Properties of Mobile Phases

Many protein isolations also involve the use of organic solvents at one or more purification steps, and thus one can often use this solvent in HPLC to maximize the recovery of protein samples. For example, apolipoproteins can be solubilized in isopropanol without loss of activity. For this reason Hancock and Sparrow 20) made extensive use of this solvent in the separation of C-apolipoproteins by reversed-phase HPLC. Glasel (//) found that neurophysins had a high solubility in aqueous methanol and used this solvent mixture in HPLC studies. 

Effect of Mobile Phase on Properties of Stationary Phase 

The silanol group (Si-OH), present at least at low concentrations in all reversed-phase columns, is capable of adsorbing a layer of a polar solvent such as methanol or water. A column conditioning procedure based on this adsorption is an essential step in normal-phase chromatography on silica columns. This process is probably also important in reversed-phase 

The initial gradient analysis (such as shown in Fig. 12) does not guarantee that the sample will be eluted from the column or be separated from other components of the mixture. For example, the sample may be bound irreversibly to the column, precipitated by the organic solvent, obscured by the solvent peaks in the breakthrough volume, or obscured by peaks from buffer or solvent impurities present in the gradient run. For these reasons it is essential that the initial gradient analysis be followed by some identification procedure to confirm that the sample of interest has in fact been eluted satisfactorily. Some widely used procedures are the following  

Fluorescent monitoring of effluent (either specific for a fluorogenic residue, e.g., Trp, or the presence of a free amino group if a fluorescent derivative is introduced) 

---

Special additives are used to improve properties of mobile phases for chromatography in biochemical separations. Antimicrobial reagents are the most often applied a list of their characteristics is given in Ref. 98 (Table 10.8) and in Ref. 6 (Table... 

Conductometric Detector Characteristics. Universal for aU anions or cations in solution (i.e., a bulk property of mobile phase detector) nondestructive concentration detector compound sensitivities differ over an order of magnitude range, useable... 

In conclusion, two distinct properties of mobile phases are to be distinguished in polymer HPLC namely their strength toward the coluiim packing and their thermodynamic quality toward both the column packing and the separated macromolecules. 

Another classification of detector is the bulk-property detector, one that measures a change in some overall property of the system of mobile phase plus sample. The most commonly used bulk-property detector is the refractive-index (RI) detector. The RI detector, the closest thing to a universal detector in lc, monitors the difference between the refractive index of the effluent from the column and pure solvent. These detectors are not very good for detection of materials at low concentrations. Moreover, they are sensitive to fluctuations in temperature. 

It is clear that tire rate of growdr of a reaction product depends upon two principal characteristics. The first of these is the thermodynamic properties of the phases which are involved in the reaction since these determine the driving force for the reaction. The second is the transport properties such as atomic and electron diffusion, as well as thermal conduction, all of which determine the mobilities of particles during the reaction within the product phase. 

In order to examine the thermal changes that take place in a column, it is necessary to derive an equation that describes the temperature change in a theoretical plate, in terms of its physical properties of the plate and the volume flow of mobile phase that passes through it. 

Another advantage of HdC is its generosity in terms of mobile-phase selection. The polymer size and solution properties of a polymer can be studied using HdC, especially OTHdC, in almost any solvent. In SEC, by comparison, the packing material and mobile phase have to be selected to prevent the nonsize exclusion effect. Because the instrumentation of HdC is similar to SEC, and the packing material and columns have become available commercially, this technique will gain in popularity. 

Supercritical fluid chromatography (SFC) refers to the use of mobile phases at temperatures and pressures above the critical point (supercritical) or just below (sub-critical). SFC shows several features that can be advantageous for its application to large-scale separations [132-135]. One of the most interesting properties of this technique is the low viscosity of the solvents used that, combined with high diffusion coefficients for solutes, leads to a higher efficiency and a shorter analysis time than in HPLC. 

In NPLC, which refers to the use of adsorption, i.e. liquid-solid chromatography (LSC), the surface of microparticulate silica (or other adsorbent) constitutes the most commonly used polar stationary phase normal bonded-phase chromatography (N-BPC) is typified by nitrile- or amino-bonded stationary phases. Silica columns with a broad range of properties are commercially available (with standard particle sizes of 3, 5 and 10 im, and pore sizes of about 6-15nm). A typical HPLC column is packed with a stationary phase of a pore size of 10 nm and contains a surface area of between 100 and 150m2 mL-1 of mobile phase volume. 

Reinitzer discovered liquid crystallinity in 1888 the so-called fourth state of matter.4 Liquid crystalline molecules combine the properties of mobility of liquids and orientational order of crystals. This phenomenon results from the anisotropy in the molecules from which the liquid crystals are built. Different factors may govern this anisotropy, for example, the presence of polar and apolar parts in the molecule, the fact that it contains flexible and rigid parts, or often a combination of both. Liquid crystals may be thermotropic, being a state of matter in between the solid and the liquid phase, or they may be lyotropic, that is, ordering induced by the solvent. In the latter case the solvent usually solvates a certain part of the molecule while the other part of the molecule helps induce aggregation, leading to mesoscopic assemblies. The first thermotropic mesophase discovered was a chiral nematic or cholesteric phase (N )4 named after the fact that it was observed in a cholesterol derivative. In hindsight, one can conclude that this was not the simplest mesophase possible. In fact, this mesophase is chiral, since the molecules are ordered in... 

In liquid-solid and liquid-liquid systems, manipulation of mobile phase properties is the dominant approach. Mixtures of poor and good solvents for the types of molecules being separated are prepared and retention observed. There are fewer stationary phases for liquid mobile phase systems for this reason and at least one other. 

Dittmann, M. M., and Rozing, G. P. (1997). Capillary electrochromatography investigation of the influence of mobile phase and stationary phase properties on electroosmotic velocity, retention, and selectivity. J. Microcolumn Sep. 9, 399-408. 

Structures made of transforming materials exhibit a striking capacity to hysteretically recover significant deformation with a controllable amount of energy absorbed m the process. The unusual properties of these materials are due to the fact that large deformations and inelastic behavior are accomplished by coordinated migration of mobile phase or domain boundaries. Intensive research in recent years has led to well-defined static continuum theories for some of the transforming materials (see Pitteri and Zanzotto (1997) for a recent review). Within the context of these theories, the main unresolved issues include history and rate sensitivity in the constitutive structure. 

Chromatography is a very versatile technique offering a wide range of solid phase materials and detector types which can deal with very complex mixtures. In practice all materials and conditions used in the instrument are carefully chosen to match the type of sample mixture involved. This includes selection of stationary phase (chemical and physical properties) column type and length sample pretreatment, operational temperatures, pressures, and flow rates physical and chemical nature of mobile phase detector type and so forth. Detection to nanogram level is quite common and some systems can detect to picogram level using very small volumes of sample. 

Some problems associated with conventional LLC (e.g., the loss of the liquid stationary phase through dissolution in the mobile phase) may be obviated by chemically bonding the liquid stationary phase to the support medium. This type of liquid-liquid chromatography is designated bonded phase chromatography (BPC)(11). Since the properties of bonded phases may differ substantially from those of coated phases, BPC separation characteristics may differ from those of conventional LLC. Many phases have exhibited increased efficiency when bonded to the support medium. Most current reverse phase HPLC work involves the use of stationary phases bonded to microparticles. 

---