Refractive index RI detection

Refractive index detection is the second most popular non-destructive method used in HPLC. RI detection utilises the principle of the change in direction of a beam of light as it passes through different matrices. Different methods have been used to monitor this change. 

---

Detection in 2DLC is the same as encountered in one-dimensional HPLC. A variety of detectors are presented in Table 5.2. The choice of detector is dependent on the molecule being detected, the problem being solved, and the separation mode used for the second dimension. If MS detection is utilized, then volatile buffers are typically used in the second-dimension separation. Ultraviolet detection is used for peptides, proteins, and any molecules that contain an appropriate chromophore. Evaporative light scattering detection has become popular for the analysis of polymers and surfactants that do not contain UV chromophores. Refractive index (RI) detection is generally used with size exclusion chromatography for the analysis of polymers. 

All reactions were run in a Parr stirred autoclave (Model 4561) at 1000 psi H2 and 200°C for 6h. A weighed quantity (0.5 g dry basis) of the catalyst, Ru/C or Ni/Re/C, was introduced into the reactor and reduced at 250 C and 200 psi H2 (Ru/C) or at 280°C and 500 psi H2 (Ni/Re/C) for 13 hours. After cooling, 100ml of solution (l.OM GO and O.l-l.OM KOH) was added to the closed reactor. For reactions in solvent mixtures, entries 1-4 in Table 2, the water/solvent ratio was 1/9 (v/v). When the solvent was either t-BuOH or 1,4-dioxane, 1.5 g of water was added to the solution to facilitate dissolution of KOH, because of its low solubility these solvents. Once steady state was achieved following heatup, samples were taken at 30 minute intervals for the first hour, and then hourly, and analyzed via HPLC. The HPLC column was a BIORAD Aminex HPX-87H run at 65 C with 5mM H2SO4 as the mobile phase at a flow rate of 0.6mFmin, using both UV (210 nm) and refractive index (RI) detection. 

Refractive index (RI) detection is based on the principle that a light beam is refracted differently depending on the substance through which passes. A beam passing through a solu-... 

A nonaqueous reversed-phase high-performance liquid chromatography (NARP-HPLC) with refractive index (RI) detection was described and used for palm olein and its fractions obtained at 12.5°C for 12-24 h by Swe et al. (101). The objective of their research was to find the optimum separation for analysis of palm olein triglycerides by NARP-HPLC, and to find a correction factor to be used in calculating CN and fatty acid composition (FAC). The NARP-HPLC method used to determine the triglyceride composition was modified from the method of Dong DiCesare (88). Palm olein was melted completely at 70°C in an oven for 30 min prior to crystal-... 

The second method, using refractive index (RI) detection, is carried out using a resin-based polymer column. Sucrose elutes first from this column, followed by glucose, fructose and then sorbitol. This type of column is generally more robust than the amino-bonded column and if handled well will last much longer however, it is around three times more expensive. The method has been collaboratively tested for the analysis of sugars and sorbitol in fruit juices by the IFU. The HPLC conditions are given below. 

Another approach is the combination of SEC with multiple concentration detectors. If the response factors of the detectors for the components of the polymer are sufficiently different, the chemical composition of each slice of the elution curve can be determined from the detector signals. Typically, a combination of ultraviolet (UV) and refractive index (RI) detection is used another possibility is the use of a diode-array detector. In the case of non-UV-absorbing polymers, a combination of RI and density detection yields information on chemical composition [29-31]. 

With the major constituents in foods the choice of LC detector is often the most important issue. Compounds such as vitamins, carbohydrates etc. may not have a strong ultraviolet (UV) chromophore. Therefore refractive index (RI) detection and, increasingly, electrochemical detection are often used. As discussed later, the choice of detector is even more important when determining the concentration of components in the foodstuff rather than the bulk constituent. 

More recently, a less sensitive detection technique, refractive index (RI) detection, has been used. The disadvantage of RI is that one is limited to isocratic elution, because RI detection is affected by changes in the pressure and temperature of the mobile phase. On the other hand, infrared (IR) detection can be used when the solvents in the mobile phase do not absorb infrared light, which can create interference. Because of these limitations, RI and IR have not been used as widely as UV detection. 

Ginkgolide A, B, C, and bilobalide in G. biloba leaves were determined simultaneously by RP-HPLC-ELSD. Methanolic extracts (10%) of the leaves were cleaned up by solid phase extraction via polyamide cartridge and silica gel cartridge, successively. RP-HPLC was carried out on a Cl 8 column with Me0H-H20 as mobile phase, eluted in gradient mode, and detected by ELSD. The poor linear response of ELSD could be compensated by multilevel calibration and logarithmic calculation. Meth-anol-water-orthophosphoric acid mobile phase was used in conjunction with refractive index (RI) detection. 

Ultraviolet (UV) spectroscopy, mass spectrometry (MS), refractive index (RI) detection, and electrochemical detection (ECD) are common online monitoring techniques for analytical chromatography. UV and RI are regularly used for monitoring preparative operations as well. To employ MS or ECD in a high-flow scheme, usually a side stream must be diverted from the main eluate line via a flow splitter so that what passes through the detector has a flow rate that is acceptable for an analytical-scale system. 

HPLC, using a reversed phase system consisting of a Zorbax NH2 column eluted with acetonitrile/water (4 1), was employed for the preparative separation of the calystegines [10], The lack of a chromophore renders the use of UV or fluorescence detection impossible without pre- or post-derivatization of the sample and the relatively low sensitivity of refractive index (RI) detection severely limits this technique for routine analysis. However, the rapid evolution of LC-MS methods may lead to their application in the future. 

In order to compare the results of critical chromatography with results of an independent method, SEC with coupled density (D) and refractive index (RI) detection was used, which has been shown to be very useful for the characterization of copolymers with respect to their chemical composition [39,40]. The MMD curve for one of the block copolymers and the mass distribution curves of the components are shown in Fig. 18. From these the overall chemical composition may be calculated. An excellent agreement between the results of critical chromatography and the SEC experiments was obtained. 

UV detection of compounds having very low values around the nonselective wavelength of 210-220 nm is a poor choice and a much better approach is, therefore, a detector that shows less variation in response factors, e.g., refractive index (RI) detection or evaporative light scattering (ELS) detection. Examples of UV/RI and UV/ELS detection of ginkolides are given in Figures 5 and 6, respectively. 

For compounds that lack a UV-vis chromophore, refractive index (RI) detection is a common substitute. An RI detector measures the difference in refractive index between the eluant and a reference cell filled with the elution solvent. Refractive index detection is significantly less sensitive than UV-vis detection, and the detector is quite sensitive to temperature changes during the chromatographic run. 

Determination of molar masses and polydispersity of /polymers is often complicated because of the limited solubility particularly of highly fluorinated polymers. To avoid different conditions for the polymers and to ensure a certain comparison, SEC with a mixture of pentafluorophenol (PFP)/chloroform (1/3 vol/vol) as eluent (even if some of the samples were soluble in chloroform or tetrahydrofurane, THF), refractive index (RI) detection, and PMMA standards for calibration and determination of relative molar masses was employed. For some samples, molar masses could not be detected due to isorefractive behavior (no signal due to same RI of sample and eluent). This was observed in the series for P(MMA-co-sfMA-H10F10) at 20-25 mol% sfMA-HlOFlO and for P(MMA-co-sfMA-H2F8) at 20-30 mol% sfMA-H2F8. The chemical characterization of selected copolymers used for the present study is given in Table 11.2. 

Finally, size-exclusion chromatography coupled with more than one detector, such as laser light-scattering (LS) and refractive-index (RI) detection, provides an excellent approach for determination of the molecular weight (MW) of proteins [10]. 

Another approach to the determination of aromatics in middle distillates is high performance liquid chromatography, (HPLC), with refractive index (RI) detection [2]. The Institute of Petroleum has standardized this technique as IP-391. ASTM is currently considering this method and may adopt it as a standard. 

---