Refractive index detector response

The differential refractive index detector response on the ordinate of the SEC chromatogram in Fig. 3-8 can be transformed into a weight fraction of total polymer while suitable calibration permits the translation of the elution volume axis into a logarithmic molecular weight scale. 

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... 

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). 

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. 

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. 

If the solute is assumed to be eluted at a (k ) of 1 from a column 15 cm long, 4.6 mm in diameter and packed with 5 m particles, then this would indicate a sensitivity in terms of concentration of approximately 2 x 10 g/ml, which is equivalent to the response of a very sensitive refractive index detector. The great advantage of the detector, however, is its catholic response and that its output is linearly related to the mass of solute present. 

The first practical refractive index detector was described by TiseUus and Claesson [1] in 1942 and, despite its limited sensitivity and its use being restricted to separations that are isocratically developed, it is stiU probably the fifth most popular detector in use today. Its survival has depended on its response, as it can be used to detect any substance that has a refractive index that differs from that of the mobile phase. It follows that it has value for monitoring the separation of such substances as aliphatic alcohols, acids, carbohydrates, and the many substances of biological origin that do not have ultraviolet (UV) chromophores, do not fluoresce, and are nonionic. 

The UV detector, fluorescence detector, electrolytic detector, refractive index detector, and other detection systems must be checked for repeatability of response signal, sensitivity, and stability. 

Nonselective detector A detector which gives a response (i.e., a change in current or voltage) upon a concentration change for any component. Ideally, the detector should be linear (i.e., its response should be proportional to the component concentration), and this proportionality coefficient, or response factor, should be the same for all components. There are no such detectors. The differential refraction index detector comes closest to being nonselective. 

Gel permeation chromatography (gpc) was performed on a Waters GPC-3 with a model 600 solvent delivery system, a 730 data module, a variable wavelength ultraviolet detector (uv), and a refractive index detector (Rl). Calculations were made on the uv detector response with the wavelength set at 325 nm. Three j/Styragel columns of porosities 105, 104, and 103 A were used and calibrated with polystyrene standards. Injection size was 50-125//I of 0.05% solutions with a flow rate of 1.4 ml/min. The solvent was HPLC grade N-methyl pyrrolidone (NMP) obtained from Aldrich Chemical Co. buffered with 0.03 M LiBr and 0.03 M H3P04.(2, 3)... 

The most commonly used detector is the refractive index detector (cf. Section 3.11.3), considered as universal since for polymers a variation of the refractive index is at first approximation independent of the molecular mass. As this detector is not very sensitive, other detectors are sometimes added to it. They are based upon the light absorption (UV detector) or fluorescence or light scattering (Figure 7.4). This last detector provides a more uniform response to structurally similar analytes than do light absorbtion detectors. Users can create a universal calibration set from a single analyte to quantify all analytes of the same class. 

The refractive index detector is applicable to all compounds, although it is not as sensitive as the uv detector. An RI detector can detect differences of about 10 RI units, which means that about 5x10 g/mL must pass through the detector for a favorable response. As a general rule, the sensitivity in mg of sample is almost equal to the reciprocal of the differences in refractive index between the solvent and the sample. Figures 19-43 and 19-44, show two basic methods of refractive index measurement. 

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