Chromatography isocratic elution

Use of Automated Column Chromatography, Isocratic Elution and Differential Refractometry... 

The acceptable separation of Am(III) and Cm(III) by countercurrent chromatography (CCC) was achieved using both isocratic elution, and a new approach to the creation of the reagent concentration gradient in the stationary phase [1]. This way allows reduce the experiment length. 

A liquid chromatography-mass spectrometry (LC-MS) method that can quantitatively analyze urinar y normal and modified nucleosides in less than 30 min with a good resolution and sufficient sensitivity has been developed. Nineteen kinds of normal and modified nucleosides were determined in urine samples from 10 healthy persons and 18 breast cancer patients. Compounds were separ ated on a reverse phase Kromasil C18 column (2.1 mm I.D.) by isocratic elution mode using 20 mg/1 ammonium acetate - acetonitrile (97 3 % v/v) at 200 p.l/min. A higher sensitivity was obtained in positive atmospheric pressure chemical ionization mode APCI(-i-). 

Chromatography is essentially a method of separation based on two phases, one stationary and one mobile. If the composition of the mobile phase is not changed during the separation, the term isocratic elution is used. For separation of complex mixtures with wide ranges of polarities, the composition of the mobile phase can be changed during separation, a process known as gradient elution. 

MacNair, J.E., Patel, K.D., and Jorgenson, J.W., Ultrahigh-pressure reversed-phase capillary liquid chromatography isocratic and gradient elution using columns packed with 1.0 pm particles, Anal. Chem., 71, 700, 1999. 

Jandera, P. and Churacek, J., Gradient elution in liquid chromatography. I. The influence of the composition of the mobile phase on the capacity ratio (retention volume, band width, and resolution) in isocratic elution — theoretical considerations, /. Chromatogr., 91, 207, 1974. 

Procedure Flavonoids are then further purified with 2 ml of methanolic HC1 (2 N), followed by centrifugation (2 min, 15 600 g), hydrolyzation of 150 il of suspension in an autoclave (15 min, 120 C). A reverse osmosis-Millipore UF Plus water purification system is used in high performance liquid chromatography (HPLC) with an autosampler. After injections of 5 pg of samples, the mobile phases flow at a rate of 1 ml/minute with isocratic elution in a column at 30 C. 

The scale-up of chromatography operated in isocratic elution can be carried out as follows. 

The investigations mentioned so far aimed at objectives of rather low molar mass. Reversed-phase chromatography of polystyrenes with 17,500 and 50,000 g/mol was performed on C 18 columns with water/tetrahydrofuran gradients or mixtures3). The latter sample was isocratically eluted from 30 nm-pore packing with 85 % THF as a broad band, 87 % THF let the polymer elute in the void volume, whereas 83 % did not produce an observable band at all. 

FF Shih, AD Kalmer. Determination of glutamine and asparagine by isocratic elution reverse phase liquid chromatography with fluorescent detection. J Liq Chromatogr 7 1169-1183, 1984. 

For flavones in citrus peel oils, separations were accomplished with isocratic mobile phases of 38% and 40% acetonitrile in H20 (1). The extracts of peel and cold-pressed peel oils were diluted in ethanol and analyzed by reversed-phase on various C18 columns with good results. For the dilute citrus oils, gradient elution was preferred, to prevent the accumulation of terpenes on the column. With normal-phase chromatography, the elution order is reversed terpenes elute with the solvent front and are not a problem. 

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