The Isocratic System

This consists of a solvent delivery for isocratic reversed phase and gel filtration chromatography. 

This isocratic system provides an economic first step into HPLC techniques. The system is huilt around a high-performance, dual-piston, pulse-free pump providing precision flow from 0.01 to 5 ml/min. 

Any of the following detectors can be used with this system  

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Another point of interest was the time required to equilibrate the system after changes were made in solvent composition. While the ChromSpher Lipids column had a column volume of ca. 3 ml, an increase in ACN concentration was not noted until the introduction of 7-8 ml of solvent (determined with refractive index detector). The problem of ACN-silver ion interaction and subsequent ACN retention is not new and may be noted in all forms of chromatography employing silver ions in the stationary phase. In the isocratic system, the column was equilibrated with the appropriate solvent mix for at least 0.5 h before sample injection. Since ACN dissolves very slowly into hexane, the ACN-hexane solvent mix was thoroughly stirred for 5 min before use. To obtain reproducible retention times, thorough mixing of the ACN and hexane is essential. 

This is a simple upgrade of the isocratic system with the facility for gradient elution techniques and greater functionality (Fig. 1.1(b)). The basic system provides for manual operating gradient techniques such as reversed phase, ion exchange and hydrophobic interaction chromatography. Any of the detectors listed above under the isocratic system can be used. 

The isocratic systems used in this study do succeed in characterizing the components of most natural dye samples. Even a sample that elutes with the solvent or remains on the column provides a clue to its identity and an indication of its relative polarity. Yet, when gradient elution is available, even better separations can be achieved. 

Four basic types of elution are used in HPLC, namely, the isocratic system, the basic gradient system, the inert system and the advanced gradient system (see Figure 1.1). The most commonly used detectors are those based on spectroscopy in the region 185-400 nm, visible-ultraviolet (UV) spectroscopy in the region 185-900 nm, post-column derivatisation with fluorescence detection (see next), conductivity [7] and multiple wavelength UV detectors using a diode array system detector (see next). Other types of detectors available are those based on electrochemical principles, refractive index, differential viscosity, and mass detection [8]. 

This consists of a solvent delivery for isocratic reversed phase and gel filtration chromatography. The isocratic system (Fig. 1.1) provides an economic first step into high performance liquid chromatography techniques. The system is built around a high performance, dual-piston, pulse-free pump providing precision flow from 0.01 to 5mL min Any of the following detectors can be used with this system ... 

A high performance Hquid chromotography (hplc) method to determine citric acid and other organic acids has been developed (46). The method is an isocratic system using sulfuric acid to elute organic acids onto a specific hplc column. The method is sensitive for citric acid down to ppm levels and is capable of quantifying citric acid in clear aqueous systems. 

Kilpatrick, L Jones, M. and Phillipson, O. A semiautomated analysis method for catecholamines, indoleamines, and some prominent metabolites in microdissected regions of the nervous system An isocratic HPLC technique employing eoulometric detection and minimal sample preparation. J Neurochem 46(6) 1865-1876, 1986. 

The isocratic reversed phase solvent system consists of water (polarity, p = 10.2), the most polar solvent in RPLC, as a primary solvent to which water-miscible organic solvents such as methanol (p = 5.1), acetonitrile (p = 5.8), or tetrahydrofuran (p = 4.0) are added. In order to optimize the speed of separation for an analyte pair, the proportions of water to nonpolar solvent are chosen such that the capacity factor of the last-eluting analyte of interest has a value of about 2.13... 

Liquid chromatography has a number of different configurations with regard to technical (instrumental) as well as separation modes. The HPLC system can be operated in either isocratic mode, i.e. the same mobile phase composition throughout the chromatographic ran, or by gradient elution (GE), i.e. the mobile phase composition varies with run time. The choice of operation... 

In chromatography, the separation efficiency of any single separation method is limited by the efficiency and selectivity of the separation mode, that is, the plate count of the column and the phase of the selected system. Adding more columns will not overcome the need to identify more components in a complex sample, due to the limitation of peak capacities. The peak capacity in an isocratic separation can be described, following Grushka (1970), as given in Equation (17.1) ... 

Some LC/MS users adhere to isocratic separation because of the myths around gradient elution (it is complex to develop and transfer between instruments and laboratories, it is inherently slower than isocratic methods because of re-equilibration, and other reasons summarized by Carr and Schelling6). A researcher may have a very good reason to use an isocratic method, for example, for a well defined mixture containing only a few compounds. The isocratic method would certainly not be useful in an open access LC/MS system processing varying samples from injection to injection. 

Natural extracts generally contain molecules with highly different retention characteristics which cannot be separated under isocratic conditions. The application of gradient elution is a necessity for these types of natural samples. However, the optimization of gradient elution on the base of isocratic data is cumbersome and the prediction of retention in gradient elution from isocratic data is difficult. Retention in an isocratic system can be described by a polynomial function ... 

The method has been proposed for the prediction of retention data in isocratic systems from data measured in gradient elution and vice versa [84], Similar calculation methods may be very important in the analysis of natural extracts containing pigments with highly different chemical structure and retention characteristics. The calculations make possible the rational design of optimal separation conditions with a minimal number of experimental runs. 

RP-HPLC has also been used for the analysis of flavan-3-ols and theaflavins during the study of the oxidation of flavan-3-ols in an immobilized enzyme system. Powdered tea leaves (20Qmg) were extracted with 3 X 5 ml of 70 per cent aqueous methanol at 70°C for lQmin. The combined supernatants were filtered and used for HPLC analysis. Flavan-3-ols were separated in a phenyl hexyl column (250 X 4.6 mm i.d. particle size 5 /im) at 30°C. Solvents A and B were 2 per cent acetic acid in ACN and 2 per cent acetic acid in water, respectively. Gradient elution was 0-lQmin, 95 per cent B 10-4Qmin, to 82 per cent B to 40-5Qmin 82 per cent B. The flow rate was 1 ml/min. Theaflavins were determined in an ODS column (100 X 4.6 mm i.d. particle size 3pm) at 30°C. The flow rate was 1.8 ml/min and solvent B was the isocratic mobile phase. The data demonstrated that flavan-3-ols disappear during the oxidation process while the amount of theaflavins with different chemical structures increases [177],... 

The decolourization of the azo dye amaranth was also investigated using atomic hydrogen permeating through a Pt-modified palladized Pd sheet electrode. The decolouration products were separated by RP-HPLC in an ODS column. The isocratic mobile phase was 0.1 M aqueous orthophosphoric acid. The flow rate was 1.2 X 10 2 cm3/s and decomposition products were detected at 236 nm. The RP-HPLC system separated two analytes with retention times of 3.4 and 4.5 min, as demonstrated in Fig. 3.47. The peaks were... 

A similar heterogeneous photocatalytic system was applied for the study of the decomposition of the anthraquinone dye, Acid blue 25 (AB25). The chemical structure of the dye and those of the first intermediates tentatively identified by HPLC-MS are shown in Fig. 3.55. RP-HPLC-DAD analysis of AB25 was carried out in a C4 column (250 X 4 mm i.d. particle size 5 //m) at ambient temperature. The isocratic mobile phase was composed of ACN (solvent A)-water (pH adjusted to 4.5 with acetic acid and ammonium acetate) (42 58, v/v). 

Zweigenbaum and co-workers [11] used high flow rates and an isocratic system using a Mac-Mod Rapid Resolution column (15 mm x 2.1 mm, 3 pm) to perform the fast separation of six benzodiazepines isolated from human urine using a 96-well liquid-liquid extraction (LLE). 

The HPLC system used a mobile phase consisting of CH3CN/H2O/HCOOH (20 80 1, v/ v/v). Quantitation of standards and samples was achieved by isocratic elution over a C18,5 pm, Econosil HPLC column at a flow rate of 0.4 mL min. HPLC retention volumes and detection wavelengths for standards were as follows vanrllyl alcohol, 1.7 mL and 277 nm vanillic acid, 2.5 mL and 260 nm and vanillin, 4.1 mL and 284 nm. 

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