Mobile phases amine modifiers

Beyond the density changes that can be used to control method modifications in SFC, the mobile phase composition can also be adjusted. Typical LC solvents are the first choice, most likely because of their availability, but also because of their compatibility with analytical detectors. The most common mobile phase modifiers, which have been used, are methanol, acetonitrile and tetrahydrofuran (THF). Additives, defined as solutes added to the mobile phase in addition to the modifier to counteract any specific analyte-column interactions, are frequently included also to overcome the low polarity of the carbon dioxide mobile phase. Amines are among the most common additives. 

In a similar way, the use of formic acid/ammonium formate was shown to be superior to the use of acetic acid/ammonium acetate in the RP HPLC-ESI MS/MS analysis of a number of heterocyclic aromatic amines [7]. It is clear from numerous other examples in the literature that HPLC resolution and MS sensitivity can be dramatically influenced by the correct selection of mobile phases, organic modifiers, and ion-pairing additives. 

The type 11 CSPs have been used to resolve a wide variety of pharmacologically active compounds, both with and without derivatization (9-11). Some of the compounds that have been resolved without derivatization are hexobarbital (OA-CSP) (51), glutethimide (OB, OC, OK) (51), warfarin (OB, OC) (51), compounds containing a chiral sulfur atom (OB, OC) (52), verapamil (AD) (53), propranolol (OD) (54), and flurbiprofen (AD) (55), The resolution of the latter two compounds, propranolol, an ot, p-amino-alcohd, and flurbiprofen, a carboxylic acid, is representative of new applications for the cellulosic CSPs that are primarily used with hexane alcohol mobile phases. In the case of propranolol, the mobile phase was modified with N,N-dimethyIoctyl amine, and when flurbiprofen was chromatographed, trifluoroacetic acid was used as the modifier. 

Retention and stereoselectivity on the BSA columns can be changed by the use of additives to the aqueous mobile phase (30). Hydrophobic compounds generally are highly retained on the BSA, and a mobile-phase modifier such as 1-propanol can be added to obtain reasonable retention times. The retention and optical resolution of charged solutes such as carboxyUc acids or amines can be controlled by pH and ionic strength of the mobile phase. 

Competing amines such as triethylamine and di-rc-butylamine have been added to the mobile phase in reversed-phase separations of basic compounds. Acetic acid can serve a similar purpose for acidic compounds. These modifiers, by competing with the analyte for residual active sites, cause retention time and peak tailing to be reduced. Other examples are the addition of silver ions to separate geometric isomers and the inclusion of metal ions with chelating agents to separate racemic mixtures. 

The efficacy of CE separation depends considerably on the type of capillary. Fused-silica capillaries without pretreatment are used most frequently. Its outside is coated with a polymer layer to make it flexible and to lessen the occurrence of breakage. The polymer coating has to be dissolved with acid or burned away at the detection point. Capillaries with an optically transparent outer coating have also found application in CE. The objectives of the development of chemically modified capillary walls were the elimination of electro-osmotic flow and the prevention of adsorption on the inner wall of the capillary. Another method to prevent the adsorption of cationic analyses and proteins is the use of mobile phase additives. The modification of the pH of the buffer, the addition of salts, amines and polymers have all been successfully employed for the improvement of separation. 

In packed column SFC, polar solutes such as amines and carboxylic acids often have too much retention or elute with poor peak shapes when neat carbon dioxide is used as a mobile phase [28, 92]. This is mainly due to the weak solvent strength of neat carbon dioxide compared to a liquid solvent. The use of modifiers is often necessary to enhance the solvating power of the mobile phase in SFC. Various alcohols such as methanol and isopropanol are commonly used modifiers in SFC, but other solvents such as acetonitrile was also utilized [92]. The concentrations of modifiers are usually less than 50%. The technique in which the concentrations of modifiers are greater than 50% is often called enhanced-fluidity liquid chromatography [93]. 

FIGURE 9.18 Separation of 1, methyl benzyl amine 2, tartaric acid 3, diastereomer 4, drug candidate BMS-X 5, oxidation product and 6, vehicle polymers. Using a ES-PFP column (250 x 4. 6 mm with 5 p.m particles) with a mobile phase gradient starting with 95% modifier/5%C02 held for 25 minutes and ramped to 15%C02 for 5 minutes at 1.5mL/min, 311 K and 18.0 MPa modifier 43% H20/56% methanol). (Unpublished data from S. L. Phillips et al., unpublished data. With permission.)... 

What is the reason for the overwhelming acceptance of stationary phases based on high-purity silicas in the pharmaceutical industry The answer is simple superior peak shapes for analytes with basic functional groups, which has been a problem with older phases. The older, low-purity silicas contain metal ions buried in the matrix of the silica. These contaminants acidify the surface silanols, and the consequence is a strong and non-uniform interaction with basic analytes. This in turn results in tailing peaks, which is an impediment for accurate peak integration and peak resolution. Of course, adding appropriate additives, such as amine modifiers, to the mobile phase can solve these difficulties. But this is an unnecessary and undesired complication in methods development. Therefore, silicas that are free from this complication are much preferred. 

When amine-modified silica-gel columns are eluted with a mobile phase of acetonitrile-water, carbohydrates are separate on the basis of normal, phase partitioning - increasing the water content of the mobile phase... 

Amine modifiers in the mobile phase have been used in conjunction with silica-gel columns to provide more-stable columns. In addition, the use of diol-type silica-gel columns for the separation of food sugars has been reported. These columns provide separations similar to those afforded by aminopropyl silica gel, but they are much more stable. The use of these diol columns, whenever practical, is therefore recommended. 

Bushey, M.M., Jorgenson, J.W. (1989). Separation of dansylated methyl-d3-amine by micellar electrokinetic capillary chromatography with methanol modified mobile phase. Anal. Chem. 61 491—493. 

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