Flow cells, detectors

Check mixing spaces, detector flow cell, and diameter of tubing Adapt flow rate... 

In Fig. 1.1 (d) the hydrodynamic behaviour is simplified in order to explain the mixing process. Let us assume that there is no axial dispersion and that radial dispersion is complete when the sampler reaches the detector. The volume of the sample zone is thus 200pl after the merging point (lOOpl sample+lOOpl-reagent as flow rates are equal). The total flow rate is 2.0ml min-1. Simple mathematics then gives a residence time of 6s for the sample in the detector flow cell. In reality, response curves reflect... 

An air pocket in the pump can cause low or no pressure or flow, erratic pressure, and changes in retention time data. It may be necessary to bleed air from the pump or prime the pump according to system startup procedures. Air pockets in the column will mean decreased contact with the stationary phase and thus shorter retention times and decreased resolution. Tailing and peak splitting on the chromatogram may also occur due to air in the column. Air bubbles in the detector flow cell are usually manifested on... 

We have already briefly described a popular application of amperometry in Chapter 13. This was the electrochemical detector used in HPLC methods. In this application, the eluting mobile phase flows across the working electrode embedded in the wall of the detector flow cell. With a constant potential applied to the electrode (one sufficient to cause oxidation or reduction of mixture components), a current is detected when a mixture component elutes. This current translates into the chromatography peak... 

UV detector—replace deuterium source and clean/rebuild detector flow cell if contaminated. 

In contrast with UV methods where the linear range is approximately 1-2 orders of magnitude at best, the HPLC method with UV detection typically has a linear range of 3-4 orders of magnitude due to the narrow path length of the detector flow cell. For example, a simple... 

By assuming that a proportional increase in the amount of sample injected results in a proportional increase in the detector response for the solute band of interest, the detector response for chromatogram I in Figure 7 will increase 14 times when the maximum sample volume of 7 /xL is injected. However, for the 4.6-mm i.d. column, the detector response will increase 400 times when the maximum sample volume of 200 (lL is injected. By taking into account the relative detector responses for the 0.5-/xL injection, at the maximum sample injection volumes, the 4.6-mm i.d. column with the 20-/liL detector flow cell will produce approximately five times the detector response of the 1-mm i.d. column with the 5-/zL flow cell. In most cases, studies can be designed to provide excess sample because aqueous environmental samples are seldom limited with respect to volume. 

Pulse polarographic studies have been described using a microcell of 0.5 mL capacity, which analyzed two 1,4-benzodiazepines, with the lowest detection limit reported to date being 10-20 ng/mL of blood [199]. Detailed construction of the cell and electrode assembly was also described (shown in Fig. 26.16). Further miniaturization of this type of three-electrode cell is not practical hence further increases in sensitivity will have to rely on electrochemical detector flow cells of microliter capacity such as those used in conjunction with liquid chromatography (see Chap. 27). 

Detector traces for the removal of high-activity fission products in the wash step and elution of "Tc are shown in Figure 9.4. "Tc is present at much lower activities than the more abundant fission products that pass unretained through the column and detector flow cell. Nevertheless, during elution of the column with the 6 M HN03, the "Tc is readily seen in the vertically expanded inset. The SI separation procedure provided a decontamination factor of 104 for removal of 137Cs or 90Sr from the "Tc. 

Because the system was set up with a diverter valve as shown in Figure 9.2, samples could be processed onto the column and washed, with solutions going to waste, while the previous sample was stopped in the detector for counting. On completion of the counting interval, the detector flow cell could be washed, and then the sample already processed and separated on the column could be eluted to the detector. Using a count time of 15 minutes, the analysis time was 40 minutes for the first sample and 20 minutes for each subsequent sample or blank. 

On a reverse-phase column, separation occurs because each compound has different partition rates between the solvent and the packing material. Left alone, each compound would reach its own equilibrium concentration in the solvent and on the solid support. However, we upset conditions by pumping fresh solvent down the column. The result is that components with the highest affinity for the column packing stick the longest and wash out last. This differential washout or elution of compounds is the basis for the HPLC separation. The separated, or partially separated, discs of each component dissolved in solvent move down the column, slowly moving farther apart, and elute in turn from the column into the detector flow cell. These separated compounds appear in the detector as peaks that rise and fall when the detector signal is sent to a recorder or computer. This peak data can be used either to quantitate, with standard calibration, the amounts of each material present or to control the collection of purified material in a fraction collector. 

There are always two outputs from a detector, one electrical and one liquid. The electrical signal is sent to the recorder for display and quantitation (analytical mode). The liquid flow from the detector flow cell consists of concentration bands in the mobile phase. The liquid output from nondestructive detectors can be collected and concentrated to recover the separated materials (preparative mode). 

SFC is a relatively new technique using a silica-packed column in which the mobile phase is a gas, typically carbon dioxide, which has been converted to a supercritical fluid under controlled pressure and temperature. Sample is injected as in a GLC system, carried by the working fluid onto the packed column where separation occurs by either adsorption or partition. The separated components then wash into a high-pressure UV detector flow cell. At... 

It is critically important to understand this last point. There are two tubing volumes that can dramatically affect the appearance of your separation the one coming from the injector to the column and from the column to the detector flow cell. It is important to keep this volume as small as possible. The smaller the column diameter and the smaller the packing material diameter, the more effect these tubing volumes will have on the separation s appearance (peak sharpness). 

Connect one end of your column blank to the tubing from the injector outlet the other end is connected to the line leading to the detector flow cell. We have one more fluid line to connect to complete our fluidics. A piece of 0.02-in tubing can be fitted to the detector flow cell outlet port to carry waste to a container. In some systems, this line will be replaced with small-diameter Teflon tubing. 

Stop the flow and connect the column outlet with a short piece of 0.10-in tubing to the inlet of the detector flow cell. Resume flow to the column. Turn the detector on and start the recorder chart speed or computer data acquisition at 0.5cm/min. You should have a flat baseline. If the baseline continues to drift up or down, the column still hasn t finished its wash out and equilibration, or the detector has not fully warmed up. 

Using the HPLC system with a mass spectrometer as a detector forces the use of volatile buffers to avoid contamination of the analyzer. The buffers are still needed in many cases to control sample or column ionization to improve the chromatography, but must be removed in some way before they reach the detector flow cell. A table of volatile buffers and their pKa s is listed in Appendix C. 

The next step in the pathway is the column. The compression fitting on the inlet end-cap leads to the stainless steel frit at the top of the column. The column itself is a heavy-walled stainless steel tube filled with packing and mobile phase. The outlet end is identical to the inlet. Moving on down the wetted surface, we find 0.01-in tubing leading to the detector flow cell. 

Detector flow cell (breakage and hazing) and lamp (aging). 

Pacification after removing the column is an excellent cleaning technique for the whole system. It tends to remove buffer precipitation from check valves, organics from the injector rotor, and deposits on detector flow cell windows. Such a treatment is recommended to protect the system from halide attack on stainless steel. Experienced protein people have told me that pacification will protect a system against 200 mM NaCl for a month. This assumes you wash out the NaCl before you shut the system down. 

If it takes a while to get replacements, double the amounts of the preceding parts and add a detector flow cell, another Ci8 column, and a full pump head. If you are going to Antarctica for the season, an extra injector, pump, detector, strip chart recorder, and a case each of strip chart paper and pens might be nice. One of my customers found that his back-order time in Little America (Antarctica) was 14 months. 

Commercially available HPLC instrumentation was originally designed for use with standard-bore columns (4.6 mm I.D.). Detector flow cells were optimized for maximum sensitivity with these analytical columns, injectors were designed to introduce microliter quantities of sample, and pumps were designed to be accurate and reproducible in the milliliter flow-rate ranges commonly employed with standard-bore columns. However, these instruments are not well suited for use with small-bore columns, as the dispersion introduced by the large volumes is detrimental to the separation. In addition, the reproducibility and accuracy of the pumping system at the low flow rates required are questionable. 

Testing apparatus should be designed to minimize band spreading external to the column (e.g., short, narrow connecting tubing between the column and injector and detector, low dead-volume detector flow cell, etc.). 

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). 

The detector flow-cell, the contribution of which to ctv is approximately equal to its volume [707], represents a considerable and recognizable contribution to the extra-column band broadening. Typical conventional flow-cells have a volume of 8 pi, which is quite substantial compared with the maximum allowable extra-column dispersion. 

Table 7.3a lists the maximum allowable extra-column dispersion for the first three columns listed in table 7.1, using three different internal diameters. It is seen that the contribution from the detector flow-cell (as well as other contributions) will have to be reduced considerably if short columns packed with 3 pm particles are to be used. An even larger reduction in the extra-column dispersion is required for the use of columns with a reduced inner diameter. 

Response volume — In case of flow-through detectors (- flow-cell) the volume vr that flows through the detector with a flow rate / within the time interval corresponding to the time constant r (- response time) vr = t/. The response volume is a measure of the quality of a detector. 

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