Detector sensitivities

For ultraviolet and visible spectroscopic detectors, a standard solution of a compound whose molar absorption constant is known must be prepared, and placed in the flow cell. The absorbance obtained is then compared with the value measured by a standard spectrophotometer. 

For a fluorescence detector, quinine sulfate is used as the standard compound. The flow cell is filled with a standard solution and the fluorescence intensity is measured. The value is compared with that measured by a fluorescence spectrophotometer. This standard solution is also used for fixing the wavelength and position of the flow cell. The Raman spectrum of water can also be used for this purpose. 

For refractive index detectors, 13.33 g of sucrose is dissolved in 100 ml of pure water and then diluted 200 times. The refractive index of the final solution is 1 x 10-4 RI, which is used to calibrate the instrument. 

Another potential problem concerns the selection of the range of substrate concentrations to be used throughout the study. Considering the sensitivity of most HPLC detectors and the apparent Km values of most enzyme activities, the selection of the upper limit of concentration is usually not a problem. A problem will develop, however, when rate determinations are made at low substrate concentrations, since at these concentrations the amount of product formed during the course of the reaction will be small and may be below the monitor s level of detection. 

Therefore, prior to executing any experimental protocols dealing with low substrate concentrations, it is prudent to determine what product concentra- 

The choice of the stationary phase can best be made after the analysis of both the primary and secondary reactions has been completed and the compounds to be separated have been listed. The selection of. the stationary phase is guided by the number of compounds in the reactions, and the kinds present. With an understanding of the differences between these compounds, be it their size, solubility, or charge, the stationary and mobile phases can be selected. 

The use of the HPLC method to assay the activity of an enzyme may require some modifications in the composition of the reaction mixture. For example, the presence of metals in the reaction mixture can cause problems, since a metal complex may form and produce new peaks on the chromatogram. Complications associated with the requirement for termination of the reaction and for dealing with the small amounts of product that appear during early stages of the reaction may require changes in the reaction conditions as well. 

Termination of a reaction is best accomplished by using heat to inactivate the enzymes. Numerous procedures have been employed to heat the incubation mixture one of the most convenient is a sand bath. Heating will result 

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Detectivity. Detector sensitivity (1,2) is expressed in terms of the minimum detectable signal power or noise equivalent power (NEP) given in units of watts or W. The reciprocal function when normalized for detector area, M, and noise bandwidth, is defined as detectivity, D, in units of /W. Thus,... 

Fig. 3. Ideal photon detector sensitivity as a function of cutoff wavelength. Lower background flux generates less photon-induced noise giving higher sensitivity. The sensitivity limit for the condition of 300 K background temperature and hemispherical (27T) field of view is shown.

Fig. 16. Resistance area (R ) product for HgCdTe photodiodes cooled to 77 K. The soHd line represents the theoretical limit, the dashed lines (—) and (- -) high and low performance, respectively. Dark current caused by defects lowers R and detector sensitivity. In the high performance range dark...

Although performance varies with the isotopes for which they are intended, and with the balance in the design between resolution and efficiency, the overall sensitivity of a y-camera collimator is on the order of 5000 counts/(MBqmin) (several hundred counts/(/iCi-min)). In terms of photons detected per photon emitted, this is equivalent to about 2 x lO ". In other words, about two photons out of 10,000 emitted arrives at the crystal. This necessitates exposure times that range from several minutes to the better part of an hour. Fortunately, the large number of photons available from a modest injected radioactive dose more than offsets the poor detector sensitivity. The camera s abiUty to resolve small objects, however, is ultimately limited by the collimator inefficiency. 

Improvements in technology will shape developments in PL in the near future. PL will be essential for demonstrating the achievement of new low-dimensional quantum microstructures. Data collection will become easier and ter with the continuing development of advanced focusing holographic gratir, array and imaging detectors, sensitive near infiared detectors, and tunable laser sources. 

To unambiguously identify the presence of a peak and, in addition, be able to give some proximate estimation of its size for quantitative purposes, the peak height needs to be at least 5 times the noise level. The detector sensitivity, or the minimum detectable concentration, (Xd), is defined as that concentration of solute that will give a signal equivalent to twice the noise level and, consequently, the concentration of solute at the limiting (k ) value must be 2.5Xd. 

Equation (33) shows that the maximum capacity ratio of the last eluted solute is inversely proportional to the detector sensitivity or minimum detectable concentration. Consequently, it is the detector sensitivity that determines the maximum peak capacity attainable from the column. Using equation (33), the peak capacity was calculated for three different detector sensitivities for a column having an efficiency of 10,000 theoretical plates, a dead volume of 6.7 ml and a sample concentration of l%v/v. The results are shown in Table 1, and it is seen that the limiting peak capacity is fairly large. 

Detector Sensitivity Maximum (k ) Retention Time (min.) Peak Capacity... 

FIGURE 16.23 Inulln isolated from small (—). medium ( ) and large (A) tubers separated on P-6 (140 X 1.5 cm) flow rate 0.33 ml/min eluenf. H20(dest) + 0.002% NaNa mass detection Waters 403 R differential refractive index detector, sensitivity 8X applied sample solution volume I ml of a 20-mg/ml aqueous inulin solution. 

SEC measurements were made using a Waters Alliance 2690 separation module with a 410 differential refractometer. Typical chromatographic conditions were 30°C, a 0.5-ml/min flow rate, and a detector sensitivity at 4 with a sample injection volume of 80 fil, respectively, for a sample concentration of 0.075%. All or a combination of PEO standards at 0.05% concentration each were used to generate a linear first-order polynomial fit for each run throughout this work. Polymer Laboratories Caliber GPC/SEC software version 6.0 was used for all SEC collection, analysis, and molecular weight distribution overlays. 

Prepare a mixture, by weight, of A with compound (IV). Inject a 0.3 pL sample of this mixture, measure the various peak areas and, after making appropriate corrections for differences in detector sensitivity, determine the percentage composition of A. 

Detector Sensitivity, or Minimum Detectable Concentration Pressure Sensitivity Flow Sensitivity Temperature Sensitivity... 

Detector Sensitivity or the Minimum Detectable Concentration has been defined as the minimum concentration of an eluted solute that can be differentiated unambiguously from the noise. The ratio of the signal to the noise for a peak that is considered decisively identifiable has been arbitrarily chosen to be two. This ratio originated from electronic theory and has been transposed to LC. Nevertheless, the ratio is realistic and any peak having a signal to noise ratio of less than two is seriously obscured by the noise. Thus, the minimum detectable concentration is that concentration that provides a signal equivalent to twice the noise level. Unfortunately, the concentration that will provide a signal equivalent to twice the noise level will usually depend on the physical properties of the solute used for measurement. Consequently, the detector sensitivity, or minimum detectable concentration, must be quoted in conjunction with the solute that is used for measurement. 

As a result of limited detector sensitivity, there is often a need for sample concentration when determining trace materials contained in a bulk matrix. The need for such procedures frequently arises in forensic work, environmental samples, blood testing etc. A number of methods have been developed for this purpose and some of those in common use will be described. 

An excellent discussion on derivatization techniques has been given by Lawrence (17) including a detailed discussion on pre-column derivatization (18) and post-column derivatization (19). Probably, the more popular procedures are those that produce fluorescing derivatives to improve detector sensitivity. One of the more commonly used reagents is dansyl chloride (20), 5-dimethylamino-naphthalene-1-sulphonyl chloride (sometimes called DNS-chloride or DNS-C1). The reagent reacts with phenols and primary and secondary amines under slightly basic conditions forming sulphonate esters or sulphonamides. 

Where Q, is the minimum detectable amount, R the detector noise level and s the detector sensitivity [135,146,151,152]. For a concentration sensitive detector the minimum detectable concentration is the product of Q, and the volumetric gas flow rate through the detector. The minimum detectable amount or concentration is proportional to the retention time, and therefore, directly proportional to the column radius for large values of n. it follows, then, that very small quantities can be detected on narrow-bore columns. 

The signal tram gas chroaatographic detectors can be further characterized as nass or concentration dependent. For concentration-dependent detectors the aost notable feature is that the detector response is dependent upon the flow rate through the detector (carrier gas and makeup gas, if any) and, therefore, the sensitivity of the detector is usually defined as the product of the pe dc area and flow rate divided by the wei t of the sanple. For B ss-dependent detectors sensitivity is defined as the product of the peak area divided by the sanple wel t in grans or noles and is independent of flow rate through the detector. 

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