Isocratic HPLC elution

Mobile phase storage The figure shows four reservoirs, one for each of up to four pure solvents [e.g., water (perhaps with pH controUing buffer), acetonitrile, methanol, tetrahydrofuran, etc.]. Note the use of inlet filters. Alternatively one could prepare the mobile phase mixture to the desired composition manually, and store it in a single reservoir. Operation at a single, constant mobile phase composition is called isocratic HPLC elution. 

Bernhard et al., on the other hand, selected 5-fim spherical Nucleosil-NH2 as the stationary phase (43). In order to improve the resolution of the early-eluting components and to reduce the overall time needed per analysis, they set up an isocratic HPLC system, including two separate columns. By means of a switch valve, the second 175-mm column could be excluded after the elution of PC, SPH, LPC, /V-methyl PE, PG, and PE. This enabled the rapid elution of the acidic phospholipids PI and PS from the 50-mm column and hence significantly decreased the total run time (Fig. 3). The mobile phase contained 1460 ml A, 500 ml M, 30 ml W, and 600 jj, 1... 

Figure 9.67 Isocratic HPLC separation and UV absorption detection of UMP, UDP-Gal, and UDP as sequentially eluted. A mixture containing 500 pmol of each compound was injected. (From Hymes and Mullinax, 1984.)...

Figure 9.140 HPLC elution profile of a sample taken from a discontinuous assay of crude mouse spleen extract, using iV,-fluorenylmethoxycarbonyl-EEY(P)AA [Fmoc-EEY(P)AA] as substrate. Chromatography was performed on a C g Novapak column (10 cm X 8 mm) eluted isocratically with 36% acetonitrile-water (0.1% TFA) (v/v) at a flow rate of 2 mL/min. Peaks are due to (A) methanol and methanol-soluble compounds derived from the sample of crude homogenate, (B) Fmoc-EEY(P)AA (1238 pmol), and (C) Fmoc-EEYAA (195 pmol). Appropriate controls showed that no interfering compounds eluted in the position of the peptides. Inset Fluorescence monitoring of HPLC of Fmoc-EEYAA (75 fmol) eluted isocratically with 36% acetonitrile-water (0.1% TFA). Excitation and emission wavelengths, 268 and 307 nm, respectively, with gain X 100 and 10 mV chart scale. (From Nash et al., 1993.)...

The HPLC enzyme assay method provides an alternative procedure with which to approach this problem. Clearly, to be able to assay these activities by the HPLC method, it is necessary to separate ATP, ADP, and AMP. This separation can be easily accomplished by ion-exchange HPLC eluted isocratically with a mobile phase containing a phosphate buffer and sufficient concentration of salt to elute the ATP. Under these conditions, the order of elution of the compounds would be AMP first, ADP next, and ATP last. 

Isocratic HPLC systems utilise a single mobile phase and are often preferred on the basis of cost, convenience and improved detector responses. This is especially true for refractive index or electrochemical detectors where the use of gradient systems is usually precluded. Additionally, isocratic elutions do not require column re-equilibration after each run, providing savings both in time and solvent. 

Most chromatographers have a good understanding of the basis and application of isocratic HPLC. Workers in the life sciences have adapted these principles to the special requirements of their individual samples, as described elsewhere in this book. Separations that use gradient elution, however, are inherently more complicated. All the variables present in isocratic elution... 

PLA was determined by reversed-phase isocratic HPLC. The HPLC system consisted of a JASCO (Tokyo, Japan) AS-2055 autosampler, a PU-2080 pump, a CO-2060 column thermostat, an FP-2025 fluorescence detector and 807-IT integrator. The column used was of Cosmosil 5C18MS-II column (250 cm X 4.6 cm Nacalai Tesque, Kyoto, Japan). The mobile phase buffer was 20 mM potassium phosphate, pH 7.0, containing 10% (v/v) methanol. The flow rate, sample volume and column temperature were 0.5mL/min, 100 pL and 20 °C, respectively. The fluorescence intensity of the eluted 4-PLA was monitored at 430 nm (excitation at 360 nm). 

The concentration of the individual surfactants in the aqueous samples from the columns was measured by using isocratic HPLC equipped with a refractive index detector (30°C), and a RP-8 column (5 pm, 250 x 4 mm ID). Aqueous samples (20 pi) were injected either directly or after a preconcentration step on SPE octadecyl (Cl8) disposable columns and then eluted with acetonitrile/water (80/20). 

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