Instrument, autosampler configuration

Sample solution instability or incomplete extraction/separation would show up if several aliquots from the same sample work-up were put in a series of vials that would be run in sequence that would cover at least the duration of the longest sequence that could be accommodated on the autosample/instrument configuration. For example, if an individual chromatogram is acquired for 5.5 minutes, postrun reequilibration and injection take another 2.75 minutes, and 10 repeat injections are performed for each sample vial in the autosampler, then at least 15 60/(5.5 -I- 2.75)/10 = 11 vials would have to be prepared for a 5 P.M. to 8 A.M. (=15 hour) overnight run. If there is any appreciable trend, then the method will have to be modified or the allowable standing time limited. 

A simple system is comprised of an isocratic pump, a manual injector, a UV detector, and a strip-chart recorder. A schematic diagram of an HPLC instrument is shown in Fig. 15.4. This simple configuration is rarely used in most modern laboratories. A typical HPLC system is likely to consist of a multi-solvent pump, an autosampler, an on-line degasser, a column oven, and a UV/Vis or photodiode array detector all connected to and controlled by a data-handling workstation. Examples of modular and integrated systems are shown in Fig. 15.5. Some of the important instrumental requirements are summarized in Table 15.2. 

Configure the LC instrument with a 5-pL injection loop (or autosampler) a precolumn (for endogenous samples) a column (ca. 2.1x30 mm, 3.5 pm particle size C18 column) two solvent reservoirs, Reservoir A 0.05% TFA in H20 and Reservoir B 95% MeCN and 5% a soln of 0.05% TFA in H20 a mobile phase gradient (e.g., 5% A for 3min, increase to 90% A over 8min, hold at 90% A for 3min, at 0.5 mL-min 1 flow rate) and an MS inlet flow appropriate for the mass spectrometer. Dissolve sample (preferably 1 pmol-pL-1) in 20% MeCN with 0.05% TFA. Inject 10 pL of sample into LC-MS and aquire the spectra. 

Different capillary columns are available for organic acid separation and analysis. In our laboratory, the gas chromatography column in all GC-MS applications is crosslinked 5% phenyl (poly)methyl silicone, 25 m internal diameter 0.20 mm stationary phase film thickness 0.33 pm (Agilent HP-5, DB-5, or equivalent). Several instrument configurations are commercially available, which allow for positive identification of compounds by their mass spectra obtained in the electron impact ionization mode. A commercially available bench-top GC-MS system with autosampler (Agilent 6890/5973, or equivalent) is suitable. Software for data analysis is available and recommended. The use of a computer library of mass spectra for comparison and visualization of the printed spectra is required for definitive identification and interpretation of each patient specimen. 

In modern instrument configurations, derivatization can take place in a conventional autosampler the resultant derivatives are separated on the analytical column. Detection limits at the femtomole level are achieved, and the resolution of polar amino acids is greatly enhanced. 

Seiko offers the EXSTAR6000 TGA/DTA series with dual-beam, horizontally oriented balance configuration. The TG/DTA6200 has an ambient-1100 °C range, and the TG/DTA6300 has an ambient-1500 °C range. Both systems have a maximum sample capacity of 200 mg, can heat from 0.01 to 100°C/min, and will cool from 1000 °C to 50 °C in less than 15 min. A 30-pan autosampler is optional. For more information, see wvw.sii.co.jp or the North American distributor RT Instruments at vww.rtinstruments.com. RT Instruments also sells previously owned, refurbished thermal analysis systems. 

Commonly, in a SIA instrument the commutation device is a multiposition selection valve (with 6-14 peripheral positions, any one of which can be internally connected to the central position) operating in synchronization with the pump. The peripheral positions of the multiposition valve are coupled to various sample and reagent reservoirs as well as other flow devices via Teflon tubing. Common autosamplers can also be used as commutating units. A major conceptual and instrumental breakthrough associated with the commutation device is the introduction of the LOV configuration (described in detail in Section 2.7). 

System volume is one of the parameters that instrument manufacturers have striven to reduce when the engineering of UHPLC systems began. Some reductions were simple. In others, for instance, in autosamplers, the volume reduction was more complicated. For this reason, total system volumes are not uniform across all available instrumentation. Some manufacturers strictly define their system volume, and the qualification of the instruments for use in regulated environments hinges on this volume being fixed. Other manufacturers offer more flexible options. Virtually all UHPLC systems are modular and can have different components (column switcher vs. single column, binary pump vs. quaternary pump, multiple detectors), and for some manufacturers, two systems may have the same components but be plumbed differently. All of these configurations have an effect on the system volume. Because some of the benefits of UHPLC are the speed of analysis, sharpness of the peaks, and retention of resolution, extra dead volume is undesirable, as it reduces these benefits. To ease method transfer problems, one must account for these types of differences. 

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