Response index,detectors

A common error is to confuse the GPC distribution with the weight distribution. The response of a refractive index detector is proportional to the mass of polymer. The GPC elution volume (V) typically scales according to the logarithm of the degree of polymerization (or the logarithm of the molecular... 

It is seen that, for a truly linear detector, the response index (a) will be unity and the experimentally determined value of (a) will be an accurate measure of the proximity of the response to strict linearity. It is also clear that (a) could be used to correct for any non-linearity that might occur in the detector and thus improve the accuracy of an analysis. 

The curves relating detector output to solute concentration for detectors having different response indexes are shown in figure 2. 

It is seen that errors in the smaller component can be as great as 12.5% (1.25% absolute) when the response index is 0.94. Yet on examining the curve for a response index of 0.94 in figure 2 the non-linearity is scarcely apparent. When the response index is 1.05 the error is 9.5% (0.95% absolute) and again the poor linearity is not obvious in figure 2. As already stated, to obtain accurate results without employing a correction factor, the response index should lie between 0.98 and 1.02. Most LC detectors can be designed to meet this linearity criteria. 

The refractive index detector, in general, is a choice of last resort and is used for those applications where, for one reason or another, all other detectors are inappropriate or impractical. However, the detector has one particular area of application for which it is unique and that is in the separation and analysis of polymers. In general, for those polymers that contain more than six monomer units, the refractive index is directly proportional to the concentration of the polymer and is practically independent of the molecular weight. Thus, a quantitative analysis of a polymer mixture can be obtained by the simple normalization of the peak areas in the chromatogram, there being no need for the use of individual response factors. Some typical specifications for the refractive index detector are as follows ... 

The normalization method is the easiest and most straightforward to use but, unfortunately, it is also the least likely to be appropriate for most LC analyses. To be applicable, the detector must have the same response to all the components of the sample. An exceptional example, where the normalization procedure is frequently used, is in the analysis of polymers by exclusion chromatography using the refractive index detector. The refractive index of a specific polymer is a constant for all polymers of that type having more than 6 monomer units. Under these conditions normalization is the obvious quantitative method to use. 

Detection requirements in preparative-scale chromatography also differ from analytical erations where detectors are selected for their sensitivity. Sensitivity is not of overriding importance in preparative-scale chromatography the ability to accommodate large column flow rates and a wide linear response range are more useful. The sensitivity of the refractive index detector is usually quite adequate for prqtaratlve work but the ... 

Sample preparation, injection, calibration, and data collection, must be automated for process analysis. Methods used for flow injection analysis (FLA) are also useful for reliable sampling for process LC systems.1 Dynamic dilution is a technique that is used extensively in FIA.13 In this technique, sample from a loop or slot of a valve is diluted as it is transferred to a HPLC injection valve for analysis. As the diluted sample plug passes through the HPLC valve it is switched and the sample is injected onto the HPLC column for separation. The sample transfer time typically is determined with a refractive index detector and valve switching, which can be controlled by an integrator or computer. The transfer time is very reproducible. Calibration is typically done by external standardization using normalization by response factor. Internal standardization has also been used. To detect upsets or for process optimization, absolute numbers are not always needed. An alternative to... 

Detection is also frequently a key issue in polymer analysis, so much so that a section below is devoted to detectors. Only two detectors, the ultra-violet-visible spectrophotometer (UV-VIS) and the differential refractive index (DRI), are commonly in use as concentration-sensitive detectors in GPC. Many of the common polymer solvents absorb in the UV, so UV detection is the exception rather than the rule. Refractive index detectors have improved markedly in the last decade, but the limit of detection remains a common problem. Also, it is quite common that one component may have a positive RI response, while a second has a zero or negative response. This can be particularly problematic in co-polymer analysis. Although such problems can often be solved by changing or blending solvents, a third detector, the evaporative light-scattering detector, has found some favor. 

Radioactive label, 330 Raman diffusion, 184 Raman scattering, 227 Ratio fluorimeter, 228 Rayleigh scattering, 226 Real mean, 385 Red-shift, 196 Reference electrode, 347 Reflectron, 298 Refractive index detector, 59 Relative response factor, 78 Relative standard deviation, 387 Reliability, 389 Resolving power, 282 Response factor, 77 Restrictor, 98 Retardation factor, 88 Retention factor, 14 Retention index, 41 Retention time, 7 Retention volume, 14 RP-18, 53 RSD, 387 Ruhemann, 112... 

Refractive index detectors are useless in gradient elution because it is impossible to match exactly the sample and the reference while the solvent composition is changing. Refractive index detectors are sensitive to changes in pressure and temperature (—0.01 °C). Because of their low sensitivity, refractive index detectors are not useful for trace analysis. They also have a small linear range, spanning only a factor of 500 in solute concentration. The primary appeal of this detector is its nearly universal response to all solutes, including those that have little ultraviolet absorption. 

In the beginning, the refractive index detector was the most used detection system, although it has two important drawbacks (1) solvent gradients cannot be used, and (2) it has low sensitivity and different responses to saturated and highly unsaturated TGs (112). Moreover, use of the ultraviolet (UV) detector is difficult, because the most adequate solvents also absorb in the same range and therefore cause an important baseline drift with gradient elution systems (106). 

Reduced parameters, 66-69 Refractive index (RI) detector, 206-207 Regular solution, 49 Relative retention, 20-21, 22, 77 Repeatability, see Precision Reproducibility, see Precision Resolution, 17-19, 55 Response factors (detector), 104, 125 Response time, 94 Retardation factor, Rf, 71 Retention index of Kovats, 78 Retention ratio, 11, 12, 71 Retention time, 6, 9 Retention volume, 9, 75 adjusted, 10, 75 corrected, 62-63, 75 net, 63, 75 specific, 110 Reverse phase LC, 158 Rohrschneider/McReynolds constants, 137-140... 

Chromatographic System (See Chromatography, Appendix IIA.) Use a liquid chromatograph equipped with a refractive index detector that can be maintained at a constant temperature of 25°, a 25-cm x 4.6-mm (id) column packed with 10- im porous silica gel bonded with aminopropylsilane (Alltech 35643, or equivalent), and a guard column that contains the same packing. Maintain the column at a constant temperature of 25° 2°, and the flow rate at about 2.0 mL/min. Inject 20 pL of System Suitability Preparation into the chromatograph, and record the peak responses as directed under Procedure. The relative standard deviation for replicate injections is not more than 2.0%, and the alpha-Cyclodextrin and beta-Cyclodextrin peaks exhibit baseline separation, the relative retention times being about 0.8 and 1.0, respectively. 

Separately inject about 50-p.L portions of the Assay Solution and Standard Solutions into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The elution order for the standards is maltose, maltitol, dextrose, and sorbitol. The differential refractive index detector should show similar response factors. 

Detectors can be mass flow-sensitive (refractive index detectors, ELSDs) or concentration-sensitive (UV-Vis and fluorescence detectors). The former respond to the amount (mass) of analyte passing through the detector per unit of time (calculated by multiplying the eluent flow rate by the analyte concentration in the eluent), while the latter respond to analyte concentration. In mass flow-sensitive detectors, the response (signal amplitude) is proportional to the amount of sample component reaching the detector per unit of time. 

A refractive index (RI) detector is probably the most widely used in GPC. The main advantage of an RI detector is that any polymer solution will generate a response. This detector has several disadvantages ... 

This method for defining detector linearity is perfectly satisfactory and ensures a minimum linearity from the detector and consequently an acceptable quantitative accuracy. However, the specification is significantly looser than that given above and there is no means of correcting for any non-linearity that may exist as there is no correction factor given that is equivalent to the response index. It is strongly advised that the response index of all detectors (CiC and LC)... 

There are two methods that can be used to measure the response index of a detector, the incremental method of measurement and the logarithmic dilution method of measurement. The former requires no special apparatus but the latter requires a log-dilution vessel which fortunately is relatively easy to fabricate. 

Thus the slope of the Log/Log curve will give the value of the response index (r). If the detector is truly linear, r = 1 i.e. the slope of the curve will be sin 7r/4 =1). Alternatively, if suitable software is available, the data can be curved fitted to a power function and the value of (r) extracted from the results. The same data can be employed to determine the linear range as defined by the ASTM E19 committee. In this case, however, a linear plot of detector output against solute concentration at the peak maximum should be used and the point where the line deviates from 45° by 5% determines the limit of the linear dynamic range. 

Thus if the logarithm of the detector output is plotted against time, then for a truly linear detector, a straight line will be produced having a slope (-QA )- If the detector has a response index of (r) and the... 

Thus, the response index can be easily determined. However the accuracy of the measurement will depend on the flow rate remaining constant throughout the calibration, and consequently for a GC detector a precision flow controller must be employed and for an LC detector, a good quality solvent pump. Manufacturers do not usually provide the response indices for their detectors and so it is left to the analysts to measure it for themselves. 

The noise level of detectors that are particularly susceptible to variations in column pressure or flow rate (e.g. the katherometer and the refractive index detector) are often measured under static conditions (i.e. no flow of mobile phase). Such specifications are not really useful, as the analyst can never use the detector without a column flow. It could be argued that the manufacturer of the detector should not be held responsible for the precise control of the mobile phase, beitmay a gas flow controller or a solvent pump. However, all mobile phase delivery systems show some variation in flow rates (and consequently pressure) and it is the responsibility of the detector manufacturer to design devices that are as insensitive to pressure and flow changes as possible. 

The Response Index - (r) - The response index of detector is a measure of its linearity and for a truly linear detector would take the value of unity. In practice the value of (r) should lie between 0.98 and 1.02. If (r) is known, quantitative results can be corrected for any nonlinearity. 

Linear Dynamic Range - (D ) - The linear dynamic range of a detector is that concentration range over which the detector response is linear within defined response index limits. It is also dimensionless and is taken as the ratio of the concentration at which the response index falls outside its defined limits, to the minimum detectable concentration or sensitivity. The linear dynamic range is important when the components of a mixture being analyzed cover a wide concentration range. 

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