Chromatography fraction collection

Fig. 1. SP Sepharose FF cation exchange chromatography. Fractions collected from a SP Sepharose FF column were resolved in a 15-25% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and stained with Coomassie blue.

Use of Simple Column Chromatography, Fraction Collection and Liquid Scintillation Counting... 

Accelerated solvent extraction (ASE), focused microwave soxhiet extraction (FMSE), immuno affinity cleanup (im-Cu), liquid-liquid extraction (LLE), low-temperature lipid precipitation (LTLP), matrix solid-phase dispersion (MSPD), microwave-assisted extraction (MAE), nanofiltration (NF), pressurized fluid extraction (PEE), single drop microextraction (SOME), solid-phase extraction (SPE), solid-phase microextraction (SPME), steam distillation (SD), stir bar sorptive extraction (SBSE), surpercritical fluid extraction (SFE), subcritical fluid extraction (ScFE), supported liquid membrane extraction (SLME), ultra-sonication (US), size exclusion chromatography (SEC), liquid chromatography-fraction collection (LC)... 

A, acaricide AV, avicide I, insecticide F, fungicide H, herbicide GR, growth regulator N, nematocide R, rodenticide US, ultrasonication LLE, liquid-liquid extraction CU, cleanup ImCU, immuno cleanup SPE, solid-phase extraction MSPD, matrix solid-phase dispersion SBSE, stir bar sorptive extraction SD, steam distillation FMSE, focused microwave Soxhiet extraction PFE, pressurized fluid extraction ASE, assisted solvent extraction ScFE, subcritical fluid extraction SFE, supercritical fluid extraction SEC, size-exclusion chromatography LC, liquid chromatography (fraction collection) LTLP, low temperature lipid precipitation. 

Figures 2 through 9 are infrared spectra of fractions collected from partition columns, gas chromatography, thin-layer chromatography, or a combination of these separation techniques. Figure 10 is the infrared spectrum of a compound isolated by gas chromatography after hydrolysis of a pyrethrum concentrate. In this case the compound is a long-chain ester. All the infrared spectra were made with a Perkin-Elmer Model 221 instrument. The following operating parameters were used. A liquid demountable cell with a 0.01-mm path length was employed.

If simple sample pretreatment procedures are insufficient to simplify the complex matrix often observed in process mixtures, multidimensional chromatography may be required. Manual fraction collection from one separation mode and re-injection into a second mode are impractical, so automatic collection and reinjection techniques are preferred. For example, a programmed temperature vaporizer has been used to transfer fractions of sterols such as cholesterol and stigmasterol from a reversed phase HPLC system to a gas chromatographic system.11 Interfacing gel permeation HPLC and supercritical fluid chromatography is useful for nonvolatile or thermally unstable analytes and was demonstrated to be extremely useful for separation of compounds such as pentaerythritol tetrastearate and a C36 hydrocarbon standard.12... 

Immediately purify the oxidized enzyme by gel filtration using a column of Sephadex G-25. The chromatography buffer is 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2. Collect 0.5 ml fractions and monitor for protein at 280 nm. HRP also may be detected by its absorbance at 403 nm. In oxidizing large quantities of HRP, the fraction collection process may be done visually—just pooling the colored HRP peak as it comes off the column. 

Adsorption chromatography. The concentrated sample in 30 ml eluent (n-butanol/acetic acid/water = 4 1 1 by vol. (Bu0H/HAc/H20)) was applied to a column of 5 g CFl cellulose preswollen in eluent. The column was first eluted with Bu0H/HAc/H20 and three 100-ml fractions collected, then with water and four additional 50-ml fractions collected. 

Separation of a purified hydrolyzate of demineralized bovine dentin by cation exchange chromatography at pH 5.25. Gradient 0.0 - 0.5 M NaCl in 0.05 M HAc/NaAc, 1 mM NaNs, pH 5.25. Column SP Sephadex C25 (34 x2.6cm). Flow rate approx. 30 ml/h. The 4-ml fractions were assayed for amino acids by ninhydrin reaction (—) and for NaCl by electric conductivity measurement after 75-fold dilution (—). Fractions collected for further characterization are denoted by bars and Roman numerals. 

Pipet 0.2 ml of Soln. C into tubes for fraction collection. If chromatography is performed in the cold, the phosphate buffer Soln. C tends to crystallize therefore, add phosphate buffer immediately before use. 

Lipids can be identified and quantified using thin-layer chromatography (TEC) and gas chromatography (GC) (Galliard, 1968). Extraction of lipids is achieved by homogenizing potato tubers with isopropanol in a blender, followed by a series of filtrations and extractions with chloroform-methanol (2 1). Chloroform is removed by rotary evaporation and the residue is redissolved in benzene-ethanol (4 1). This extract is passed through a DEAE-cellulose column, and the fractions collected are subjected to TEC on 250 p,m layers of silica gel G, using three solvent systems. Fatty acid methyl esters for GC analysis are prepared by transmethylation of the parent lipids, or by diazomethane treatment of the free fatty acids released by acid... 

Figure 5. Composite MWD diagram showing the calculated distributions of four of the five master fractions obtained from preparative chromatography of organosolv aspen lignin (fraction number 4 to 1 from left to right). The Mw values for the master fractions from left to right are 1,110, 1,310, 2,420, and 8,050, respectively. The insert shows the elution profile of organosolv aspen lignin from the YMC preparative /z-Styragel column (5 x 200 cm). The 30 fractions collected were pooled into the five master fractions shown.

Solid-phase extraction columns offer a rough cleanup of the crude extract, which might nevertheless not be sufficient for some detection systems such as mass spectrometry. Some authors have proposed a combination of solid-phase extraction and liquid chromatography columns for extract cleanup (440). Other methods appeal to liquid chromatography on Cig columns with automated fraction collection. Fractions containing the analyte of interest were evaporated to dryness, yielding a residue that in most cases was suitable for gas chromatographic detection after suitable derivatization (445, 437). 

OR Larroque, MC Gianibelli, IL Batey, F MacRitchie. Electrophoretic characterization of fractions collected from gluten protein extracts subjected to size-exclusion high-performance liquid chromatography. Electrophoresis 18 1064-1067, 1997. 

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