Sample valve injectors, injection volume

Even with all these chromatographic parameters optimised, sensitivity may not be enough. The simplest way to increase sensitivity further is to inject more sample. Valve injectors are usually supplied with 20-pl loops but there are few assays for which 100-200 pi of sample cannot be injected simply by changing the loop size. A further way is to use minibore (1 mm or 2mm i.d.) or even microbore (< 1 mm) columns. Such columns give increases in sensitivity due to the narrower peaks eluted but this may reduce column loading volumes and with some equipment the extra sensitivity gained may be lost due to extra column band broadening. 

Although the problems associated with septum injectors can be eliminated by using stop-flow septumless injection, currently the most widely used devices in commercial chromatographs are the microvolume sampling valves (Fig. 8.3) which enable samples to be introduced reproducibly into pressurised columns without significant interruption of the mobile phase flow. The sample is loaded at atmospheric pressure into an external loop in the valve and introduced into the mobile phase by an appropriate rotation of the valve. The volume of sample introduced, ranging from 2 piL to over 100 /iL, may be varied by changing... 

The maximum injection volume depends on the volume of the sample loop in the injection valve. The reproducibility of manual injection depends on the skill of the operator. The use of a small sample loop and an overflow injection of the sample solution so that the loop is fully flushed with sample are basic requirements for quantitative analysis. The highest injection reproducibility can be obtained by an auto-injector with a fixed sample loop. The smallest reasonable injection volume is 1 (A. A nl-scale injection valve can be constructed however, the memory effect at the surface of contact parts affects quantitative analysis compared with the use of a /d-scale injection valve. For a semi-micro system, a low hold-up volume injection valve is desired. The minimum injection volume is 80 nl. For a preparative-scale injection, the sample loop can be easily replaced with a larger-volume loop, such as a 200 jA, instead of the standard 20 /A loop. 

An HPTC injector allows the introduction of a precise sample volume onto the column. A typical manual injector consists of a 6-port valve with a rotor, a sample loop and a needle port (Eigure 9). A sample solution is introduced into the sample loop using a 22-gauge blunt tip syringe in the TOAD position. The sample is then injected into the column by switching the valve to INJECT. The typical external sample loop size ranges from 6 pT to 2 mT. For many years, the Rheodyne 7125 injector was the industry-standard. In the early 1990s, it was replaced by the Rheodyne 7725 injector, which injects samples without momentary flow disruptions. ... 

Application of a potential between reservoirs 1 (sample) and 4 (injection waste) electrokinetically pumps sample solution as indicated in Fig. 3. In this way, a geometrically defined 150 pm (90 pi) section of the separation channel can be filled [19]. If the injection potential is applied long enough to ensure that even the slowest sample component has completely filled the injection volume, a representative aliquot of sample can be analyzed (so-called volume defined injection). This is in contrast to electrokinetic sample injection in conventional capillaries, which is known to bias the sample according to the respective ionic mobilities [61]. These characteristic differences are shown schematically in Fig. 4. It should be noted that this picoliter sample injector is exclusively controlled by the application of electric fields and does not require any active elements with moving parts such as valves and external pumps. The reproducibility of the peak height of the injected sample plugs has been reported to be within 2 % RSD (relative standard deviation) and less [19,23]. 

The D4 content of the samples was determined by reverse-phase high-pressure liquid chromatography (HPLC) with a Varian 5500 liquid chromatograph. A DuPont Zorbax ODS (Cis) column was used with a Wilmad infrared detector set at 12.45 xm to monitor the Si-CHa vibration. The mobile phase was an 83 17 mixture of acetonitrile and acetone at a flow rate of 0.8 mL/min. A Rheodyne injector valve operating on compressed air was used with a 10-p,L sample loop for reproducible injection volumes. Ethyl acetate was used to dissolve the samples for analysis. 

The sample is injected into the system via a valve injector, as schematically shown in Fig. 1-2. A three-way valve is required, with two ports being connected to the sample loop. The sample loading is carried out at atmospheric pressure. After switching the injection valve, the sample is transported to the separator by the mobile phase. Typical injection volumes are between 10 pL and 100 pL. 

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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