Chromatographic Dilution

Another important phenomenon that varies strongly with column i.d. is that of chromatographic dilution. As a result of all the dispersive processes, an analyte will be progressively diluted as it proceeds along the column. The dilution factor D for analyte A can be estimated (Vissers 1997)  

Evaluation of Equation [3.50] shows, for example, that if all other parameters are the same, changing from a column with i.d. 2.1 mm to a 300 jxm capillary will result in a 50-fold increase in the concentration of the analyte as it elutes from the column into the detector, with corresponding increases in peak height (and also peak area depending on whether the detector is concentration or mass flow dependent, see Section 4.4.8). Note, however, that such a comparison is valid only if aU parameters other than i.d. are maintained the same in particnlar this comparison assumes that it is possible to load the same amount of analyte in both cases, a real limitation since the sample size that can be injected without overloading a column is proportional to the amount of stationary phase 

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Another factor that needs to be considered is the dilution of the chromatographic peak during the separation process. The extent of chromatographic dilution can be... 

Microbore columns are of advantage due to the low mobile phase volumetric flow rates involved, the reduced on-column samples and the reduced chromatographic dilution, conferring high efficiency. Microbore columns with ELD were applied to the analysis of antioxidants, which are usually electroactive compounds. This combination led to highly selective and sensitive analyses. A micro ELD was designed and tested with... 

Reducing the ID of the LC column offers several advantages, among which are most importantly (1) increased mass sensitivity for concentration sensitive detectors due to reduced chromatographic dilution, (2) easier coupling to MS interfaces [ESI and matrix-assisted laser desorption/ionization (MALDI)] due to the lower flow rates, and (3) a lower stationary phase and solvent consumption. 

D Chromatographic dilution as a result of the dispersive processes accounted for in the van Deemter equation an analyte will be progressively diluted as it proceeds along the column D gives the ratio of the initial sample concentration to the concentration at the peak maximum (caP ) as it elutes D = = (e.irdp /4Vjnj).(l -I-... 

There are advantages and disadvantages in the use of packed columns with smaller i.d. As discussed further below, the most important advantages are the small sample sizes that can be accommodated, small volume flow rates (environmental and cost considerations), reduced chromatographic dilution of the analyte... 

The positive effects include a reduction in plate height (mainly via reduction in the A-term, multipath dispersion), reduction in chromatographic dilution and thus increased detection sensitivity, and reduction in volume flow rate and thus in solvent consumption. However, offsetting disadvantages include limitations on the volume of sample solution and the amount of analyte that can be injected without overloading the column, and the meticulous attention that is required to reduce the extra-column dead volume (injectors, detectors etc.) to avoid excessive band broadening. As noted above, these columns do not appear to have been used in vahdated quantitative methods. 

The chief uses of chromatographic adsorption include (i) resolution of mixtures into their components (Li) purification of substances (including technical products from their contaminants) (iii) determination of the homogeneity of chemical substances (iv) comparison of substances suspected of being identical (v) concentration of materials from dilute solutions (e.g., from a natural source) (vi) quantita tive separation of one or more constituents from a complex mixture and (vii) identi-1 ig- II, 16, 3. gcajjQij and control of technical products. For further details, the student is referred to specialised works on the subject. ... 

Gyclodextrins. As indicated previously, the native cyclodextrins, which are thermally stable, have been used extensively in Hquid chromatographic chiral separations, but their utihty in gc appHcations was hampered because their highly crystallinity and insolubiUty in most organic solvents made them difficult to formulate into a gc stationary phase. However, some functionali2ed cyclodextrins form viscous oils suitable for gc stationary-phase coatings and have been used either neat or diluted in a polysiloxane polymer as chiral stationary phases for gc (119). Some of the derivati2ed cyclodextrins which have been adapted to gc phases are 3-0-acetyl-2,6-di-0-pentyl, 3-0-butyryl-2,6-di-0-pentyl,... 

Chromatographic separation of diluted molasses streams into a high purity fraction suitable for concentration and crystallization and a second low purity by-product, which can be concentrated and sold as an animal feed product, has been employed in Finland since the 1970s and in the United States since the mid-1980s. Since the early 1990s, production of sugar from beet molasses has almost tripled, and the trend is expected to continue for the next two years to consume most of the domestic beet molasses (Fig. 7) (3,9). 

Preferably, high pressure Hquid chromatography (hplc) is used to separate the active pre- and cis-isomers of vitamin D from other isomers and allows their analysis by comparison with the chromatograph of a sample of pure reference i j -vitainin D, which is equiUbrated to a mixture of pre- and cis-isomers (82,84,85). This method is more sensitive and provides information on isomer distribution as well as the active pre- and cis-isomer content of a vitamin D sample. It is appHcable to most forms of vitamin D, including the more dilute formulations, ie, multivitamin preparations containing at least 1 lU/g (AOAC Methods 979.24 980.26 981.17 982.29 985.27) (82). The practical problem of isolation of the vitamin material from interfering and extraneous components is the limiting factor in the assay of low level formulations. 

Operational temperatures of 4—27°C are maintained. In this process the flavor components are concentrated in the retentate. A reduced alcohol product is obtained by adding back water to give the desired flavor impact. Typical gas chromatographic results, comparing unprocessed 80° proof whiskey with reverse osmosis processed 54° proof whiskey and diluted 54° proof whiskey, indicate good congener retention in the alcohol-reduced (RO) processed whiskey (Table 7). 

Conventional elution chromatography has the serious disadvantage of dilution, and usually a concentration step must follow. The technique of displacement chromatography circumvents dilution and may even result in an eluant more concentrated than the feed. A displacer compound breaks the desired product from the chromatographic material sharply, and a column heavily loaded with several biochemicals will release them one at a time depending on their adsorption equilibria. However, the displacers tena to be expensive and can be troublesome to remove from the product. 

This type of chromatographic development will only be briefly described as it is rarely used and probably is of academic interest only. This method of development can only be effectively employed in a column distribution system. The sample is fed continuously onto the column, usually as a dilute solution in the mobile phase. This is in contrast to displacement development and elution development, where discrete samples are placed on the system and the separation is subsequently processed. Frontal analysis only separates part of the first compound in a relatively pure state, each subsequent component being mixed with those previously eluted. Consider a three component mixture, containing solutes (A), (B) and (C) as a dilute solution in the mobile phase that is fed continuously onto a column. The first component to elute, (A), will be that solute held least strongly in the stationary phase. Then the... 

Pereirine, CjoHjgONj. O-SH O. According to Bertho and Moog , this amorphous alkaloid, m.p. 134-5°, [a]ff° + 137-5° (EtOH), gives a methiodide decomposing at 233-5° and a methyl ether, m.p. 195-7° (dec.), and is stable to dilute mineral acid. Bertho and Sarx state that chromatographic examination shows the alkaloid has not been obtained pure. 

A solution of 1 g of the dione in 200 ml of methanol at 0° is treated with 75 mg of sodium borohydride and the mixture is kept for 2 hr. After addition of 0.1 ml of acetic acid the mixture is concentrated to ca. 20 ml. Dilution with water gives 0.9 g of crystals which are chromatographed on 20 g of unwashed alumina. Elution with benzene-ether (40 60) yields 0.73 g of the methyl-hydroxytestosterone, mp 245-249°, which after crystallization from acetone has mp 255-256° [a] 111° (CHCI3). 

To a mixture of ethyl 5a-cholestan-3-one 2a-xanthate (2 g, 3.95 mmol) and 100 ml methanol is added sufficient ether to completely dissolve the solids. Sodium borohydride (90 mg, 2.36 mmol) is added directly to the reaction flask and the solution is stirred at room temperature for 4 hr. (The use of an excess of sodium borohydride and an extended reaction time produces 5oc-cholestan-2a,3a-thiirane.) The reaction is diluted with 200 ml ether and washed several times with ca. 100 ml water, dried (MgS04) and the solvent is removed under vacuum. The crude sticky gum is chromatographed on a column of 85 g silicic acid. The hexane eluates contain 5a-cholest-2-ene. Ethyl 5a-cholestan-3a-ol 2a-xanthate is obtained in ca. 30% yield by subsequent elution with benzene hexane (1 7) and the desired ethyl 5a-cholestan-3 -ol 2a-xanthate is eluted with ether hexane (1 3) in ca. 30% yield. 

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