Product elution

Elution with a petroleum ether-benzene (1 1) mixture (2.2 liters) affords 5.8 g of starting material. Further elution with petroleum ether-benzene (1 1) (400 ml) gives initially 0.57 g of a mixed fraction, followed by 0.47 g of a crude product (eluted with another 400 ml of the same solvent mixture). Crystallization of the latter fraction from methylene dichloride-petroleum ether gives (20 R)-18,20-cyclo-5a-pregnane-3/5,20-diol 3-acetate mp 164-166.5° [ ]q 9°. Elution with benzene (400 ml) gives 1.57 g of an oil. Continued elution with benzene (600 ml) and benzene-ether (9 1) (800 ml) provides 1.81 g of crude (20 S)-18,20-cyclo-5a-pregnane-3j9,20-diol 3-acetate mp 139-140° [a]o 15°. 

Extract the residue with ethyl ether and filter. Concentrate the ethereal extract to a residue. Dissolve the residue in benzene and chromatograph on 300 g of alumina contained In a glass column 1.5 inches in diameter to give the crude product. Elute with benzene. Crystallize this product from acetone-petroleum ether to obtain the product. 

TLC analysis of the crude product (elution with 50 1 pentane ether, visualization with iodine) showed three non-baseline spots Rf 0.65 (cis isomer), Rf 0.52 (unknown impurity), and Rf 0.32 (trans isomer). The unknown impurity is intensely sensitive to iodine and largely coelutes with the cw-isomer in the subsequent column chromatography. However, the ll NMR spectrum of this isomer shows excellent purity despite the presence of this spot on TLC. In 100 1 pentane ether, Rf values of the cis and trans isomers are about 0.50 and 0.15, respectively. 

Glycerol monostearate can also be determined by LC-MS. Detection by positive APCI is possible. Figure 15 shows a typical chromatogram for the technical product eluting from a Phenomenex Aqua Ci8 column with methanol-water gradient (70-100% methanol). The molecular ion (M + H)+ mass spectra of the two main peaks detected are shown in Figures 16 and 17. 

B. 2,2-(Trimethylenedithio)cyclohexanone. A solution of 3.02 g. (0.02 mole) of freshly distilled 1-pyrrolidinocyclohexene, 8.32 g. (0.02 mole) of trimethylene dithiotosylate4 (Note 2), and 5 ml. of triethylamine (Note 3) in 40 ml. of anhydrous acetonitrile (Note 4), is refluxed for 12 hours in a 100-ml., round-bottom flask under a nitrogen atmosphere. The solvent is removed under reduced pressure on a rotary evaporator, and the residue is treated with 100 ml. of aqueous 0.1 N hydrochloric acid for 30 minutes at 50° (Note 5). The mixture is cooled to ambient temperature and extracted with three 50-ml. portions of ether. The combined ether extracts are washed with aqueous 10% potassium bicarbonate solution (Note 6) until the aqueous layer remains basic to litmus, and then with saturated sodium chloride solution. The ethereal solution is dried over anhydrous sodium sulfate, filtered, and concentrated on a rotary evaporator. The resulting oily residue is diluted with 1 ml. of benzene and then with 3 ml. of cyclohexane. The solution is poured into a chromatographic column (13 x 2.5 cm.), prepared with 50 g. of alumina (Note 7) and a 3 1 mixture of cyclohexane and benzene. With this solvent system, the desired product moves with the solvent front, and the first 250 ml. of eluent contains 95% of the total product. Elution with a further 175 ml. of solvent removes the remainder. The combined fractions are evaporated, and the pale yellow, oily residue crystallizes readily on standing. Recrystallization of this material from pentane gives 1.82 g. of white crystalline 2,2-(trimethylenedithio)cyclo-hexanone, m.p. 52-55° (45% yield) (Note 8). 

Denovan-Wright EM, Howlett SE, Robertson HA. 1999. Direct cloning of differential display products eluted from Northern blots. Biotechniques 26 1046. 

Silica gel (275 g) was packed to form a column of dimensions 16 cm x 6.5 cm. Elution was accomplished with technical grade hexanes ethyI acetate (7 3), both of which were used as purchased from Mallinckrodt Inc. The flow rate was 4 drops/sec. After collection of 150 mL of eluant, 20-mL fractions were collected. The UV-active product eluted in fractions 17-34 (R = 0.30 developed with /SiCte). 

Product Elution. Several salt solutions, including LiCl, Na2S04, NH4NO3, and Ca(N03)2, have been used as eluents. In some cases a low concentration of formate or acetate was added to buffer the solution at pH 5-5.5. All the solutions were about equally effective as long as the concentration was >2-4M for the monovalent cations and 0.5M for the Ca-(N03)2 at lower concentrations the fraction eluted decreased. Concentrated LiCl was used initially because it was a suitable medium for subsequent processing of the product, but more dilute salt solutions are less viscous and more convenient to work with. 

To a soln of Fmoc-TOAC-OH (0.437 g, 1.0 mmol) in CH2C12 at —10 °C were added cyanuric fluoride (0.085 mL, lmmol) and pyridine (0.081 mL, lmmol) under nitrogen. After stirring for lh at 0 °C the mixture was directly applied to a silica gel column and eluted with EtOAc/light petroleum ether (1 5). The product eluted first was identified as the oxazol-5(4T/)-one l 51 (0.110 g, 0.026 mmol yield 26%). The fluoride, which was eluted from the column immediately after the oxazolone, was obtained as a thick, orange oil yield 0.308 g (70%) IR (film) 1839,1715 cmA... 

Fig. 45 Reversed-phase HPLC of autoxidized trilinolenin (peroxide value = 236.4 meq/kg). Nova-Pak C18 cartridge column (Waters, Milford, MA) (3.9 X 150 mm, 60 A, 4 yam), mobile phase acetonitrile/ dichloromethane/methanol (80 10 10). Ultraviolet (UV) detector (235 nm) and evaporative light-scattering detector (ELSD). Primary oxidation products, double peak at 3.6 min secondary oxidation products elute before primary oxidation products.

Fig. 46 Reversed-phase HPLC of autoxidized triolein (peroxide value = 149.7 meq/kg). See Fig. 45 for abbreviations and chromatographic conditions. Primary oxidation products, peak at 18.6 min, secondary oxidation products elute before primary oxidation products.

Spectrum of reaction product eluted from chroma- 225° 289 —... 

Results of the previously described semiquantitative method of analysis, applied to methyl enters, are reported in Table V. Yields of products eluting before methyl benzoate have not been included in this table since these should be included in the results presented in Table III. Total yield of methyl esters was largest in the neutral oxygen oxidation, slightly lower under alkaline oxygen oxidation, and substantially lower in acidic oxygen, nitric acid, and alkaline nitrobenzene oxidations. The differences in yields may be attributed in a large part to three major peaks phthalate, a component at a relative retention time of 0.490, and benzoate. 

In a 50-ml three-necked flask are placed the carboxylic acid (0.01 mol), ethyl polyphosphate (6g, PPE) and purified chloroform (5 ml). The mixture is cooled in an ice bath and the flask is connected to a balloon containing ammonia gas ( 3 litres). Air in the flask is replaced with ammonia and the mixture is mechanically stirred at 0-5 °C for 30 minutes and then at room temperature for one and a half hours whereupon the mixture turns very viscous (1). The balloon is removed and PPE (10 g) is added. The stirring is continued at 80 °C until the reaction is complete (usually within several hours) the dehydration is monitored by t.l.c. analysis (1). The mixture is stirred with aqueous 25 per cent sodium carbonate solution (150 ml), and then extracted with benzene (3 x 40 ml CAUTION). The combined organic extracts are dried with sodium sulphate and evaporated. The residual oil is passed through a short column packed with silica gel ( 20g) and the product eluted with benzene. The eluate is evaporated and the residue purified by short path distillation under reduced pressure (Kugelrohr apparatus). 

---