Polar analytes, separation

Separation in column 1 (C-1) removes early-eluting interference compounds, and so considerably increases the selectivity. The fraction of interest separated in C-1 is then transferred to column 2 (C-2) where the analytes of the fraction are separated. These transfers can be carried out either in forward mode or backflush mode. The forward mode is preferred because the backflush mode has two disadvantages for polar to moderately polar analytes. For most polar compounds, it leads to additional band broadening, while for more retained analytes there is a decrease in the separation obtained earlier in the process (31). 

Another important issue that must be considered in the development of CSPs for preparative separations is the solubility of enantiomers in the mobile phase. For example, the mixtures of hexane and polar solvents such as tetrahydrofuran, ethyl acetate, and 2-propanol typically used for normal-phase HPLC may not dissolve enough compound to overload the column. Since the selectivity of chiral recognition is strongly mobile phase-dependent, the development and optimization of the selector must be carried out in such a solvent that is well suited for the analytes. In contrast to analytical separations, separations on process scale do not require selectivity for a broad variety of racemates, since the unit often separates only a unique mixture of enantiomers. Therefore, a very high key-and-lock type selectivity, well known in the recognition of biosystems, would be most advantageous for the separation of a specific pair of enantiomers in large-scale production. 

HPLC requires a mobile phase in which the analytes are soluble. The majority of HPLC separations which are carried out utilize reversed-phase chromatography, i.e. the mobile phase is more polar then the stationary phase. In these systems, the more polar analytes elute more rapidly than the less polar ones. 

Supercritical fluid extraction (SEE) is another modern separation technology usually employed to extract lipophilic compounds such as cranberry seed oil, lycopene, coumarins, and other seed oils. Anthocyanins generally and glycosylated anthocyanins in particular were considered unsuitable for SEE due to their hydrophilic properties, since SEE is applicable for non-polar analytes. However, a small amount of methanol was applied as co-solvent to increase CO2 polarity in anthocyanin extraction from grape pomace. New applications of SEE for anthocyanin purification have been reported for cosmetic applications from red fruits. ... 

A further extension of the DFG S19 method was achieved when polar analytes and those unsuitable for GC were determined by LC/MS or more preferably by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Triple-quadrupole MS/MS and ion trap MS" have become more affordable and acceptable in the recent past. These techniques provide multiple analyte methods by employing modes such as time segments, scan events or multiple injections. By improving the selectivity and sensitivity of detection after HPLC separation, the DFG S19 extraction and cleanup scheme can be applied to polar or high molecular weight analytes, and cleanup steps such as Si02 fractionation or even GPC become unnecessary. 

Until this point, the sample preparation techniques under discussion have relied upon differences in polarity to separate the analyte and the sample matrix in contrast, ultraflltration and on-line dialysis rely upon differences in molecular size between the analyte and matrix components to effect a separation. In ultrafiltration, a centrifugal force is applied across a membrane filter which has a molecular weight cut-off intended to isolate the analyte from larger matrix components. Furusawa incorporated an ultrafiltration step into his separation of sulfadimethoxine from chicken tissue extracts. Some cleanup of the sample extract may be necessary prior to ultrafiltration, or the ultrafiltration membranes can become clogged and ineffective. Also, one must ensure that the choice of membrane filter for ultrafiltration is appropriate in terms of both the molecular weight cut-off and compatibility with the extraction solvent used. 

Fused silica capillary columns of various internal bores and of lengths in the range 25 to 50 m are mainly employed for analytical separations. A variety of polar and non-polar column types are available including those open tubular types with simple wall coatings (WCOT), those with coatings dispersed on porous solid-supports to increase adsorbent surface area (SCOT) and porous layer open tubular (PLOT) columns. Important stationary phases include polyethylene glycol, dimethylpolysiloxane and different siloxane copolymers. Various sample introduction procedures are employed including ... 

In matrix solid-phase dispersion (MSPD) the sample is mixed with a suitable powdered solid-phase until a homogeneous dry, free flowing powder is obtained with the sample dispersed over the entire material. A wide variety of solid-phase materials can be used, but for the non-ionic surfactants usually a reversed-phase C18 type of sorbent is applied. The mixture is subsequently (usually dry) packed into a glass column. Next, the analytes of interest are eluted with a suitable solvent or solvent mixture. The competition between reversed-phase hydrophobic chains in the dispersed solid-phase and the solvents results in separation of lipids from analytes. Separation of analytes and interfering substances can also be achieved if polarity differences are present. The MSPD technique has been proven to be successful for a variety of matrices and a wide range of compounds [43], thanks to its sequential extraction matrices analysed include fish tissues [44,45] as well as other diverse materials [46,47]. 

The separation of polar analytes can also be optimized by applying ternary eluent mixtures. In this case, the retention is adjusted with binary mixtures with methanol-water for k in the desired range (2polar analytes resolution [57]. 

Polar analytes can also form adducts with various ions. A typical adduct ion that is formed in the positive ion mode is [M + nNaJ L but many other adduct ions with K+, NHJ, CL, acetate are frequently observed (Figure 8.4). Their formation normally occurs in the bulk solution before the charge separation process, in the electrospray droplets during evaporation, or in the gas phase. If the analyte is weakly basic or polar, salts bearing cations may be intentionally added to the sample to stimulate the formation of positive ions [10,11]. 

Octadecylsilyl stationary phases with hydrophilic endcapping have been developed for the separation of very polar analytes, which are not sufficiently retained on conventional reversed-phase columns. Among numerous other applications, they have been demonstrated to be suitable for the separation of flavonol and xanthone glycosides from mango Mangifera indica, Anacardiaceae) peels. °... 

To ensure overall extraction of a polar solute, the pressure or density must be increased or a modifier must be added to the extraction fluid after an initial low-pressure extraction. In this way, the analyte of interest can be collected separately from the oily interference. Polar solutes may be removed more easily from matrices with higher moisture content. It is believed that the analyte is solubilized in the entrained water and removed from the matrix with physical removal of water. Since polar analytes benefit from the presence of water in samples, water has been added as a mobile phase modifier to enhance extraction recoveries (152). 

With the advent of improved column deactivation, elution of more polar analytes has become possible. For this type of analytes, columns coated with selectively separating stationary phases may provide the most rapid analysis. This is the background to the increasing demand for moderately polar and polar stationary phases. Users want universal columns, that is, columns that can solve a number of analytical tasks. For that purpose, tire new generation of stationary phases should include phases in which different types of functional groups such as phenyl and cyanopropyl are present simultaneously (48). 

---