Split flow gradient

P. Palma and A. CappieUo, Micro-HPLC split flow gradient elution in the analysis of peptides, Annali di Chinica, 82 (1992), 371. 

A modest example of the sensitivity possible for the integral column/tip LC-MS configuration is shown in Figure 1.5. A 75-pm-ID column that terminates in a 15- am-ID tip was packed with RP material to a length of 10 cm. A split-flow gradient mobile phase of approximately 1000 1 was delivered... 

High tray efficiency is achieved when all the contactor units are delivering gas uniformly into liquid that is evenly distributed over the entire tray surface. The beneficial effects of a liquid concentration gradient are obtained if liquid crossflow is used, where the liquid enters on one side of the tray and makes one pass across the tray. For tower diameters larger than 4 ft, better liquid distribution and less change in liquid head can often be achieved by using split flow, radial flow, or cascade flow, as illustrated in Fig. 16-5. 

Two solutions to this problem are feasible one is to purchase a low-dead-volume pump. Pumps with dead volumes as low as 10 oL are available for low-flow-gradient work. A second solution is to construct and introduce a splitter before the column or the ion source. In this way, the pump can be operated at 1.0 mL/min and the lag time shortened to 1 min. By using a 5 1 split, the ion source will be exposed to 0.2 mL/min, a typical flow rate for an electrospray source. 

In the direct splitter, the ratio of the spht changes as the temperature is ramped upwards in the commonly used gradient mode. The relative quantities of different analytes vary, making absolute quantitation impossible without tedious measurements of the enrichment or split flow ratios. 

Several approaches are used to analyze and/or fractionate the rotor effluent. The simplest is to split the gradient into fractions according to volume. Alternatively, the effluent may be routed through one or more in-line flow cells to monitor a selected gradient property, e.g., density, absorbance, refractive index, fluorescence, etc. Automated fractionators that switch collection vessels according to effluent volume or to feedback from an in-line detector are available. 

There is one major problem associated with working at very high loading which is the determination of cut points. Ultraviolet detectors become overloaded at relatively low concentrations and, whilst refractive index detectors are better in this respect, they suffer from other disadvantages such as an inability to cope with solvent gradients. Split flow to the detector may be helpful, but can cause problems. Where displacement is taking place, the boundary between adjacent peaks can be very sharp, and if the materieils are closely related, may not show on a detector suffi-... 

Continuum theory has also been applied to analyse tire dynamics of flow of nematics [77, 80, 81 and 82]. The equations provide tire time-dependent velocity, director and pressure fields. These can be detennined from equations for tire fluid acceleration (in tenns of tire total stress tensor split into reversible and viscous parts), tire rate of change of director in tenns of tire velocity gradients and tire molecular field and tire incompressibility condition [20]. 

The second is associated with the irreproducibility of the mobile phase gradients that may be formed. The latter is overcome by forming the gradient at a conventional flow rate and then splitting prior to the injector. 

Countercurrent electrophoresis can be nsed to split a mixtnre of mobile species into two fractions by the electrical analog of elntria-tion. In such countercurrent electrophoresis, sometimes termed an ion still, a flow of the suspending flnid is maintained parallel to the direction of the voltage gradient. Species which do not migrate fast enough in the applied electric field will be physically swept out of the apparatus. An apparatus based mainly on this principle bnt nsing also natural convection currents has been developed (Bier, Electrophoresis, vol. II, Academic, New York, 1967). 

Column temperature Flow rate Post-column split Injection volume Mobile phase A Mobile phase B Gradient... 

FIGURE 16.5 Schematic of instrumental setup for 2D micro-RPLC-CZE. A split injection/ flow system is used to deliver a nanoliter per second flow rate to the micro-RP-HPLC column from the gradient LC pump. The HPLC microcolumn has 50 pm i.d. and 76 cm length, and the electrophoresis capillary has 17 pm i.d., L — 25 cm, and/= 15 cm. The valve is air-actuated and controls the flow of flush buffer (reprinted with permission from Analytical Chemistry). 

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