Cyano bonded phase, HPLC

Variations in retention and selectivity have been studied in cyano, phenyl, and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline, and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile, or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns were correlated with polar group selectivities of mobile-phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers [84],... 

Riggin and Howard (1979, 1982), Matsui et al. (1983), Fabre et al. (1984), and Ahuja et al. (1988) reported that High Performance Liquid Chromatography (HPLC) with UV or electrochemical detection is capable of analyzing 1,2-diphenylhydrazine. Reversed phase chromatographic columns have been used most often (Ahuja et al. 1988 Fabre et al. 1984 Riggin and Howard 1979, 1982). Cyano-amino polar bonded phase columns also have been used (Matsui et al. 1983). Using a reversed phase and UV detection, the minimum amount detected (on column amounts) is approximately 6-7 ng and the minimum amount quantifiable is less than 1 pg (Ahuja et al. 1988 ... 

The nomenclature of the RP is not consequent. The RP most often used contains octyl (RP C8) or octadecyl (RP C18) groups. There is no differentiation even when two methyl groups are introduced additionally with the silane (as with monofunctional silanes) or only one (difunctional) or none (trifunctional silane). Some manufacturer use silanes with bulky side groups (e.g., isopropyl groups) to improve the hydrolytic stability of the bonded phases, but here also, only the longest alkyl group is used in nomenclature. RP C8 and RP C18 are the work horses in HPLC. Shorter chains (RP4) are used in protein separations, and special selectivity can be obtained with bonded phenyl, cyano, amino or fluoro groups. 

In general, there is a wide variety of chromatographic modes (types) that can be employed for the HPLC determination of food components, but only a few have been used for the determination of NOC. These include partition/adsorption on silica gel, liquid-liquid partition on polar-bonded phase (e.g., cyano, amino) or nonpolar hydrophobic-bonded phase (e.g., reversed-phase), and anion-exchange chromatography. Macrae (61) discussed the theories behind the various modes of chromatography. 

The IUPAC Compendium of Chemical Technology defines Normal Phase as an elution procedure in which the stationary phase is more polar than the mobile phase . In practice, the most widely used stationary phases for preparative HPLC are based on silica and the polarity of the underlying silyl ether and silanol provides the required hydrophilic surface. Amino and cyano bonded silica are also commonly used in normal phase mode though the latter also has some reversed phase properties. The predominant mechanism of interaction is hydrogen bonding. However, the silanol is mildly acidic so the silica surface will also have mild cation exchange properties. 

The disadvantage of normal phase HPLC for the separation of carbonyl dinitrophenylhydrazones is the difficulty in maintaining reproducible retention times due to absorption of water from the solvent and interaction with the active groups on the column (Fig. 5.10). To overcome this problem, a cyano-propyl bonded phase (Exsil 100, 5 /im Chromtech, U.K.) column may be used for isolation of hy-droxyalkenals and a mixture of alkanals and alk-2-enals from rat liver microsomes. The less polar carbonyls can be separated under isocratic conditions as follows. 

In normal phase HPLC, the cyano and amino columns, in conjunction with the diol bonded phase columns, are now often used in preference to unmodified silica or alumina. Whilst silica can demonstrate remarkable selectivity for the separation of closely related isomers (e.g. E/Z isomers are commonly separated on silica), some of the operational demands of silica limit its usefulness. The difficulty of manufacturing reproducible microcrystalline silica and the possible effect of trace amounts of water on... 

Early PG analysis using HPLC techniques was carried out as adsorption chromatography on normal-phase (NP) columns packed with silica or alumina. The nonpolar mobile phase comprizing of organic solvents (hexane, toluene, ethyl acetate, and HOAc) allows separation of PGs which are unstable in aqueous media (e.g., PGH2 on cyano- or phenyl-bonded phases). Usually, the injection medium must be fairly polar to dissolve the PGs. This is achieved by the addition of... 

Near-critical pSFC applications can be described as those where the mobile phase is solvent-modified CO2, pressurized oniy enough to maintain a single phase, with temperatures near (typically less than) the critical temperature. Many commercially available HPLC bonded silica phases have been used with modified-CO2 mobile phases to achieve normal-phase separations, the choice of stationary phase being dictated by sample polarity. The modifiers added to CO2 accept-abfy overcome the unwanted analyte-silica interactions obeserved with neat CO2 mobile phases. For structural separation of polar compounds such as pharmaceuticals [typically weak acids or bases of molecular weight (MW) <1000], polar phases such as diol-, amino-, and cyano-bonded silica (or bare silica)... 

Packed column SFC stationary phases are very similar or identical to those used for HPLC. With neat CO2 mobile phases, polymer or polymer-coated silica stationary phases have typically been used. With modified-C02 mobile phases, bonded-phase silica columns are typically used. For structural separations, diol, amino, or cyano stationary phases are most often used. For stereochemical separations, derivatized polysaccharide-bonded silica columns are most often the stationary phases of choice. A powerful feature of modified-C02 pSFC is the ability to serially connect different stationary phases to obtain enhanced or mul-... 

Several reversed-phase methods were also developed which do not use a C18 column. A reversed-phase method using a C8 Spherisorb column has been reported (54) to quantitate diltiazem and two of its metabolites (N-monodemethyl diltiazem and desacetyl diltiazem). A 10 pm particle size PRP-1 column (55), mobile phase of 60% acetonitrile and 0.01 M aqueous KH2PO4, 40% 0.005M aqueous tetrabutylammonium hydroxide and UV absorbance detection at 254 nm was used to determine diltiazem present in plasma. Several HPLC methods have been developed which use a cyano-bonded column. One such method was developed for the determination of diltiazem and its metabolite desacetyl diltiazem in human plasma (56). The analytes are extracted from plasma made basic with 0.5M aqueous dibasic sodium phosphate (pH 7.4) using 1% 2-propanol in hexane. The method uses a cyanopropylsilane column with a mobile phase of 45% acetonitrile and 55% 0.05M aqueous acetate buffer (pH 4.0). The minimum detectable limit was 2 ng/mL in plasma. A similar HPLC method was developed by Johnson and Pieper (57) for the determination of diltiazem and three of its metabolites. Also, an HPLC method was developed (58) for the analysis of diltiazem and desacetyl diltiazem in plasma using UV detection at 237 nm, a Zorbax CN 6 pm particle size column and a mobile phase of 45% methanol, 55% 0.05M aqueous ammonium dihydrogen phosphate and 0.25% triethylamine adjusted to pH 5. 

Reversed-phase high-performance liquid chromatography (RP-HPLC) is more popular for analysis of carotenoids than is normal-phase HPLC because (1) retention is very little affected by small variations in the mobile-phase composition, and (2) the risk of artifact formation on passage through the column is minimal as solute-support interactions on non-polar-bonded phases only involve weak forces. A variety of stationary phases of various polarities are available, such as C18, C8, C4, C2, Cl, phenyl, and cyano derivatives the C18 phase is the most popular. 

Analytical screens are performed with both reverse-phase RP-HPLC and SFC isolation techniques. Analytical SFC should be screened first unless instrumentation availability or project background specifics dictate otherwise. Screening achiral column bonded phases varying in polarity and functionality against different mobile-phase solvent choices is effective for identifying analytical methods for the purpose of impurity isolation. There are currently many unique achiral SFC bonded phase column choices commercially available (2-ethyl pyridine, diethyl amino, dinitrophenyl, pyridine urea, diol, cyano, etc.). SFC column choice provides the most impact in manipulation of relative selectivity for individual... 

Only the silica-based stationary phases with covalently bonded alkyl chain, cyano and propylamino ligands have found practical applications in HPLC. Besides these common ligands, the experimental use of naphthalene, pyrene and nitroaromatic as ligands has also been reported. Silica-based stationary phases with covalently bonded cyclodextrins or cyclodextrin derivatives have been frequently employed in the separation and quantitative determination of isomer pairs. 

Benzalkonium chloride ( ) is used as an antimicrobial preservative in Nasonex. It is quantifiable using a HPLC method with a column that has a cyano group chemically bonded to porous silica particles. The HPLC was equipped with a UV-Vis detector, a 150 mm x 4.6 mm, 3- jm Spherisorb S3 CN column. The mobile phase consisted of 45% acetonitrile in 0.05 M phosphate buffer (pH 6.0). The flow rate was set at 1 mL/min. The following procedure was used for sample and standard preparations. About 2.5 g of Nasonex was transferred into a 25-mL volumetric flask. This was diluted to volume with acetonitrile and... 

A Perkin-Elmer MPF-2A Fluorescence Spectrophotometer was used to determine the excitation and emission wavelengths required for achieving maximum fluorescence intensity for the pesticides studied. The MPF-2A contained a 150 watt xenon arc and an excitation monochromator with a grating blazed at 300 nm as the excitation unit a Hamamatsu R 777 photomultiplier tube (sensitivity range 185 - 850 nm) and an emission monochromator grating blazed at 300 nm as the emission detection unit. A DuPont Model 848 Liquid Chromatograph was used for HPLC (Figure 2). The accessory injection device included a Rheodyne Model 70-10 six-port sample injection valve fitted with a 20 y liter sample loop. A Whatman HPLC column 4.6 mm x 25 cm that contained Partisil PXS 1025 PAC (a bonded cyano-amino polar phase unspecified by the manufacturer) was used with various mobile phases at ambient temperature and a flowrate of 1.25 ml/minute. 

An alternative to liquid-liquid extraction is solid-phase extraction (SPE). With SPE a liquid sample is introduced into the top of a plastic syringe shape column containing a small amount (often 100- 500 mg) of a selective adsorbent (Figure 8.1). The adsorbents are of the same types as used for HPLC, typically silica, or bonded silica such as Cl 8, C8, C5, C2, cyano, phenyl, diol and ion-exchange materials. The properties of the adsorbents are similar to HPLC columns and so the same principles apply for retention and desorption of analytes. 

Over 500 HPLC packings have been described in the Hterature. Nevertheless, as the result of years of development, only a limited number of types of stationary phases remain on the market. Most of the conventional HPLC separations today are performed using monodisperse silica gel 3 or 5 Xm microbeads, especially those grafted with C4, C8, or C18 alkyl chains, as well as with cyano-propyl or amino-propyl groups. The last two bonded silicas and bare silica are used in normal phase (NP) HPLC, where the mobile phase (usually hexane with small amounts of isopropyl alcohol) is less polar than the stationary phase. Even more popular is the reversed phase (RP) mode, which uses polar eluents (mosdy water or methanol with such additives as acetonitrile, methanol, or tetrahydrofuran (THE)) in combination with nonpolar alkyl-bonded stationary phases. 

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