Reversed phase adsorbents

Recovery and Purification. The dalbaheptides are present in both the fermentation broth and the mycelial mass, from which they can be extracted with acetone or methanol, or by raising the pH of the harvested material, eg, to a pH of 10.5—11 for A47934 (16) (44) and A41030 (41) and actaplanin (Table 2) (28). A detailed review on the isolation of dalbaheptides has been written (14). Recovery from aqueous solution is made by ion pair (avoparcin) or butanol (teicoplanin) extraction. The described isolation schemes use ion-exchange matrices such as Dowex and Amberlite IR, acidic alumina, cross-linked polymeric adsorbents such as Diaion HP and Amberlite XAD, cation-exchange dextran gel (Sephadex), and polyamides in various sequences. Reverse-phase hplc, ion-exchange, or affinity resins may be used for further purification (14,89). 

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. 

L. V. Poucke, D. Rousseau, C. V. Peteghem and B. M. J. Spiegeleer, Two-dimensional HPTLC of sulphonamides on cyanoplates, using the sti aight-phase and reversed-phase character of the adsorbent , 7. Planar Chromatogr. 2 395-397 (1989). 

Quality of the adsorbent layer. Layers for HPTLC are prepared using specially purified silica gel with average particle diameter of 5-15 /mi and a narrow particle size distribution. The silica gel may be modified if necessary, e.g. chemically bonded layers are available commercially as reverse-phase plates. Layers prepared using these improved adsorbents give up to about 5000 theoretical plates and so provide a much improved performance over conventional TLC this enables more difficult separations to be effected using HPTLC, and also enables separations to be achieved in much shorter times. 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

These sorbents may be used either for selective fixation of biological molecules, which must be isolated and purified, or for selective retention of contaminants. Selective fixation of biopolymers may be easily attained by regulation of eluent polarity on the basis of reversed-phase chromatography methods. Effective isolation of different nucleic acids (RNA, DNA-plasmid) was carried out [115, 116]. Adsorption of nucleosides, nucleotides, tRN A and DNA was investigated. It was shown that nucleosides and nucleotides were reversibly adsorbed on... 

Many racemic mixtures can be separated by ordinary reverse phase columns by adding a suitable chiral reagent to the mobile phase. If the material is adsorbed strongly on the stationary phase then selectivity will reside in the stationary phase, if the reagent is predominantly in the mobile phase then the chiral selectivity will remain in the mobile phase. Examples of some suitable additives are camphor sulphonic acid (10) and quinine (11). Chiral selectivity can also be achieved by bonding chirally selective compounds to silica in much the same way as a reverse phase. A example of this type of chiral stationary phase is afforded by the cyclodextrins. 

If the mixture to be separated contains fairly polar materials, the silica may need to be deactivated by a more polar solvent such as ethyl acetate, propanol or even methanol. As already discussed, polar solutes are avidly adsorbed by silica gel and thus the optimum concentration is likely to be low, e.g. l-4%v/v and consequently, a little difficult to control in a reproducible manner. Ethyl acetate is the most useful moderator as it is significantly less polar than propanol or methanol and thus, more controllable, but unfortunately adsorbs in the UV range and can only be used in the mobile phase at concentrations up to about 5%v/v. Above this concentration the mobile phase may be opaque to the detector and thus, the solutes will not be discernible against the background adsorption of the mobile phase. If a detector such as the refractive index detector is employed then there is no restriction on the concentration of the moderator. Propanol and methanol are transparent in the UV so their presence does not effect the performance of a UV detector. However, their polarity is much greater than that of ethyl acetate and thus, the adjustment of the optimum moderator concentration is more difficult and not easy to reproduce accurately. For more polar mixtures it is better to explore the possibility of a reverse phase (which will be discussed shortly) than attempt to utilize silica gel out of the range of solutes for which it is appropriate. 

Solvents interact with reverse phases in very much the same way as they do with the surface of silica gel. However, in this case it is the more dispersive component of the mobile phase that is adsorbed on the surface as opposed to silica gel, which being a polar stationary phase, adsorbs the more polar solvent onto its surface. 

The more dispersive solvent from an aqueous solvent mixture is adsorbed onto the surface of a reverse phase according to Langmuir equation and an example of the adsorption isotherms of the lower series of aliphatic alcohols onto the surface of a reverse phase (9) is shown in figure 9. It is seen that the alcohol with the longest chain, and thus the most dispersive in character, is avidly adsorbed onto the highly dispersive stationary phase, much like the polar ethyl acetate is adsorbed onto the highly polar surface of silica gel. It is also seen that... 

Solutes will interact with the reverse phase surface in much the same way as they do with the silica gel surface. There will be basically two forms of interaction, by sorption and by displacement. Sorption interaction has been experimentally confirmed by Scott and Kucera (10) by measuring the adsorption isotherm of acetophenone on the reverse phase RP18 from a 40%w/v acetonitrile mixture in water. The authors noted that there was no change in the acetonitrile concentration, as the solute was adsorbed. Displacement interactions, although certain to occur, do not appear to have been experimentally demonstrated to date. 

As the solvent concentration increases, the PIC reagents will interact more strongly with the mobile phase and will be less strongly adsorbed on the reverse phase surface. As a consequence, there will be less ion exchange material on the stationary phase surface. This is clearly demonstrated by the adsorption isotherm of octane sulfonate shown in figure 10. 

This precipitation process can be carried out rather cleverly on the surface of a reverse phase. If the protein solution is brought into contact with a reversed phase, and the protein has dispersive groups that allow dispersive interactions with the bonded phase, a layer of protein will be adsorbed onto the surface. This is similar to the adsorption of a long chain alcohol on the surface of a reverse phase according to the Langmuir Adsorption Isotherm which has been discussed in an earlier chapter. Now the surface will be covered by a relatively small amount of protein. If, however, the salt concentration is now increased, then the protein already on the surface acts as deposition or seeding sites for the rest of the protein. Removal of the reverse phase will separate the protein from the bulk matrix and the original protein can be recovered from the reverse phase by a separate procedure. 

The solid phase extraction cartridge (SPEC) is another somewhat vainglorious name given to a short inert plastic tube packed with an adsorbent, usually a reversed phase or an ion exchange resin. The particle size of the packing is often significantly larger than that used... 

The tetrahydrocannabinol carboxylic acid was extracted from the urine by means of a solid state extraction cartridge packed with a Cl 8 reverse phase (octyldecyldimethyl chains). As the urine sample was used direct, and contained no added solvent, the materials of interest were irreversibly adsorbed on the reverse phase solely by dispersive interactions. 

The analysis of a pharmaceutical tablet (6) requires sample preparation that is little more complex as most tablets contain excipients (a solid diluent) that may be starch, chalk, silica gel, cellulose or some other physiologically inert material. This sample preparation procedure depends on the insolubility of the excipient in methanol. As the components of interest are both acidic and neutral, the separation was achieved by exploiting both the ionic interactions between the organic acids and the adsorbed ion exchanger and the dispersive interactions with the remaining exposed reverse phase. 

As the solvent mixture also contained 225 mg of tetramethyl ammonium hydroxide pentahydrate per liter at a high water content (75%), the surface of the reverse phase would have been largely covered with the tetramethyl ammonium hydroxide pentahydrate. This would have acted as an adsorbed ion exchange stationary phase. It is clear that the free acids, salicylic acid, acetylsalicylic acid (aspirin) and benzoic acid were retained largely by ionic interactions with adsorbed basic ion exchanger and partly by dispersive interactions with the exposed reversed phase. The acetaminophen and the caffeine, on the other hand, being unionized substances, were retained only by dispersive interactions with the exposed reversed phase. 

Complete resolution was not achieved due to the carryover of interfering substances which frequently occurs when separating the components of biological samples. The column carried a reverse phase, but as the mobile phase contained low concentrations of lauryl sulfate, some would have adsorbed on the surface of the stationary phase and significantly modified its interacting properties. The retention mechanism is likely to have involved both ionic interactions with the adsorbed ion exchanger together with dispersive interactions with any exposed areas of the reverse phase. 

There are two basic procedures that have been successfully used for the separation of isomers. The first is to add a chiral agent to the mobile phase such that it is adsorbed, for example, on the surface of a reverse phase, producing a chirally active surface. This approach has been discussed on page (38) in chapter 2. The alternative is to employ a stationary phase that has been produced with chiral groups bonded to the surface. 

The peak capacity is not pertinent as the separation was developed by a solvent program. The expected efficiency of the column when operated at the optimum velocity would be about 5,500 theoretical plates. This is not a particularly high efficiency and so the separation depended heavily on the phases selected and the gradient employed. The separation was achieved by a complex mixture of ionic and dispersive interactions between the solutes and the stationary phase and ionic, polar and dispersive forces between the solutes and the mobile phase. The initial solvent was a 1% acetic acid and 1 mM tetrabutyl ammonium phosphate buffered to a pH of 2.8. Initially the tetrabutyl ammonium salt would be adsorbed strongly on the reverse phase and thus acted as an adsorbed ion exchanger. During the program, acetonitrile was added to the solvent and initially this increased the dispersive interactions between the solute and the mobile phase. 

As the acetonitrile concentration increased, however, the concentration of adsorbed tetrabutyl ammonium salt would also be reduced and it would be desorbed from the reverse phase with a resulting reduction in the ionic interactions of the solutes with the stationary phase. At higher concentrations of acetonitrile, the tetrabutyl ammonium salt would be completely desorbed from the stationary phase and the interactions of the solutes with the stationary phase would become almost exclusively dispersive. This is an example... 

Sixteen solid-phase materials were tested on a laboratory scale and the antho-cyanin and sugar content of collected fractions were determined. Among these, reverse-phase silica gels and macroreticular non-ionic acrylic polymer adsorbents such as Serdolit PAD IV or Amberlite XAD-7 turned out to be most suitable. SPE was used to investigate these materials on an enlarged scale, improving elution gradient and column purification. Amberlite XAD-7 was successfully applied in a middle-scale separation. ... 

A large range of stationary phases is available, and according to their polarity they can be divided into normal phase and reversed phase types. Silica gel, aluminium oxide, and a nitrile-bonded-phase are normal adsorbents used to separate carotenoids... 

Solvent strength determines the value, but not the selectivity. The mobile phase can be established by using the polarity index P proposed by Snyder. The highest values of P represent the strongest solute adsorbed in conventional TLC but represent the weakest for the separation in reversed phases. Sometimes aqueous polar mixtures cannot totally wet the chemically bonded layer. For this reason, checking... 

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