Manual sample application

Sample application manual automated One analyst performs the full test using manual and automated sample applications from the same sample solution with 5 parallel runs on the same plate System precision Resolution (/ min) RSD variation... 

Semiautomatic devices suited for preparative purposes are the CAMAG Linomat 5, the Desaga HPTLC applicator AS 30, and the Alltech TLC sample streaker. For all devices, the syringe has to be filled manually with sample solution and rinsed after sample application. Except for the Alltech TLC sample streaker, each of these instruments can be employed either as software-controlled or as a stand-alone device. The former is more convenient for creation, editing, and saving of the application pattern and instrument parameters. 

Sample Application Methods ol Application, Manual AND Instrumental, Sample Load... 

Manual devices for sample application are inappropriate if scanning densitometry is to be used for detection. First of all, the starting position of each spot must be known precisely. This f is most easily achieved with mechanical devices operating to a precise grid mechanism. The sample must be applied to the layer without disturbing the surface, something that is near iaqposslble r... 

The first "real-sample" application is given in Table IX, where chlorpyrifos was extracted from treated grass supplied by an outside collaborating laboratory. Two instruments were used for this application, extracting 11 samples on 4/26/89 and 8 samples on 10/13/90. The relative standard deviations were 3.9% and 1.7% respectively. The latter value is extraordinarily good since, in the work reported, precision due to the chromatographic analysis variability was between 1% and 3%. Further, the values of precision reported by the supplier for manual extraction of chlorpyrifos from similar matrices, leaves and roots, were 8% and 4% respectively. 

Optimal resolution for planar methods are only obtained when the application spot size or width at the origin is as small or narrow as possible. As with any chromatographic procedure, sample and solvent overloading will decrease resolution. Studies show that in most instances automated sample application is preferred over manual application especially when applications are greater than 15 /d [28]. Inadequate manual application of a sample will cause diffusion and double peaking. Depending on the purpose of the analysis, various sample amounts are recommended [29] and listed in Table 3.3. The design of commercially available automatic spotters has been reviewed [30]. 

A manual sample application of 0.5 /d is the smallest volume that can be reproducibly applied 128]. For volume applications greater than 2-10 jd, the sample should be applied stepwise with drying between each step. 

The method used for application of sample solutions is determined by whether HPTLC, TLC, or preparative layer chromatography (PLC) and qualitative or quantitative analysis are being performed. Sample volumes of 0.5-5 pi for TLC and 0.1-1 pi for HPTLC are applied manually to the layer origin as spots using fixed volume glass micropipets, such as Drummond Microcaps or selectable volume 10 or 25 pi digital microdispensers. In addition, many manual and automated instruments are available for sample application, especially for quantitative HPTLC. 

One other application is the automation of a method for the determination of total vitamin C in foods [51]. Here, the robotic station is used for homogenization of the sample, weighing, addition of an extractant, centrifugation, filtration and clean-up through a C j( column. After this treatment, the sample is manually transferred to the FI autosampler. A derivatizing reaction is implemented along the FI manifold to obtain a fluorescent product prior to insertion into the spectrofluorimeter. Although not specifically stated, the information produced is also transferred manually between both systems. 

Manual sample application employs various microcapillary pipettes and microsyringes. The microcapillary is one of the simplest and most useful methods for application of small sample volumes onto thin-layer plates. The capillary has a fixed volume of 0.5, 1, 2, or 5 pL, and accuracy is often better than 1%. The capillaries are supplied by the manufacturer in color-coded vials containing 100 pieces. The capillaries are handheld and can be positioned with a multipurpose spotting guide. 

Automatic sample applicators and automatic scanners exist. The changing of the plates in the scanner is done manually. A complete automatization would be difficult and expensive and will therefore not be realized in the near future. 

Personnel requirements depend on whether the sample application and the measurement is done manually or automatically. It is still common practice to apply the samples manually with microcaps or nanocapillaries and the automatic devices have not yet reached the same accuracy and speed as the manual application. 

Discontinuities in sample application may be caused by dirty applicators in cellulose acetate or inclusion of an air bubble in AGE. In practice, caution must be used in cleaning applicators because they are easily bent. Twin-wire applicators are best cleaned merely by agitating in water followed by gently pressing the applicators against absorbent paper. It is inadvisable to clean wires by manual wiping. The pipette tip should be checked for air bubbles before application of the sample to the agarose gel template. 

Manual application of samples is performed by touching a capillary tube containing the sample to the plate or by use of a hypodermic syringe. A number of mechanical dispensers, which increase the precision and accuracy of sample application, arc now offered commercially. 

Automated or semiautomated sample applicators are available as well. These devices apply consistent and reproducible sample spots, but it is a misconception that they are necessary for quantitative work. With proper technique, manual methods of sample application can provide results entirely comparable to those from automatic devices. Commercial automated units employ syringes or rows of microcapillaries to apply a spot or band of sample, and some actually .spray the sample onto the layer. Many are designed for preparative separations, applying large amounts of sample as streaks across the sorbent layer. 

U sually samples can be applied on TLC plates without extensive pretreatment, if any at all. The sample is dissolved in an appropriate solvent, which needs to be volatile. A small volume (typically between 1 and 5 pi) is applied as a spot or band, preferably in repetitive steps, when applying the sample manually. This should result in spots with diameter between 2 and 4 mm for conventional TLC and below 1 mm for HPTLC. To avoid damage of the stationary phase, the sample applicator should preferably not be in contact with the TLC plate. It is of importance that the size of the spot/band is minimized during sample application the larger the initial size, the... 

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