Eluent conditions, variation

Most of the observed parameter effects under variation of the mobile phase can be explained on a physico-chemical basis, but the information gained about different stahonaiy phases as a result of variation of eluent conditions was found to be rather Hmited. Thus, a variation of the eluent is not mandatory for meaningful phase characterization with parameters from the solvation equation. However, it appears favorable to make such measurements at higher water content, whereby the individual characteristics of different phases prove to be more pronounced, but shll imder conditions where the alkyl chains are solvated (wetted) (-30% organic content). 

In the first publication [16], the experimental strategy was applied to just ten different stationary phases, in part different modifications of the same silica-based material. Phases with incorporated polar fimctions were not included. The eluent conditions were kept constant. The chromatographic experiments with all of the neutral substances were carried out with a H2O/CH3CN (1 1, vjv) eluent with buffer additives, while basic and acidic solutes were eluted with a 31.2 mM phosphate buffer at pH 2.8. The selected pH value does not imply very critical conditions, as it suppresses dissociation of most residual silanol groups and of weakly acidic solutes. A variation of the eluent conditions wiU be discussed in a later section. 

Certain SEC applications solicit specific experimental conditions. The most common reason is the limited sample solubility. In this case, special solvents or increased temperature are inavoid-able. A possibility to improve sample solubility and quality of eluent offer multicomponent solvents (Sections 16.2.2 and 16.8.2). The selectivity of polymer separation by SEC drops with the deteriorating eluent quality due to decreasing differences in the hydrodynamic volume of macromolecules with different molar masses. The system peaks appear on the chromatograms obtained with mixed eluents due to preferential solvation of sample molecules (Sections 16.3.2 and 16.3.3). The multicomponent eluents may create system peaks also as a result of the (preferential) sorption of their components within column packing [144,145]. The extent of preferential sorption is often sensitive toward pressure variations [69,70,146-149]. Even if the specific detectors are used, which do not see the eluent composition changes, it is necessary to discriminate the bulk sample solvent from the SEC separated macromolecules otherwise the determined molecular characteristics can be affected. This is especially important if the analyzed polymer contains a tail of fractions possessing lower molar masses (Sections 16.4.4 and 16.4.5). 

Both radial and axial temperature gradients may appear. As shown is Section 16.3.5, adsorption of polymers depends on temperature. Given the temperature and pressure dependence of the preferential sorption of the mixed eluent components within column packing [146-149], one can expect also considerable changes in the column interactivity with the temperature and pressure variations that may result in a possible gradual departure from the critical conditions. 

Variation of solute retention as a function of the eluent pH. Conditions beta-cyclodextrin-silica pure aqueous eluent pH adjusted by HC1. Column temperature 30 C, eluent flow-rate 1 mL/min. 

For the separation of ionogenic (ionizable) solutes, the variations of mobile-phase pH can lead to extreme changes in selectivity. The mobile-phase (eluent) pH affects the ionization of ionogenic species and consequently their HPLC retention. However, the pH of the aqueous phase is not equivalent to the pH of the aqueous/organic eluent, and consequently the variation of the mobile-phase composition leads to the variation in pH under both isocratic and gradient conditions [58-60]. Therefore the pH shift of the mobile phase upon the addition of the organic modiher is imperative for a proper description of the... 

On the other hand, the steeper the gradient, the further the separation conditions are from the equilibrium, and reproducibility of these methods could be challenging. Most of the modern HPLC equipment can reproduce gradient profile with high accuracy, but permanent variation of the eluent composition... 

The R[ value is a constant for a particular substance and eluent system on a specific stationary phase, but variations in chromatographic conditions adsorbant, eluent (in particular solvent mixtures), temperature and atmosphere make the application of Rf values to absolute identification rather problematical. Usually an authentic sample is run alongside the unknowns in the mixture (Fig. 32.13) or on top of the mixture - double spotting - as shown in Fig. 32.14, to enable identification. The general procedure for running a TLC plate is described in Box 32.3. 

Another thing to consider with gradient elution is changes in the eluent viscosity. When gradient elution with a hydro-organic mobile phase is used (e.g., methanol-water), systematic variations in the flow rate are expected under conditions of constant-pressure operation, and systematic variations in the operating pressure will be found when a constant flow rate is used [1]. The compressibility of the solvent is species-specific. 

Fig. 7.5. Effect of variation of the eluent pH upon elution volume. Chromatographic conditions column, pBondapak Cjg (10 pM) (300 mmX4 mm I.D.) mobile phase, eluent solutions (pH 3.0, 5.0, 7.0 and 9.0) were made up from 0.025 M NaH2P04 and/or 0.025 M Na2HP04 solutions plus 40% methanol (solutions were adjusted to final pH by the addition of 5% sodium hydroxide or phosphoric acid solution) detection, UV at 220 nm. , salicylic acid a, phenobarbitone a, phenacetin O, nicotine , methylamphetamine. Reproduced from Twitchett and Moffat (1975), with...

The extremely high sensitivity of critical conditions to the eluent composition and possibly also to the continuous variations in the column interactivity, for example due to the irreversible retention of some sample components. The sensitivity of critical conditions rapidly increases with the molar mass of macromolecules concerned. It may be very difficult to repeatably prepare mixed eluents with exactly the same composition, as well as to prevent preferential evaporation from-, and absorption of humidity into- the eluent container. The colurtms should be frequently cleaned by flushing with an efficient displacer and then (laboriously) reequilibrated with the original eluent. Often, the log M versus dependence is not perfectly vertical and the exact critical conditions cannot be reached at all. Therefore, many measurements are done near the LC CC conditions, not at the proper critical point but in the critical range. This brings about additional imcertainty on accuracy of results. 

Since the columns must be interchangeable, the full set of connections must be provided on them all, and this involves a fairly extensive set of control valves. Since the start of one feed corresponds precisely with the cessation of another, it is convenient to employ multi-port valves, and various mechanical designs are available. These may be manually operated or may be driven by motors controlled by time switches if operating on a constant time cycle and with constant flow rates. A poppet valve assembly of this type is shown in Fig. 3.5. If each cycle must be unique to cope with variations of feed concentrations, etc., it is possible in some processes to control the motorized valve assemblies by means of automatic analysers on the effluent, eluent and wash streams. Instrumentation of this type can only be installed after very thorough development trials with process solutions under a wide variety of anticipated operating conditions, if reliable operating results are to be obtained. 

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