Isocratic and Gradient Elution

Gradient linear from 22% to 73% B in 16 min. Peaks (1) fluoride. (2) acetate, (3) formate. (4) chlorite, (5) bromate, (6) chloride, (7) carbonate. (8) nitrite, (9) bromide, (10) chlorate, 

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Dias, N. C., Nawas, M. I., Poole, C. F. Evaluation of a reversed-phase column (Supelcosil LC-ABZ) under isocratic and gradient elution conditions for estimating octanol-water partition coeffidents. 

Schellinger, A. P., Carr, P. W. Isocratic and gradient elution chromatography a comparison in terms of speed, retention reprodudbility and quantitation. /. Chromatogr. A 2006, 1109, 253-255. 

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. 

The conceptual basis for understanding the connection between isocratic and gradient elution is well established and is called "linear solvent strength theory".22 27 Linear solvent strength theory proposes that, for a given solute, mobile phase, and column, if one measures the retention time of an analyte at two organic component concentrations, it will be possible to predict the retention time with any other mobile phase composition. The k value that would be observed in pure water, kw, is related to the actual k by the relationship... 

The mobile phases used to provide separations that interface cleanly with the MS are of great importance. Both isocratic and gradient elution can be used. High purity (HPLC grade) water, acetonitrile, and Ci to C4 alcohols are compatible with APTelectrospray and APCI. Less polar solvents such as hexane, cyclohexane, toluene, and ethyl acetate are also compatible with APCI. In general, it is advisable to always have an organic solvent present in the mobile phase to reduce surface tension, which enhances the formation of smaller, more uniform droplets and also aids vaporization and ionization and hence provides greater sensitivity. 

Kutter, J. P., Jacobson, S. C., and Ramsey, J. M. (1997). Integrated microchip device with electrokinetically controlled solvent mixing for isocratic and gradient elution in micellar electrokinetic chromatography. Anal. Chem. 69, 5165-5171. 

HAAs HPLC with (i) UV detection at 263 nm (ii) ED at 950 mV (iii) fluori-metric detection at 450 nm (Aex = 360 nm). LiChrosorb RP-Select B column. Mobile phase NH4 (acetate) (pH = 4.5), MeOH, ACN in isocratic and gradient elution. Food flavors, meat extract Cleaning up using the Gross method (94). The spray-dried product was Soxhlet extracted, placed on Kie-selgur, and extracted with ethyl ether. Cleaning up by affinity chromatography on Cu-phthalocya-nine complex. 201... 

Workers at Perkin-Elmer [34] have studied the high-speed separation of PAHs using Cis-bonded phase packings (5pm particles) using both isocratic and gradient elution. The analysis of several PAH standards was performed using the 5 pm bonded phase column with gradient elution... 

Recently, Alexander et al. [44] reported the use of an automated separation system developed for micro-LC and CEC using both isocratic and gradient elutions. The complete system is shown in Fig. 2.15. An enlarged view of the coupling of the column to the injection valve presents Fig. 2.16. The mobile phase was delivered by two micro-LC pumps at a flow rate of 30 pL/min to a post injection splitter that houses the column inlet. In the CEC mode, pressure was not applied (no restriction on... 

Fig. 2.15. Schematic automated isocratic and gradient elution nemo-liquid chromatograph/ capillary electrochromatograph according Alexander et al. (reproduced from Ref. [44] with permission of the publisher). 1, high-voltage power supply (negative polarity) 2, platinum electrode 3, outlet reservoir vial 4, UV detector with on-column flow cell 5, nanocolumn 6, two-position switching valve 7, jack stand 8, fused-silica make-up adapter (split device) 9, ground cable 10, internal loop micro-injection valve 11, plexiglas compartment 12, autosampler 13, dynamic mixer 14, micro-LC pumps.

Fig. 8.2. Schematic illustration of a CEC instrument capable of isocratic and gradient elution and ESI-MS detection.

Separations of complex steroid mixtures were achieved recently by Que et al. [76] using both isocratic and gradient elution. Mass spectrometric detection gave femto-mole detection limits while laser-induced fluorescence of dansylated ketosteroids ranged in attomole levels (Fig. 10.16). Monolithic column packings were used with a 35 cm (25 cm packed bed) x 100 pm i.d. capillary packed with a polymer prepared from 5% T (total monomer concentration), 60% C (total crosslinker concentration), 3% polyethylene glycol, 10% vinylsulfonic acid and 15% lauryl acrylate. Details of the monolithic column preparation can be found in refs. 36,76, and 193. Similar monolithic columns can be used for the separation of bile acids [194],... 

As noted, the retention of a polypeptide or protein with HP-IEX sorbents primarily arises from electrostatic interactions between the ionized surface of the polypeptide or protein and the charged surface of the HPLC sorbent. Various theoretical models based on empirical relationships or thermodynamic considerations have been used to describe polypeptide and protein retention, and the involvement of the different ions, in HP-IEC under isocratic and gradient elution conditions (cf. Refs.6,19 33 40,78-90). Over a limited range of ionic strength conditions, the following empirical dependencies derived from the stoichiometric retention model can be used to describe the isocratic and gradient elution relationships between the capacity factor In and the corresponding salt concentration [C,] or the median capacity factor In k ex, and the median salt concentration [C,] of a polypeptide or protein solute, namely,... 

The separation of the substrate testosterone from the numerous reaction products was accomplished by reversed-phase HPLC on a Ci8 column preceded by a guard column. The compounds were separated by a combined isocratic and gradient elution procedure. The eluent was monitored at 240 nm. The separations obtained with these HPLC conditions are shown in Figure 9.127. 

Reverse phase liquid chromatography has typically been used for the separation of PFCs, employing either Cg or Cig columns [96], although the use of perfluoroalkyl columns has also been reported [115]. Mobile phases are typically mixtures of methanol-water or acetonitrile-water and are often modified with ammonium acetate to improve chromatographic separation and MS sensitivity. Both isocratic and gradient elution methodologies have been employed [96]. LC-MS/MS methods [116, 117] have also been developed for the separation of PFSA and PFCA isomers and generally employ linear perfluorooctyl stationary phases and acidified mobile phases. 

In this context it should further be pointed out that a commercial method development computer software package (DRYLAB) has been successfully applied to optimize enantiomeric separations of a multi-component amino acid sample utilizing isocratic and gradient elution techniques 389. ... 

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