Aminopropyl-bonded phase columns

Veuthey and Haerdi reported the separation of amphetamines using packed-column SFC [26]. The amphetamines were derivatized with 9-fluorenylmethyl chloroformate and chromatographed with a methanol or 2-propanol-modified carbon dioxide as the mobil phase. The separations were compared on bare silica and aminopropyl-bonded silica columns. Both columns gave comparable results and the separation of all five amphetamines (methylamphetamine, amphetamine, phenethylamine, ephed-rine, and norephedrine) was achieved in less than 5 min. Both methanol and 2-propanol-modified carbon dioxide gave comparable results. It was observed that the modifier concentration had more effect on the solvating power than the mobile-phase density. 

Later, Klemm and co-workers [86,87] achieved partial resolution of aromatic compounds by low-pressure chromatography on silica gel impregnated with TAPA. The separation was attributed to n-n complexation between TAPA and the enantiomers. Mikes et al. [88] used a column packed with an (i )-(-)-TAPA aminopropyl-bonded silica support to accomplish the full resolution of helicenes. The authors extended their study to other homologues of TAPA (Figure 22-19). These compounds were coated on silica gel or ion-paired to an aminopropyl-bonded phase, and they were used in the HPLC separation of helicenes. To describe the selective interactions that occur between the stationary phase and the helicenes, the authors assumed that the 2,4,5,7-tetranitro-9-fluorenylidene moieties of the selector are laying down on the silica surface, while the X groups point away from the surface and above the plane of the fluorenyl ring. 

Fignc 11.1 Sqnration of sugars on an aminopropyl bonded phase. Peak identification (1) fiructose (2) glucose (3) sucrose (4) maltose (S) lactose. (Chromatographic conditions Column Waters High-Performance Carbohydrate Column. Mobile phase aoetonitrile/water 7S-2S v/v.) Chromatogram courtesy of D. J. Phillips, Waters Corp.)... 

The primary amine in aminopropyl bonded phases reacts easily with aldehydes and ketones in both the mobile phase and samples. Therefore the exposure of these columns to aldehydes and ketones should be limited. A guard column can be used to protect the analytical column from small amounts of contaminants in the sample. 

Polymers developed specifically for HPLC include the macroporous crosslinked vinylpyridinium type, which has proved very effective in HILIC of sugars in aqueous acetonitrile. These polymers can be used at column temperatures up to 70°C, under which conditions, with the resin in the phosphate or sulfate form, baseline resolution of mixtures of the common monosaccharides and some disaccharides, such as maltose and lactose, can be achieved. Stationary phases in which a polymer replaces silica as the support for the aminopropyl bonded phase much used in HILIC of carbohydrates have also been successfully applied in separations of mono- and disaccharides in this case precolumn derivatization with 4-amino-benzoic acid ethyl ether (ABEE) has been recommended to overcome the problem of glycosylamine formation that occurs with imderivatized sugars. 

Ion-exchange and reversed-phase media are most commonly used as stationary phases in the LC analysis of water-soluble vitamins. Aminopropyl-bonded silica columns (e.g., LiChrosorb NH2, /tBondapak NH2) have been used as weak anion exchangers for... 

Conversely, alcohols should not be used in the separation of sugars. First, the resultant peak shapes generated on the typical column of choice, aminopropyl, are considerably worse than those generated using acetonitrile. Second, the reaction of the reducing sugars with the aminopropyl bonded phase, via a Schiff base formation, is accelerated by alcohols [459]. 

Ascorbic acid and dehydroascorbic acid have been determined by reversed-phase h.p.l.c., post-column reduction of dehydroascorbic acid to ascorbic acid with dithiothreitol, reaction of excess reagent with JV-ethylmaleimide, and electrochemical detection. Ascorbic acid and its 2-phosphate were determined by h.p.l.c. on an aminopropyl bonded-phase silica column. Dehydroascorbic acid could also be determined by the increase in the ascorbic acid content after reduction with dithiothreitol The method was applied to raw apple and potato to which these compounds are added to prevent browning. ... 

Sugar analysis by hplc has advanced greatly as a result of the development of columns specifically designed for carbohydrate separation. These columns fall into several categories. (/) Aminopropyl-bonded siHca used in reverse-phase mode with acetonitrile—water as the eluent. (2) Ion-moderated cation-exchange resins using water as the eluent. Efficiency of these columns is enhanced at elevated temperature, ca 80—90°C. Calcium is the usual counterion for carbohydrate analysis, but lead, silver, hydrogen, sodium, and potassium are used to confer specific selectivities for mono-, di-, and... 

Surfactants are separated according to adsorption or partitioning differences with a polar stationary phase in NPLC. This retention of the polar surfactant moiety allows for the separation of the ethylene oxide distribution. Of all the NPLC packings that have been utilized to separate nonionic surfactants, the aminopropyl-bonded stationary phases have been shown to give the best resolution (Jandera et al., 1990). The separation of the octylphenol ethoxylate oligomers on an amino silica column is shown in Fig. 18.4. Similar to the capabilities of CE for ionic surfactants, the ethylene oxide distribution can be quantitatively determined by NPLC if identity and response factors for each oligomer are known. 

Many of the l.c. stationary-phases available may be packed into columns by the user at a fraction of the cost of a commercial column (see Section II,l,f). The most notable of these are the cation-exchange res-ins8.89 and the aminopropyl-bonded silica gels, which can be readily packed to provide high-resolution separations. The Cis-bonded silica gel and cation-exchange resin stationary-phases are especially useful, as large columns (2 x 25 cm) can be accommodated on analytical chromatographs, at flow rates of 1 to 5 mL/min, without any modifications of equipment. 

New advances in the l.c. of carbohydrates are likely to come from three general areas. The first is in the development of more-durable and stable, stationary phases. At present, a major limitation on the use of commercial columns, especially those of the aminopropyl-bonded silica-gel variety, is their short life-time and ease of fouling. More-durable, resin-based columns that operate with the same solvent system and selectivity as aminopropyl silica-gel columns are currently available, and will see further use and development. The development of improved phases for supercritical, fluid-type l.c. will allow this method to be of use for analysis of various carbohydrates. ... 

Basic solutes such as amines, ethers, esters, and ketones are preferentially retained on amino and diol columns when compared to cyano columns [14,15]. The amino phase is a good alternative to the cyano column for a change in selectivity. When analyzing ketones and aldehydes, the aminopropyl phase should be carefully used due to the possible reactivity of the amino group with these substances. This may lead to the formation of imines and the bonded phase maybe easily oxidized [13],... 

Early work in SFC was carried out using absorbants such as alumina or silica, or stationary phases insoluble in supercritical CO2, such as polyethylene glycol. Now, bonded non-extractable stationary phases such as octadecyl-silane and aminopropyl-bonded silicas, are usually used in packed columns. 

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