Manual Application of Samples

You have now carried out the necessary steps of sample preparation, and your samples are now in solution and ready to be applied. Before you take the selected plate material out of the package or the desiccator, please consider how your chromatograms should appear later on, e.g. with respect to the positioning of the samples, migration distance etc. 

Practical Tip for positioning the samples Fill out the form TLC III (see Section 9.3 GMP/GLP-Conforming Raw Data Sheets ) and place this or fix it with a clothes peg or similar device at your laboratory work bench. 

For beginners or for people less experienced in TLC, it is often difficult to carry out the preliminary work correctly before applying the samples. Beginners have often been seen to mark with a pencil not only the desired position of the front but also that 

Only absolutely essential markings are written on the layer with a soft pencil (e.g. 2B). These can include the starting point and, on the right hand edge (ca. 0.5 cm wide), the desired position of the front. By now you will certainly have made a note of the apph-cation scheme on the data sheet, making it is unnecessary to transfer it to the layer. 

The commercially available application templates are useful aids to the positioning of the starting points, and make it imnecessary to mark the plate directly with a lead pencil. The use of these templates goes back to the time when apphcation of samples for quantitative analysis was done by hand, and where the exact position of the spot was very important. However, modem precoated layers are more abrasion resistant than they were in the early days. Moreover, the distance apart of the spots when using application stencils is inflexible, and some proprietary stencils give a pattern of spots 5 mm apart, while with others the distance is 15 mm. 

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Starting time dependence in case of manual application of sample No No Yes ... 

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. 

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]. 

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. 

Manual application of a large number of samples is time-consuming and, obviously, leads to poor reproducibility. Speed and reproducibility are often achieved by using automatic applicators. Such automatic samplers combine precision, accuracy, speed, and versatility. 

Prepare Immobilized NeutrAvidin protein column well ahead so that it would stabilize as per manufacturer s (Pierce) manual for at least 30 min of application of samples. 

Accurate and consistent application of samples and standards to the plate in small, equally sized zones is critical for successful densitometry [79], This is best accomplished manually using Drummond disposable microcap micropipets [80] (Fig. 

Fig. 1 VersaFlash high throughput flash purification (HTTP) system shown with two stacked prepacked 40 x 75 mm (51 g) cartridges inserted in the support stand and 110 x 300 mm (1.35 kg), 80 X 150 mm (410 g), and 23 X 110 mm (23 g) cartridges on the right side of the eluent pump. The 3-way valve injector at the upper right allows direct application of sample onto cartridges using a syringe. The outlet tubing can be used for manual sample collection or connection to an automated fraction collector or UV detector.

This entry describes the general considerations, procedures, and instruments that are important for the correct application of sample and standard zones in thin-layer chromatography (TLC) and high-performance TLC (HPTLC). The application of spots and bands manually and by semiautomated and completely automated instrumental techniques is covered. 

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. 

Using Micropipettes for Manual Application of Analytical Samples. Care should be taken to ensure that separate spots are placed on a straight line horizontally across the plate. With hard-layer plates a guideline can be drawn on the plate sur-... 

Approved techniques for manual and mechanical sampling are often documented for various commodities handled in commerce by industiy groups. Examples are the International Standards Organization (ISO), British Standards Association (BSA), Japan Institute of Standards (JIS), American Society for Testing Materi s (ASTM), and the Fertihzer Institute. Sampling standards developed for use in specified industry applications frequently include instructions for labora-toiy work in sample preparation and analysis—steps (2) and (3) above. 

Moreover, such solutions can also canse the sample to crystallize out in the syringe. In this case it is recommended to dilnte the sample and apply larger volumes. Of course, a further possibility is the application of highly concentrated solutions manually with a pipette or, if the sample does not crystallize out in the syringe, semiautomated by the Alltech TLC sample streaker. 

FIGURE 5.6 Example of improper manual application resulting in an inhomogeneous distribution of the sample over the entire length of the unacceptable band, documentation after chromatography at UV 366 nm. 

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... 

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