Aminopropyl bonded phase

Kim and Salem (33) found that the acidic PL were eluted from an aminopropyl bonded phase with over 95% recovery with 4 ml of a mixture of H/2-P/Ethanol/0.1 M aqueous NH4-ac-etate/formic acid (420/350/100/50/0.5) containing 5% phosphoric acid (Table 2). Neither the solvent mixture without phosphoric acid nor methanol containing 5% phosphoric acid was able to elute the acidic PL. Actually, Table 2 indicates that a fractionation of both neutral and acidic PL is enabled by the sequential elution with methanol and the aforementioned solvent mixture. In order to remove the phosphoric acid from the acidic PL fraction, it is first dried under N2 for 10 min to remove the hexane and then extracted three times with 1 ml of chloroform after the addition of 1 ml of water. 

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. 

Diol phases arc about as retentive as silica or aminopropyl bonded phases (17). Compared to silica and alumina, diol phases are much less sensitive to the water content of the mobile phase. They arc also less reactive than aminopropyl phases. Furthermore, they can be washed with water without difficulty. Thus they are the best choice among the stationary phases for normal-phase chromatography. Although the stationary phase is readUy available from many sources, it is most commonly found as a packing for aqueous size-exclusion dromatography. 

If the analytes are very polar and neutral, the best analytical technique is likely to be hydrophilic interaction chromatography. It has proved its value for the analysis of carbohydrates. Hydrophilic interaction chromatography is carried out using very polar stationary phases in conjunction with aqueous mobile phases with a high organic content. Useful stationary phases are silica, alumina, polar bonded phases such as aminopropyl or diol, and even ion exchangers. The stability of the aminopropyl bonded phase in aqueous eluents is limited, but the other stationary phases can be recommended without reservation. For a detailed discussion, see Chapter 11. 

As expected, the aminopropyl bonded phase is more retentive toward acidic samples than either silica or cyanopropyl and diol bonded phases. For example, steroids with phenolic functional groups are more retained on the aminopropyl bonded phase (16). 

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. 

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]. 

Doner, L.W. and Biller, L.M. 1984. High-performance thin-layer chromatographic separation of sugars Preparation and application of aminopropyl bonded-phase silica plates impregnated with monosodium phosphate, J. Chromatogr. 287 391-398. 

Normal Phase Chromatography. Normal phase mode involves the polar interaction of sample molecules with a polar stationary phase, commonly silica-based packings with an aminopropyl bonded phase. Alternatively, pure silica gel modified in situ by equilibration (and simultaneous regeneration) with a mobile phase containing a polyfunctional amine that is adsorbed could be used (47). 

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. ... 

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