Stationary-phase fraction

Stationary phase fraction. e. The volume of the stationary phase per unit volume of the packed column. 

The column is swept continuously by a carrier gas such as helium, hydrogen, nitrogen or argon. The sample is injected into the head of the column where it is vaporized and picked up by the carrier gas. In packed columns, the injected volume is on the order of a microliter, whereas in a capillary column a flow divider (split) is installed at the head of the column and only a tiny fraction of the volume injected, about one per cent, is carried into the column. The different components migrate through the length of the column by a continuous succession of equilibria between the stationary and mobile phases. The components are held up by their attraction for the stationary phase and their vaporization temperatures. 

The distribution coefficient, represents the fractional volume of a specific stationary phase explored by a given solute, represented by equation 3 ... 

In reversed-pViase chromatography (RPC), the mobile phase modulator is typically a water-miscible organic solvent, and the stationary phase is a hydrophobic adsorbent. In this case, the logarithm of solute retention factor is commonly found to be linearly related to the volume fraction of the organic solvent. 

Q) is that fraction of the pore volume accessible to the solute, and ( ) is the fraction of the stationary phase accessible to the solute. 

Concentrations of moderator at or above that which causes the surface of a stationary phase to be completely covered can only govern the interactions that take place in the mobile phase. It follows that retention can be modified by using different mixtures of solvents as the mobile phase, or in GC by using mixed stationary phases. The theory behind solute retention by mixed stationary phases was first examined by Purnell and, at the time, his discoveries were met with considerable criticism and disbelief. Purnell et al. [5], Laub and Purnell [6] and Laub [7], examined the effect of mixed phases on solute retention and concluded that, for a wide range of binary mixtures, the corrected retention volume of a solute was linearly related to the volume fraction of either one of the two phases. This was quite an unexpected relationship, as at that time it was tentatively (although not rationally) assumed that the retention volume would be some form of the exponent of the stationary phase composition. It was also found that certain mixtures did not obey this rule and these will be discussed later. In terms of an expression for solute retention, the results of Purnell and his co-workers can be given as follows,... 

Vs) is the total volume of stationary phase in the column and (a) is the volume fraction of phase (A) in the stationary phase mixture... 

This remarkably simple relationship is depicted in Figure 14. It was apparent from his results that the volume fraction of the solvent determined the probability of interaction with the solute in much the same way that the partial pressure of a gas determines the probability of collision. It also indicated that the influence of each stationary phase component was independent and unaffected by presence of the other. 

Figure 14. Graph of Corrected Retention Volume against Volume Fraction of Stationary Phase...

Figure 15. Alternative Methods for Combining Volume Fractions of Stationary Phase in GC...

However, in this case, over the time period (tr), a fraction of time spent by the solute in the stationary phase and, thus,... 

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

S. Blomherg and J. Roeraade, Preparative capillary gas cliromatography. IF Fraction collection on rtaps coated with a very tliick-film of immobilized stationary phase , J. Chromatogr. 394 443-453 (1987). 

In addition to the development of the powerful chiral additive, this study also demonstrated that the often tedious deconvolution process can be accelerated using HPLC separation. As a result, only 15 libraries had to be synthesized instead of 64 libraries that would be required for the full-scale deconvolution. A somewhat similar approach also involving HPLC fractionations has recently been demonstrated by Griffey for the deconvolution of libraries screened for biological activity [76]. Although demonstrated only for CE, the cyclic hexapeptides might also be useful selectors for the preparation of chiral stationary phases for HPLC. However, this would require the development of non-trivial additional chemistry to appropriately link the peptide to a porous solid support. 

The same concepts apply to column chromatography, where the stationary phase is normally small particles of silica, Si02, or alumina, A1,0 . These substances are not very reactive and have specially prepared surfaces to increase their adsorption ability. The column is saturated with solvent, and a small volume of solution containing the solutes is poured onto the top. As soon as it has soaked in, more solvent is added. The solutes travel slowly down the column and are eluted (removed as fractions) at the bottom (Fig. 2). If the mobile phase is less polar than the stationary phase, the less polar solutes will be eluted first and the more polar ones last. 

Figure 4-1. Components of a simple liquid chromatography apparatus. R Reservoir of mobile phase liquid, delivered either by gravity or using a pump. C Glass or plastic column containing stationary phase. F Fraction collector for collecting portions, called fractions, of the eluant liquid in separate test tubes.

Adsorption and ion exchange chromatography are well-known methods of LC. In adsorption, one frequently selects either silica or alumina as stationary phase for separation of nonionic, moderately polar substances (e.g. alcohols, aromatic heterocycles, etc.). This mode works best in the fractionation of classes of compounds and the resolution of isomeric substances (J). Ion exchange, on the other hand, is applicable to the separation of ionic substances. As to be discussed later, this mode has been well developed as a tool for analysis of urine constituents (8). 

Other modes of LC operation include liquid-liquid partition chromatography (LLC) and bonded phase chromatography. In the former, a stationary liquid phase which is immiscible with the mobile phase is coated on a porous support, with separation based on partition equilibrium differences of components between the two liquid phases. This mode offers an alternative to ion exchange in the fractionation of polar, water soluble substances. While quite useful, the danger exists in LLC that the stationary phase can be stripped from the column, if proper precautions are not taken. Hence, it is typical to pre-equil-ibrate carefully the mobile and stationary phases and to use a forecolimn, heavily loaded with stationary phase 9). 

A biphasic solvent system composed of tert-butyl methyl ether, -butanol, acetonitrile, and water (2 2 1 5) acidified with triflnoroacetic acid has been applied to fractionate anthocyanins. The npper (organic) phase acts as the stationary phase and the lower (aqneons) as the mobile phase. HSCCC has been applied to obtain several anthocyanin fractions from wine, red cabbage, black cnrrants, chokeber-ries, " bilberries (Vaccinium myrtillus) acylated anthocyanins, and also isolate individnal anthocyanins from wine. ... 

---