Reverse polarity mode

Reversal of the direction of the EOF using the cationic surfactant didodecyldimethylammonium hydroxide (DDAOH) in reversed polarity mode has also been reported (16). The bromide form of this surfactant (DDAB (didodecyldimethylammonium bromide)) was converted to the hydroxide form to eliminate an undesirable system peak caused by bromide. Using 10 mM phenylphosphonic acid as the probe and a buffer consisting of 200 mM borate, 0.35 mM DDAOH, and 0.03% Trition X-100 at pH 4.0, the separation of 8 alkylphosphonates was achieved in less than 3 min. Eimits of detection were in the 100 xg/L range. 

The second typical technology applied for d. of water is -> electrodialysis. After appropriate pretreatment (as above), the feed solution is pumped through the unit of one or more stacks in series or parallel. The concentrated and depleted process streams leaving the last stack are recycled, or finally collected in storage tanks. The plants operate unidirectionally, as explained, or in reverse polarity mode, i.e., the current polarity is changed at specific time intervals (minutes to hours), and the hydraulic flow streams are reversed simultaneously, thus preventing the precipitation in the brine cells. 

Capillary electrophoresis of PCR-amplified products is usually performed in the reverse polarity mode (negative potential at the injection end of the capillary). A coated capillary (100 mm i.d., 37-57 cm total length) is filled with a gel buffer system. PCR samples are introduced hydrodynamically or, after desalting, electrokinetically. The PCR sample and a DNA marker of known size may be injected sequentially and allowed to comigrate in the capillary. With a capillary temperature set at 20 to 30°C, separation of PCR products is accomplished at field strengths of 200 to 500 V/cm. Detection is on-line, measuring either UV absorbance at 260 nm, or LIF. 

Special techniques were developed for the analysis of nucleotide phosphates. For the separation of these compounds, either dynamic pH gradient (Sustacek et al., 1989) or linear polyacrylamide-coated capillaries (Takigiku and Schneider, 1991) were used. In the latter case separations were done in the reversed-polarity mode (i.e., from cathode to anode). 

Glycopeptide antibiotics have been found to be very effective chiral selectors in the enantiomeric separation of racemic pharmaceutical compounds. Vancomycin, ristocetin A, rifamycins, teicoplanin, kanamycin, streptomycin, and avoparcin have been added to the running buffer to obtain enantioseparation (161,203— 207). A few technical modifications, such as coated capillaries and separation conditions in the reverse polarity mode (as opposed to normal polarity mode, where the flow is from anode to cathode) were found to improve sensitivity and increase efficiency (116,208). 

In the electrode-positive (reverse) polarity mode, cleaning action takes place on the work surface by the impact of gas ions. This removes a thin oxide layer while the surface is under the cover of an inert gas, allowing molten metal to wet the surface before more oxide can form. 

Sulfated CF6 showed excellent enantioselectivity toward primary, secondary, tertiary, and quaternary amines [44]. Enantiomeric separations can be obtained in both normal and reverse polarity modes, although the reversed polarity mode usually produced electropherograms with better peak shapes [44]. In addition, 25 native amino acids were also separated in CE with the reversed polarity mode [44]. 

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. 

Statistically, of the compounds enantioresolved by macrocyclic glycopeptide CSPs, new polar organic mode accounts for more than 40 %, balanced by reversed-phase mode, while typical normal-phase operation resulted in approximately 5 % of separations. Some categories of racemic compounds that are resolved on the glycopeptide CSPs at different operating modes are listed in Table 2-4. 

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

Another example of the use of small particle silica is in the analysis of theophylline in plasma, as shown in Figure 5 (40). The clean-up procedure is simply a single extraction of the plasma with an organic solvent. This analysis has also been achieved by reverse phase chromatography (41), and this points out the fact that in some separations (e.g. with components of moderate polarity) either the adsorption or reverse phase mode can be used. 

Distilled or deionised water contains small amounts of organic impurities which can cause problems in long term use with bonded phase columns in the reverse phase mode. The non-polar stationary phase will collect these organics, which can alter the nature of the stationary phase or sometimes produce spurious peaks (Fig. 4.3c is an example of this). Water purification can be done by distillation from permanganate, by passage of the water through bonded phase columns, or by means of commercial systems, eg the Milli-... 

Additionally, the inj ected matrix must also be miscible with the solvents used in the separations. For normal phase mode separations, all water must be removed from the injected matrix. Since many of the complex matrixes, such as plasma, urine, and other biological fluids contain a large amount of water, this requires more time consuming sample preparation. However, water can be injected into a polar organic or reverse phase mode separation. Even within the same mode, mobile phases that are very different can cause large disturbances in the baseline. Oda et al., (1991) solved this problem by inserting a dilution tube followed by a trap column in order to dilute the mobile phase used on the achiral column. Following the dilution tube, a trap column was used to reconcentrate the analyte of interest before the enantiomeric separation. 

Because plasma and urine are both aqueous matrixes, reverse-phase or polar organic mode enantiomeric separations are usually preferred as these approaches usually requires less elaborate sample preparation. Protein-, cyclodextrin-, and macrocyclic glycopeptide-based chiral stationary phases are the most commonly employed CSPs in the reverse phase mode. Also reverse phase and polar organic mode are more compatible mobile phases for mass spectrometers using electrospray ionization. Normal phase enantiomeric separations require more sample preparation (usually with at least one evaporation-to-dryness step). Therefore, normal phase CSPs are only used when a satisfactory enantiomeric separation cannot be obtained in reverse phase or polar organic mode. 

Ekgorg-Ott et al. (1997). An interesting trend was discovered when considering the relative amount of D-theanine present in the samples. The teas of the highest grades consistently contained the lowest amounts of D-theanine. The theanine achiral-chiral system configuration included a C18 column operated in the reverse-phase mode and a y-cyclodextrin CSP in the polar organic mode. 

There are two commonly used ways to elute a given compound in HPLC the normal-phase mode (t)s><5m) and the reversed-phase mode (<5m><5s). Reversed-phase systems offer superior general selectivity. Solutes are eluted in ascending order of polarity in normal-phase systems and in descending order of polarity in reversed-phase systems. 

Although stationary phases of intermediate polarity (alumina, silica, carbon) provide only moderate general selectivity, they are potentially most powerful for very polar solutes when operated in the reversed-phase mode. 

---