HPLC detector cells

Commercially available cells with rate constant of 500 s and a cell volume of about 5 pi assure coulometric efficiency for typical HPLC flow rates with minimal extra-column band broadening. Each electrochemical unit has a central porous carbon electrode, on either side of which is situated a reference electrode and an auxiliary electrode. The characteristics of porous graphitic carbon electrode facilitate the construction of electrode arrays, lypical commercial systems include two units placed in series but arrays of up to 16 units are commercially available (Thermo Scientific, formerly ESA/Dionex). These cells have some degree of resistance to flow and with use can develop a significant back pressure. To minimize such back pressure changes, they need to be protected from particulate materials. Their intrinsic back pressure should also be borne in mind when connecting other types of HPLC detector cell in series. 

Electrochemical Detectors Another common group of HPLC detectors are those based on electrochemical measurements such as amperometry, voltammetry, coulometry, and conductivity. Figure 12.29b, for example, shows an amperometric flow cell. Effluent from the column passes over the working electrode, which is held at a potential favorable for oxidizing or reducing the analytes. The potential is held constant relative to a downstream reference electrode, and the current flowing between the working and auxiliary electrodes is measured. Detection limits for amperometric electrochemical detection are 10 pg-1 ng of injected analyte. 

The detection of the migrating sample boundary in CE can be accomplished by UV, fluorescent, electrochemical, radiochemical, conductivity, and mass spectrometry (MS) means. The use of high-sensitivity detection systems is always a key issue in CE applications. The sensitivity of HPCE detectors may be at least 2 to 3 orders of magnitude better than that of HPLC detectors. Since the detection cell volume is very small, the concentration sensitivity... 

LC-NMR hyphenation consists of a liquid chromatograph (autosampler, pump, column and oven) and a classical HPLC detector. The flow of the detector is brought via an interface to the flow-cell NMR probe. Using commercial NMR flow-cells with volumes between 40 and 180 p,L, in connection with microbore columns or packed capillaries, complete spectra have been provided from 1 nmol of sample. These micro-cells allow expensive deuterated solvents to be used, and thus eliminate solvent interference without excessive cost. The HPLC eluent can be split in order to allow simultaneous MS detection. 

An HPLC detector is often a modified spectrophotometer equipped with a small flow cell, which monitors the concentration (or mass) of eluting sample components. A number of detectors used in HPLC are discussed below. Most applications utilize absorbance detectors such as UV/Vis or... 

The ultraviolet (UV) absorption HPLC detector is basically a UV spectrophotometer that measures a flowing solution rather than a static solution. It has a light source, a wavelength selector, and a phototube like an ordinary spectrophotometer. The cuvette is a flow cell, through which the column effluent flows. As the mobile phase elutes, the chromatogram traces a line at zero absorbance, but when a mixture... 

Worked Example 7.3. A redox-active dye is eluting from an HPLC column. Since the analyte is redox-active, the HPLC detector is unusual in that it consists of a small annulus of silver, mounted within a short Teflon tube. Eluent from the column contains analyte, trickling at a constant rate, V, through the cell and over the electrode while the current is monitored. It is assumed that the silver ring only sees the redox-active dye, i.e. the current is wholly faradaic. 

For comparison of impurity levels quoted as % area/area, the normalized PA [area divided by the respective MTs, often stated as corrected PA (Ac)] must be used in CE to compensate for the residence time difference of the species in the detector. In HPLC, the separation takes place on the column. After the column, all analytes travel through the detector at the same speed (that of the mobile phase) and hence have the same residence time in the detector cell. However, in CE, the electrical field also takes effect in the detection cell. Therefore, the residence time of the species that have a higher apparent mobility (as shorter Jm) will give a lower response than species with a lower mobility, for species with the same absorptivity and concentration. ... 

A simphstic view of a FIA analytical system is outfined in Fig. 2.19. The liquid flow can be obtained in a number of ways, most commonly by using a peristaltic pump. In addition, gravity-feed systems, overpressure systems on liquid vessels and simple and double piston pumps normally associated with HPLC systems, are often used. The interrelation of the pumping system and the bore size of the transport tubing to a great extent modify the theoretical considerations involved and the operation of the FIA regime. In common with CFA, the minimization of dead volumes—in detector cells and between T pieces for example—is particularly important. Injections of the sample into... 

The detection cell must be designed with its volume small enough to prevent additional peak broadening in the detector in practice, this means the detection cell volume should be at least 10 times smaller than the volume of the first, most narrow, chromatographic peak. For nano-flow HPLC systems, in which the peaks can be sub-microliter, detection cells with a volume on the order of 100 uL or less are appropriate. For conventional HPLC systems, in which the peak volume is tens of microliters, there is no benefit to having a detection volume smaller than a few microliters, and indeed most conventional HPLC flow cells are around 5-12 pL. It is best to ensure that the detection cell is well matched to the sample and peak volumes, as making the detection volume too small will unnecessarily decrease the sensitivity of the detector. 

In flow microcalorimetry a small sample is put into the cell of the calorimeter and the probe molecule passes through it in an appropriate solvent. Adsorption of the probe results in an increase in temperature and integration of the area under the signal gives the heat of adsorption [70]. This quantity can be used for the calculation of the reversible work of adhesion according to Eq. 13. The capabilities of the technique can be further increased if a HPLC detector is attached to... 

An ultraviolet detector using a flow cell such as that. in Figure 25-19 is the most common HPLC detector, because many solutes absorb ultraviolet light. Simple systems employ the intense 254-nm emission of a mercury lamp. More versatile instruments have deuterium, xenon, or tungsten lamps and a monochromator, so you can choose the optimum ultraviolet... 

Three types of inline HPLC detector have been used to measure fat-soluble vitamin concentrations in food sample extracts absorbance, fluorescence, and electrochemical detectors. Each of these detectors provides a continuous electrical output that is a function of the concentration of solute in the column effluent passing through the flow cell. 

Flow Cell—Low volume (8-20 juL) detector cell designed to accept eluant output from an HPLC or an ion chromatography. 

Fig. 15. Schematic layout of the HPLC chip a layout of the channel system, b cross section along the channel axis, c cross section along the detector cell axis. IS sample and mobile phase inlet, S split injector, OS outlet for rejected sample and mobile phase, C separation channel, F frit, D optical detector cell, OD outlet to waste, P positioning grooves for optical fibers (reprinted with permission from [84]. Copyright 1995 John Wiley)...

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