Reversed-phase chromatography operation

Stationary phases used for achiral, small molecule separations are often further separated into two major classifications normal and reversed phase. Normal-phase chromatography uses a polar stationary phase with a less polar mobile phase, while reversed-phase chromatography operates using the exact opposite conditions. Additionally, there are several functionalized stationary phases that can operate in either normal or reversed phase, e.g., cyano and amine resins. 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Operational Considerations with the Reversed-Phase Chromatography of Peptides... 

There remains little more for the operator to decide. Sometimes, alternative but similar solvent mixtures that have a lower viscosity or higher solute diffusivity could be selected. For example, a n-hexane/methanol mixture might be chosen as an alternative to the more viscous n-heptane/isopropyl alcohol mixture as it has similar elution properties. However, it will be shown later, that if a fully optimized column is employed the viscosity of the mobile phase does not seem to effect the column performance as it is taken into account in the optimization procedure. The operator would, under some circumstances, be free to choose less toxic or less costly solvents for example, in reverse phase chromatography the operator could select methanol/ water solvent mixtures as opposed to acetonitrile/water mixture on the basis of lower cost or less... 

The growing popularity of reversed phase chromatography in particular has prompted polymer manufacturers to investigate the use of polymeric media for this mode of operation. Macroporous copolymers of styrene and divinylbenzene have similar properties to silica based stationary phases bonded with alkyl chains. However, the absence of leachables and stability at high pH can offer advantages under certain circumstances. High quality, mechanically stable macroporous polymeries are now manufactured at much larger scales than the... 

The following are suggested supplemental experiments which will further increase your understanding of the use and characteristics of reverse-phase chromatography. They may be done under the operating conditions previously established, depending on the lab time available and course requirements. 

Kubeczka (J58) utilized this general approach to separate a terpene mixture Into oxygenated terpenes, monoterpene hydrocarbons and sesquiterpene hydrocarbons fractions before a final analytical separation. He used a C-18 reverse phase system operated in the semi preparative mode to obtain h1s preliminary separations. The sample fractions were then analyzed by liquid-solid chromatography using deactivated silica gel (4.8% water/n-pentane) at -15 °C. 

Finally, HPLC is used for the analyses of explosives and in some forensic laboratories it is being introduced as a routine procedure. Chromatographic separation of explosives can be achieved by reversed-phase chromatography on alkyl-bonded silicas but a major problem has been in finding a technique capable of detecting nanogram quantities of materials. To date, the most sensitive method has been reductive electrochemical detection and provided fairly extensive operational precautions are taken (in particular the exclusion of oxygen), the required detection levels can be achieved. 

An extract of the sample is separated by reversed phase chromatography. The interfering polymeric matrix is removed by switching columns. A UV detector operating at 235 nm, calibrated with an external standard, is used to determine the level of monomer. 

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