Application of Sample

Samples must be applied to TLC plates with extreme care and minimal disturbance of the adsorbent layer. Normally samples are manually applied via a capillary tube, a micropipette or a calibrated glass microsyringe such that the emerging drop just touches the surface of the plate with the appliance tip remaining just above the sorbent layer. A hole in the sorbent causes an obstruction to uniform solvent flow. The resultant channelling of solvent causes distortion of the moving spots and culminates in a loss of resolution. 

The solvent in which the sample is dissolved for spotting should be as volatile as possible, and also have as low a polarity as possible. If the spotting solvent is strongly adsorbed by the layer, marked irregularities may be observed as the mobile phase passes the position of the spots, and the separated spots may be seriously distorted. 

1 Autospotters. There are a wide variety of semiautomated and fully automated devices commercially available for spotting samples. These devices have improved the reproducibility of the technique as they can deliver exact volumes as precisely defined spots on the plate, thus removing the greatest source of error in quantitative work. In their simplest form a sample vial is presented by hand the sample is drawn into the syringe which is driven by a stepper motor and is then applied to the plate. More sophisticated, fully automated systems are available where the spotter is 

selection of a solvent for application of the sample can be a critical factor in achieving reproducible chromatography with distortion-free zones. In general, the application solvent should be a good solvent for the sample and should be as volatile and weak as possible. For silica gel TLC, a weak solvent is nonpolar for reversed-phase TLC, it is polar. High volatility promotes solvent 

Marcel Dekker, Inc. 270 Madison Avranie. New Ycnk, New York 10016 

Precoated plates exposed to high humidity or kept on hand for long periods may be activated by placing them in an oven at 70-110°C for 30 min prior to spotting. Trial and error will be necessary to determine if prewashing and activation procedures are needed for a particular separation (see Chapter 11). 

Sample application is a critical step for obtaining good resolution and quantification in TLC (Kaiser, 1988). The sample should be completely transferred from the applicator to the layer and form a compact initial zone, and the application procedure must not damage the layer. It is important to rinse the application device thoroughly with pure solvent between the various samples and standards to avoid cross-contamination, and to then rinse with the solution to be spotted to avoid dilution with solvent. 

At this point, there usually follows a chapter about the pretreatment of the samples. However, in contrast to HPLC/GC, sample preparation for TLC is not considered to be quite as critical. As well as the use of precoated layers with a concentration zone (e.g. an application zone consisting of silica 50 000 and a separation zone of sihca gel 60 or RP-18 material) upon which the matrix constituents can often be held back by suitable choice of solvent system, a chromatogram that is imusable for lack of sample preparation is more rapidly rectified (use a different preparation method and a new plate ) than an irreversibly destroyed column. A detailed treatment of the subject of sample preparation would exceed the scope of the present book. In Section 9.4, rm-der the title Examples of GMP/GLP-Conforming Testing Procedures , we describe the extraction of a pharmaceutically active substance from a tablet and the working up of plant components from dry extracts. The reader is referred to other TLC textbooks [2,21] and to literature and brochures produced by manufacturers of articles for sample preparation [28, 29]. 

Special care is necessary in TLC when working with light-sensitive samples. As special precautions must be taken when dealing with these throughout all stages of the work from receipt of the material to the documentation of the chromatogram, a special chapter is devoted to sensitive samples (see Section 10.2 Substances Sensitive to TLC ). 

Up to this point in the description of TLC, we have not differentiated between the differing experimental requirements associated with quahtative, quantitative or preparative results. However, very different attitudes lie behind these three concepts and the consequent demands made on the separation techniques used. 

Preparative work is not the main topic of this book, so that classification must be in accordance with the other areas of TLC, i.e. identity testing, purity testing and assaying. The demands made on these and hence the differences between them begin with the choice of the layers and end only with the documentation. 

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Plates with 0.5- to 2-mm layer thickness are normally nsed for increased loading capacity. Layers can be self-made in the laboratory, or commercially precoated preparative plates are available with silica gel, alumina, cellulose, C-2 or C-18 bonded siliea gel, and other sorbents. Resolution is lower than on thinner analytical layers having a smaller average partiele size and particle size range. Precoated plates with a preadsorbent or eoneentrating zone faeilitate application of sample bands. 

In paper or gel electrophoresis, the sample may be applied with a syringe or a micropipette similar to the application of samples to thin-layer plates. In some cases, there may be wells in the gel that accept the solution containing the species to be separated. In CE, samples may be applied using electromigration, hydrostatic, or pneumatic injection. In all cases, the ions to be separated must be soluble in and compatible with the stationary phases and buffers used. 

Loops and capillaries were employed earlier for the application of samples onto the plates. This method does not allow the exact determination of the sample volume, consequently it was not suitable for reliable quantitative work. Syringes have been developed and commercialized for the accurate application of microlitre and nanolitre volumes. A wide variety of automated application devices have been developed and are available on... 

Example of Application of Sampling Theory to Pesticide Analysis... 

All numerical techniques require application of sampling theory. Briefly stated, one chooses a representative sample of points within the region of interest and at each point attempts to calculate iteratively the most accurate solution possible,... 

The best column design for GPC as well as for affinity chromatography is a column with adaptors at both ends (Fig. 3.3C). This column type guarantees even surfaces of the gel bed, symmetrical application of sample, and regular flow of the eluent. 

Problems associated with use of this method involve production of the apparatus and lack of reproducibility of the test conditions, specifically, application of samples in reproducible thickness to the wet filter paper (16). 

Figure 5.7—Thu different steps in the TLC procedure. 1. Application of sample. 2. Chromatogram development. 3. Atomiser operating from a rubber pump. 4. Spray cabinet. 5. Cabinet with a video camera for acquiring image of TLC (Adapted from Camag). 6. Integration of chromatogram.

The isolation and cleanup of biological macromolecules by means of affinity chromatography form another typical example of specific application of sample treatment. Affinity chromatography exploits specific functional properties of molecules as retardation of specific groups of solutes in the adsorption step takes place. Later on, adsorbed... 

Separation of molecules by gel filtration. A Application of sample containing large and small molecules. B Large mole cules cannot enter gel matrix, so they move more rapidly through the column. C Elution of the large molecules. 

Glycerol is a dense, viscous chemical that aids in the application of samples to the gel. Bromophenol blue dye acts as a marker. It migrates very rapidly in electrophoresis. 

Minkkinen, P. 2004. Practical applications of sampling theory. Chemom. Intell. Lab. Syst. 74 85-94. 

Integration of sample preparation and analysis [46] is one of the prime objectives of /i-TAS. PCR on a chip is one of the earliest applications of sample preparation. It has been carried out in the sample reservoir of the electrophoretic chip shown in Figure 8.22a. The nucleotides, primers, and other chemicals are added into the sample reservoir, and the entire device is introduced into a conventional PCR thermal cycler. The PCR products from the sample reservoir are then injected into the separation channel and analyzed. A more complex chip with multiple PCR chambers is shown in Figure 8.22 b. 

Application of samples. An aliquot (about one to ten yl) of solution of the sample is spotted on the plate with a capillary dropper or micropipette 1.5 cm. from one end of the plate. Care must be taken to avoid diffusion of the spot moreover, the area of the spot should be restricted to a two- or three millimeter-diameter. To ensure this, the solution should be applied in stages and dried in a stream of warm air. The layer on the plate should not be penetrated during spotting. Multiple spots should be applied on the plate at least one centimeter apart. 

There are two exceptions where the application of sample and hold circuits is not useful or even senseless. 

As in all chromatographic processes, the sample should occupy as small a volume on the bed as possible. Solutions of the sample can be applied as spots or streaks at one end of the bed. In neither case should the application of sample disturb the bed, so that for regular TLC plates the sampling device cannot touch the surface. The harder layers on some commercial plates are preferred for this reason. 

Deshmukh, R., Leitch, W. E., and Cole, D. L. (1998). Application of sample displacement techniques to the purification of synthetic oligonucleotides and nucleic acids A mini-review with experimental results. J. Chromatogr. 806, 77-92. 

Manhita, A.C. Teixeira, D.M. da Costa, C.T. 2006. Application of sample disruption methods in the extraction of anthocyanins from solid or semi-solid vegetable samples. J. Chromatogr. A 1129 14-20. 

We have developed an LC-based sensor which relies upon the displacement of a drug-dye conjugate from a CAP-imprinted polymer upon application of samples containing the original template molecule (Fig. 20.15). This is essentially a competitive binding assay in a flow injection format. The increased number of theoretical plates available during the separation serves to greatly enhance the selectivity of the assay. 

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. 

Separation in the first dimension Cleaning of the glass plates, assembly of the cell, pouring of the slabs, loading of the sample and autoradiography are performed as described ( 8.4.2.1). The slabs are subjected to a pre-run of about 1 hr before application of samples. The ethanol-precipitated, dried RNA is dissolved in 6 pi 6 M urea solution. The density is increased by addition of 3 pi of an aqueous solution containing per ml 500 mg sucrose or 300 g urea, and if required the dye markers, 5 pg trypan red. 2 pg xylene cyanol FF, and 2 pg bromophenol blue/ml of solution. The sample forms a layer of approximately 1 mm in the 5 mm-wide loading slot. The separations are carried out in the cold room at 4°C in the conditions described. 

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