Preparative layer chromatography chambers

Chromatographic development chambers for analytical pirrposes are commercially available in several different sizes. The most commonly used ones are rectangiflar glass tanks with inner dimensions of 21 X21 X9 cm, and they can be used to develop two plates simultaneously in the preparative scale. Even bigger tanks are available for much larger plates, for preparative layer chromatography. The width of the chamber should be varied depending on the size and the number of plates to be developed. 

RPC involves the use of centrifugal force to accelerate the flow of solvent from the feed-point at the center to the periphery of a rotating plate. Up to 72 samples can be separated and quantified in situ by analytical RPC. One sample is applied as a circle for micropreparative and preparative RPC, for which separations can be carried out off-line or on-line with elution from the layer and recovery in a fraction collector (48). A variety of N- and S-type chambers with the prefix designations N (normal), M (micro), U (ultramicro), and C (column) are used for RPC, differing mostly in the size of the vapor space (153). Nyiredy (45) has described commercial instruments (Chromatotron and Rotachrom) and the various modes of RPC and covers preparative layer chromatography, including RPC, in Chapter 11 of this Handbook. 

Isocratic linear development is the most popular mode of chromatogram development in analytical and preparative planar chromatography. It can be easily performed in horizontal chambers of all types. The mobile phase in the reservoir is brought into contact with the adsorbent layer, and then the movement of the eluent front takes place. Chromatogram development is stopped when the mobile phase front reaches the desired position. Usually 20 X 20 cm and 10 X 20 cm plates are applied for preparative separations, and this makes the migration distance equal to about 18 cm. Due to the fact that the migration distance varies with time according to the equation Z, = (Z, c, and t are the distance of the solvent front traveled, constant,... 

Each column fraction is analyzed for lipid material by spotting on a 5 X 10 cm silica gel thin-layer plate and exposing it to iodine vapor as follows. Prepare seven tapered capillary tubes and use these to place a spot of each solution on the TLC plate. Put at least 10 capillary applications from a single fraction on a spot. Your final plate should then have seven spots, one for each fraction. Set the plate in an iodine chamber and allow it to remain for about 15 minutes or until some spots are yellow or red-brown. The presence of lipid in a fraction is indicated by the red-brown color. Retain all the column fractions that appear to have lipid. Each of these fractions will be analyzed in part B by thin-layer chromatography. 

Thin-Layer Chromatography Coupled with Fluorometry. Aliquots of the prepared samples stored in the pear-shaped flasks are spotted on the thin-layer plates. The development of these plates for separation of the individual compounds can be done in one or two directions which takes place in an equilibrated chamber first with a mixture of toluene/n-hexane/n-pentane (5 90 5) to a distance of 15-16 cm. After drying, the plates are developed in the second direction with a mixture of methanol/diethyl ether/water (6 4 1). A typical two dimensional TLC of a PAH standard is shown in Figure 3. 

Fig. 167. Rapid testing of the influence of heavy metals on steroids on adsorbent layers. I silica gel G layer, prepared with a 0.6% solution of Pb(N03)2 6 variously substituted oestrenols applied nitrogen atmosphere in the chromatography chamber plate kept 12 weeks at room temperature in this chamber, under exclusion of light then chromatographed with heptane-acetone (80 -f- 40) dried, sprayed with sulphuric acid (Rgt. No. 241B) and inspected in 366 nm UV-light. II as / but using layers prepared with 10% HgCh solution...

The slurry for TLC is prepared from 10 g cellulose-B (Firm 33) and 65 ml distilled water and is spread in a layer 0.5 mm thick. 10 p.1 urine (A), 10 p,l urine/ distilled water (1 1) (B) and 10 (xl creatinine standard (= 10 p.g creatinine) (S) are applied to the air-dried layer. Chromatography is carried out with n-butanol-acetic acid-water (60 15 15) at chamber saturation. The layers are then sprayed with 5% alcoholic picric acid solution and 10% sodium hydroxide (lower limit of detection is 0.4 (xg) and the outlines of the orange spots immediately marked round with a needle. The areas are then measured planimetrically (five measurements per spot) or by counting squares on millimeter paper. 

Modihcation of the ES chamber was reported by Ruminski [8], who applied a glass rod (3) to obtain even distribution of solvent to the adsorbent layer (Eigure 6.5). This modihcation was employed for preparahve planar chromatography, especially with plates of large width. Preparative chromatograms can be developed even on 40-cm-wide plates. 

---