Mobile-Phase Considerations

The HPLC pump draws the mobile phase from the reservoir via vacuum action. In the process, air dissolved in the mobile phase may withdraw from the liquid and form bubbles in the flow stream unless such air is removed from the liquid in advance. Air in the flow stream is undesirable because it can cause a wide variety of problems, such as poor pump performance or poor detector response. Removing air from the mobile phase, called degassing, in advance of the chromatography is a routine matter, however, and can be done in one of several ways 1) helium sparging, 2) ultrasonic agitation, 3) drawing a vacuum over the surface of the liquid, or 4) a combination of numbers 2 and 3. 

Helium sparging refers to the vigorous bubbling of helium gas through the mobile phase. This can be done while it is contained in the reservoir by using a metal bubbler with tubing attached to a cylinder 

FIGURE 13.2 The analytical strategy flow chart for HPLC. 

FIGURE 13.3 Mobile phase reservoir, shown on the right, with vented cap and a coarse filter on the tip of the flow tube shown inside a well on the bottom of the reservoir. 

Charlie Focht of the Nebraska State Agriculture Laboratory prepares the mobile phase for an atrazine assay. Note that the vacuum flask is positioned in an ultrasonic cleaner bath. Simultaneous vacuum filtration and sonication provide a more efficient means for degassing. Right, Charlie adjusts the flow rate setting on the HPLC pump. 

1 Choice of pH. If analytes are ionizable, the proper mobile-phase pH must be chosen based on the analyte pKa so the target analyte is in one predominate ionization state ionized or neutral. If possible, method development at both of these dehned mobile-phase pH values is encouraged to maximize the potential gains that may be obtained in regard to selectivity (for the neutral and ionized forms of the target analyte and related substances). 

Optimum buffering capacity occurs at a pH equal to the pA of the buffer. In general, you can expect most buffers to provide adequate buffering capacity for controlling mobile-phase pH only within +1 unit of their respective pA values. Beyond that, buffering capacity may be inadequate. 

buffers are great media for growing bacteria. It is recommended to have at least 10 v/v% of organic in the aqueous phase to prevent bacterial growth. 

3 General Considerations for Buffers. The type of buffer that is chosen will depend on the wavelength of the method and the concentration of organic in the mobile phase. A judicious choice of type and concentration of buffer must be made to ensure mobile-phase compatibility. 

Citrate buffers can attack stainless steel. When using these buffers, be sure to flush them out of the system as soon as the analysis is completed, but this is a recommendation for any buffer system. 

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In supercritical fluid chromatography, fluids above their critical point are used as mobile phases. This chapter discusses the principles of operation, mobile phase considerations, parameters that can be adjusted in method development as well as an overview of instrumentation required and a few pertinent examples from current literature. Not everything can be illustrated, but the advantages of this diverse technology will be highlighted. 

Sorbent and Mobile-Phase Considerations for Reversed-Phase Chromatography and Hydrophobic Interaction Chromatography... 

A mobile phase is primarily chosen for its effectiveness in solubilizing and stabilizing the sample. Because of the short contact time related to the isocratic conditions, proteins remain stable if the appropriate mobile phase and column are used. As discussed earlier, nonideal SEC behavior may be observed on silica-based columns. Mobile phase considerations therefore play an important role in SEC. Elimination of protein adsorption is crucial, but the effect of the eluant on protein structure must also be considered. Additionally, polyelectrolytes expand and condense with changes in macroion concentration within the buffer (118). 

Certain other additives to the mobile phase have been tested in MIC. It was found that the inclusion of urea or detergents such as sodium dodecyl sulfate and Tween 80 in the mobile phase considerably decreased the adsorption of proteins on Ni +-TED agarose [16]. In the separation of glycophorins, 0.05% SDS was required in the mobile phase to achieve elution [54]. Likewise. 7M urea was required for the analysis of proinsulin variants on a Ni -IDA MIC column [35]. 

The complexity of the system increases with the number of solvents used and, of course, their relative concentrations. The process can be simplified considerably by pre-conditioning the plate with solvent vapor from the mobile phase before the separation is started. Unfortunately, this only partly reduces the adsorption effect, as the equilibrium between the solvent vapor and the adsorbent surface will not be the... 

Silica gel, per se, is not so frequently used in LC as the reversed phases or the bonded phases, because silica separates substances largely by polar interactions with the silanol groups on the silica surface. In contrast, the reversed and bonded phases separate material largely by interactions with the dispersive components of the solute. As the dispersive character of substances, in general, vary more subtly than does their polar character, the reversed and bonded phases are usually preferred. In addition, silica has a significant solubility in many solvents, particularly aqueous solvents and, thus, silica columns can be less stable than those packed with bonded phases. The analytical procedure can be a little more complex and costly with silica gel columns as, in general, a wider variety of more expensive solvents are required. Reversed and bonded phases utilize blended solvents such as hexane/ethanol, methanol/water or acetonitrile/water mixtures as the mobile phase and, consequently, are considerably more economical. Nevertheless, silica gel has certain areas of application for which it is particularly useful and is very effective for separating polarizable substances such as the polynuclear aromatic hydrocarbons and substances... 

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,... 

The major advantage of the capillary hydrodynamic chromatography is that the mobile phase does not need to have similar solubility parameter as the sample and packing material. (In SEC, nonsize exclusion effects may be observed if the solubility parameter of the sample, packing material, or mobile phase is considerably different.) Therefore, the hydrodynamic size of polymers can be studied in a 0 solvent and even in a solvent that is not compatible with any currently available SEC packing material (9). Figure 22.4 is an example of polystyrene separation in both THF and diethyl malonate. Diethyl malonate is the 0 solvent of polystyrene at 31-36 C. 

Recently, Chester has described (21) how a consideration of the phase diagram of the mobile phase shows that a one-phase region (Figure 1.1) is available for the selection of the mobile phase parameters, and that the boundaries separating... 

However, in LC solutes are partitioned according to a more complicated balance among various attractive forces solutes interact with both mobile-phase molecules and stationary-phase molecules (or stationary-phase pendant groups), the stationary-phase interacts with mobile-phase molecules, parts of the stationary phase may interact with each other, and mobile-phase molecules interact with each other. Cavity formation in the mobile phase, overcoming the attractive forces of the mobile-phase molecules for each other, is an important consideration in LC but not in GC. Therefore, even though LC and GC share a considerable amount of basic theory, the mechanisms are very different on a molecular level. This translates into conditions that are very different on a practical level so different, in fact, that separate instruments are required in modern practice. 

If some fields may be empty in the sublevels, all the fields in the main level are required for each entry. A new chiral separation record can be added in CHIRBASE solely if the authors correctly identify both sample and CSP. Since the beginning of the project, our policy has been to contact the authors of all publications containing incomplete, ambiguous or inconsistent data and to ask for additional information. Providing the separations with unique case numbers helps us considerably in this essential task, and also facilitates avoiding redundancies in the database. When chiral separations are reported for the second time in a new publication with exactly the same chromatographic conditions, this is stated in a footnote added in the field comments . In this field, miscellaneous information that cannot appear elsewhere are listed (detection limit, description of a reported chromatogram, racemization study, mobile phase limitations, etc.). 

Volume overload can be treated in a simple way by the plate theory (8,9). In contrast, the theory of mass overload is complicated (10-12) and requires a considerable amount of basic physical chemical data, such as the adsorption isotherms of the solutes, before it can be applied to a practical problem. Volume overload is useful where the solutes of interest are relatively insoluble in the mobile phase and thus, to apply a sample of sufficient size onto the column, a large sample volume is necessary. If the sample is very soluble in the mobile phase then mass overload might be appropriate. 

The dimensions of the exit tube from the detector are not critical for analytical separations but they can be for preparative chromatography if fractions are to be collected for subsequent tests or examination. The dispersion that occurs in the detector exit tube is more difficult to measure. Another sample valve can be connected to the detector exit and the mobile phase passed backwards through the detecting system. The same experiment is performed, the same measurements made and the same calculations carried out. The dispersion that occurs in the exit tube is normally considerably greater than that between the column and the detector. However, providing the dispersion is known, the preparative separation can be adjusted to accommodate the exit tube dispersion and allow an accurate collection of each solute band. 

The mobile phase was an aqueous solution containing 50 mM sodium phosphate and 150 mM sodium chloride at a pH 7.0. Although ionic interactions are likely to constitute the major contribution to retention and selectivity, there would also be significant polar interactions and some dispersive interactions between the aromatic nuclei of the solutes and the aromatic nuclei and the aliphatic side chains of resin respectively. Under these circumstances, without considerable experimental work, it is impossible to identify the relative magnitude of the different contributions from each type of interaction. 

The effect of the mobile-phase composition on the operation of the different interfaces is an important consideration which will be discussed in the appropriate chapter of this book but mobile-phase parameters which affect the operation of the interface include its boiling point, surface tension and conductivity. The importance of degassing solvents to prevent the formation of bubbles within the LC-MS interface must be stressed. 

The selectivity (separation capability) of an HPLC system is dependent upon the combination of mobile and stationary phases. Since ions are being generated directly from the mobile phase by electrospray, its composition, including the identity and concentration of any buffer used, and its flow rate are important considerations. 

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