should be checked. In all analytical investigations of proteins on SEC columns it is desirable to be able to monitor the eluted peaks at a very high sensitivity of the ultraviolet detector. Therefore, very pure (analytical grade) salts and buffers should be used. 

Despite their higher sensitivity and relative cheapness compared with ultraviolet detectors, amperometric detectors have a more limited range of applications, being often used for trace analyses where the ultraviolet detector does not have sufficient sensitivity. 

By far, the most frequently used device is the ultraviolet detector. Three types are employed (23) — single wavelength with low pressure mercury source, multiwavelength filter photomer with medium pressure mercury source, and spectrophotometer. 

It must also be noted that since the ultraviolet detector is an optical device, objects that are able to block its view cannot be allowed to come between the detector and the area to be protected. 

All of the ionizing air systems at Longhorn are located in areas where ultraviolet sensors are used in conjunction with deluge systems for fire protection. Care must be taken to shield the ultraviolet detectors from the ion generating corona source. The systems used at Longhorn are individually shielded with PVC tubing or with hoods. 

In liquid chromatographic analysis of macrolides and lincosamides, most popular is the ultraviolet detector (Table 29.4). Tylosin, tilmicosin, spiramycin, sedecamycin, and josamycin exhibit relatively strong ultraviolet absorption, but erythromycin, lincomycin, pirlimycin, and oleandomycin show extremely weak absorption in the ultraviolet region. Hence, detection at 200-210 nm has been reported for the determination of lincomycin (146). However, a combination of poor sensitivity and interference from coextractives necessitated extensive cleanup and concentration of the extract. Precolumn derivatization of pirlimycin with 9-fluorenylmethyl chloroformate has also been described to impart a chromophore for ultraviolet detection at 264 nm (140). 

Since, as has already been said, the separation of alcohols is performed very often together with other analytes, such as organic acids and sugars, the choice of the type of detector also takes into account their chemical-physical properties. Usually, the choice is the ultraviolet detector (UV), the refractive index detector (RI), or the electrochemical detector (EC). Of the last, various types exist, which we shall describe briefly. 

Fig. 2.7 shows a separation of seven common anions monitored using the conductivity detector. Fig. 2.7(b) was obtained with the ultraviolet detector after the suppressor column. As is illustrated in Fig. 2.7, nitrite, bromide, and nitrate absorb strongly in the ultraviolet, while fluoride, phosphate and sulphate do not show appreciable absorption above 190nm. Chloride absorbs weakly in the ultraviolet region below 200nm. Note that... 

Arsenous acid is a veiy weak acid and cannot be detected at low levels by the conductivity detector. However, like sulphide, it is easily detected with the ultraviolet detector. The simultaneous determination of arsenite and arsenate is possible. 

Arsenite also can be separated by ion chromatography using standard conditions. It elutes early, near the carbonate dip. For this application the ultraviolet detector should be placed between the separator and suppressor columns. 

The analytical procedure is outlined in Fig. 4.6. Reproducible retention times (which varied less than 2% over a nine-month period) eliminated the need for column reequilibrium. Although some coeluting PAHs (eg anthracene and phenanthrene) had been placed into different fractions, it was clear that no single ultraviolet wavelength was capable of resolving all of the PAHs within a fraction. The sensitivities of the ultraviolet detectors, defined as a signal-to-noise ration of 2, ranged from 0.25 to lng IT1. 

The ion chromatographic determination of weak acid anions is complicated by ion exclusion in the suppressor column, resulting in faster elution and sharper peaks, directly proportional to the degree of exhaustion of the suppressor column [7], A lOmg L 1 nitrite standard showed a 37% increase in peak height over an 8h period when monitored with the conductivity detector while on only a minor 2% increase in peak height was observed over the same time period by using the ultraviolet detector after the separator column. 

The ultraviolet detector can also be used in some cases to resolve overlapping peaks. Determination of the nitrite peak by using the conductivity detector is complicated by both the ion exclusion effect and the incomplete resolution between the large chloride peak and the much smaller nitrite peak. 

The ion exclusion interference can be eliminated for ultraviolet active anions by placing the ultraviolet detector between the separator and suppressor columns (position 1). In addition, the problem of overlapping peaks can sometimes be resolved spectrophotometrically by proper choice of wavelength. 

Hydrogen sulphide acid is a veiy weak acid that does not ionise sufficiently to be detected with the conductivity detector. The ultraviolet detector, however, is able to detect sulphide at low levels. 

Figure 9 shows the purification of another l c-compound in a partially purified extract of goat milk. At this stage at least seven other anions are visible to the ultraviolet detector as the components are separated on Permaphase AAX anion exchanger. Even now the strong UV band almost directly above the radioactive peak appears to belong to a contaminant rather than the C-compound itself, indicating that further work will be required. 

Dappen et al. studied the performance of a commercial (Polarmonitor, IBZ, Hannover, Germany) and a laboratory designed polarimetric detector in conjunction with a HPLC method based on a Nucleosil 100 RP-18 column, an ultraviolet detector, and aqueous 58% methanol as the mobile phase [33]. The polarimetric signal was proportional to the excess concentration of one enantiomer in a mixture of two, and measurement of the total concentration of both enantiomers using the ultraviolet detector then permitted the enantiomer ratio to be calculated. The unexpectedly strong polarimetric signals produced by some compounds... 

The individual fractions can be collected and subjected to further examination, for example, by fast atom bombardment (see the next section on mass spectrometry) or assayed for biological activity, especially if platelet activating factor is present in the original sample. Interestingly, in the latter instance PAF would not be detected by the ultraviolet detector since it usually contains very low amounts of olefinic double bonds. 

The solid lines show the ultraviolet detector response, and the squares with the regression line (slope 0.94 and F = 0.9993) represent peak area versus injection volume. The sample was first diluted in order not to exceed the overloading point. (From Ref. 7.)... 

The ultraviolet detector is suitable for compounds containing rc-bonding and nonbonded electrons. 

The fluorescence detector is available as either a filter fluorimeter or as a continuous wavelength fluorimeter. The filter fluorimeters are less expensive, but in most cases low wavelength excitation is not possible with these instruments, and this makes, e.g., the determination of indoles and catecholamines by their native fluorescence impossible. The selectivity of the fluorescence detector is much better than that of the ultraviolet detector, and for favorable compounds the sensitivity may also be better. 

The ultraviolet detector only observes the phenyl groups on the polystyrene, which fluoresce. After subtracting out the polystyrene component, the poly(methyl methacrylate) component remains (bottom curve. Figure 3.22). The results in Table 3.13 show that the result is approximately 30% in error, but not so bad considering that poly(methyl methacrylate) is the less sensitive polymer in the measurements. 

When necessary, the liquid flow can be split between the analytical colunm and the reference colunm. A valve placed before the reference column permits the flow rate through that column to be completely shut-off or varied depending on the requirements of the system. This is particularly useful when using the refractive index monitor at higher flow rates. For column flow rates up to 2 cm min good stability could be obtained by careful balance of flow. The reference column was either filled with the same liquid-liquid support employed in the analytical column or merely filled with uncoated glass beads. With the ultraviolet detector the reference column is not used. Column head pressures are read on 1 - 3,000 psi or 0 - 5,000 psi standard gauges. 

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