Normal mobile phase

Nonpolar organic mobile phases, such as hexane with ethanol or 2-propanol as typical polar modifiers, are most commonly used with these types of phases. Under these conditions, retention seems to foUow normal phase-type behavior (eg, increased mobile phase polarity produces decreased retention). The normal mobile-phase components only weakly interact with the stationary phase and are easily displaced by the chiral analytes thereby promoting enantiospecific interactions. Some of the Pirkle-types of phases have also been used, to a lesser extent, in the reversed phase mode. 

Operate the column at half of the maximum recommended flow rate, taking special care to monitor the pressure, because the cleaning solution may be of different viscosity than the normal mobile phase. 

If the column is contaminated with basic compounds, clean it with a concentrated salt in the normal mobile phase, e.g., 0.5-1.0 M K2SO4. Avoid the use of halides, as they will corrode stainless steel over time. Buffered solutions at low pH (2-3) or high pH (11-12) can also be used. 

FIGURE 5 Chromatograms of (a) fm 1s,-2,3-cyclopropanedicarboxylic anilide, (b) benzoin, and (c) norfluoxetine (A) and fluoxetine (B) on fi-CD phases using the normal mobile phase mode. (From Refs. 49,108.)... 

The packing in the form of a slurry passes directly into the column between the two frits. The column exit is closed, and the slurry solvent passes through the outer frit and exits via the normal mobile phase inlet port. The columns is easily unpacked by adopting the reverse procedure. An example of the use of the radial flow column to separate some large biomolecules is shown in figure 12.18. 

Fig. 11 Comparison of enantiomeric separation of l-(2-naphthalenethioyl)-2-propanol on (a) Chiralcel OD-H and (b) LUX CeUulose-1 in the normal mobile phase (20/80 iso-propanol/heptane). The sample was extracted from a reaction mixture. Please note that a chemical impurity co-eluted with (/ )-enantiomer in (a) was well resolved in (b) (adapted from [148])...

Kovat s retention index (p. 575) liquid-solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554)... 

As described above, the mobile phase carrying mixture components along a gas chromatographic column is a gas, usually nitrogen or helium. This gas flows at or near atmospheric pressure at a rate generally about 0,5 to 3.0 ml/min and evenmally flows out of the end of the capillary column into the ion source of the mass spectrometer. The ion sources in GC/MS systems normally operate at about 10 mbar for electron ionization to about 10 mbar for chemical ionization. This large pressure... 

Fig. 12. Tryptic map of it-PA (mol wt = 66,000) showing peptides formed from hydrolysis of reduced, alkylated rt-PA. Separation by reversed-phase octadecyl (C g) column using aqueous acetonitrile with an added acidic agent to the mobile phase. Arrows show the difference between A, normal, and B, mutant rt-PA where the glutamic acid residue, D, has replaced the normal arginine residue, C, at position 275.

The column was operated in the normal phase mode using mixtures of n-hexane and ethanol as the mobile phase. Equation (13) is validated by the curves relating the corrected retention volume to the reciprocal of the volume fraction of ethanol in Figure 19. It is seen that an excellent linear relationship is obtained between the corrected retention volume and the reciprocal of the volume fraction of ethanol. 

The theoretical treatment given above assumes that the presence of a relatively low concentration of solute in the mobile phase does not influence the retentive characteristics of the stationary phase. That is, the presence of a small concentration of solute does not influence either the nature or the magnitude of the solute/phase interactions that determine the extent of retention. The concentration of solute in the eluted peak does not fall to zero until the sample volume is in excess of 100 plate volumes and, at this sample volume, the peak width has become about five times the standard deviation of the normally loaded peak. 

The basically correct equation appears to be that of Giddings but, over the range of mobile phase velocities normally employed i.e., velocities in the neighborhood of the optimum velocity), the Van Deem ter equation is the simplest and most appropriate to use. 

The problem is made more difficult because these different dispersion processes are interactive and the extent to which one process affects the peak shape is modified by the presence of another. It follows if the processes that causes dispersion in mass overload are not random, but interactive, the normal procedures for mathematically analyzing peak dispersion can not be applied. These complex interacting effects can, however, be demonstrated experimentally, if not by rigorous theoretical treatment, and examples of mass overload were included in the work of Scott and Kucera [1]. The authors employed the same chromatographic system that they used to examine volume overload, but they employed two mobile phases of different polarity. In the first experiments, the mobile phase n-heptane was used and the sample volume was kept constant at 200 pi. The masses of naphthalene and anthracene were kept... 

Samples and reference substances should be dissolved in the same solvents to ensure that comparable substance distribution occurs in all the starting zones. In order to keep the size of the starting zones down to a minimum (diameter TLC 2 to 4 mm, HPTLC 0.5 to 1 mm) the application volumes are normally limited to a maximum of 5 xl for TLC and 500 nl for HPTLC when the samples are applied as spots. Particularly in the case of adsorption-chromatographic systems layers with concentrating zones offer another possibility of producing small starting zones. Here the applied zones are compressed to narrow bands at the solvent front before the mobile phase reaches the active chromatographic layer. 

The chromatogram is freed from mobile phase and evenly sprayed with the spray solution or immersed for 1 s in the dipping solution. After drying the TLC plate is heated to 85 —120°C normally for 10 to 15 min but in exceptional cases for 60 min. It is advisable to observe the chromatogram during the reaction period, because the temperature and duration of heating strongly affect color development. 

Column manufacturers normally provide basic information about their columns, such as plate count, particle size, exclusion limit, and calibration curve. This information is necessary and fundamental, however, it is not sufficient to allow users to make an intelligent decision about a column for a specific application. For example, separation efficiency, the dependence of separation efficiency on the mobile phase, the ability to separate the system peaks from the polymer peak, the symmetry of the polymer peak, and the possible interaction with polymers are seldom provided. 

---