Hexane mobile phase

Silica has often been modified with silver for argentation chromatography because of the additional selectivity conferred by the interactions between silver and Jt-bonds of unsaturated hydrocarbons. In a recent example, methyl linoleate was separated from methyl linolenate on silver-modified silica in a dioxane-hexane mixture.23 Bonded phases using amino or cyano groups have proved to be of great utility. In a recent application on a 250 x 1-mm Deltabond (Keystone Scientific Belief onte, PA) Cyano cyanopropyl column, carbon dioxide was dissolved under pressure into the hexane mobile phase, serving to reduce the viscosity from 6.2 to 1 MPa and improve efficiency and peak symmetry.24 It was proposed that the carbon dioxide served to suppress the effect of residual surface silanols on retention. 

Eor the analysis of petroleum hydrocarbons, a moderately polar material stationary phase works well. The plate is placed in a sealed chamber with a solvent (mobile phase). The solvent travels up the plate, carrying compounds present in the sample. The distance a compound travels is a function of the affinity of the compound to the stationary phase relative to the mobile phase. Compounds with chemical structure and polarity similar to those of the solvent travel well in the mobile phase. For example, the saturated hydrocarbons seen in diesel fuel travel readily up a plate in a hexane mobile phase. Polar compounds such as ketones or alcohols travel a smaller distance in hexane than do saturated hydrocarbons. 

The fluorescent intensities of the E vitamers are highly dependent on the solvent. Polar solvents such as diethyl ether and alcohols provide greater intensities compared with hexane. The fluorescence is negligible when the compounds are dissolved in chlorinated hydrocarbons (137). The inclusion of an ether or an alcohol in the hexane mobile phase increases the sensitivity of vitamin E detection measurably in normal-phase HPLC. 

Reversed-phase HPLC with fluorescence detection is the preferred system for the routine determination of total a-tocopherol in vitamin E-supplemented foods after saponification. The use of NARP chromatography with a predominantly hexane mobile phase allows aliquots of hexane extracts of the unsaponifiable matter to be injected directly onto the column, thus avoiding the evaporation step necessary when a semiaqueous mobile phase is used (234). 

If I know that the compound is not soluble in aqueous solvents, I will probably select a silica column and a chloroform/hexane mobile phase. Separations of proteins will take me first to a TSK-3000sw column and a lOOmM Tris-phosphate pH 7.2 mobile phase unless I am separating soluble enzymes then I use a TSK-2000sw column. 

Three variants of one protocol are used to separate 17a-hydroxypregneno-lone and its metabolites, 17 cr-hydroxyprogesterone and its metabolites, and pregnenolone and its metabolites. The compounds have been separated on a silica gel column using a THF-hexane mobile phase in conjunction with a Flo-One Model HS radiometric detector from Radiomatic Instruments. 

Vo is defined versus pure hexane mobile phase as standard state for each phase. 

The use of water in oil microemulsions (L2 type) was also termed normal phase MLC. Dorsey was the first to use such reversed micellar mobile phases with two polar silica stationary phases, an unbonded and NH2 bonded phase [6]. His goal was to suppress the retention and selectivity variations caused by the water content of apolar solvents. It was shown that the retention of phenol, naphthol and dinitrotoluene was not sensitive to the water content (range 0.1-1% v/v) of an AOT-hexane mobile phase. In this concentration range, water molecules are bound tightly to the ions (AOT sulfonate polar heads and sodium counterions) with practically no free water molecules left to adsorb on the polar stationary phase. 

Fig. 5.1. Partial Ag+-HPLC chromatogram of the f8,c10-18 2 FAME isomer after iodine isomerization. A small quantity of c9,f11 was included for reference. Operating conditions 3 analytical ChromSpher 5 Lipids columns in series, isocratic elution with 0.1% MeCN/0.5% DE/hexane mobile phase at 1.0 mL/ min.UV spectra of each peak were acquired by an HPLC-PDA detector.The wavelength of maximum absorbance is indicated for every geometric isomer. The chromatogram was extracted at 233 nm.The wavelength maxima for the different geometric isomers is given for f,f, 229.8 nm, c,f, and f,c, 232.1 nm and forc,c, 234.5 nm.

The only substantial way in which the above conditions differ from parameters previously reported in this series (1-3) is that DE has been added to the elution solvent (9, 25, 26, 31). The theory for adding the DE was to prevent the selective loss of MeCN from the stored mobile phase. DE by itself, or combined with hexane, is a poor solvent to remove CLA from the strong Ag bonding. Adding a small amount of DE with the MeCN/hexane mobile phase provided a faster separation of all CLA... 

Fig. 5.4. Separation of a commercial CLA mixture (Nu-Chek Prep. Inc, Elysian, MN) by Ag+-HPLC acquired at the beginning (lower graph) and towards the end (upper graph) of the same batch of freshly prepared mobile phase of about 2 L.The relative decrease in the MeCN content in hexane on consecutive runs resulted in an increase in the retention times of all the CLA-FAMEs. Chromatographic conditions Three ChromSpher 5 Lipids analytical columns, 0.1% MeCN/0.5% DE/hexane mobile phase at 1.0 ml/min., UV detection at 233 nm.

Acetic Acid (HOAc) /Hexane Mobile Phase for Ag -HPLC at or Near Ambient Temperature... 

Fig. 5.5. Partial Ag+-HPLC chromatograms with a relative retention volume (RRV) scale, of the iodine-isomerized solutions containing each CLA positional isomer from 6,8- to 13,15-18 2 as FAME. A small quantity of c9,fl 1-18 2 was co-injected with each positional mixture for reference. Chromatographic conditions three ChromSpher 5 Lipids columns in series at 25°C, 0.1% MeCN/0.5% DE/hexane mobile phase at 1.0 mL/min, UV detection at 233 nm.

Fig. 5.6. Plot of relative retention volumes (RRV) vs. double bond positions of CLA isomers. Data were acquired under the same conditions of chromatograms reported in Fig. 5.5.Three ChromSpher 5 Lipids columns maintained at 25X were used in series, with a 0.1% MeCN/0.5% DE/99.4% hexane mobile phase at 1.0 mL/min, UV detection of 233 nm.

C, with a 2% HOAc/hexane mobile phase at 1.0 mL/min, has been found to be optimal condition for the separation of CLA FAME. 

Eight perchlorinated polycyclic aromatic hydrocarbons (benzene, acenaphthylene, naphthalene, biphenyl, anthracene, fluoranthene, phenanthrene, pyrene) were separated on a Cjg column (A = 250 nm) using an 80/20 methanol/hexane mobile phase [162], The authors noted that other mobile phase combinations such as ethanol/cyclohexane, acetonitrile/THF, and ethanol/Jiexane were not suitable for this separation. Elution was complete in 42 min. The pyrene and acenaphthylene peaks coeluted. A standard of 1 mg/mL of each component generated detectable peaks. 

Technical grade toxaphene was chromatographically fractionated and 20 chloro-boranes (hexa to nona) and five chlorocamphenes (hexa to octa) were identified [633]. Two normal-phase columns, silica and aminopropyl, were used with a hexane mobile phase (A = 220 nm). Elution fractions were collected over a 20-min period... 

Hexane and a Cig column [654] were conditions also used for this separation. A triphenyl colunm (A = 320 run) and a hexane mobile phase were used to study the temperature effects on the retention of fullerenes. For the range of 30-70°C, little or no change in the retention profile occurred [655]. Unlike the study presented above, mixtures of 5-15% ethyl elher in pentane on a phenyl colutiui (A = 330run) successfully separated Cgo and C70 fullerenes but could not separate the higher molecular weight species [656]. Plots of bA versus percent ethyl er in pentane were shown for Cso and C70 fullerenes. 

The four 3-methylcyclohexanone [2 + 2] photoadducts of Ceo were baseline resolved on a chiral bonded phase (2 = 300 nm) using a 2/1 toluene/hexane mobile phase [659]. In this study, a 100 pL aliquot of a 5mg/mL standard was injected. Three monoadducts of the reaction of C70 lullerene with l,2-bis(bromomethyl)-4,5-dimethoxybenzene were resolved on a Buckyclutcher I column (2 = 310 nm) using either a 2/3 toluene/hexane or 2/3 hexane/dichloromethane mobile phase [660]. Peak shapes were good but resolution was incomplete. Elution was complete in 70 min. 

Abidi and Mounts [338] studied a-, P-, y, and 5-tocopherol and 5,7-dimethyltocol retention on j5- and y-cyclodextrin columns (A = 298 nm, ex 345 nm, em) using both cyclohexane and hexane mobile phases modified with alcohols (ethanol, IPA, n-propyl alcohol, 1-butanol, and 2-methyl-2-propanol), ethers (dioxane, THE, diisopropyl ether, MrBE, or tetrahydropyran) or ethyl acetate. A k versus percent hexane and percent cyclohexane plot was shown for each modifier, Selected chromatograms and extensive tables of k and a values are presented. Most elutions were complete in less than 45 min. In general, peak shapes were excellent with the notable exception of when MtBE was used as the modifier. In this instance, very broad peaks were generated. Why this occurred for MrBE and not for diisopropyl ether is not explained, nor is it readily explainable. Also observed was a significant decrease in fluorescence intensity when ediyl acetate was the mobile phase modifier. The authors ascribed this result to the decreased solubility of the analytes in the solvent, since the effect was not observed with any other solvent system. Anotho-possibility is that ethyl acetate may effectively quench the fluorescence (it is the only carbonyl-containing solvent used in the study). 

Sotolon, a flavor component in French sherry, was analyzed using a diol column (X = 232 nm) and a 60/40 DCM/hexane mobile phase. Soloton eluted at 17min and was nearly baseline resolved from a large number of co-extracted analytes [786]. Results from various samples were tabluated with levels down to 3 pg/L reported. 

The retention behavior of retinol, retinal, ergocalciferol, cholecalciferol, a-, p-, y-, and (J-tocopherol, menadione, and phylloquinone was studied on a silica column (A = 254 nm or 292 nm) using a THF-modified hexane mobile phase [677]. A plot of versus percent THF in hexane (from 5% to 20%) is shown and is a good resource for method development work. THF provided superior selectivity for these solutes as compared with IPA. Conversely, IPA yielded sharper more symmetric peaks, especially for those solutes that have accessible hydroxyl functional groups. 

Besler et al. [671] studied retinal and retinol isomer retention on a silica column (A = 325nm or 371 nm) with hexane/MlBE (97/3 and 93/7), hexane/dioxane (93/7 and 94/6), and heptane/MrBE (94/6 and 93/7) mobile phases. The purpose of this work was to find an accq)table replacement for dioxane, namely, MrBE. The best overall chromatography resulted when 94/6 hexane/dioxane resolved 11,13-di-cis-, 13-CIS-, 9,11-13-tri-cis-, 9,13-di-cis-, 11-cis-, 7,1 l-di-c , 9-cis-, 7,9-di-cis, and all-tra s retinol in 15 min. Broader peaks but better resolution were obtained for the same solute set when 93/7 hexane/MtBE was used (elution was complete in 21 min), MlBE in hexane also gave excellent resolution of 13-c/s-retinoic acid and z -trans retinoic acid methyl esters on a silica column (A = 340 nm) in <5 min, whereas dichloromethane/hexane (35/65) required 17 min and toluene/hexane (45/55) required 10 min. It was found that for the toluene/hexane mobile phase, very small changes in the water content of the system dramatically and adversely affected the chromatography. The MrBE/hexane mobile phase provided more overall stability with respect to these changes. 

Poly(methyl methacrylate) and polytetrahydrofuran polymers were studied at the critical point of adsorption. This critical point of adsorption occurs where the retention of a given polymer is governed strictly hy the number and types of functional groups on the polymer [858]. The authors show plots of log MW vs. retention time for various mobile phase compositions on a given column. The critical point is reached when the retention time becomes independent of the molecular weight of the polymer. For poly(methyl methacrylate) that point was reached on a silica column (RI detector) with a 73/27 methyl ethyl ketone/cyclohexane mobile phase. For polytetrahydrofuran, the silica column and a 95/5 acetone/hexane mobile phase created the critical conditions. This approach has enabled the individual blocks within the co-polymer to be studied (i.e., the portion of the polymer that can make contact with the support surface). 

The isomers of 4-amino-3-(4-chlorophenyl)butyric acid were separated on a preparative scale using a silica column (A = 260nm) and 80/20 ethyl acetate/ hexane mobile phase [882], Up to 375 mg of sample was injected and baseline resolution was achieved in 6 min. 

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