Stationary Phases for HILIC

According to McCalley [15], stationary phases used for HILIC [16] can be classified into five different groups  

Although the number of commercially available stationary phases designed specifically for HILIC is rapidly growing, there is still no versatile phase like C18 in reversed-phase liquid chromatography. 

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There is a great selection of stationary phases for HILIC silica, derivatized silica with diol, nitrile, amino or amide groups, silica with a hydrophilic polymeric coating, and various polymers. They all were especially developed for HILIC whereas... 

Stationary phases for HILIC, in Hydrophilic Interaction Chromatography (eds BA. Olson and B.W. Pack), John Wiley Sons, Inc., New Jersey. 

Conventional LC with CIS-modified silica stationary-phase or HILIC columns has been widely used in antibiotic residue analysis as discussed above. Since the 1980s, the need for faster and more powerful separations for the analyses of constituents, such as protein digests, in complex matrices led to the development of highly mechanically stable (to high back-pressures) columns and... 

To better distinguish the contributions of polar interactions to retention, the LEER model was transformed into the so-called hydrophobic subtraction model (HSM) for RPLC, where the hydrophobic contribution to retention is compensated for by relating the solute retention to a standard nonpolar reference compound. This approach was applied to characterize more than 300 stationary phases for RPLC, including silica gel supports with bonded alkyl-, cyanopropyl-, phenylalkyl-, and fluoro-substituted stationary phases and columns with embedded or end-capping polar groups. The QSRR models can be used to characterize and compare the suitabihty of columns not only for reversed-phase, but also for NP and HILIC systems. 

Stationary phases for hydrophilic interaction chromatography, such as the amino and ZIC-HILIC phases in Table 22-3, are strongly polar. They are thought to be coated with a thin layer of water inside the column. Polar solutes are retained by the polar bonded phase and by the thin aqueous layer. The mobile phase typically contains (25-97 vol%) CH3CN or other polar organic solvent mixed with aqueous buffer. The higher the concentration of organic solvent, the less soluble is the polar solute in the mobile phase. 

Water is the essential component of a typical mobile phase for HILIC, because it maintains the stagnant aqueous layer on the surface of the stationary phase that... 

Hydrophilic Interaction LC-MS-Based Approaches Hydrophilic interaction LC (HILIC) is a variant of normal-phase LC. HILIC uses hydrophilic stationary phases, but employs reversed-phase type eluents. Any polar chromatographic surface can be used for HILIC separations, even nonpolar bonded silicas. A typical mobile phase for HILIC includes acetonitrile with a small amount of water. However, any aprotic solvent miscible with water (e.g., tetrahydrofunan or dioxane) can be used. Alcohols can also be used with a higher concentration. Ionic additives, such as ammonium acetate and ammonium formate, are usually used to serve as the modifiers for controlling the pH and ion strength of the mobile phase. 

Additional modes of HPTC include normal phase, where the stationary phase is relatively polar and the mobile phase is relatively nonpolar. Silica, diol, cyano, or amino bonded phases are typically used as the stationary phase and hexane (weak solvent) in combination with ethyl acetate, propanol, or butanol (strong solvent) as the mobile phase. The retention and separation of solutes are achieved through adsorp-tion/desorption. Normal phase systems usually show better selectivity for positional isomers and can provide orthogonal selectivity compared with classical RPLC. Hydrophilic interaction chromatography (HILIC), first reported by Alpert in 1990, is potentially another viable approach for developing separations that are orthogonal to RPLC. In the HILIC mode, an aqueous-organic mobile phase is used with a polar stationary phase to provide normal phase retention behavior. Typical stationary phases include silica, diol, or amino phases. Diluted acid or a buffer usually is needed in the mobile phase to control the pH and ensure the reproducibility of retention times. The use of HILIC is currently limited to the separation of very polar small molecules. Examples of applications... 

One weakness of the dominant reverse phase separations mechanism has been the poor retention of highly polar analytes, and hydrophilic interaction liquid chromatography (HILIC) has emerged as an alternative. In HILIC, a polar stationary phase such as silica gel is used to retain highly polar analytes. Mobile phases components similar to those described above for reverse phase separations are used, but the proportions of aqueous vs. organic are changed. Analytes are retained under conditions of relatively low water content, and eluted using increased water content. 

Antidepressant separation was usually performed by reversed-phase chromatography with typical C8 or C18 alkyl chain columns, although phenyl [30, 59] or cyano [48,64, 84] stationary phases were also employed. As an exception, hydrophilic interaction liquid chromatography (HILIC), a variation of normal phase chromatography, was employed in two analytical methods for duloxetine [38] and... 

Different developed analytical method are discussed in this chapter related to the determination of illicit substances in blood (either whole blood, plasma, or serum), OF, urine, and hair. These methods take into consideration the particular chemical and physical composition of the matrix and applies each time a suitable pretreatment to remove interfering and matrix effect, to maximize recoveries and to achieve a suitable enrichment if necessary. For liquid matrices the applications of the most common techniques are considered from simple PPT to SPE and LLE the results of recent works from literature are reported and new trends as online SPE, pSPE, automated LLE (SLE) or MAE are examined. Several stationary phases have been shown to be suitable for determination of illicit drugs Cl8, pentafluorophenyl, strong cation-exchange, and HILIC columns. The trend toward fast chromatography is investigated, both UHPLC and HPLC with appropriate arrangements moreover, results obtained with different ion sources, ESI, A PCI, and APPI are compared. 

HILIC uses a polar stationary phase to separate proteins and peptides. Samples are loaded onto a column using a low aqueous mobile phase and are eluted by increasing the water content of the mobile phase (152). HILIC is very suitable for separation of polar species such as sialic acid-containing glycopeptides, which have limited retention on most RP materials (153). A variant of HILIC is electrostatic repulsion-hydrophobic interactions chromatography and it utilizes IEX stationary phase and a high organic mobile phase (152,154). 

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