Refractive index gradient detection

Renn, C. N. and Synovec, R. E., Refractive index gradient detection of biopolymers separated by high-temperature liquid chromatography, /. Chromatogr., 536, 289, 1991. 

Hancock DO, Synovec RE. Microbore liquid-chromatography and refractive-index gradient detection of low-nanogram and low-ppm quantities of carbohydrates. Journal of Chromatography 464, 83-91, 1989. 

Costin, C.D., Synovec, R.E., A microscale-molecular weight sensor probing molecular diffusion between adjacent laminar flows by refractive index gradient detection. Anal. Chem. 2002, 74, 4558 1565. 

Determination of Potymer Molecular Weight by Flow Injection afysis and Refractive Index Gradient Detection... 

Figure 3 Instrument setup of whole-column imaging detection for CIEF. Absorption or refractive index gradient mode with camera (I) placed in the direction of illuminated light fluorescence mode with camera (II) placed vertically to the direction of illuminated light. (Reproduced from Mao, Q., Pawliszyn, J J. Biochem. Bio-phys. Meth. 39, 1999, 93-110, with permission of Elsevier Science Publishers.)...

Peptides are not as commonly analyzed by CIEF as are proteins one reason is their lower resolution, another their lower (or lack of) detectability at 280 nm (the wavelength mostly used). The separation of tryptic digests (peptide mapping) of proteins have been performed by using absorption detection at 280 nm and refractive index gradient imaging detection no exact correlations were observed between measured and calculated p7 values [1,5]. Refractive index detection is a universal detection method (i.e., independent of chromophores like tyrosine and tryptophan) but suffers from low sensitivity. Assays of trypsin activity have also been performed with laser-induced fluorescence detection for enhanced sensitivity, with detectability down to picomolal concentrations [5,6]. 

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. 

Adsorption chromatography using small particle silica or alumina has also been employed in the separation of biologically meaningful substances. Phospholipids, for example, have been separated on silica (38). One of the big problems for such substances is detection, since many of the compounds are not U.V. active. Generally, the refractive index detector is employed for isocratic operation, and the moving wire detector for gradient operation. Formation of U.V.-active derivatives is also possible (39). 

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