Isocratic systems

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. 

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

The isocratic system (Fig. 1.2 (a)) 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-1. 

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. 

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). 

Fig. 6.1.6 HPLC of the pterins standard mixture using an isocratic system. Bio biopterin, Ixa isoxanthopterin, Mon monapterin, Neo neopterin, Pte pterin...

Consider an HPLC method for the separation of 11 priority pollutant phenols using an isocratic system. The aqueous mobile phase contains acetic acid, methanol and citric acid. From preliminary studies, it was established that the mobile phase composition was critical to ensure maximum resolution and to minimise tailing. The overall response factor, CRF, was measured by summing the individual resolutions between pairs of peaks. Hence, the CRF will increase as analytical performance improves. 

High performance liquid chromatography (HPLC) apparatus (isocratic system) fitted with a 2-mL sample loop. 

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. 

Earlier work in the HPLC analysis of TGs used a differential refractometer as the detector a number of papers have detailed isocratic systems combined with refractive index (RI) detectors, often with acetonitrile/acetone mobile phases. Although aqueous mobile phases were generally used with alkyl-bonded phase columns, due to the lipophilicity of TGs, water could not be used in the mobile phase for this particular application therefore the mobile phases generally employed consisted of mixtures of acetone and acetonitrile and occasionally tetrahydrofuran, methylene chloride, or hexane (the conspicuous absence of water in the mobile phase prompted the term nonaqueous reverse phase, or NARP, to describe these systems). 

There are four basic system types. Type I are basic isocratic systems used for simple, routine analysis in a QA/QC environment often for fingerprinting mixtures or final product for impurity/yield checking. Type II systems are flexible research gradient systems used for methods development, complex gradients, and dial-mix isocratics for routine analysis and standards preparation. They fit the most common need for an HPLC system. Type III systems are fully automated, dedicated systems used for cost-per-test, round-the-clock analysis of a variety of gradient and isocratic samples typical of clinical and environmental analysis laboratories. Type TV systems are fully automated gra-... 

An isocratic system is used with single solvents, a premixed solvent mixture, or step gradients. It has the advantage of needing only a single pump, no mixer, and no gradient controller. Because of this, isocratic systems are simpler and... 

Figure 9.8 Gradient to parallel isocratic systems, (a) Gradient system (b) Parallel isocratic system.

The detector of choice for preparative work, the RI detector can be used only in isocratic systems. It is very sensitive to turbulence, temperature, or... 

Gradient systems let you control flow rate and solvent/buffer changes to improve chromatographic separations. They can be used to sharpen separations and to speed column re-equilibration. A four-solvent gradient system is useful for methods development when equipped with methanol, acetonitrile, ammonium acetate buffer, and formic acid solution. But, many quality control laboratories prefer to use inexpensive isocratic systems that run a constant-composition premixed mobile phase for rapid separations. 

Isocratic elution is commonly used for the elution of analytes from the column. In isocratic elution, the mobile phase is kept constant throughout the analysis. The mobile phase can be a single solvent or a solution of two or more miscible solvents. The major requirements of isocratic pumps are accuracy and smoothness of flow. Because the pump delivers only one solvent system, simple, inexpensive pulse dampeners and rudimentary flow or pressure feedback control circuits can be used. The basic setup of an isocratic system is illustrated in Figure 3.10. 

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. 

For the above reasons, 1,4-dioxane (UV cutoff of 220 nm) was substituted for isopropyl alcohol in the mobile phase. An isocratic system consisting of 0.4% 1,4-dioxane in heptane, flow rate of 120 ml/hr, a 10 micron silica gel column, and monitoring at 209 nm, proved to be satisfactory for separating I-IV. With this system 20 ng of I could be accurately quantitated but levels lower than that became a problem due to a poor signal to noise ratio. This poor signal to noise ratio was apparently due to the 220 nm cutoff of 1,4-... 

There are times when a sample will contain components that have a wide range of polarities. In this situation, the peaks will have a wide range of retention times in an isocratic system and, often, the early eluting peaks will be sharp and the late eluting peaks will be quite broad. The general disadvan-... 

Natural dyes are almost entirely napthoquinoid, anthraquinoid, or flavonoid compounds having one or more hydroxyls as the primary functional group. Considerable variation in structure occurs among natural dyes, and a single HPLC system that could separate all natural dye samples was not found. Nevertheless, several workable systems were found in which all dye components eluted except for indigo-carmine, which eluted with the solvent, and indigo and turmeric, which remained on the column. For an isocratic system, we consider this situation to be very satisfactory. 

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. 

Isocratic systems require no long equilibration times and thus are preferred in such analysis. 

Another means of controlling eluent strength is the use of ternary or quaternary solvent mixtures instead of the more common binary approach. Each solvent has its own unique properties that can be used to improve the separation of difficult-to-resolve analytes or to shorten the analysis time without sacrificing resolution. Although gradients and more complex solvent matrices are more difficult to model than binary isocratic systems, software exists for such purposes and can assist in method development. 

In a series of papers, Segal 1 and co-workers described various HPLC methods for the analysis of pyrrolizidine alkaloids in plant material. A cyano-type column in combination with tetrahydrofuran - 0.01 M ammonium carbonate was used (Fig.3.3) for the isolation and identification of some senecio alkaloids11,13 13,3 . A gradient of 13% tetrahydrofuran, increasing to 26%, gave good separations. Similar results could be obtained with an isocratic system containing 16% tetrahydrofuran. 

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