Refractive index detectors experimental conditions

Fig. 4.3. High performance liquid chromatography (HPLC) of the monosaccharides obtained from a partially purified preparation of microbubble glycopeptide surfactant from forest soil. Following hydrolysis (in 2 N HC1 for 6 hr at 100°C) and filtration, the carbohydrate mixture was charged on a Bio-Rad HPX-87 cation exchange column. For comparison, part A shows the chromatogram (using the same HPLC column) of a standard solution, which contained 4 pg of each of three different monosaccharides (i.e., the last three peaks shown are glucose, xylose and fiicose, in the order of increasing retention times). Part B shows the chromatogram obtained from hydrolysis of the partially purified (see text) microbubble surfactant (approximately 30 pg). All other experimental conditions were identical in the two cases, i.e., water eluent, 0.5 ml/min flow rate, 85°C, refractive index detector attenuation -2x. (Taken from ref. 322.)...

Experimental. The differential refractive indexes of polymer solutions were measured at 25°C with a Waters Scientific R-403 differential refractometer connected on-line with a size exclusion chromatograph. The refractometer was calibrated to refractive index units (Riu) with benzene/carbon tetrachloride solutions. The rationale behind using the refractometer on-line with the chromatograph is the elimination of impurities in the sample (water, residual monomer etc.) which affect the refractive index measurements particularly at low polymer concentrations and to calibrate the detectors at the flow conditions at which they were normally operated. Polymer solutions of several concentrations (0.015-0.0025 wt %) were injected repeatedly to verify the reproducibility of the measurements, which was typically An 0.5 x 10-6 for replicates on the same solutions. 

For a common SEC system with one column of (250 to 300) mm length and (7.5 to 10) mm diameter, typical v, is (50 -100) pL and c. equals 1-5 mg.mL. c. should be decreased as M rises over 100 kg.mol , and especially over 1,000 kg.mol. Correspondingly, v. is to be increased. Generally, c. is to be raised for broad molar mass distributed samples. The latter specifications are only approximate and the actual optimum experimental conditions depend mainly on the sample detectability. For example, in the case of refractometric detector, sample detectabihty is dictated by the refractive index increment, dn/dc for the given polymer in eluent. In aity case, the c and v should be kept as low as possible. It is useful to start preliminary experiments with a higher sample concentration and to decrease it gradually down to the experimentally bearable limit, which is mainly dependent on the base line stability of chromatogram. 

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