Valve injector. HPLC

Figure A2.1 Waters ProMonix On-Line HPLC analyzer. The upper compartment door contains a keypad for programming and operation of the analyzer. The upper window allows viewing of indicator lights and a liquid crystal display that provides the operator with analyzer interface, programmed parameters, and instrument status results. The lower chamber contains the pumps, valves, injector, and detector(s) required for the chromatographic separation. The sample conditioning plate for online process monitoring is to the right of the analyzer. This is a typical process HPLC. (From Cotter, R.L. and Li, J.B., Lab Rob Autom., 1, 251,1989. With permission of VCH Publishers.)...

In theory, combining two HPLC modes sequentially would provide an online LC/LC/MS/MS and speed the analytical procedure. Bands from the first separations could be detected and collected with an automated loop-and-valve injector, and then individual bands could be passed to the second LC for... 

Sample injector The sample injector is located between the solvent pump and the HPLC column, and serves to deliver or inject the sample dissolved in solvent onto the column. The most common form of sample injector is the so-called loop-and-valve injector. Sample is injected from a specialized syringe (manually) or delivered by means of an autosampler (automatically) into a small diameter loop. The loop-and-valve injector allows switching by means of a valve between initial delivery of solvent alone to the column (so-called column equilibration), to subsequent delivery of the sample onto the column. 

Sample injection in hplc is a more critical operation than in gc. Samples may be injected either by syringe or with a valve injector although the former is now rarely used. Valves, which can be used at pressures up to about 7 000 psi (500 bar), give very reproducible results for replicate injections (<0.2% relative precision) and are therefore ideal for quantitative work (p. 129). They consist of a stainless steel body and rotating central block into which are cut grooves to channel the mobile phase. from the pump to the column (figure 4.27). The sample is loaded into a stainless steel loop incorporated... 

On-line coupling of SFC with gas chromatography has sporadically been used for the investigation of volatiles from aromatic herbs and spices. The requirements for instrumentation regarding the pumps, the restrictors, and the detectors are similar to those of SFE-GC. Additional parts of the device are the separation column and the injector, to introduce the sample into the mobile phase and successively into the column. The most common injector type in SFC is the high-pressure valve injector, similar to those used in HPLC. With this valve, the sample is loaded at ambient pressure into a sample loop of defined size and can be swept into the column after switching the valve to the injection position. The separation columns used in SFC may be either packed or open tubular... 

Sample introduction was by means of a Hamilton 701N syringe in combination with a septum injector (HETP Components, Macclesfield, Great Britain) or alternatively a Rheodyne valve injector with 20 pi sample loop was used (HPLC Technology Ltd., Macclesfield, Great Britain). 

Figure 2.2 Schematic representation of an on-column interface. The eluent leaving the HPLC detector enters the valve and in the stand-hy position, leaves it to go to waste. When the valve is switched on, the eluent is pumped through the transfer line into the inlet of the on-column injector. The liquid floods the capillary wall, thus creating a layer that will retain the solutes. Evaporation occurs from the rear pait of the solvent so refocusing the chromatographic hand. At the end of the transfer, the valve is switched off, and the eluent again flows to waste.

The basic SFC system comprises a mobile phase delivery system, an injector (as in HPLC), oven, restrictor, detector and a control/data system. In SFC the mobile phase is supplied to the LC pump where the pressure of the fluid is raised above the critical pressure. Pressure control is the primary variable in SFC. In SFC temperature is also important, but more as a supplementary parameter to pressure programming. Samples are introduced into the fluid stream via an LC injection valve and separated on a column placed in a GC oven thermostatted above the critical temperature of the mobile phase. A postcolumn restrictor ensures that the fluid is maintained above its critical pressure throughout the separation process. Detectors positioned either before or after the postcolumn restrictor monitor analytes eluting from the column. The key feature differentiating SFC from conventional techniques is the use of the significantly elevated pressure at the column outlet. This allows not only to use mobile phases that are either impossible or impractical under conventional LC and GC conditions but also to use more ordinary... 

For HPLC, the injector is a valve. In the charge position, a 50- jL syringe is used to fill the sample loop that holds a specific volume of sample solution. The valve is switched to the run position, and the eluent carries the sample out of the sample loop and into the column (auto samplers are also available for HPLC). A recording of the detector output is automatically started at the time of injection and produces a chromatogram of the separated components. 

Injectors introduce the sample into the mobile phase under high pressure. There are several approaches to injection in HPLC, such as syringe injection via septum, a combination of a septum and syringe or a valve injection. Valve injection is the method of preference in up-to-date HPLC instrumentation. 

The injector, columns and valves reside in a low temperature chamber to minimize the loss of deuterium by back exchange (Fig. 12.2). The quenched protein solution is pumped in series through a column containing an immobilized protease and a trap column to capture the peptide fragments. The gradient pump is activated following digestion and the peptides captured on the trap column are eluted and separated over an analytical reverse-phase HPLC column directly into the mass spectrometer. 

Pacification is a technique for removing organics and buffers from HPLC metal and Teflon surfaces and protecting them from salt corrosion with 6 N nitric acid (see Chapter 4). First, remove the HPLC column and replace it with a column bridge. Do not flush this wash into the mass spectrometer. Wash the system with water. Remove the column and replace it with a column blank. Flush with 6 N nitric acid for at least 30min, then overnight with water. Ensure the effluent pH is back to that of lab water. Replace the column and flush with mobile phase. This should be done at least once a month to clean check valves, line, and injectors. Under no circumstances should this wash be done with an HPLC column in place or into the mass spectrometer ... 

FIGURE 13.10 Generic setup for (a) on-line SPE-HPLC implemented with a simple valve-switching system (G. Maio, R. Morello, F. Arnold, and K.-S. Boos, Analysis of Antimycotic Drugs in Biofluids by On-Line SPE-LC Application note LPN 1859-01 06/06 Diones Corporation, Sunnyvale, CA 94088-3603 Figure 2, p. 1, 2006. With permission.) and for (b) on-line SPE-GC implemented with a large volume injector with a solvent venting option. 

Sample introduction is a major hardware problem for SFC. The sample solvent composition and the injection pressure and temperature can all affect sample introduction. The high solute diffusion and lower viscosity which favor supercritical fluids over liquid mobile phases can cause problems in injection. Back-diffusion can occur, causing broad solvent peaks and poor solute peak shape. There can also be a complex phase behavior as well as a solubility phenomenon taking place due to the fact that one may have combinations of supercritical fluid (neat or mixed with sample solvent), a subcritical liquified gas, sample solvents, and solute present simultaneously in the injector and column head [2]. All of these can contribute individually to reproducibility problems in SFC. Both dynamic and timed split modes are used for sample introduction in capillary SFC. Dynamic split injectors have a microvalve and splitter assembly. The amount of injection is based on the size of a fused silica restrictor. In the timed split mode, the SFC column is directly connected to the injection valve. Highspeed pneumatics and electronics are used along with a standard injection valve and actuator. Rapid actuation of the valve from the load to the inject position and back occurs in milliseconds. In this mode, one can program the time of injection on a computer and thus control the amount of injection. In packed-column SFC, an injector similar to HPLC is used and whole loop is injected on the column. The valve is switched either manually or automatically through a remote injector port. The injection is done under pressure. 

HPLC Waters, Model WISP 712 equipped with sample injector and automated pump (Model 6000A). A column switching valve (Waters Model 60057) is an advisable optional component. 

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