Microdialysis sampling flow rates

Calibration is necessary to allow correlation between collected dialysis concentrations to external sample concentrations surrounding the microdialysis probe. Extraction efficiency (EE) is used to relate the dialysis concentration to the sample concentration. The steady-state EE equation is shown in equation (6.1), where Coutiet is the analyte concentration exiting the microdialysis probe, Ci iet is the analyte concentration entering the microdialysis probe, CtiSSue> is the analyte tissue concentration far away from the probe, Qd is the perfusion fluid flow rate and Rd, Rm, Re, and Rt are a series of mass transport resistances for the dialysate, membrane, external... 

Compared to basic research microdialysis sampling devices, those used for clinical studies are much longer with typical membrane lengths between 10 and 30 mm. Flow rates of the perfusion fluid through these devices are also much lower (0.3 pL/min) than typically applied in basic research studies. This combination results in high EE% values for low molecular weight and hydrophilic analytes such as glucose. 

In practice, a microdialysis probe is implanted into the tissue with tubing connecting it to external components a perfusion pump and a fraction collector. The probe is perfused continually at low flow rate (0.1-10 /uL/min) with an artificial physiological solution. As the perfusate emerges from the probe, fractions are collected and samples of each fraction analyzed. 

Since the first reports on microdialysis in living animals, there have been efforts to estimate true (absolute) extracellular concentrations of recovered substances (ZetterstrOm et al., 1983 Tossman et al., 1986). Microdialysis sampling, however, is a dynamic process, and because of a relatively high liquid flow and small membrane area, it does not lead to the complete equilibration of concentrations in the two compartments. Rather, under steady state conditions, only a fraction of any total concentration is recovered. This recovery is referred to as relative or concentration recovery, as opposed to the diffusion flux expressed as absolute or mass recovery. The dependence of recovery on the perfusion flow rate is illustrated in Figure 6.2. As seen, relative recovery will exponentially decrease with increasing flow as the samples become more... 

A miniaturized thermal flow-injection analysis biosensor was coupled with a microdialysis probe for continuous subcutaneous monitoring of glucose [34]. The system (Scheme 1) consisted of a miniaturized thermal biosensor with a small column containing co-immobilized glucose oxidase and catalase. The analysis buffer passed through the column at a flow rate of 60 pl/min via a 1-pl sample loop connected to a microdialysis probe (Fig. 11). 

The flow rates of the microdialysis experiment are such that samples of 1-10 J,L are typically obtained. At typical perfusion rates, the perfusate is not at equilibrium with the extracellular fluid. As such, the concentration of sample in the dialysate is some fraction of that in the surrounding tissue. This is termed the extraction efficiency and is a function of the delivery or recovery of the probe. Not only are the sample volumes small but also the concentration in the dialysate may be low, typically ranging from 1 pM to 1 lM. Because the recovery is typically less than 100%, the limit of detection of the method should be lower than the lowest concentration expected in the dialysate. This presents a tremendous challenge for the analyst. 

Several characteristics of chromatography impact on the microdialysis experiment. The chromatography process inherently dilutes the sample. If we assume that a typical microdialysis experiment will involve a perfusion rate of 1 lL/min, with sampling for 5 min, 5 4L of sample will be obtained for the assay. A typical analytical column (15 cm X 4.6 mm) with a mobile-phase flow rate of 1 mL/min may have a peak width of 30 sec and would correspond to a 500-... 

The microdialysis sampling process which allows the monitoring of small molecules in circulation within an animal, is an example. An artificial capillary is placed in the tissue region of interest, and a sample is collected via dialysis. In the case of a laboratory animal such as a rat, a probe is placed in the jugular vein under anesthesia. Flow rates are of the order of 1 ).iL/min. 

With in vivo microdialysis (Delgado et al., 1972 Ungerstedt, 1984), the extracellular fluid sample is ftirther qualified by a small sack or loop of semipermeable dialysis membrane (Fig. 51B). Artifidal CSF is slowly circulated into and out of the dialysis r on at flow rates of 1 -5 il/min, so that recovery of small molecules across the membrane from the extracellular space is effective. In essence the probe acts as an artificial blood vessel which may be sampled externally as a function of time during basal behavioral periods, drug administration (via the probe or elsewhere), external stimuli, etc. 

In microdialysis, the recovery of analyte from the sample depends on a number of factors, including the chemistry of the analyte, temperature, perfusion rate, membrane surface area, membrane characteristics, and the nature of the sample (including its fluid volume percentage and whether it is in motion). MiCTodialysis is typically done at low perfusion flow rates (0.5 to 2 /d/min). As flow rate increases, relative recovery decreases, but absolute recovery increases. Relative recoveries from small membrane probes are in the 1-20% range. SubstantiaUy higher recoveries in the 50-80% range can be obtained with longer loop-type probes. 

There have only been a few reports of coupling microdialysis to microchip-based separation systems. Ideally, the microchip system should allow the injection of discrete sample plugs from a continuously flowing stream of dialysate without disturbing the separation element of the analysis. This allows maximal temporal resolution and limits the effect of perfusion flow rate on system performance. 

In order to conduct microdialysis experiments, several other components are required. Syringe pumps are often used to control the perfusate flow rate. The pump has to be able to deliver flow rates precisely in the microliter per minute range. Tubing is needed to connect between the probe and the pump which drives the perfusion flow and, in some cases, between the probe and a sample collector as well. The total dead volume of tubing should also be maintained as small as possible to have better time resolution. The perfusion fluid is a medium resembling the composition of extracellular fluid with minimal or zero concentration of the molecules of interest. Dial-ysate exiting from the outlet of the microdialysis probe is usually collected in a vial for later analysis. It is also possible to coimect the outlet directly to an analysis instrument without using a collector, which is usually preferred, if possible, for its convenience and usually faster analysis results. 

By coupling microdialysis to capillary electrophoresis, much shorter sampling times can be used, since the separating step can be very fast. As mentioned in Chapter 6, injection volumes are in the nanoliter range. This allows not only short sampling times but also a low perfusate flow rate that vhll give better detection limits (Section 9.6.1). 

As the relative recovery will never reach 100%, the dialysate concentrations are only a fraction of the true concentration of the analyte of interest in the surrounding fluid. Before using a microdialysis probe for continuous sampling or monitoring, the trae concentration of the analyte of interest in the surrounding environment and the recoveries at certain perfusate flow rates have to be obtained. There are many different methods for calibration, which are discussed in the following [1]. 

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