Elution order

The elution order for neutral species in MEKC depends on the extent to which they partition into the micelles. Hydrophilic neutrals are insoluble in the micelle s hydrophobic inner environment and elute as a single band as they would in CZE. Neutral solutes that are extremely hydrophobic are completely soluble in the micelle, eluting with the micelles as a single band. Those neutral species that exist in a partition equilibrium between the buffer solution and the micelles elute between the completely hydrophilic and completely hydrophobic neutrals. Those neutral species favoring the buffer solution elute before those favoring the micelles. Micellar electrokinetic chromatography has been used to separate a wide variety of samples, including mixtures of pharmaceutical compounds, vitamins, and explosives. 

A mixture of n-heptane, tetrahydrofuran, 2-butanone, and n-propanol elutes in this order when using a polar stationary phase such as Carbowax. The elution order is exactly the opposite when using a nonpolar stationary phase such as polydimethyl siloxane. Explain the order of elution in each case. 

Ion exchange (qv see also Chromatography) is an important procedure for the separation and chemical identification of curium and higher elements. This technique is selective and rapid and has been the key to the discovery of the transcurium elements, in that the elution order and approximate peak position for the undiscovered elements were predicted with considerable confidence (9). Thus the first experimental observation of the chemical behavior of a new actinide element has often been its ion-exchange behavior—an observation coincident with its identification. Further exploration of the chemistry of the element often depended on the production of larger amounts by this method. Solvent extraction is another useful method for separating and purifying actinide elements. 

In an ideal SEC separation, the mechanism is purely sieving, with no chemical interaction between the column matrix and the sample molecules. In practice, however, a small number of weakly charged groups on the surface of all TSK-GEL PW type packings can cause changes in elution order from that of an ideal system. Fortunately, the eluent composition can be varied greatly with TSK-GEL PW columns to be compatible with a wide range of neutral, polar, anionic, and cationic samples. Table 4.8 lists appropriate eluents for GFC of all polymer types on TSK-GEL PW type columns (11). 

Electrostatic effects have long been recognized in commercial HPLC columns for SEC of proteins (15,21,22). The usual remedy is to add 100 mM salt to the mobile phase. This works here too the Lys and Asp peaks collapse into the Gly peak with 100 mM salt (Eig. 8.8). High concentrations of sodium sulfate were added to determine the role played in SEC by hydrophobic interactions (sodium sulfate, a structure-forming salt, strengthens such interactions). Sodium sulfate increased the retention only of the most hydrophobic amino acids to any extent, and then only when the concentration approached 1 M. Clearly, hydrophobic interaction cannot account for the elution order of amino acids on PolyHEA. 

These controlled experiments eliminate adsorption as an explanation for the elution order of neutral amino acids from PolyHEA. Perhaps this order does... 

FIGURE 9.2 Different modes of chromatography as seen in the elution order of samples with different molar masses. 

Giddings pointed out (32) that separated compounds must remain resolved throughout the whole process. This situation is illustrated in Figure 1.5, where two secondary columns are coupled to a primary column, and each secondary column is fed a fraction of duration Ar from the eluent from the first column. The peak capacity of the coupled system then depends on the plate number of each individual separation and on At. The primary column eliminates sample components that would otherwise interfere with the resolution of the components of interest in the secondary columns. An efficient primary separation may be wasted, however, if At is greater than the average peak width produced by the primary column, because of the recombination of resolved peaks after transfer into a secondary column. As At increases, the system approaches that of a tandem arrangement, and the resolution gained in one column may be nullified by the elution order in a subsequent column. 

When columns of the same polarity are used, the elution order of components in GC are not changed and there is no need for trapping. However, when columns of different polarities are used trapping or heart-cutting must be employed. Trapping can be used in trace analysis for enrichment of samples by repetitive preseparation before the main separation is initiated and the total amount or part of a mixture can then be effectively and quantitatively transferred to a second column. The main considerations for a trap are that it should attain either very high or very low temperatures over a short period of time and be chemically inactive. The enrichment is usually carried out with a cold trap, plus an open vent after this, where the trace components are held within the trap and the excess carrier gas is vented. Then, in the re-injection mode the vent behind the trap is closed, the trap is heated and the trapped compounds can be rapidly flushed from the trap and introduced into the second column. Peak broadening and peak distortion, which could occur in the preseparation, are suppressed or eliminated by this re-injection procedure (18). 

Similar to the new polar organic mode, the retention of analytes in normal phase is not difficult to predict. For all the compounds, the average of the retention on individual columns is fairly close to the retention on the coupled columns. The selectivity of most compounds on coupled columns is an average of the selectivities of individual columns (Fig. 2-9). However, it was found that the elution order for some compounds was reversed on ristocetin A and teieoplanin or vancomycin. As a result. 

Temperature can also be used to optimize enantioselectivity in SFC. The selectivity of most CSPs increases as temperature decreases. For this reason, most chiral separations in SFC are performed at ambient or subambient temperatures [50, 74]. Subambient temperatures are particularly useful for compounds having low conformational stability [75]. Stringham and Blackwell explored the concept of entropically driven separations [76]. As temperature increased, enantioselectivity decreased until the enantiomers co-eluted at the isoelution temperature. Further increases in temperature resulted in reversal of elution order of the enantiomers. The temperature limitations of the CSP should be considered before working at elevated temperatures. 

Table 11.1. Fragmentation and elution order of underivatized drugs ...

Table I. Elution Order of Pyrethrum Constituents from Gas Chromatograph as Compared with That from Partition Column...

A third nonabromo isomer (ref. 4) was absent from the BCL material. This may have been a breakdown product in their Deca sample, which has been noted to be unstable to light (ref. 5). The elution order of the Octa and Nona isomers on GC was 6,7,5,9,8 (numbering according to Fig. 1). 

A calcium hydroxide column slurry packed in the laboratory was used to evaluate the distribution of all-trans-, l3-cis-, and 9-cis- isomers of P-carotene in fresh and processed vegetables and fruits. Elution order was reported to be l5-cis-, 3-cis-, sil-trans-, and 9-cw-P-carotene, using 2% p-methylanisole or 2% acetone in hexane as mobile phase, in a 35-min run. However, a column packed with calcium hydroxide as the stationary phase is not commercially available. 

Figure 6.2.2 shows the separations of mixtures of standards on a monomeric C,g column and also on a polymeric C30 column. The elution order on the monomeric C18 column is, as expected, first the dihydroxy xanthophyUs (lutein and zeaxanthin), followed by the monohydroxy compounds (rubixanthin and P-cryptoxanthin), and finally by the carotenes (y-, a-, and p-carotene). However, on the C30 column, rubixanthin and y-carotene, both with 1 acyclic /-end group, eluted after a- and P-carotene, with two cyclic end groups. 

The porous glass packed columns did not yield high resolution separations, but the major species present in a latex were adequately separated. Figure 1. Insoluble polymer, when present, was excluded from the pores and eluted at interstitial volume. Elution order of remaining species was soluble polymer, unreacted monomers, and water. In both types of resins studied, no separation of the two unreacted monomers was achieved. A single chromatographic peak, that included both monomers, was obtained. 

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