Mobile phase mixed solvents

Another type of nonideal SEC behavior, which will not be covered in this chapter, is related to the use of mixed mobile phases (multiple solvents). Because solute-solvent interactions play a critical role in controlling the hydrodynamic volume of a macromolecule, the use of mixed mobile phases may lead to deviations from ideal behavior. Depending on the solubility parameter differences of the solvents and the solubility parameter of the packing, the mobile phase composition within the pores of the packing may be different from that in the interstitial volume. As a result, the hydrodynamic volume of the polymer may change when it enters the packing leading to unexpected elution results. Preferential solvation of the polymer in mixed solvent systems may also lead to deviations from ideal behavior (11). 

Incomplete mobile phase mixing. 3. Mix mobile phase by hand or use less viscous solvent. 

In this case, the TLC system most commonly employed uses silica gel plates and a mobile phase of ethyl acetate/methanol/25% ammonia (85 10 5, by volume). The plates are prepared and the chromatogram developed in the standard way. After development, the plate is removed from the mobile phase, the solvent front marked, and the plate dried. Visualization of barbiturates is best achieved by the use of a mercuric chloride-diphenylcarbazone reagent. The latter is prepared as two component solutions, i.e. (i) 0.1 g of diphenylcarbazone in 50 ml of methanol, and (ii) 0.1 g of mercuric chloride in 50 ml of ethanol. These solutions should be freshly prepared and mixed just before use. The presence of barbiturates will give rise to blue-violet spots on a pink background when using this reagent system. 

The introduction of the chemically bonded apolar reversed phase materials as stationary phases is responsible for the common use of HPLC in routine laboratory analysis, because the sample can be applied directly from the aqueous solution into the HPLC-system. For the mobile phase, polar solvents such as water or aqueous buffer solutions or gradient mixes of aqueous solvents with methanol or acetonitrile are used mainly. 

Liquid chromatography (Hostettman et al., 1986) in its many forms is a separation technique based on the polarity of the analytes and their partition between the mobile and stationary phases, and is therefore complementary to fractional distillation, which separates materials by their boiling point. The usual sequence for fractionating an essential oil or extract is to distil it first and then apply liquid chromatography to the distillation fractions as a further fractionation procedure, rather than as an analytical tool. The selectivity of the technique is achieved by choosing a stationary phase, usually from the various activities of silica gel, and varying the polarity of the mobile phase, the solvent, by mixing a non-polar component (such as hexane or pentane) with different amounts of a more polar component (such as diethyl ether, ethyl acetate or chloroform). 

Here, k is the retention factor in a binary mobile phase containing solvents A and B, which can be calculated from the retention volume of the analyte, Vr, and the column hold-up volume, Vm (determined as the elution volume of a nonretained compound, such as trichloroethylene), k = Vr/Vm — 1, fea is k in pure weak solvent. A, o is the activity of the adsorbent in the column. As is the specific surface of the adsorbent, and Sa and Sab are the solvent strength (polarity) parameters of the weak (nonpolar) solvent and of the mixed binary mobile phase, respectively. 

Uptake of water and polar solvents by the column From mixed mobile phases, polar solvent(s) are preferentially adsorbed on the surface of polar adsorbents, sometimes giving rise to multilayer solvent adsorption on the adsorbent support. In such a case, the retention is contributed to by a liquid-liquid partition mechanism between the adsorbed liquid layer and the bulk mobile phase, in addition to the adsorption. Such a mixed-mode mechanism can be intentionally utilized for separation of strongly polar or even ionic compounds. 

As a rule, in a mixed mobile phase a solvent peak appears near the void volume of the column. The appearance of the solvent peak may due to one of several effects, the first of which is the preferential solvation of polymers [172]. After dissolution in a mixed solvent, the polymer binds into its solvation shell one part of mixture to a larger extent. After the separation of the solvated polymer from rest of injected solvent, the solvent peak appears on chromatogram as was demonstrated by SEC [172] and under suitable condition [173] its area, or height, may be correlated with coefficient of preferential solvation [ 172]. An evaporation of one component from the sample bottle or displacement effects may also lead to appearance of a solvent peak [173]. The solvent peak represents a local change of composition of the mobile phase. Under critical conditions small changes of the mobile phase composition (for example, 0.1 % wt.) have a large influence on polymer retention, thus the solvent peak could influence the elution of the macromolecules. If so, this could imply that a tabulated critical composition is not precisely that, which really correspond to the critical conditions. The real, acting critical composition of eluent may be, and likely is, the composition somewhere, in the middle, of the solvent peak. The presence of the solvent peak influences especially pronouncedly the elu-... 

In many examples, with little regard of the sample type, the analyses of flavonoids are usually done in the reversed-phase HPLC mode using nonpolar Cig (in few cases, Cg) columns and polar mobile phase (mixed aqueous-organic solvents) due to their structural and physicochemical properties. Conversely, with more regard of the sample type, different extractions and sample pretreatments including Soxhlet, LLE, SPE, USAE, ASE, and SEE have been used prior to HPLC flavonoid analysis. 

Numerous mobile phases (development solvents) are available for lipid work (see Table 1). They often consist of solvent mixtures that vary in polarity, along with small amounts of salts or acids. Because a mixed solvent system allows for an undefined gradient in solvent composition during movement on the silica gel layer, samples with varying polarity can be developed on a single plate in TLC the velocity of the solvent movement is reduced as the solvent front nears the top of the plate optimal separation is obtained with bands or spots with Revalues between 0.1 and 0.6 (63). 

Choosing a Mobile Phase Several indices have been developed to assist in selecting a mobile phase, the most useful of which is the polarity index. Table 12.3 provides values for the polarity index, P, of several commonly used mobile phases, in which larger values of P correspond to more polar solvents. Mobile phases of intermediate polarity can be fashioned by mixing together two or more of the mobile phases in Table 12.3. For example, a binary mobile phase made by combining solvents A and B has a polarity index, of... 

For LC, temperature is not as important as in GC because volatility is not important. The columns are usually metal, and they are operated at or near ambient temperatures, so the temperature-controlled oven used for GC is unnecessary. An LC mobile phase is a solvent such as water, methanol, or acetonitrile, and, if only a single solvent is used for analysis, the chromatography is said to be isocratic. Alternatively, mixtures of solvents can be employed. In fact, chromatography may start with one single solvent or mixture of solvents and gradually change to a different mix of solvents as analysis proceeds (gradient elution). 

The most commonly used gradients are linear gradients where the starting solvent is gradually mixed with a second gradient-forming solvent at the column entrance to yield a volume fraction

mobile phase modulator that increases huearly with time ... 

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,... 

This is an oversimplified treatment of the concentration effect that can occur on a thin layer plate when using mixed solvents. Nevertheless, despite the complex nature of the surface that is considered, the treatment is sufficiently representative to disclose that a concentration effect does, indeed, take place. The concentration effect arises from the frontal analysis of the mobile phase which not only provides unique and complex modes of solute interaction and, thus, enhanced selectivity, but also causes the solutes to be concentrated as they pass along the TLC plate. This concentration process will oppose the dilution that results from band dispersion and thus, provides greater sensitivity to the spots close to the solvent front. This concealed concentration process, often not recognized, is another property of TLC development that helps make it so practical and generally useful and often provides unexpected sensitivities. 

When prepanng mobile phase mixtures each individual component should be measured out separately and only then placed in the mixing vessel This prevents not only contamination of the solvent stock by vapors from the already partially filled mixing vessel (e g ammonia ) but also volumetnc errors caused by volume expansions or contractions on mixing... 

Figures 13.30-13.53 demonstrate the use of various mobile phases for polymer SEC using standard mixed-bed DVB columns. Once again these applications demonstrate that PDVB gels will easily tolerate virtually any solvent or mixed solvent system.

Even with mobile-phase modifiers, however, certain polymer types cannot be run due to their lack of solubility in organic solvents. In order to run aqueous or mixed aqueous/organic mobile phases, Jordi Associates has developed several polar-bonded phase versions of the PDVB gels as discussed earlier. Figures 13.60 thru 13.99 detail examples of some polar and ionic polymers that we have been able to run SEC analysis of using the newer bonded PDVB resins. 

There are two types of gradient programmer. In the first type, the solvent mixing occurs at high pressure and in the second the solvents are premixed at low pressure and then passed to the pump. The high pressure programmer is the simplest but most expensive as it requires a pump for each solvent supply. There can be any number of solvents involved in a mobile phase program, however, the majority of LC analyses usually require only two solvents but up to four solvents can... 

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