Isocratic column performance

Fig. 2.5.11. (a) APCI-LC-MS(+), (b) ESI-LC-MS(-t-), (c) ESI-LC-MS(+), (d) ESI-LC-MS(+), (e) APCI-LC-MS(—) and (f) ESI-LC-MS(—) reconstructed ion chromatograms (RIC) of methanolic solution of the household detergent mixture as in Fig. 2.5.2. Chromatographic conditions (a), (b), (e), and (f) RP-Cig, methanol/water gradient elution (c) ion-pairing RP-Cla using trifluoro acetic acid (TFA) (5 mmol), methanol/water gradient elution (d) isocratic elution performed on PLRP-column, eluent methanol/water methane...

First, we look at isocratic separations. Let us assume that the analysis can be accomplished within a retention factor of 10. We also suppose that the analysis is carried out with a typical reversed-phase solvent and a sample with a typical molecular weight of a pharmaceutical entity. In order to manipulate the analysis time, we will keep the mobile phase composition the same and vary the flow rate. The maximum backpressure that we will be able to apply is 25MPa (250 bar, 4000psi). In Figure 1, we have plotted the plate count as a function of the analysis time for a 5 J,m 15-cm column. We see that the column plate count is low at short analysis times and reaches a maximum at an analysis time of about 1 h. A further increase in analysis time is not useful, since the column plate count declines again. This is the point where longitudinal diffusion limits the column performance. The graph also stops at an analysis time of just under 5 min. This is the point when the maximum allowable pressure drop has been reached. 

The apparatus used for SEC is relatively simple isocratic high-performance liquid chromatography (HPLC) equipment, as shown in Figure 8. The principal components are a precision high-pressure solvent delivery system, detector, and a column with particles having appropriate pore size. Detection is conveniently accomplished with a UV photometer or spectro-... 

Song, D. Au, J.L.-S. Isocratic high-performance liquid chromatographic assay of taxol in biological fluids and tissues using automated column switching. J.Chromatogr.B, 1995, 663, 337-344... 

Copolymers with units of EO and units of PO are highly polar [79,80]. Some years ago, a group of researchers at University of Alberta (Canada) noted that the relative intensity of MALDI peaks [81] varies when laser fluence is varied and also when the matrix-to-analyte ratio is varied. More or less implicitly, they suggested fractionating EO-PO copolymers by LC before MALDI analysis. Following these indications, an EO-PO copolymer was injected [82] in an isocratic high performance Uquid chromatography (HPLC) column, in order to study the separation mechanism. The authors col-... 

The principles of how to operate an HPLC column under optimal isocratic conditions were outlined a long time ago by Guiochon and coworkers [4]. The maximum column performance at the lowest pressure is always achieved around the minimum of the van Deemter curve. If we reduce the particle size, the back-pressure increases - at equal velocity - inversely proportionally to the square of the particle diameter. At the same time, the velocity at the minimum of the van Deemter curve increases with decreasing particle diameter. Thus, the pressure at the optimum rises with the third power of the reduction in particle diameter dp. 

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. 

Figure 4.10 Typical routine column test chromatogram for a 30 cm X 4.6 mm I. D. column pacXed with an octadecylsiloxane bonded silica packing of lO micrometers particle diameter. The test mixture consisted of resorcinol (0.55 mg/ml), acetophenone (0.025 mg/ml), naphthalene (0.20 mg/ml) and anthracene (0.01 mg/ml) in acetonitrile, 10 microliters injected. The separation was performed isocratically at 23 C with acetonitrile-water (55 45) as the mobile phase at a flow rate of 1.5 ml/min. Detection was by UV at 254 nm (0.1 AUFS).

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. 

When we talk about optimization of serial LC/MS operations, we consider the genuinely serial sequences of actions necessary to perform such analyses including equilibration of columns, sample aspiration, sample injection, isocratic or solvent gradient sample separation, detection, and column washout. 

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