Eluent components

Eluent components should be volatile. Solvents such as ethyl acetate, isopropyl ether, diethylketone, chloroform, dichloromethane, and toluene as modifiers and n-hexane as diluent are recommended for normal phase chromatography. For reversed-phase systems, methanol or acetonitrile are used as modifiers. Such components as acetic acid or buffers, as well as ion association reagents, should be avoided. 

A particular column can be used for different types of LC by changing the eluent components. For example, a column packed with RP-18 bonded silica gel can be used for SEC with THF, NPLC with n-hexane, and RPLC with aqueous acetonitrile. When separation cannot be achieved by improving the theoretical plate number of a column, it may be achieved by selection of an appropriate stationary phase material and/or eluent. 

In addition to the above-mentioned restrictions, eluent selection for LC and HPLC is especially important. While the gas used in GC will not interfere with analysis, it is possible for eluent components used in LC or HPLC to interfere with follow-on analysis. This will be true for both MS and IR analysis. Usually, however, all samples can be accommodated if sufficient thought is exercised in selecting both the method of separation and the method of introduction into the follow-on analytical procedure. 

The physical and chemical aspects of liquid chromatography, in addition to mechanical aspects, are briefly described in this chapter. Theoretical approaches are explained in detail in later chapters. The effect of stationary phase materials on the chemical selectivity is described in Chapter 3, and the influence of the eluent components is covered in Chapter 4. The plate number theory is discussed in Chapter 5. Quantitative optimization is explained in Chapter 6. 

Figure 1.2 Effect on eluent component of flow rate. Column 3 pm Cjg-bonded silica gel, 10 cm x 4.6 mm i.d. eluent, 80% aqueous acetonitrile flow rate A, 1, B, 2 ml min-1 detection, UV 254 nm. Peaks 1, benzene, 2, acetophenone 3, toluene, 4, naphthalene.

The physical and chemical properties of stationary phase materials are described in Chapter 3 (including methods for their synthesis) to clarify the differences in similar stationary phase materials supplied from different manufacturers. A detailed selection guide to solvents is given in Chapter 4. The unlimited selection of eluent components and their concentrations is a powerful force in developing separations in liquid chromatography. Although this area seems rather complicated, it is easy to understand the selection of a suitable eluent when you first identify the molecular properties of the analytes and solvents. 

The mass spectrometer is a very sensitive and selective instrument. However, the introduction of the eluent into the vacuum chamber and the resulting significant pressure drop reduces the sensitivity. The gas exhaust power of a normal vacuum pump is some 10 ml min-1 so high capacity or turbo vacuum pumps are usually needed. The gas-phase volume corresponding to 1 ml of liquid is 176 ml for -hexane, 384 ml for ethanol, 429 ml for acetonitrile, 554 ml for methanol, and 1245 ml for water under standard conditions (0°C, 1 atmosphere). The elimination of the mobile phase solvent is therefore important, otherwise the expanding eluent will destroy the vacuum in the detector. Several methods to accomplish this have been developed. The commercialized interfaces are thermo-spray, moving-belt, electrospray ionization, ion-spray, and atmospheric pressure ionization. The influence of the eluent is very complex, and the modification of eluent components and the selection of an interface are therefore important. Micro-liquid chromatography is suitable for this detector, due to its very small flow rate (usually only 10 p min - ). 

Apply b above clean flow cell Replace lamp if using an unsuitable eluent component of absorbance at detection wavelength, change eluent Fix or change earth connection... 

Figure 3.8 A, Schematic structure of porous stationary phase a, support b, bonded phase and c, adsorbed solvent layer. B, detailed view of eluent components in surface layer , lower phase molecules Q, upper phase molecules.

Even the elution volume of acetonitrile was not constant in these eluents and varied from 1.25 to 1.95 ml. The volumes of ionized sodium nitrate (e) and 2,4-dinitronaphthol (d) were smaller than the exclusion limit (1.0 ml) of this column. The dramatic change for 2,4-dinitronaphthol in Figure 3.9A indicates that the volume depended strongly upon the ratio of eluent components.5... 

Ion-pair liquid chromatography can be applied to the separation of a wide variety of compounds. However, the flexibility of the selection of eluent components can confuse the operation. Trouble-shooting solutions are summarized in Table 4.6. 

A discussion on the theories of retention mechanisms and the type of forces generating retention and selectivity is not intended in this chapter. The interactions between solute, eluent components, and stationary phases with inhomogeneous surfaces are based on molecular recognition, where our understanding is still very limited. Therefore, an empirical description of retention and selectivity is preferred. 

Eqnation 16.10 holds for mixed eluents A plus B with not very low concentration of stronger component B. The role of intermolecular interactions between eluent components A and B is not considered. The solvent strength of a binary elnent can be roughly estimated also from the relation... 

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. 

The elution pattern in IEC results from the charge distribution on the folded chain. Therefore, IEC was used for indication, whether the native structure of the protein had been affected by previous RPC or not. Ribonuclease was found to retain its native structure, whereas bovine serum albumin, horse radish peroxidase, and ovalbumin were much altered through RPC on a C 18 column with a gradient water/ (ethanol-butanol 80 20) containing 0.012 M HC1 in both eluent components 59>. 

Table t. Solvent composition at sample elution (volume percent of eluent component B)... 

One of the rare drawbacks of the detector is its total lack of response for any volatile material. Accordingly, the classical method of the determination of the void volume by elution of an isotopic analog of one of the eluent components cannot be used. 

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