Mobile phases — eluents

Severe requirements are laid on the gel chromatographic mobile phases. They must dissolve the sample analyzed, must be compatible with both the detector and the gel, must be chemically and biologically inert toward the sample, and non-corrosive for the constructing materials of the instrument. Further on, the eluents should suppress the interactions between the sample and the gel, should exhibit low viscosity, low coefficient of compressibility, and low vapour tension. Finally, they should be minimally poisonous and explosive as well as cheap and readily available in rather high purity. 

In gel chromatography of biological systems, water-based eluents are dominant. The main reason for using the aqueous miUeu is both the low solubility and susceptibility to the irreversible denaturation of many biologically active substances in organic solvents. However, pure water is applied only exceptionally, e.g., when separating saccharides. In the majority of applications, one works with the aqueous solutions of various substances, which means with the mixed eluents, and therefore the above mentioned problems are often encountered. 

Further additives to the mobile phase involve the structure-breaking, solubilizing and dissociation promoting agents that prevent the excessive solute-solute interactions or ensure the appropriate and conformationally stable structure of biological macromolecules, e.g., random coil conformation. These additives are most often urea 

The above reference materials are available from the following companies Boehringer. Mannheim. FRG BDH Chemicals Ltd., Poole, UK Calbiochem, Lucerne, Switzerland Fluka AG, Buchs, Switzerland Intermed-Export-Import, Berlin GDR Koch-Light, Laboratories, CoInbrook, UK E. Merck, Darmstadt, FRG Pharmacia Fine Chemicals, AB, Uppsala, Sweden Reanal Fine Chemicals, Budapest, Hungary Serva Feinbiochemica, Heidelberg, FRG Sigma London Chemical Company, Ltd., Poole. UK. 

Suppliers 1, Pressure Company, Pittsburgh, PA, USA 2, Toyo Soda Manufacturing Co., Ltd., Tokyo, Japan 3, Waters Assoc. Inc., Milford, MA, USA 4, National Bureau of Standards, Washington DC, USA 5, Chrompack Nederland B.V., Middelburg, The Netherlands 6, Polysciences Inc., Warrington, PA, USA 7, National Physical Laboratory, Teddington, UK 8, Pharmacia Fine Chemicals AB, Uppsala, Sweden. 

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Fig. 2.19. Chromatogram of carotenoid solution in THF. 1 lutein, 2 canthaxanthin, 3 /Tcryptoxan-thin, 4 lycopene, 5 /1-carotene. ODS column, 5/an, 150mm X 4.6mm. Mobile phase eluent A (methanol-acetonitrile 6 11), eluent B (THF) gradient profile 0-5min, isocratic conditions 95 per cent A 5-20min, gradient to 20 per cent A. Flow rate lml/min. Detection 450 nm. Reprinted with permission from C. Tricard el al. [45].

In chromatography the components of a mixture are separated as they pass through a column. The column contains a stationary phase which may be a packed bed of solid particles or a liquid with which the packing is impregnated. The mixture is carried through the column dissolved in a gas or liquid stream known as the mobile phase, eluent or carrier. Separation occurs because the differing distribution coefficients of the components of the mixture between the stationary and mobile phases result in differing velocities of travel. 

Solute ions X that compete weakly with mobile phase eluent ions Y for ion exchanger sites R will be retained only slightly on the column. Solute ions that interact strongly with the ion-exchanger elute later in the chromatogram. 

Mobile phase Eluent (A) 70 mM KH2P04, pH 2.5 eluent (B) methanol... 

Carey and Caruso [126] also summarised the two main approaches to interfacing the SFC restrictor with the ICP torch. The first method, used with packed SFC columns, introduces the restrictor into a heated cross-flow nebuliser and the nebulised sample is subsequently swept into the torch by the nebuliser gas flow. Where capillary SFC systems are used, a second interface design is commonly employed where the restrictor is directly introduced into the central channel of the torch. This interface is more widely used with SFC-ICP-MS coupling [20]. The restrictor is passed through a heated transfer line which connects the SFC oven with the ICP torch. The restrictor is positioned so that it is flush with the inner tube of the ICP torch. This position may, however, be optimised to yield improved resolution. The connection between the transfer line and the torch connection must be heated to prevent freezing of the mobile phase eluent after decompression when exiting the restrictor. A make-up gas flow is introduced to transport the analyte to the plasma. This... 

In liquid chromatography the mobile phase (eluent) transports the dissolved sample through the stationary phase. Depending upon the polarity of the sta-... 

The function of the solvent delivery system is to deliver the mobile phase (eluent) through the chromatograph, accurately and reproducibly. The solvent delivery system comprises the pump, check valves, flow control-... 

Each separation requires 5-10 mL of mobile phase (eluent) and can be performed in a few minutes. If time permits, students should repeat each experiment three times so that the reproducibility of the method can be checked. Calculation of a mean and standard deviation for selected data and / or calculated values would be a useful exercise. The cartridges will not perform properly if the required pretreatment and between-injection washings are ignored. This is particularly true for a new Cig column, which will show little retentivity toward the dyes unless thoroughly prewetted with isopropanol. 

Distance moved by mobile phase (eluent or solvent front) along the stationary phase... 

Example Chromatography was used to separate a mixture of biomolecules. The mixture was added to the stationary phase - paper - at a point that was marked as the origin. The mobile phase (eluent or solvent) - ethanol - was then added, and after several hours a number of spots were visible along the paper at distances of (i) 5 cm, (ii) 10 cm and (iii) 15 cm from the origin. During this time the mobile phase moved a total distance of 20 cm (the solvent front). From this, calculate the R/ values of each separated biomolecule ... 

Molecules set in motion by the mobile phase (eluent) move through the stationary phase, suitably immobilized on a medium. The higher the affinity for the stationary phase and the lower the affinity for the mobile phase, the slower the analyte. As in a race, the fastest chemical species cover a prearranged distance in the shortest time, arrive at the finish line, and produce a detector signal proportional to the amount of analyte. The aggregation state of the mobile phase enables us to differentiate liquid, gas, and supercritical chromatographic techniques. 

Mobile phase (eluent) is by far the major tool for the control of analyte retention in RPLC. Variations of the eluent composition, type of organic mod-iher, pH, and buffer concentration provide the chromatographer with a valuable set of variables for successful development of a separation method. 

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... 

Analytical HPLC is based on the elution development, which means that a small volume of the sample to be analysed is introduced into the flowing mobile phase — eluent — at the top of the chromatographic column. Various sample compounds have different equilibrium distributions between the stationary and the mobile phases, so that each compound spends a different time in the stationary phase and zones containing individual sample components move along the column at different velocities. This leads to the separation of the sample components in the chromatographic column and eventually the individual compounds are eluted from the column at different times from the introduction (injection) of the sample. 

Before a chromatographic system is selected, its attributes have to be defined. As described in Chapter 2.1 a chromatographic system consists of the adsorbent (stationary phase), mobile phase (eluent, solvent, desorbent) and solutes (samples, analyte etc.). Figure 4.5 illustrates the interrelationship of these three constituents. The selection of the chromatographic system is influenced by their properties and their interaction. These properties are described in this section, while rules and criteria for the selection of the chromatographic system are explained in Section 4.3. 

The general principles for separation are perhaps best illustrated by a specific example. Suppose that chloride and bromide are to be separated on an anion-exchange column. The sample contains 8 x 10 M sodium chloride and 8 x 1(H M sodium bromide and the mobile phase (eluent) contains 10 x M sodium hydroxide. 

Developing solvent, mobile phase, eluent (only used in OPLC)... 

Let us consider the bed of randomly packed porous gel particles within the column that is percolated by the mobile phase (eluent) carrying the sample components. The sizes of gel pores and separated molecules have mutually matched distribution so that the largest sample molecules are totally excluded from all pores of the gel. These molecules will not be retained in the column and are eluted by the volume of the eluent equal to the interstitial volume of the column, 1, . This means that the retention volume, of excluded molecules equals V . On the other hand, the smallest sample molecules with the sizes that are comparable with the size of eluent molecules permeate all pores of gel and diffuse back into the mobile phase. The consequence of this repeated process is the retention of the small molecules in the column and, for their elution, such volume of eluent is necessary that equals the interstitial volume, kj, plus pore volume, V,. The resulting retention volume is VR = yn.= K+yc... 

If using a stronger solvent for the sample than the mobile phase eluent to dissolve the sample, care should be taken and a maximum of 5-10 p.1 should be injected. 

The major contrast with GC is that the mobile phase eluent strength (affecting k ) and the relative selectivity between analytes (a) can both be readUy varied. In GC the carrier gas composition has practically no effect on selectivity, and varying temperature affects only k. The large number and range of parameters to be evaluated to optimize the eluent composition makes HPLC method development more complex. Every benefit comes with its own price ... 

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...

Chromatography is a physico-chemical method of separation and analysis of mixtures and solutions, based on distribution of their components between two phases - the motionless phase (sorbent) and the mobile phase (eluent) that flows through the motionless phase. Depending on the nature of interaction that determines the phase distribution of components, one can identify the following types of chromatography adsorption, partition, ion exchange, exclusion, and precipitation. 

As mentioned before, ion chromatography (IC) is the method to analyze ionic species in solution. Basically, in IC systems, the stationary phase is an ion-exchange resin and the mobile phase (eluent) contains an electrolyte providing... 

Mobile Phase Eluent Mass Spectrometer References ... 

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