Amino bonded phases

Achiral-chiral chromatography has also been accomplished using subcritical fluid chromatography (Phinney et al., 1998). In this work, the structurally related [3-blockers, 1,4-benzodiazepines, and two cold medicines were separated using methanol or ethanol modified carbon dioxide mobile phases. The (3-blockers were separated using cyanopropyl and Chiracel OD columns connected in series. Likewise, an amino bonded phase and Chiracel OD column were used for the separation of the 1,4-benzodiazepines. Guaifenesin and phenylpropanolamine from cough syrup were separated on cyanopropyl and Chiralpak AD columns in series. 

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... 

The most important point during sample preparation is to prevent oxidation of ascorbic acid. Indeed, it is easily oxidized by an alkaline pH, heavy metal ions (Cu and Fe ), the presence of halogens compounds, and hydrogen peroxide. The most suitable solvent for this purpose is metaphosphoric acid, which inhibits L-ascorbic oxidase and metal catalysis, and it causes the precipitation of proteins. However, it can cause serious analytical interactions with silica-based column, e.g., C18 or amino bonded-phases [542] and it could co-elute with AA. 

Remove the guard column before washing the analytical column, so that impurities from the guard column are not washed into the analytical column. Bare silica and cyano- and diol-bonded phases are washed (in order) with heptane, chloroform, ethyl acetate, acetone, ethanol, and water. Then the order is reversed, using dried solvents, to reactivate the column. Use 10 empty column volumes of each solvent. Amino-bonded phases are washed in the same manner as silica, but a 0.5 M ammonia wash is used after water. C18 and other nonpolar phases are washed with water, acetonitrile, and chloroform, and then the order is reversed. If this is insufficient, wash with 0.5 M sulfuric acid, and then water. 

Of the polar bonded-phase packings that have been investigated, the interactions between carotenes and nitrile stationary phases are very weak thus the limitations described for silica apply (164). Amino-bonded phases eluted with iso-octane containing 0.5% stabilized tetrahydrofu-ran separate a- and /3-carotene (unresolved) from y-carotcnc canthaxanthin and /3-cryptoxanthin are not eluted (161). The cis isomers of /3-carotene are separated from the all-trans isomer thus amino columns offer an alternative option to alumina columns for determining all-trans-/3-carotene without its cis isomers interfering. 

We also saw in figure 3.8 that moderately polar stationary phases may be most useful in the reversed phase mode for the separation of very polar solutes, which cannot be sufficiently retained on reversed phase (alkyl) materials. A good example of this is the separation of carbohydrates on amino bonded phases. 

Different polar functional groups bonded onto silica can also be used for normal phase. The most frequently used bonded phases are amino-, diol-, cyano-, and nitro-groups. These phases, when used in the normal mode, are similar to adsorption onto silica gel. The use of bonded-phase columns in the normal phase mode is shown in Figure 5-44. There is a different order of elution on the amino-bonded phase compared to the cyano phase and different elution than on silica (ortho < meta < para), which is not shown. This is due to interactions with the functional groups on the packing rather than solely with the hydroxyl groups on the silica gel. 

Quantification of five different drugs in pigs kidney Separation of carboxylic polyether antibiotics (monensin, salinomycin, and narasin) with an amino bonded phase and 15% methanol in carbon dioxide using light-scattering detector Analysis of hydroxylated metabolites of dialkyldithio-carbamates... 

Equation (11) has been verified for numerous mobile phases and alumina, silica, or amino-bonded phase as adsorbent, using nonlocalizing solvents A and B (/. 14-17). 

Site-competition delocalization of solutes X has been observed for adsorption of localizing solutes X onto silica and amino-bonded-phase packings, but not for alumina (/, 17). We will comment on this later. 

Fig. 5. Plot of log k versus e° for various solutes on an amino-bonded-phase column (17). Mobile phases are tetrahydrofuran/hexane mixtures solutes are substituted aromatic compounds. Taken from Ref. (17).

The expected proportionality of log A versus c° plots with the solute As value has also been demonstrated in numerous LSC systems. For example, Fig. 6 shows a plot of experimental As values (slopes of log k versus e° plots) versus values calculated from the solute molecular dimensions, for various nonlocalizing solutes on an amino-bonded-phase column (aromatic hydrocarbons and ethoxylated nonylphenols). Another study (34) with alumina as adsorbent examined the ratio (kjki) [Eq. (8)) for various pairs (1 and 2) of pure solvents as mobile phase pentane/CCl, CCUAjenzene, benzene/CHzClj, and 52 different solutes (aliphatics, aromatics, polar, nonpolar). The deviation of individual experimental and calculated values of A was 1.5 units (1 standard deviation) for the range 5.1 < As s 21.2. 

For silica and amino-bonded phases as adsorbent, the experimental As value of Eq. (8) is larger than the calculated value (molecular size) for solute molecules that localize. This is summarized by Eq. (14), where... 

Fig. 6. Experimental versus calculated Values of As for nonlocalizing solutes on amino-bonded-phase (/7) ( ) aromatic hydrocarbons (O) ethoxylated nonyl phenols system of Fig. 5. Data replotted from Ref. (17).

Here the asterisk indicates Cig-deactivated silica. There is a regular trend from left to right as intramolecular delocalization (value of j3) decreases, site-competition delocalization (value of y) becomes more important. This is predicted by the above discussion. Furthermore, there seems to be a regular transition in the nature of surface sites in the order alumina Cig-silica. silica. amino-bonded phase. This will be discussed further when we consider localization as a function of the nature of adsorbent sites (Section III,A,3). 

L. R. Snyder and T. C. Schunk, Retention mechanism and the role of the mobile phase in normal-phase separation on amino-bonded-phase columns. Anal. Chem. 54 (1982), 1764-1772. 

Normal-phase HPLC, using an amino-bonded phase, was used for determination of the PAHs of up to 7 rings. This type of separation results in elution by the number of n bonds. A special reversed-phase octadecyl column was used for PAHs of 7 through 12 rings. This HPLC packing, Vydac 201TP5, is well known for its orderly structure and separates the PAHs by their overall shapes. It has been compared to the liquid-crystal phases used in gas chromatography. It provides the best isomer specific separation of PAHs. 

With normal-phase HPLC, oil samples were analyzed as is by simple dilution in n-hexane. A Du Pont Zorbax amino-bonded phase column, 25 cm x 0.46 cm ID, was used, with n-hexane and dichloromethane as solvents. For reversed-phase HPLC, Vydac 201TP5 columns were used (25 cm x 0.46 cm ID for analytical scale and 25 cm x 1 cm ID for preparative scale). Samples for reversed-phase HPLC were fractionated in order to remove the saturated hydrocarbons which can interfere with the separation mechanism. The samples dissolved in n-hexane were passed Baker silica solid-phase extraction cartridges. The PAH fraction was then collected by eluting with a 1 1 mixture of dichloromethane and methanol. Acetonitrile and dichloromethane were used in the HPLC gradient. 

Fig. 4.4.8. Separation of protected oligonucleotide on a Partisil PAC column (Cyano-amino-bonded phase) (0.4x25 cm). The mixture chromatographed is the result of the synthesis of a tetramer of cytidine and guanine, protected by, e.g., dimethoxytrityl groups. Eluent Gradient from methanol/dichloromethane (5 95) to (50 50). Reprinted from Ref. 12 with permission.

Normal-phase chromatography used an amino bonded-phase column (Du Pont Zorbax, 25- X 0.48-cm i.d., 5-p,m spherical particle size) n-hexane and dichloromethane mixtures were used as the mobile phases. Reverse-phase separations used an octadecyl bonded-phase column (Vydac 201TP5, 25- X 0.46-cm i.d. for analytical scale, and 25- X 0.94-cm i.d. for preparative scale, both with 5-p,m spherical particles, Separations Group) methanol and dichloromethane were the mobile phases. As in previous work (5-7, 9), the concentration of strong solvent in the mobile phase was varied from 0% to 100% for reverse-phase studies and from 0% to 30% for normal-phase studies. The peaks isolated from preparative-scale runs were further purified by separation on the analytical-scale octadecyl bonded-phase column. 

Polar bonded phases such as propylamino-, cyano-, or diol packings used in normal-phase chromatography can be tested exactly like silica columns. Amino-bonded phases used for hydrophilic interaction chromatography are best tested with a carbohydrate sample in 70% acetonitrile 30% water. 

Figure 22-28 Separation of ascorbic acid and isoascorbic acid by hydrophilic interaction chromatography (HILIC) using the amino bonded phase in Table 22-3. Isocratic solvent is 90 10 acetonitrile H20 (containing 0.1 M ammonium acetate). [From S. Drivelos, M. E. Dasenaki, and N. S. Thomaidis, Anal. Bioanal. Chem. 2010,397, 2199.)...

As with the amino-bonded phase, the organosilane reagent used for bonding the CN phase has a trimethylene (-(CH2)3-) between the silicon atom (with its three leaving groups for bonding) and a cyano group. This thin layer can be used in the RP mode with polar mobile phases or in the NP mode with nonpolar mobile phases. As mentioned, it has less activity compared to a silica gel layer and also a less nonpolar character than C18- or C8-bonded phases. 

A two-phase Pirkle modified amino-bonded phase for enantiomer separation was prepared by partially immersing a commercial HPTLC NH2 F-plate (Merck) in a solution of the chiral selector Af-(3,5-dinitrobenzoyl)-L-leucine. The portion of the plate modified with chiral selector was used to separate the enantiomers of the model compounds 2,2,2-trifluoro-(9-anthryl)ethanol and l,l -binaphthol with the mobile phase hexane/isopropanol (80 20). The separated enantiomers were then eluted using continuous development onto the unmodified portion of the plate. The absence of the selector on this segment of the layer allowed the detection of the separated compounds by fluorescence quenching [20]. 

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