Columns refractive index

Deng YZ, Li BC. On-column refractive-index detection based on retroreflected beam interference for capillary electrophoresis. App/ierf Optics 37, 998-1005, 1998. 

Differential refractometer detectors measure the difference between the refractive index of the mobile phase alone and that of the mobile phase containing chromatographed compounds as it emerges from the column. Refractive index detectors are used to detect non-UV absorbing compounds, but they are less sensitive than UV detectors. They are sensitive to small changes in solvent composition, flow rate, and temperature, so that a reference column may be required to obtain a satisfactory baseline. 

Stearic and 12-hydroxy stearic acids were extracted from lubricating greases and then separated on a C[g column (refractive index detector) using a 70/30 THF/water (0.1% TFA) mobile phase [823]. Elution was complete in <8 min. Four grease samples were analyzed and the chromatograms were shown. The resulting sample contents (3-13% by weight) were tabulated. 

In LC, the most common means for monitoring the eluant is to pass it through a cell connected into an ultraviolet spectrometer. As substances elute from the column, their ultraviolet absorption is measured and recorded. Alternatively, the refractive index of the eluant is monitored since it varies from the value for a pure solvent when it contains organics from the column. 

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

Chromatographic conditions elution with 50 50 methanol/water solvent at the rate of 1.5 ml,/min through a DuPont Zorbax ODS column using a Waters R-401 Refractive Index Detector. 

Another classification of detector is the bulk-property detector, one that measures a change in some overall property of the system of mobile phase plus sample. The most commonly used bulk-property detector is the refractive-index (RI) detector. The RI detector, the closest thing to a universal detector in lc, monitors the difference between the refractive index of the effluent from the column and pure solvent. These detectors are not very good for detection of materials at low concentrations. Moreover, they are sensitive to fluctuations in temperature. 

Bromine (128 g., 0.80 mole) is added dropwise to the well-stirred mixture over a period of 40 minutes (Note 4). After all the bromine has been added, the molten mixture is stirred at 80-85° on a steam bath for 1 hour, or until it solidifies if that happens first (Note 5). The complex is added in portions to a well-stirred mixture of 1.3 1. of cracked ice and 100 ml. of concentrated hydrochloric acid in a 2-1. beaker (Note 6). Part of the cold aqueous layer is added to the reaction flask to decompose whatever part of the reaction mixture remains there, and the resulting mixture is added to the beaker. The dark oil that settles out is extracted from the mixture with four 150-ml. portions of ether. The extracts are combined, washed consecutively with 100 ml. of water and 100 ml. of 5% aqueous sodium bicarbonate solution, dried with anhydrous sodium sulfate, and transferred to a short-necked distillation flask. The ether is removed by distillation at atmospheric pressure, and crude 3-bromo-acetophenone is stripped from a few grams of heavy dark residue by distillation at reduced pressure. The colorless distillate is carefully fractionated in a column 20 cm. long and 1.5 cm. in diameter that is filled with Carborundum or Heli-Pak filling. 4 hc combined middle fractions of constant refractive index are taken as 3-l)romoaccto])lu iu)nc weight, 94 -100 g. (70-75%) l).p. 75 76°/0.5 mm. tif 1.57,38 1.5742 m.]). 7 8° (Notes 7 and 8). 

Degassed and preswelled Bio-Gel P-6 and Sephacryl S-200 were packed in self-made glass columns (70 X 1.5 cm 140 X 1.5 cm) and equilibrated for 20 hr with H20(dest.) -t- 0.002% NaN3 to prevent microbial growth. The mass of eluted fractions was detected with a differential refractive index detector (Waters 403 RI, sensitivity 8). 

Column set 2 TosoHaas 30 cm X 7.8 mm PW series Detection Refractive index... 

These combined HDF and GPC separations require the use of detectors such as static light scattering or viscometers to help sort out the convoluted elution profiles seen in those type of experiments. It should also be remembered in these situations that the typical refractive index or ultraviolet detector responses may not be representative of the actual mass fraction of insolubles eluting from the column because of the significant light scattering that can occur with those large particles in the detector cell. 

For acrylate polymers with higher levels of carboxylic acids, THF can be modified by the addition of acids such as acetic, phosphoric, or trifluoroacetic. Levels as high as 10% acetic acid are considered acceptable by most manufacturers for their styrene/DVB columns. If such a modified mobile phase is used, it may need to be premixed rather than generated using a dynamic mixing HPLC pump because on-line mixing often leads to much noisier baselines, particularly when using a refractive index detector. 

Synthetic, nonionic polymers generally elute with little or no adsorption on TSK-PW columns. Characterization of these polymers has been demonstrated successfully using four types of on-line detectors. These include differential refractive index (DRI), differential viscometry (DV), FALLS, and MALLS detection (4-8). Absolute molecular weight, root mean square (RMS) radius of gyration, conformational coefficients, and intrinsic viscosity distributions have... 

Figure 14.17 Schematic diagram of the on-line coupled LC-GC system VI, valve foi switcliing the LC column outlet to the GC injector V2, valve for switching the LC column to back-flush mode V3, LC injection valve RI, refractive index monitor detector UV, ulti avio-let monitor detector FID, flame-ionization detector.

Refractive index detectors. These bulk property detectors are based on the change of refractive index of the eluant from the column with respect to pure mobile phase. Although they are widely used, the refractive index detectors suffer from several disadvantages — lack of high sensitivity, lack of suitability for gradient elution, and the need for strict temperature control ( + 0.001 °C) to operate at their highest sensitivity. A pulseless pump, or a reciprocating pump equipped with a pulse dampener, must also be employed. The effect of these limitations may to some extent be overcome by the use of differential systems in which the column eluant is compared with a reference flow of pure mobile phase. The two chief types of RI detector are as follows. 

The deflection refractometer (Fig. 8.4), which measures the deflection of a beam of monochromatic light by a double prism in which the reference and sample cells are separated by a diagonal glass divide. When both cells contain solvent of the same composition, no deflection of the light beam occurs if, however, the composition of the column mobile phase is changed because of the presence of a solute, then the altered refractive index causes the beam to be deflected. The magnitude of this deflection is dependent on the concentration of the solute in the mobile phase. 

Water hydrolyzes diethyl phosphorochloridate [chloro-diethoxyphosphorus(V) oxide] readily but does not affect the diene. Alternatively, the reaction mixture can be processed by fractionation. Evaporation of the petroleum ether and fractionation of the residue through a 25-cm. x 2.2-cm. column of glass helices yields 170 g. (98.5%) of diethyl phosphorochloridate, b.p. 34-36°/0.2 mm., d 1.4210-1.4250 (the refractive index indicates that it contains 5-10% of the title compound), and 240-255 g. (90-96%) of l,2,3,4,5-pentachloro-5-ethylcyclo-pentadiene, b.p. 51-53°/0.2 mm., 1.5398. 

The separation was carried out on a TSKgel Amide-80 column 4.6 mm i.d. and 25 cm long with a mobile phase consisting of a 80% acetonitrile 20% water mixture. The flow rate was 1 ml/min and the column was operated at an elevated temperature of 80°C. The saccharides shown were 1/ rhamnose, 2/ fucose, 3/ xylose, 4/ fructose, 5/ mannose, 6/ glucose, 7/ sucrose and 8/ maltose. The analysis was completed in less than 20 minutes. These types of separations including other biomonomers, dimers and polymers are frequently carried out employing refractive index detection. 

The molecular weight distribution of cell wall polysaccharides was estimated by gel filtration with a TOSOH TSK gel G4000 PWXL (7.8 x 300 mm) column equilibrated and eluted with 0.05 M sodium acetate, 0.01 M EDTA, 0.05 M NaCl (pH 5.0) in polyuronide and 0.05 M sodium citrate, 0.1 M NaCl (pH 5.5) in the hemicellulose fraction. Samples (1 mg/ml) of 100 ml were injected. The eluate was monitored by a refractive index detector (Shimadzu R1D-6A, Kyoto, Japan) and collected at the fraction size of 0.4 ml. 

In this study, four Styragel columns were utilized one column had a nominal porosity rating of 10, two colvtmns of 10, and the fourth column of 10 A. The refractometer was maintained at 37°C. A 5 ml syphon was used to monitor a solvent flow rate of 1 ml/min. The instrviment was run at the highest sensitivity setting because the refractive index difference between our solvent and polymer was only moderate and because a number of samples analyzed had a broad molecular weight distribution (MWD). 

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