Column chromatographies

Column chromatography is another form of solid-liquid adsorption chromatography and depends on the same fundamental principles as does thin-layer chromatography (TLC, Sec. 6.2), as you will see from the discussion that follows. It has an advantage over TLC in that multigram amounts of mixtures can be separated but has the disadvantage that this technique requires considerably more time 

Alumina and silica gel are the most commonly used adsorbents for column chromatography, just as they are for TLC. The quality of these adsorbents is high in that they have uniform particle size and high specific area. The higher the specific area, the faster the equilibrium of the solute between the mobile and solid phases is established and the narrower the bands. High specific areas on the order of several hundred m /g are common for good grades of alumina and silica gel. 

As noted in the discussion of TLC, the strength of the adsorption of an organic compound to the solid support depends on the polarity and nature of the adsorbent as well as on the nature of the functional groups present in the molecule. When normal-phase column chromatography is performed, a polar stationary phase such as alumina or silica gel is used in combination with organic solvents as the mobile phase or eluant. Under these conditions, the elutropic series described for TLC in Section 6.2 applies. 

In reverse-phase column chromatography, the packing material for the stationary phase consists of glass beads coated with a nonpolar hydrocarbon film. 

The most efficient method for determining the optimal solvent system(s) for a specific column chromatographic separation often is to perform a series of trial separations using TLC. These trials can be run quickly, and the amount of material needed is small. 

Column liquid chromatography is actually the parent of aU the other types of chromatography. As stated previously, this is the type of set-up used by Tswett as he pioneered into the field of chromatography. The technique he used is now called classical open-column liquid chromatography or simply LC. 

The column size and amount of adsorbent must be selected as a function of the amount of material to be chromatographed. As a general rule, the amount of adsorbent is 25-30 times, by weight, the amount of material to be analysed and the column has a height-to-diameter ratio of about 8 1. For example, a Ig sample will require 30g of adsorbent in a 130mm long column 

The rest of this section describes techniques arising from modification of the classical LC principle. 

Reverse-phase h.p.l.c. on Wates /i-Bondapak C-18 column has been used successfully for the fairly rapid separation of mono-, di-, and tri-saccharides using water as the mobile phase and, for the separation of partially methylated sugars, 1% ammonium acetate was used as mobile phase, with ethanol being added to elute the more highly methylated sugars.  

The separation and quantitative determination of glucosamine and galacto-samine at the nanogramme level has been reported by prior reaction with a suitable sulphonyl chloride, followed by h.p.l.c. The use of toluene-p-sulphonyl chloride was more rapid (10 min) but less sensitive (0.4-50 /Ug) than that using 5-dimethylaminonaphthalene 1-sulphonyl chloride (0.02-2 jUg in 25 min).  

analysis of unsaturated disaccharides obtained by enzymic hydrolysis of heparan sulphate and heparin was found to be more rapid and much more sensitive than the previously described method which employed paper chromatography.  

Conditions for the separation of methyl ethers of methyl a-L-rhamnopyrano-side on silica gel columns have been reported and the separation of mixtures of alditols on cation-exchange resin in the La form has been described. 

At some point in the near future you should watch the video entitled Column Chromatography in the multimedia activity Practical techniques on the Experimental techniques CD-ROM that accompanies this book. There you will see a video of this technique using aluminium oxide (alumina) as the stationary phase. This activity should take approximately 5 minutes to complete. 

A schematic diagram illustrating the column chromatography process. A mixture of molecules X and Y is placed on a column of silica as the stationary phase, where Y is more polar than X and so adsorbs on the column more strongly. As the solvent passes down the column, the molecules of X are eluted more easily and so travel down the column more quickly than Y, thus effecting separation. 

A more sophisticated method of column chromatography is known as HPLC, high-performance liquid chromatography. This employs very fine solid particles which pack closely together. This increases the surface available for adsorption, and so improves the separation, but because the solid particles are packed tightly together, a pump is needed to force the mobile liquid phase through the column. 

Another variant is GLC, gas-liquid chromatography. In this case the mobile phase is a gas, known as the carrier gas, and the stationary phase in the column is a liquid — a non-volatile oil or grease. This liquid can be coated on the surface of small particles of an inert solid, which is called packed-column GLC, or alternatively it is simply coated on the inside wall of a very long narrow column. 

In GLC, the point at which the sample is introduced has to be heated so that the components in the mixture vaporize and pass down the column in the gas phase. 

This method involves passing the protein through a column filled with resins of unique characteristics. Depending on the type of the resin or beads, purification can be achieved through (i) Ion Exchange, (ii) Size Exclusion or (iii) Affinity Chromatography. 

The group of Patrikeev prepared silicas imprinted against harmine and a derivative, and employed these materials as stationary phases for zonal liquid 

Strain homogenate to remove connective tissue and blood vessels 

Ribosomes and microsomes, consisting of endoplasmic reticulum, Golgi, and plasma-membrane fragments 

The mobile phase flows over the stationary material and carries the sample to be separated along with it. The components of the sample interact with the stationary phase to different extents. Some components interact relatively strongly with the stationary phase and are therefore carried along more slowly by the mobile phase than are those that interact less strongly. The differing mobilities of the components are the basis of the separation. 

Size-exclusion chromatography, also called gel-filtration chromatography, separates molecules on the basis of size, making it a useful way to sort proteins of varied molecular weights. It is a form of column chromatography in which the stationary phase consists of cross-linked gel particles. The gel particles are usually in bead form and consist of one of two kinds of polymers. The first is a 

Effluent is collected manually or automatically and analyzed for the presence (and sometimes the amount) of solute 

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

The nitration of the 2-anilino-4-phenylselenazole (103) is much more complicated. Even careful nitration using the nitrate-sulfuric acid method leads to the formation of a mixture of variously nitrated compounds in an almost violent reaction. By the use of column chromatography as well as thin-layer chromatography a separation could be made, and the compounds could be partly identified by an independent synthesis. Scheme 33 shows a general view of the substances prepared. Ring fission was not obser ed under mild conditions. 

A typical column chromatography experiment is outlined in Figure 12.4. Although the figure depicts a liquid-solid chromatographic experiment similar to that first used by Tswett, the design of the column and the physical state of the... 

Mixtures passed through special columns (chromatography) in the gas phase (GC) or liquid phase (LC) can be separated into their individual components and analyzed qualitatively and/or quantitatively. Both GC and LC analyzers can be directly coupled to mass spectrometers, a powerful combination that can simultaneously separate and identify components of mixtures. 

Acetaldehyde can be isolated and identified by the characteristic melting points of the crystalline compounds formed with hydrazines, semicarbazides, etc these derivatives of aldehydes can be separated by paper and column chromatography (104,113). Acetaldehyde has been separated quantitatively from other carbonyl compounds on an ion-exchange resin in the bisulfite form the aldehyde is then eluted from the column with a solution of sodium chloride (114). In larger quantities, acetaldehyde may be isolated by passing the vapor into ether, then saturating with dry ammonia acetaldehyde—ammonia crystallizes from the solution. Reactions with bisulfite, hydrazines, oximes, semicarb azides, and 5,5-dimethyl-1,3-cyclohexanedione [126-81 -8] (dimedone) have also been used to isolate acetaldehyde from various solutions. 

Preparation of soil—sediment of water samples for herbicide analysis generally has consisted of solvent extraction of the sample, followed by cleanup of the extract through Uquid—Uquid or column chromatography, and finally, concentration through evaporation (285). This complex but necessary series of procedures is time-consuming and is responsible for the high cost of herbicide analyses. The advent of soUd-phase extraction techniques in which the sample is simultaneously cleaned up and concentrated has condensed these steps and thus gready simplified sample preparation (286). 

Polymeric -peroxides (3) from hydrogen peroxide and lower carbon ketones have been separated by paper or column chromatography and have been characterized by conversion to the bis(p-(nitro)peroxybenzoates). Oligomeric peroxides (3, R = methyl, R = ethyl, n = 1-4) from methyl ethyl ketone have been separated and interconverted by suitable treatment with ketone and hydrogen peroxide (44). 

Three general methods exist for the resolution of enantiomers by Hquid chromatography (qv) (47,48). Conversion of the enantiomers to diastereomers and subsequent column chromatography on an achiral stationary phase with an achiral eluant represents a classical method of resolution (49). Diastereomeric derivatization is problematic in that conversion back to the desired enantiomers can result in partial racemization. For example, (lR,23, 5R)-menthol (R)-mandelate (31) is readily separated from its diastereomer but ester hydrolysis under numerous reaction conditions produces (R)-(-)-mandehc acid (32) which is contaminated with (3)-(+)-mandehc acid (33). 

Contaminant by-products depend upon process routes to the product, so maximum impurity specifications may vary, eg, for CHA produced by aniline hydrogenation versus that made by cyclohexanol amination. Capillary column chromatography has improved resolution and quantitation of contaminants beyond the more fliUy described packed column methods (61) used historically to define specification standards. Wet chemical titrimetry for water by Kad Eisher or amine number by acid titration have changed Httle except for thein automation. Colorimetric methods remain based on APHA standards. 

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). 

Spectrophotometric deterrnination at 550 nm is relatively insensitive and is useful for the deterrnination of vitamin B 2 in high potency products such as premixes. Thin-layer chromatography and open-column chromatography have been appHed to both the direct assay of cobalamins and to the fractionation and removal of interfering substances from sample extracts prior to microbiological or radioassay. Atomic absorption spectrophotometry of cobalt has been proposed for the deterrnination of vitamin B 2 in dry feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2irnida2ole in the vitamin B 2 molecule have not been widely used. 

The separation of xanthates by ion-interaction reversed-phase column chromatography is described for the determination of eight different xanthates in reagents commonly used in flotation plants. The separated species were detected spectroscopically at a wavelength of 305 nm (96). 

In classical column chromatography the usual system consisted of a polar adsorbent, or stationary phase, and a nonpolar solvent, mobile phase, such as a hydrocarbon. In practice, the situation is often reversed, in which case the technique is known as reversed-phase Ic. 

High Pressure Liquid Chromatography. This modem version of the classical column chromatography technique is also used successfully for separation and quantitative analysis of dyes. It is generally faster than thin-layer or paper chromatography however, it requires considerably more expensive equipment. Visible and uv photometers or spectrophotometers are used to quantify the amounts of substances present. 

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