The Reverse Elution Order

One of the most interesting and useful phenomena observed with CD-based CSPs is the ability to reverse the chiral resolution in GC. The significance 

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Resolution of the three HBCD diastereomers is easily achieved by reversed-phase LC on C18 columns, often in less than 15 min. The usual elution order for the diastereomers is a-, P-, y-HBCD however, an alternate elution order (i.e., a-, y-, P-HBCD) was reported by Dodder et al. [Ill] for shape-selective columns operated with methanolic mobile phases (see Figure 13.8). The reversed elution order for P-HBCD and y-HBCD provides the basis for the development of orthogonal methods that may help eliminate matrix interferences. Yu et al. [112] also studied the influence of mobile phase composition on HBCD diastereomer selectivity and show that MS sensitivity was also affected by this parameter. 

We use the second-dimension separation from Fig. 6.6 with a 25 pL injection volume and 2.5 min sampling time the separation is an RPLC method that uses a monolithic column. Thus, 10 pL/min is the maximum flow rate in the first-dimension. Fig. 6.7 shows the development of the first-dimension column that utilizes a hydrophilic interaction (or HILIC) column for the separation of proteins at decreasing flow rates. The same proteins were separated in Fig. 6.6 (RPLC) and 6.7 (HILIC) and have a reversed elution order, which is known from the basics of HILIC (Alpert, 1990). It is believed that HILIC and RPLC separations are a good pair for 2DLC analysis of proteins as they appear to have dissimilar retention mechanisms, much like those of NPLC and RPLC it has been suggested that HILIC is similar in retention to NPLC (Alpert, 1990). Because the HILIC column used in Fig. 6.7 gave good resolution at 0.1 mL/min and no smaller diameter column was available, the flow was split 10-fold to match the second-dimension requirement... 

Nucleotides, peptides, and amino acids also differ subtly in their polarities Some are more hydro-phobic than others. Thus, separation via reverse phase HPLC is possible. A reverse phase column, such as C18 or C8, has a low- to medium-polarity stationary phase. The more hydrophobic sample components interact to a greater degree with the stationary phase, and therefore elute more slowly than the more hydrophilic components. The sample elution order is from most hydrophilic to most hydrophobic. 

An HPLC method for chlorogenic acids with lactones in six different commercial brands of roasted coffee was developed by Schrader et al. (143). Hydroxycinnamic acid derivatives, including mono- and di-caffeoylquinic acids, corresponding lactones, and feruloylquinic acids were extracted from coffee with methanol at 80°C for 1 h under reflux. An HPLC method using step-gradient elution with 2% aqueous acetic acid (eluent A) and ACN (eluent B) for a 75-min run time was developed. Determination was carried out by HPLC with UV detection at 324 nm, and further confirmation was conducted by HPLC-thermospray (TSP)-MS and HPLC-diode array detection. Elution order for mono-caffeoylquinic acid (CQA) was 3-CQA, 5-CQA, followed by 4-CQA, which was different from the usual elution order of mono-CQA (Fig. 17). These results indicate that it is currently not possible to predict the elution order of different reversed-phase packings due to the different selectivity (143). 

Melphalan has been converted to its trimethylsilyl derivative with bis(trimethylsilyl)acetamide and has been analyzed by GC on a 1.8 m x 3 mm column packed with 2.5% (w/w) SE-54 on acid-washed, silanized Chromosorb W (80-100 mesh) at 210° (injector temperature, 250° flame ionization detector temperature, 215°) using nitrogen as the carrier at 30 ml/min. The order of elution from a partly hydrolyzed mixture was melphalan, mono-hydroxy-derivative VI and di-hydroxy-derivative VII (Scheme III). The same elution order was obtained on a SE-30 column it was reversed on a more polar liquid phase (OV-17). Identification of the peaks was done by mass spectrometry [52]. 

Adsorption-desorption mechanisms of RP systems are a turnaround from NP systems. In this case the non-polar, or hydrophobic, portion of solute molecules adsorbs to the surface of the stationary phase, while the polar part of the molecule is solvated by the mobile phase solvent. The result is a reversed elution order - polar before less polar solutes (Fig. 4.9). 

In contrast to conventional cation exchangers, a reversed elution order is observed with crown ether phases, which is mainly determined by the size ratio between crown ether ring and alkali metal ion. Due to the high affinity of poly(benzo-15-crown-5) toward potassium and rubidium ions, these are more strongly retained than lithium, sodium, and cesium ions, respectively. However, the complexing properties of crown ethers also depend on the counter ion being employed. Thus, in potassium salts, for example, an increase in retention in the order KC1 < KBr < KI is observed with an increasing size of the counter ion. 

Inorganic ions are presumed to be the smallest in the mixture, and hence their elution should be expected to be retarded the most. This, however, is not always the case because there are unpredictable interactions between the compounds and column support, such that the predicted elution order can often be reversed. These interactions can hold advantages for the extractor and can be used to isolate specific t3 es of compounds. The purification of paralytic shellfish toxins such as saxitoxin and gonyautoxins was done by taking advantage of their specific adsorption on Bio-Gel P-2 of Sephadex G-10. 

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