D Column Chromatography

Column chromatography was first developed by the American petroleum chemist D. T. Day in 1900. 

Leucrose, 6-0-(a-D-glucopyranosyl)-P-D-fmctopyranose [7158-70-5] is synthesized from sucrose usiag a dextranase enzyme from l euconostoc mesenteriodes and a small proportion of fmctose (2%). Pfeifer Langen of Germany have developed a production process for leucrose that iavolves extraction of the enzyme, treatment with 65% aqueous solution of sucrose and fmctose (1 2 wt/wt) at 25°C, separation of the product from fmctose by ion-exchange column chromatography, and crystallization. The product has not yet been launched on the market as of this writing (1996). 

Liquid chromatography was performed on symmetry 5 p.m (100 X 4.6 mm i.d) column at 40°C. The mobile phase consisted of acetronitrile 0.043 M H PO (36 63, v/v) adjusted to pH 6.7 with 5 M NaOH and pumped at a flow rate of 1.2 ml/min. Detection of clarithromycin and azithromycin as an internal standard (I.S) was monitored on an electrochemical detector operated at a potential of 0.85 Volt. Each analysis required no longer than 14 min. Quantitation over the range of 0.05 - 5.0 p.g/ml was made by correlating peak area ratio of the dmg to that of the I.S versus concentration. A linear relationship was verified as indicated by a correlation coefficient, r, better than 0.999. 

Traditionally, column efficiency or plate counts in column chromatography were used to quantify how well a column was performing. This does not tell the entire story for GPC, however, because the ability of a column set to separate peaks is dependent on the molecular weight of the molecules one is trying to separate. We, therefore, chose both column efficiency and a parameter that we simply refer to as D a, where Di is the slope of the relationship between the log of the molecular weight of the narrow molecular weight polystyrene standards and the elution volume, and tris simply the band-broadening parameter (4), i.e., the square root of the peak variance. 

Figure 12.11 Coupled SEC-RPLC separation of compound Chemigum mbber stock (a) SEC ti ace (b) RPLC trace of fraction 1, dibutylphthalate (c) RPLC trace of fraction 2, elemental sulfur. Coupled SEC conditions MicroPak TSK 3000H (50 cm) X 2000H (50 cm) X 1000 H (80 cm) columns (8 mm i.d.) eluent, THE at a flow rate of 1 mL/min UV detection at 215 nm (1.0 a.u.f.s.) injection volume, 200 p-L. RPLC conditions MicroPak MCH (25 cm X 2.2 mm i.d.) column flow rate, 0.5 mL/min injection volume, lOpL gradient, acetonitrile-water (20 80 v/v) to 100% acetonitrile at 3% acetonitrile/min UV detection at 254 nm (0.05 a.u.f.s.). Reprinted from Journal of Chromatography, 149, E. L. Jolmson et al., Coupled column cliromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

The only recorded example using this method in the sugar series is the chlorination of l,2 3,4-di-0-isopropylidene-D-galactopyranose (73) which affords in addition to the expected 6-chloro-6-deoxy derivative 74a, a 5,6-unsaturated derivative 75 as well. These products were separated by silica gel column chromatography no yields were given. 

A suspension of Li metal (0.02 g, 2.9 mmol) in octan-l-ol (3.0 mL) was heated to 170 C and stirred until a homogeneous solution was obtained. The solution was cooled to 20 C and 3-neopentyloxyphthalonitrile (150 mg, 0.7 mmol) in THF (1 mL) was added. The mixture was stirred for 7 10 d, after which excess Zn(OAc)2 was added and the mixture stirred for another 2 3 d. The reaction was quenched with McOH/ H20 (1 1) and washed with MeOH. The crude products were purified by column chromatography (silica gel) and the title compound was obtained as a blue solid yield 28 mg (18%). 

To a solution of 1.44 g (3 mmol) of MAD in 10 mL of toluene are added 154 mg (1 mmol) of 4-rerr-butyl-cyclohexanone (3) at — 78°C. Butyimagnesium bromide (3 mmol) in btzO is added and the reaction mixture is stirred at — 78 C for 2 h. After quenching with 1 N HCI and extraction with Et20, the combined extract is dried and concentrated. The crude product is purified by column chromatography on silica gel (Et20/hex-ane) yield 142 mg (67%) d.r. 100 0 [determined by capillary GC (column PEG-HT, 0.25 mm x 25m temp. 130"C) by comparison with authentic samples]. 

To 158 mg (1 mmol) of 2-formyl-jVJV,3-trimcthylbutanamide in 5 ml, of CH2C12 are added at 0°C 2 mL of 1.0 M (dichloro)methylaluminum (2 mmol) in hexane. The reaction mixture is stirred at 25 °C for 2 h, then quenched with 1 M HC1. After extraction with CHCI,/EtOH (3 1), the combined organic layer is dried over Na,So4 and concentrated in vacuo. The crude product is purified by silica gel column chromatography yield 106 mg (62%) d.r. [(26, 35 )/(25, 3R )] 99 1 (determined by capillary GC). 

At 03C, a solution of 120 mg (1.2 mmol) of phenylacetylene in 2 mL of dry Et20 is treated with 0.45 mL (1.2 mmol) of 2.6 M BuLi in hexane, The mixture is stirred for 30 min, then a solution of 290 mg (1.3 mmol) of anhyd zinc bromide in 2 mL of Et20 is added. After cooling to —78 C, 100 mg (0.6 mmol) of 2-(ben-zyloxy)propanal (7) are added and the reaction mixture is allowed to warm to 0 C over a 2-h period. The reaction is quenched with sat. aq NH4C1. The organic layer is separated, dried over Na2S04 and concentrated. The residual oil is purified by column chromatography on silica gel yield 154 mg (95%) d.r. (syn/anti) 95 5 (determined by HPLC). 

The synthesis of enantiomerically pure propargylic alcohols is possible using the same methodology 43b. Thus, addition of (—)-[(l-chloro-2-phenylethyl)sulfinyl]-4-methylbenzene (14) to propan-al led to a mixture of the diastereomers 15A/15B (d.r. 44 56) which are easily separated by column chromatography. After thermal elimination of the sulfinyl group the vinyl chlorides 16A/16B were obtained as a mixture of E- and Z-oleftns. Elimination of hydrogen chloride was carried out with three equivalents of butyllithium, leading to enantiomerically pure 1 -phenyl-1-pentyn-3-ol. 

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