Sensitivity optimization

An important aspect of the chromatographic process is the sensitivity of the detection. For most common detectors, the recorded signal is directly proportional to the concentration of the solute in the effluent from the column. We have seen in chapter 1 (eqn.1.15) that the observed peak height (h0) can be related to the peak area (A) by 

Using the common equations for the plate count and the retention volume we now find 

15) is the key equation for the optimization of chromatographic sensitivity. Naturally, the peak height is proportional to the concentration of the solute in the sample and to the volume of the injected sample. However, this proportionality holds over a limited range and we cannot increase these two quantities indefinitely without having to sacrifice another vital characteristic of the system, the linearity of detection. The proportionality between cmax and the product cgV-in ends when N may no longer be considered as a constant. Consequently, the aforementioned product may be increased until the plate count starts to be affected. 

A series of tricks has been devised for the injection of large volumes of samples, all of which aim at increasing Vin- without affecting N. 

In GC the injection may take place at a temperature that is lower than that of the column oven. The solute bands will be concentrated in a small volume and may be brought into the column by a subsequent heating of the cold zone. If this zone is part of the column itself we talk about cold (on-column) injection , if it is part of a separate injector unit we talk about cold trap injection. A similar band compression effect may be achieved in a different way by leaving the first part of the capillary column uncoated (i.e. no stationary phase present). The solute band will then be compressed at the point where the stationary phase starts to be present in the column. This band compression technique is usually referred to by the unfortunate term retention gap [706]. 

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Parriaux O., Veldhuis G.J., Normalized analysis for the sensitivity optimization of integrated optical evanescent-wave sensors, J of Lightwave Technol. 1998 16 573-582. 

Steuer et al. compared supercritical fluid chromatography with capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) for its application in pharmaceutical analysis [24]. Efficiency, performance, sensitivity, optimization, sample preparation, ease of method development, technical capabilities, and orthogonality of the information were the parameters studied. They concluded that SFC is ideal for moderately polar compounds, such as excipients, for which mass detection is required. 

A sorption colloid flotation method has been developed for the separation of vanadium from sea water. The separation is based on a surfactant-collector inert gas system in which vanadate is sorbed on a positively charged colloidal iron(III) hydroxide collector. The vanadate enriched collector rises to the sea water surface and floats as a separable foam with aid of sodium dodecylsulfate as surfactant and nitrogen as inert gas. The major advantages of this method are the rapid attainment of flotation and the excellent recovery of 86 % vanadium based on spiked sea water samples. Flotation was found to be highly pH sensitive optimal values were found to be 5.00 + 0.02. In effect, at pH 4.90 a slight decline in recovery of vanadium could already be observed, whereas at pH 7 and above there was no vanadium float 53). 

Due to the genetically diverse and polymorphous parasite, sensitivity of the prototype assay was assessed with various positive specimens from donors covering many regions of Latin Americas and southern US. 228 out of 228 specimens were detected as reactive or 100% sensitivity. Optimization to further improve the assay performance is in progress. The Abbott PRISM Chagas Assay is potentially a screening test to improve the safety of the blood supply by reducing the risk of T. cruzi transfusion. 

The relative response of an analyte is influenced by two factors (equations 10 and 11) the relative retention of the solute (aj and the fractional coverage of the adsorbent exerted by the probe (0 ). A system with naphthalene-2-sulfonate as the probe is shown in Figure 10, where the dotted lines give the estimated relative response for cationic and anionic solutes [19, 57]. There is an increase from zero to a maximum value in the range 0-1, and the relative response then decreases and approaches an almost constant level. In order to achieve high detection sensitivity, optimization of the retention is more important than a high absorptivity of the probe. 

In order to obtain a perfect sensitivity, optimization of the concentration of anchored DNA is needed. Here, we use 0.2 pM ADNA as the appropriate concentration to obtain a perfect sensitivity. 

Matrix ENDOR of spin probes that reside in an internal interface has the potential to provide detailed information on the microscopic structure of this interface, but measurement techniques and data analysis are still in their infancy, so that only semiquantitative information can be obtained to date. Development of more precise and sensitivity-optimized measurement techniques and better quantification of the spectra are now in progress. 

Faijon and co-workers have introduced the sensitivity optimized SOFAST-HMBC technique, which is a phase sensitive echo/anti-echo HMBC with selective pulses applied to H. The technique allows the natural abundance measurement of Ynh oc in peptides with values lower than 1 Hz. 

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