微量柱层析操作.doc

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微量柱层析操作

Procedure for Microscale Flash Column Chromatography In microscale flash chromatography, the column does not need either a pinchclamp or a stopcock at the bottom of the column to control the flow, nor does it need air-pressure connections at the top of the column. Instead, the solvent flows very slowly through the column by gravity until you apply air pressure at the top of the column with an ordinary Pasteur pipet bulb. For more pictures of this process, see the ID Unknowns experimental procedure. (1) Prepare the column. The column is packed using a simple dry-pack method. Plug a Pasteur pipet with a small amount of cotton; use a wood applicator stick to tamp it down lightly. Take care that you do not use either too much cotton or pack it too tightly. You just need enough to prevent the adsorbent from leaking out . Add dry silica gel adsorbent, 230-400 mesh -- usually the jar is labeled for flash chromatography. One way to fill the column is to invert it into the jar of silica gel and scoop it out . . . then tamp it down before scooping more out. Another way to fill the column is to pour the gel into the column using a 10 mL beaker. Whichever method you use to fill the column, you must tamp it down on the bench top to pack the silica gel. You can also use a pipet bulb to force air into the column and pack the silica gel. When properly packed, the silica gel fills the column to just below the indent on the pipet. This leaves a space of 4–5 cm on top of the adsorbent for the addition of solvent. Clamp the filled column securely to a ring stand using a small 3-pronged clamp. (2) Pre-elute the column. The procedure for the experiment that you are doing will probably specify which solvent to use to pre-elute the column. A non-polar solvent such as hexanes is a common choice. Add hexanes (or other specified solvent) to the top of the silica gel. The solvent flows slowly down the column; on the column above, it has flowed down to the point marked by the arrow. Monitor t

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