Split-loop injection

Figure 4.28. The FI A split-loop injection technique, the concept of which is that the (external) loop of an injection valve is split into two sections a and 6), which in the LOAD position are filled with either sample (5) and reagent (/ ), respectively (a), or with a mixture of both (b). In the first variant, the dimensions of sections a and b determine the volumes, which, upon turning of the valve to position INJECT (90° turn of the figure), are propelled into the FI A system by carrier solution (C) in the latter variant the injected volume is determined by the size of section b while the premixed ratio of sample and reagent is a function of the two pumping rates Qr and Qs.

Extra column dispersion, as would result from large injection volumes > 1 pi and dead volumes introduced by frits and fittings must be reduced to obtain optimum performance. A variety of injection systems have been reported, including those of split injection, internal loop-rotary valve, microfeeder and pneumatic microsyringes [130]. Considerable advances have been made in quantitative reproducibility. Dead volumes have been minimised by locating the columns directly into the injection and detector systems. 

An alternative form of split injection is the timed split technique, Figure 6.11 [130,131,133]. In this case the column is connected directly to the valve and the valve actuator is controlled electronically to turn the valve to the inject position and back very rapidly with only a portion of the sample in the loop displaced to the column. Timed split allows variable volumes to be injected by changing the valve actuator tine and provides more reproducible splitting than the dynamic split technique. However, it suffers from many of the same problems as dynamic split, namely, poor accuracy, split ratios that depend on pressure, and high detection limits. 

The loop for the 2nd-D was loaded with the effluent of the 1 st-D at 50 pL/min for 1 min 58 s, and then the injection valve was turned to inject the 100 pL fraction for 2 s onto the 2nd-D HPLC. The flow rate was 5 mL/min, and the valve was turned back for the next loading, resulting in fractionation of the lst-D every 2 min. In this case less than 2% of the effluent from the 1 st-D was wasted during sample injection. The 2nd-D effluent eluted at 5 mL/min from the 2nd-D column, passed through a UV detector, and then was split by using a T-joint at approximately a 1/140 split ratio, resulting in a flow rate of ca. 36 pL/min going into the spray capillary for ESI-TOF-MS detection. 

Experimental (simplex and window diagram). The chromatographic system consisted of a Model 501 supercritical fluid chromatograph (Lee Scientific, Salt Lake City, Utah) with the flame ionization detector (FID) set at 375°C. The instrument was controlled with a Zenith AT computer. A pneumatically driven injector with a 200 nL or a 500 nL loop was used in conjunction with a splitter. Split ratios used were between 5 1 and 50 1, depending on sample concentration and the chosen linear velocity, while the timed injection duration ranged from 50 ms to 1 s. We found that the variation of both the split ratio and injection time allowed greater control over the... 

Samples from the 40-mL VOA vials were split for chromate and PCE analyses and typically analyzed within 48 hours of collection. Chromate concentration was determined via an HPLC method using a Gilson Model 116 UV detector set at 365 nm and a 2- by 150-mm Waters Nova-Pak C18 60A HPLC column packed with 4-pm particles. The mobile phase consisted of 5-mM tert-butylammonium hydrogen sulfate buffered to pH 4.4 with NaOH with 10% acetonitrile (v/v) as a modifier. The eluent flow rate was 0.8 mL min-i Samples were filtered through a0.45-pm filter as they were injected by an Alcott 708 autosampler with a 0.1-mL sample loop. The typical run time was 4 min with a calibration range of 0.05 to 20 mg L 1 (0.001 - 0.38mmol L 1) Cr as chromate. 

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. 

Good reproducibility has been reported for capillary supercritical fluid chromatography using a direct injection method without a split restrictor. This method (Fig. 1.2(b)) utilises a rapidly rotating internal-loop injector (Valeo Inst. Switzerland) which remains in-line with the column for only a short period of time. This then gives a reproducible method of injecting a small fraction of the loop into the column. For this method to be reproducible the valve must be able to switch very rapidly to put a small slug of sample into the column. To attain this a method called timed-split injection was developed (Lee Scientific). For timed split to operate it is essential that helium is used to... 

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.

Timed-split, where the sample loop is placed in the flow stream for short periods, and the time in the inject position determines the amount on column. This is the most popular injection method and gives good efficiency and reproducibility. It requires fast actuation and an internal sample loop. 

In order to avoid problems with sample inhomogeneity, the entire oil sample from each sample of shale was dissolved in 1.5 to 2.5 mL of CS2 (about 1 g oil to 1.5 mL solvent). One pL of this solution was injected into a Hewlett-Packard Model 5880 Gas Chromatograph equipped with capillary inlet and a 50 m x 0.25 mm Quadrex "007" methyl silicone column. Injection on the column is made with a split ratio of approximately 1 to 100. The column temperature started at 60°C and increased at 4°C/min to 280°C where it remained for a total run time of 90 min. The carrier gas was helium at a pressure of 0.27 MPa flowing at a rate of 1 cm /min. The injector temperature was 325°C and the flame ionization detector (FID) temperature was 350°C. Data reduction was done using a Hewlett-Packard Model 3354 Laboratory Automation System with a standard loop interface. Identification of various components was based on GC/MS interpretation described previously (4). For multiple runs on the same shale, the relative standard deviations of the biomarker ratios were about 10%. 

Figure 2. The modified Valeo 0.5 p 1 internal loop sampling valve for split injection in narrow-bore packed column HPLC (Reproduced with permission from Ref. 12, copyright Elsevier Sci. Publ. Co.)...

Reformed gas is compressed to 100 barg in a 27 MW split, three-stage compressor and injected into the converter loop at the suction of the 5 MW circulating compressor. Converter circulating gas plus make-up gas is heated and fed to a six-bed converter (ZnO/CuO/Al O catalyst). Part of the gas is fed to the inlet of the reactor at 210-240°C and the balance is fed, at a lower temperature, to the lower beds as quench gas. Reactor effluent is cooled both against reactor feed and saturator water feed before passing to the methanol condensers. The crude methanol is then rundown to off-site tankage and the MTG unit. 

Finally, it should be stressed that there are some specific strategies that can strongly influence sample dispersion in flow injection analysis, e.g., merging zones, zone sampling, stream splitting and closed-loop arrangements. These strategies are discussed in Chapter 7. 

The formation of junctional channels requires end-to-end association between the extracellular domains of hemichannels. This interaction is not likely to be covalent because junctional channels can be split into hemichannels by alkaline urea treatments (7, 79). However, the formation of junctional channels between mRNA-injected oocytes is critically dependent on the presence of the three cysteines in each connexin extracellular loop (78). These cysteines apparently do not form interhemichannel disulfide bonds (80-82), but may serve to stabilize the extracellular domains during the homophilic binding reaction with the apposing hemichannel. 

Figure 4.52. FIA manifold designs for sequential multidetection employing a single injection position, (a) Splitting of the sample into a number of subplugs, which are guided through individual reaction coils and finally routed to a common, single detector, (b) Use of multiple detectors located in series, the response from each of which is fed to a microcomputer, (c) Use of a closed-loop system where the injected sample is recycled around a number of times in a loop, which includes the detector, until a constant signal is emitted, whereupon the sample is directed to waste.

In the volumetric sample injection (Fig. 5.10a) the sample loop has its simplest function, that is, merely to meter the volume of the analyte to be injected. The next step is to inject the reagent and analyte simultaneously with the purposes discussed previously in Chapters 2 and 4. This can be done in two ways (1) by nesting another loop by means of a second valve in the way shown in Fig. 5.10/ , or (2) by splitting the sample loop as shown in Fig. 5.10c. 

Apparatus. High performance liquid chromatographic separations were achieved on a binary gradient microbore HPLC systems primary pump (A) Model 305, secondary pump (B) Model 306, monomclric module Model 805, and a dynamic mixer Model 811C from Gilson Inc. (Middleton. Wl). Sample injections were achieved with a 20 pL loop on a Model EQ-36 injection valve from Valeo Instruments Co. Inc. (Houston, TX). A stainless steel Y-splitter also from Valeo was used in order to achieve a post-column split of the mobile phase Dow to the CLND. A Supelcosil LC-18S analytical HPLC column was purchased from SUPELCO Inc. (Bellefonte. PA). The Y-splitter was attached to the analytical column by a SLIPFREE connector, available from Keystone Scientific Inc. (Bellefonte, PA). Analyses of nor-dihydrocapsaicin, capsaicin and dihydrocapsaicin in spices as well... 

Figure 5.2. Schematic diagram of a standard liquid chromatograph modified for use with a packed capillary column. Typical pump settings are 300-400 p,l / min with flow splitting of 1 2000 to give a column flow of 150-200 nl / min. The microinjection valve has a 40 nl internal loop and an additional T.IO split creates an injection volume of 2-3 nl (larger volumes can be injected by on-column focusing with gradient elution separations). The detector uses a U- or Z-shaped flow cell with a 3 nl volume and 8 mm path length. Fused-silica capillary tubing with an internal diameter < 20 pm and zero-dead-volume connectors are used for column connections. (From ref [8j. American Chemical Society).

The sample is loaded at atmospheric pressure into an external or internal loop, or groove in the valve core and introduced into the mobile phase stream by a short rotation of the valve. The volume of sample injected is normally varied by changing the volume of the sample loop or by partially filling a sample loop with a fraction of its nominal volume. External sample loops have volumes from about 5 p.1 up to about 5 ml, although typical injection volumes for conventional diameter columns are 10-50 xl. Injections from 1 p,l to about 40 nl require micro-injection valves equipped with replaceable internal loops [7,32-34]. Injection volumes less than about 40 nl are performed by positioning a split vent between the injector and the column. Typical injection volumes that preserve column efficiency for packed columns of different internal diameters are summarized in Table 5.1. For packed capillary columns with internal diameters < 0.2 mm direct injection will usually require the use of a split vent to minimize volume overload unless on-column focusing is possible. Injection volumes about 5 times larger than those indicated in Table 5.1 are sometimes used to increase sample detectability but with some decrease in the column separation power. 

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