Detectors flow cell volume

Despite the numerous advantages the instrumental demands of microcolumn LC are considerable, and these demands are further accentuated as the requirements vary from one column type to another. A consequence of the reduced flow rates is that the detector flow-cell volume should be reduced to <10nl for OTCs, 0.1 pi for packed microcapillaries and 1 pi for microbore columns. An additional demand of the detector is that it should have a rapid response, <0.5 s. Development of suitable detectors is paramount if the potential of micro-LC is to be realised. Study of detector systems has focused in two areas firstly, the miniaturisation of ultraviolet, fluorescence and electrochemical systems, using in the former two systems LASERS as excitation sources and ultraviolet fibre optic and on-line cells to reduce band broadening and increase sensitivity [123,124] secondly, the direct interfacing with systems which previously required transport and/or concentration of the eluant. Interfacing of HPLC with mass spectroscopy has been undertaken by Barefoot et al. [125] and Lisek et al. [126] and flame systems (FPD and TSD) have been reviewed by Kientz et al. [127]. Jinno has reviewed the interfacing of micro-LC with ICP [128]. 

Consideration should be given to the flow rate of the sample through the detection cell. Shultz and co-workers have demonstrated the wide variability in reaction kinetics between ECL reactions, and hence the influence of flow rate on ECL intensity [60], For example, the rate constants (k) of the Ru(bpy)32+ ECL reactions of oxalate, tripropylamine, and proline were calculated to be 1.482, 0.071, and 0.011/s, respectively. Maximum ECL emission was obtained at low linear velocities for slow reactions ranging up to high linear velocities for fast reactions. That is, the flow rate and flow cell volume should be optimized such that the light-emitting species produced is still resident within the flow cell, in view of the light detector, when emission occurs. 

Detection Inlet heat exchangers Flow cell volume and geometry MS ion sources Sprayers (e.g., in evaporative light scattering detectors) Data filtering effects in high speed applications... 

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... 

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. 

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). 

Figure 1.17 Effect of detector time constant on resolution, system efficiency, and sensitivity (a) 100 msec, (b) 200-msec. Flow cell volume was 2.4 ml, and both chromatograms were recorded at the same sensitivity. (Reprinted from Ref. 41 with permission.)...

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. 

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. 

Another useful rule is that the volume of the detector flow cell should be less than the estimated injection volume. Flow cell volume could be considered as the hypothetical peak slice used for digital quantitation. The amount of analyte in the flow cell at the specific moment of time is equal to the average concentration in the cell multiplied by its volume. The smaller the peak slice, the more accurate the quantitation. At least 15-20 points needed for the proper description of symmetrical Gaussian peak. In the example above, we estimated the optimum injection volume as one-tenth of the earliest peak, and the flow cell volume should be even smaller, 5 pL for the above example. 

Spreading for some commercial instruments. While instrument bandspreading is related to the extra-column volume, it does not correlate exactly due to the complexity of flow through tubing, connections, injection systems, and detector flow cells. 

One area for increases in sensitivity that has not been widely exploited is in the PTH detection system. Most commercial instruments use HPLC columns with internal cross sections of 2.1 mm, allowing flow rates of 200-350 pL/min. The introduction of the use of these columns provided an increase in sensitivity over the use of 4.6 mm columns, which were run at higher flow rates. This was a result of delivering the PTH analyte to the detector flow cell in a smaller volume, hence higher local concentration, and an increase in signal to noise. Reports have been made of PTH separations on smaller diameter columns both in the published literature (9,10), as well as commercial brochures, but extensive reports of reduction to practice in actual sequencer runs have not been made. 

Detector flow cells are the link between the chromatographic system and the detector system. The cell cuvettes are made of quartz, with either cylindrical or square shapes and volumes between 5 and 20 /tL. The sensitivity is directly proportional to the volume. However, resolution decreases with increasing volume. Fluorescence is normally measured at an angle perpendicular to the incident light. An angle of 90° has the lowest scatter of incident light. However, fluorescence from the flow cell is isotropic and can be collected from the entire 360°. 

It is interesting to note that the peak shape is altered for detectors characterised by a large flow cell volume and /or time constant. In this situation, the peak height resembles the peak area. 

The detector itself consists of a small liquid flow cell through which the eluent from the column flows. UV light passes through the cell and hits the UV photodetector. The cell is usually made of quartz which is UV-transparent. To avoid band broadening, the flow cell volume is minimised about 10-15 pL in the case of a standard flowcell or 6-8 pL in the case of a semimicro flowcell, the choice of which depends, among other factors on the column and the application. 

Detectors to be used in FIA should idealiy be endowed with a number of attributes such as low flow-cell volume and noise, flow-rate-independent signal, fast and linear response over a wide concentration range and high sensitivity. FIA methodology utilizes a variety of analytical detection techniques such as optical (spectroscopic and non-spectroscopic), electric (amperometric, po-tentiometrlc, conductimetric, coulometric) and thermochemical. 

With narrow bore columns the flow-cell volume must be < 3 pi in order that the resolution achieved on the column is not lost due to analyte spreading in the detector cell. 

Careful consideration must be given to the design of the detector flow cell as it forms an integral part of both the chromatographic and optical systems. A compromise between the need to miniaturize the cell volume to reduce extracolumn band broadening... 

In the normal FI extraction mode the enrichment factor is limited by the phase ratio which, in turn, is restricted by practical factors. A relatively complex extraction system was described by Atallah et al.[26] in which the continuously pumped sample is extracted by a small volume of oiganic phase trapped in a closed loop which incorporates the segmentor, extraction coil, phase separator and detector flow-cell. A simplified schematic diagram of the circulated extraction part of the manifold in shown in Fig. 

The sensitivity of UV detectors is generally in the low nanogram range although this can be increased by sample derivatisation. The sensitivity of detection may also be increased by alterations in the design of the detector flow cell and it is important to emphasise that the volume of the flow cell should be chosen to match the HPLC system thus a very low volume flow ceU (e.g. 1-2 /il) should be used with microbore, or very small particle columns where very high column efficiencies are anticipated, whereas larger flow cells (e.g. 5 jttl) should be used with more conventional systems. 

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