Stationary phase porous

Besides silica, silica-based and polymeric stationary phases, porous graphitized carbon (PGC), zirconium oxide and its derivatives, alumina and its derivatives have been used for the solution of special separation problems which cannot be easily solved by using traditional HPLC stationary phases. 

Stationary phase Porous glass sheet (5 cm x 5 cm x 0.5 mm) pore diameter 700 nm mean pore volume 0.46 cm /g specific surface area 6.6 M /g). Mobile phase M = n-Butanol-benzene-1.0 M HNO3-I.O M HCl (75 69 4 2) Afj = acetone-3.0 M HCl (99 1). Detection (a) fluorescent reaction by heating (3 il solution of each cation was spotted on the glass sheet, dried and heated in an electric muffle furnace at 100 - 700°C for 15 min., irradiated in the dark with a UV lamp at 254 nm to reveal the fluorescence of the spots), (b) 0.05% solution of dithirone in chloroform or 1% solution of 8-hydroxy-quinoline in methanol followed by exposure to ammonia gas. The spots detected by (a) overlapped the triplicate spots detected by (b), confirming that the fluorescent compounds were not due to impurities. Remarks Examination of fluorescence reactions of inorganic cations e.g. Al ,... 

The most widely used particulate support is diatomaceous earth, which is composed of the silica skeletons of diatoms. These particles are quite porous, with surface areas of 0.5-7.5 m /g, which provides ample contact between the mobile phase and stationary phase. When hydrolyzed, the surface of a diatomaceous earth contains silanol groups (-SiOH), providing active sites that absorb solute molecules in gas-solid chromatography. 

In liquid-liquid chromatography the stationary phase is a liquid film coated on a packing material consisting of 3-10 pm porous silica particles. The stationary phase may be partially soluble in the mobile phase, causing it to bleed from the column... 

In liquid-solid adsorption chromatography (LSC) the column packing also serves as the stationary phase. In Tswett s original work the stationary phase was finely divided CaCOa, but modern columns employ porous 3-10-)J,m particles of silica or alumina. Since the stationary phase is polar, the mobile phase is usually a nonpolar or moderately polar solvent. Typical mobile phases include hexane, isooctane, and methylene chloride. The usual order of elution, from shorter to longer retention times, is... 

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

A form of liquid chromatography in which the stationary phase is a porous material and in which separations are based on the size of the solutes. 

Gas chromatography, depending on the stationary phase, can be either gas—Hquid chromatography (glc) or gas—soHd chromatography (gsc). The former is the most commonly used. Separation in a gas—Hquid chromatograph arises from differential partitioning of the sample s components between the stationary Hquid phase adsorbed on a porous soHd, and the gas phase. Separation in a gas—soHd chromatograph is the result of preferential adsorption on the soHd or exclusion of materials by size. 

It is clear that the separation ratio is simply the ratio of the distribution coefficients of the two solutes, which only depend on the operating temperature and the nature of the two phases. More importantly, they are independent of the mobile phase flow rate and the phase ratio of the column. This means, for example, that the same separation ratios will be obtained for two solutes chromatographed on either a packed column or a capillary column, providing the temperature is the same and the same phase system is employed. This does, however, assume that there are no exclusion effects from the support or stationary phase. If the support or stationary phase is porous, as, for example, silica gel or silica gel based materials, and a pair of solutes differ in size, then the stationary phase available to one solute may not be available to the other. In which case, unless both stationary phases have exactly the same pore distribution, if separated on another column, the separation ratios may not be the same, even if the same phase system and temperature are employed. This will become more evident when the measurement of dead volume is discussed and the importance of pore distribution is considered. 

The stationary phase can be apportioned in a similar manner. For example, with a bonded phase, due to the porous nature of the support, some of the pores will become blocked with stationary phase and so the total amount of stationary phase can be divided into that which is chromatographically available (Vs(A)) and that which is chromatographically unavailable (Vs(u)). 

Interactions in the stationary phase employing a porous stationary phase or support must also involve the mobile phase trapped in a static form inside the pores. It follows that the diffusivity of the solute in the stationary phase (Ds) will be similar to that in the mobile phase (Dm). Thus, to a first approximation, it can be assumed that Ds = coDm, where (co) is a constant probably close to unity. Thus, equation... 

FIGURE 1.5 Cumulative pore volume curves of 5-/j.m monosized porous particles. [Reprinted from T. Ellingsen et al. (1990). Monosized stationary phases for chromatography. J. Chromatogr. 535, 147-161 with kind permission from Elsevier Science-NL, Amsterdam, The Netherlands.]... 

Other specifications of the porous materials that affect the performance of HOPC include pore volume. A larger pore volume, or equivalently closer packing, of the porous materials increases the ratio of the volume of the stationary phase to the volume of the mobile phase. The difference causes a shift in the segregation boundary in the partitioning and a change in the resolution. 

G. Gastello and G. D Amato, Gomparison of the polarity of porous polymer-bead stationary phases with that of some liquid phases , J. Chromatogr. 366 51-57 (1986). 

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 support materials for the stationary phase can be relatively inactive supports, e.g. glass beads, or adsorbents similar to those used in LSC. It is important, however, that the support surface should not interact with the solute, as this can result in a mixed mechanism (partition and adsorption) rather than true partition. This complicates the chromatographic process and may give non-reproducible separations. For this reason, high loadings of liquid phase are required to cover the active sites when using high surface area porous adsorbents. 

The materials originally used as stationary phases for GPC were the xerogels of the polyacrylamide (Bio-Gel) and cross-linked dextran (Sephadex) type. However, these semi-rigid gels are unable to withstand the high pressures used in HPLC, and modern stationary phases consist of microparticles of styrene-divinylbenzene copolymers (Ultrastyragel, manufactured by Waters Associates), silica, or porous glass. 

Finally, the useful life of an analytical column is increased by introducing a guard column. This is a short column which is placed between the injector and the HPLC column to protect the latter from damage or loss of efficiency caused by particulate matter or strongly adsorbed substances in samples or solvents. It may also be used to saturate the eluting solvent with soluble stationary phase [see Section 8.2(2)]. Guard columns may be packed with microparticulate stationary phases or with porous-layer beads the latter are cheaper and easier to pack than the microparticulates, but have lower capacities and therefore require changing more frequently. 

The volume of stationary phase with which the solutes in a mixture can interact (Vs in equation (11)) will depend on the physical nature of the stationary phase or support. If the stationary phase is a porous solid, and the sizes of the pores are commensurate with the molecular diameter of the sample components, then the stationary phase becomes... 

The mixed retention mechanism described above has a parallel in the effect of exclusion on retention when using porous stationary phases. The smaller molecules can enter more pores and thus interact with more stationary phase than the larger molecules that are excluded from many pores and, consequently, interact with less stationary phase. Assuming the smaller molecules are more strongly retained, the exclusion of large molecules augments the difference in retention between molecules of different size. 

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). 

The precision in retention from injection to injection will often be better than 1%. Over longer periods of time such precision requires the following (a) good flow control from the pump (b) constant mobile and stationary phases and (c) temperature control of the column. The critical question of reproducibility from column to column is still a matter of concern, especially when dealing with the more sophisticated packing materials, e.g. small porous particles, bonded phases While frequently this reproducibility is quite good, workers should recognize that care must be exercised to achieve and/or maintain reproducible columns. Undoubtedly, with experience, this need not be a severe problem. 

The two techniques differ in that HDC employs a nonporous stationary phase. Separation is affected as a result of particles of different size sampling different velocities in the interstitial spaces. Size exclusion chromatography is accomplished by superimposing a steric selection mechanism which results from the use of a porous bed. The pore sizes may vary over a wide range and the separation occurs as a result of essentially the same processes present in the gel permeation chromatography of macromolecules. 

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