Sample valve injection automation

Sample preparation, injection, calibration, and data collection, must be automated for process analysis. Methods used for flow injection analysis (FLA) are also useful for reliable sampling for process LC systems.1 Dynamic dilution is a technique that is used extensively in FIA.13 In this technique, sample from a loop or slot of a valve is diluted as it is transferred to a HPLC injection valve for analysis. As the diluted sample plug passes through the HPLC valve it is switched and the sample is injected onto the HPLC column for separation. The sample transfer time typically is determined with a refractive index detector and valve switching, which can be controlled by an integrator or computer. The transfer time is very reproducible. Calibration is typically done by external standardization using normalization by response factor. Internal standardization has also been used. To detect upsets or for process optimization, absolute numbers are not always needed. An alternative to... 

Burns [27] described a fully automated approach for HPLC analysis of vitamin tablets. A sample valve provides the injection interface in this apphcation. Tablets direct from the production plant are dispensed into the sample cups on a Technicon Sohd Prep sampler, they are dissolved, and the fat-soluble vitamins are extracted. The solution is concentrated... 

Grudpan, K., Jakmunee, J., and Sooksamiti, R, Flow-injection in-valve solid-phase extraction spectrophotometric determination of uranium in geological samples, Lab. Rob. Autom., 10, 25-31, 1998. 

Autosamplers take this same loop and valve principle and automate the filling and handle-turning sequence. The major differences between models on the market are in the way they get sample into the loop and the method of cleaning between injections. Most autoinjectors use a carousel loaded with sample valves to hold samples until their turn for injection occurs. Sample vials are usually capped with a screw cap fitted with a septum, although some recent autosamplers replace the carousel with microtiter plates having 96-364 wells containing the samples for use with robotic workstations. Conical vials are available for limited samples and 1-jUL injections are possible with some... 

Column switching in HPLC offers an alternative for the processing of liquid samples such as biological fluids or water. Samples are injected directly onto one HPLC column and the analytes retained. After a set time a second solvent elutes the analytes onto a second analytical column for separation and detection. There are several alternatives in column switching such as the back-flushing of retained components. It is also possible to use more than two columns. Columns can be of the same type or contain different stationary phases. The valves involved in column switching are automated so the technique offers the possibility of complete automation. 

Samples can be introduced manually into the valve with a syringe to fill the sample loop. Automated sampling valves are routinely used today in which samples are taken from an autosampler for unattended operation. The major limitation of valve injectors is that the sample size is fixed, and the loop must be changed in order to vary the injected sample size. There are automated motor-driven adjustable syringes that provide enough pressure to inject the sample past a check valve that prevents backflow. 

Valve injection. Valve injection of the sample is now the preferred and accepted technique. Sample application is rapid, the solvent flow from the pump does not have to be stopped and these systems are easy to use, readily adapted for automated injection and can operate at pressures up to 6000psi (41.4MPa) with reproducibility >0.2%. Six-port valves are commonly used, either fitted with an internal or an external sample loop and are an integral component of an HPLC system. 

Septum injection, pneumatic high-pressure syringe injection and v.alve injection are the usual kinds of sampling devices. The stop-flow injection technique is also used. Automated sampling devices based on high-pressure syringe injection or valve injection are already available. 

The sample introduction system must be capable of introducing a known and variable volume of sample solution reproducibly into the pressurized mobile phase as a sharp plug without adversely affecting the efficiency of the column. The superiority of valve injection has been adequately demonstrated for this purpose and is now universally used in virtually all modern instruments for both manual and automated sample introduction systems [1,2,7,31,32]. Earlier approaches using septum-equipped injectors have passed into disuse for a several reasons, such as limited pressure capability, poor resealability, contamination of the mobile phase, disruption of the column packing, etc., but mainly because they were awkward and inconvenient to use compared with valves. For dilute sample solutions volume overload restricts the maximum sample volume that can be introduced onto the column without a dramatic loss of performance. On-column or precolumn sample focusing mechanisms can be exploited as a trace enrichment technique to enhance sample detectability. Solid-phase extraction and in-column solid-phase microextraction provide a convenient mechanism for isolation, concentration and matrix simplification that are easily interfaced to a liquid chromatograph for fully or semi-automated analysis of complex samples (section 5.3.2). 

Full automation of separations by liquid chromatography requires automation of the sample introduction process. This includes automation of scheduled injections and automation of sample processing when required to isolate the analytes of interest from the sample in a form suitable for separation and detection. An example of the latter approach is on-line solid-phase extraction-liquid chromatography (SPE-LC) discussed in section 5.3.2. Automation of time scheduled injections increases accuracy and precision by removing human error [1,18,39]. Typical precision for manual valve injections is about 0.5% for complete-fill and 1-2% for partial-fill loop injections. Typical precision for automated injection is about 0.25% for complete-fill and 0.5% for partial-fill loop techniques. 

Ding et al. described an automated on-line SPE-LC-MS/MS method for the determination of macrolide antibiotics, including erythromycin, roxithromycin, tylosin, and tilmicosin in environmental water samples. A Capcell Pak ME Ph-1 packed-column RAM was used as SPE column for the concentration of the analytes and clean-up of the sample. One millilitre of a water sample was injected into the conditioned SPE column, and the matrix was washed out with 3 ml high-purity water. By rotation of the switching valve (see Fig. 4.2), macrolides were eluted in the back-flush mode and transferred to the analytical column. The limits of detection and quantification obtained were 2-6 and 7-20 ng/1, respectively, which is suitable for trace analysis of macrolides. The intra- and inter-day precisions ranged within 2.9-12% and 3.3-8.9%, respectively. At the three fortification concentrations tested (20, 200, and 2000 ng/1), recoveries of macrolides ranged from 86.5% to 98.3%. 

Figure 8. Photograph of the fully automated total Tc analyzer instrument in the laboratory. The labeled components are (A) robotic autosampler (B) microwave digestion unit (C) fluid handling components for sample injection, automated standard addition, sample acidification/digestion (D) separation fluidics including syringe pumps, flow reversal, and diversion valves (E) separation column (F) flow scintillation detector and (G) control computer with automation software. Reproduced with permission from the Handbook of Radioactivity Analysis, Second Edition Chapter 14, page 1152. Copyright...

To illustrate the hyphenation between FIA and HPLC, a system for food additives (acesulfame-k, saccharin, caffeine, benzoic acid and sorbic acid) determination [40] is shown in Figure 3.14. The system includes a dialysis cell in order to eliminate the sample matrix. Thus, a peristaltic pump dispenses the sample, which is loaded into a holding coil of an injection valve (IVl) and later injected into the donor stream. Both, donor and acceptor streams are also propelled by the peristaltic pump. Once the dialysis has been performed, analytes are loaded into a holding coil of a second injection valve (IV2). At this point, the sample passes to the HPLC system. The sample is injected into a mobile phase stream dispensed by the HPLC pump. So, the pretreated sample passes through the precolumn and column, and the analytes are separated and detected. Thus, the treatment, separation and detection steps are carried out in an efficient and high automated way. 

On-line techniques are easily automated, but are more expensive as they require additional valves with associated switching equipment. Off-line methods are rather easier to carry out, but, because of the sample collection and re-injection steps, they are slower and tend... 

An eight-port or a ten-port valve with dual matching sample loops is generally used for automated comprehensive 2DLC so that solute is injected repetitively into the... 

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