Microscale columns

The sample and standards are spotted on a TLC plate and developed with each of the developing solvents, dried, and examined under uv radiation. The / /s are determined, and the solvent providing an between 0.2 and 0.35 is selected. A microscale column is prepared, and the sample added. 15 to 18 fractions are collected and spotted on two TLC plates, dried, examined with uv radiation and stained. The fractions containing the desired compound are combined, the solvent is evaporated and the compounds are collected. 

Dry Pack Method 2. An alternative dry pack method for microscale columns is to fill the Pasteur pipette with dry adsorbent, without any solvent. Position a plug of cotton in the bottom of the Pasteur pipette. The desired amount of adsorbent is added slowly, and the pipette tapped constantly, until the level of adsorbent has reached the desired height. Figure 19.7 can be used as a guide to judge the correct height of the column of adsorbent. When the column is packed, added solvent is allowed to percolate through the adsorbent until the entire column is moistened. The solvent is not added until just before the column is to be used. 

As with microscale columns, the procedures described in this section should be followed carefully in preparing a semimicroscale or conventional-scale chromatography column. Failure to pay close attention to the details of these procedures may adversely affect the quality of the separation. 

Slurry Method. The slurry method is not recommended as a microscale method for use with Pasteur pipettes. On a very small scale, it is too difficult to pack the column with the slurry without losing the solvent before the packing has been completed. Microscale columns should be packed by the dry pack method, as described in Section 19.6. 

The flow of solvent through the column should not be too rapid or the solutes will not have time to equilibrate with the adsorbent as they pass down the column. If the rate of flow is too low or stopped for a period, diffusion can become a problem—the solute band will diffuse, or spread out, in all directions. In either of these cases, separation will be poor. As a general rule (and only an approximate one), most macroscale columns are run with flow rates ranging from 5 to 50 drops of effluent per minute a steady flow of solvent is usually avoided. Microscale columns made from Pasteur pipettes do not have a means of controlling the solvent flow rate, but commercial microscale columns are equipped with stopcocks. The solvent flow rate in this type of column can be adjusted in a marmer similar to that used with larger columns. To avoid diffusion of the bands, do not stop the column and do not set it aside overnight. 

Exposure of 76 to HF in H20/MeCN for 20 hours was followed by a short microscale flash column. Analysis by H NMR spectroscopy (500 MHz, CD3CN, 16 hours) of the global deprotection product showed it to be a complex mixture, however there were some encouraging signals. Purification of this mixture was attempted by HPLC-MS (25 75... 

Chromatographic and electrophoretic separations are truly orthogonal, which makes them excellent techniques to couple in a multidimensional system. Capillary electrophoresis separates analytes based on differences in the electrophoretic mobilities of analytes, while chromatographic separations discriminate based on differences in partition function, adsorption, or other properties unrelated to charge (with some clear exceptions). Typically in multidimensional techniques, the more orthogonal two methods are, then the more difficult it is to interface them. Microscale liquid chromatography (p.LC) has been comparatively easy to couple to capillary electrophoresis due to the fact that both techniques involve narrow-bore columns and liquid-phase eluents. 

A direct injection nebuliser (DIN) was used to interface LC with ICP-MS (Shum et al., 1992a). The DIN transferred all of the sample into the inductively coupled plasma. Microscale LC separations in small packed columns were studied because the column flow rates of about 30 ml min 1 were compatible with the DIN. The low dead volume (less than 1 ml) of the interface prevented excessive band broadening. Eluents containing up to 85% methanol were accommodated. The analyte signal varied by about 20% as the eluent changed from 20% to 80% methanol in water. Detection limits for arsenic and tin species using the HPLC-DIN-ICP-MS system were 0.2-0.6 and 8-10pg, respectively. 

Chemistry Video Consortium, Practieal Laboratory Chemistry, Educational Media Film and Video Ltd, Harrow, Essex, UK - Microscale ehromatography (TLC, column chromatography, gas chromatography and preparation of a Grignard reagent). 

M.T. Davis, T.D. Lee, M. Ronk, S.A. Hefta, Microscale IMER columns for peptide mapping byLC-MS analysis. Anal. Biochem., 224 (1995) 235. 

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