Hydrophilic bonded phases

Both polymeric and silica-based columns are in common use.The polymeric columns are heavily used in the analysis of synthetic polymers and plastics where organic solvents are required. Silica-based columns with hydrophilic bonded phases are used to separate aqueous solutions of macromolecules. Finally, polymeric size-separation columns with hydrophilic phases are available for separation of polysaccharides, peptides, and very small proteins. 

Examples of hydrophilic bonded phases are NH2, diol, and CN. Using these stationary phases, separations are achieved on the basis of normal-phase interactions. These bonded phases can also be used in the reversed-phase mode, depending on the mobile phase composition. 

A new material based on a particle size 10 /rm, pore size 20 nm silica coated with a hydrophilic bonded phase is SOTA Phase GF200 (SOTA Chromatography Inc., Crompond, USA). When used with 100-150 mmol salts at neutral or slightly acidic pH, GF200 columns, like the Bio-Sil TSK series, provide high recoveries of proteins, and in most cases biological activity is retained. Figure 5 compares similar separations on both columns. 

The above results proved the potential viability of the adsorbed hydrophilic macromolecules as bonded phases in chromatography of biopolymers but it must be admitted that additional crosslinking of previously adsorbed macromolecules is usually needed in order to obtain stable composites. The cross-linked bonded polymeric phases, however, may suffer from the restricted flexibility of the chain segment and their steric repellency may be diminished. Moreover, the conformational adaptivity of cross-linked chains for binding with solutes is poorer than that of grafted or chemically bound macromolecules. 

Indeed, the polymeric interface seems to be highly diffuse and hydrophilic because copolymers of N-vinylpyrrolidone and N- (2-hydroxyethyl) acrylamide are readily soluble in water [53]. Besides, aminopropyl-glass adsorbs the acryloyl chloride copolymer so that only 10% of its active functions become amidated. The rest is located on the loops and tails of the attached macromolecules [51]. Thus the steric repulsion of the bonded phase is a probable reason for the high inertness of the packing towards viruses. 

It is likely that small molecules such as short oligopeptides have almost equal access to the hydrocarbonaceous sublayer at the surface of the bonded phases and thus their retention behavior is not affected significantly by the size of the hydrophilic polyether moieties. 

Hydrophilic interaction chromatography on Asahipak NH2P or Excel-pak CHA-P44 with pulsed amperometric detection has been used to fractionate malto-oligosaccharides.266 The Asahipak NH2P is a polyvinyl alcohol support with a polyamine bonded phase, and the Excelpak is a sulfonated polystyrene in the Zn+2 form. Amine adsorption of sialic acid-containing oligosaccharides was performed on a Micropak AX-5 column (Varian) using acetonitrile-water-acetic acid-triethylamine.267... 

Many other types of solid phase adsorbents, including those based on conventional and specialty materials like restricted access media (RAM), can increase analysis speed and improve assay performance. These types of materials, also known as internal reversed-phase packings, are especially useful for assaying target compounds in biological samples such as serum and plasma. They are chemically modified porous silicas that have hydrophilic external surfaces and restricted-access hydrophobic internal surfaces. The ratio of interior to external surface areas is large. Macromolecules such as proteins cannot enter the pores of the RAM (they are excluded from the hydrophobic internal surface) and they elute quickly through the column. However, the smaller analyte molecules that can enter the pores are retained via interactions with the hydrophobic bonded phase within... 

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... 

Derivatized silica gel that is used for size-exclusion HPLC contains a proprietary bonded phase that is defined as hydrophilic to minimize nonspecific hydrophobic and ionic interactions. Polymeric supports consist of highly cross-linked agarose beads, with or without bonded dextran or cross-linked copolymers of allyl dextran and AyV -methylenebisacryl-amide. All supports are available with a variety of particle and pore sizes and distribution. 

The sample is disrupted completely and distributed over the surface as a function of interactions with the support, the bonded phase, and the tissue matrix components themselves. The solid support acts as an abrasive that promotes sample disruption, whereas the bonded phase acts as a lipophilic, bound solvent that assists in sample disruption and lysis of cell membranes. The MSPD process disrupts cell membranes through solubilization of the component phospholipids and cholesterol into the Cis polymer matrix, with more polar substituents directed outward, perhaps forming a hydrophilic outer surface on the bead. Thus, the process could be viewed as essentially turning the cells inside out and forming an inverted membrane with the polymer bound to the solid support. This process would create a pseudo-ion exchange-reversed-phase for the separation of added components. Therefore, the Cis polymer would be modified by cell membrane phospholipids, interstitial fluid components, intracellular components and cholesterol, and would possess elution properties that would be dependent on the tissue used, the ratio of Cis to tissue employed and the elution profile performed (99-104). 

Hydrophilic size separation columns for use with aqueous samples are very popular choices for purifying proteins and carbohydrates. Protein separation columns are available on both silica and polymeric supports. It is surprising that the best of these protein purification columns in terms of resolution and in recovery of native protein are silica-based columns. One would expect that protein release from silica would be a real problem. It certainly is in many other silica columns. These columns, however, especially the TSK family of columns, give excellent recovery of enzymatic activity. I have talked to other column manufacturers who have investigated the problem. They say that when you remove the bonded phases from these columns they appear to be identical to bonded phases from a number of other, less successful, columns designed for protein purification. All of these bonded phases are primarily diol ether polymers, very hydrophilic, but of intermediate polarity. Some modification of... 

The results obtained by the first method especially those of recent studies performed with a commercially available stable 0(-, fj- and )f-CD bonded phases ( 8 -15) and with their newest improved modifications (16T demonstrate the great practical value of the sorbents and procedure.These studies dealt with structural and geometrical isomers and diastereoisomers as well as enantiomers of numerous compounds of various hydrophobic or hydrophilic nature. 

Besides the bonded diol or epoxy type of hydrophilic stationary phase, a number of other types of bonded silica supports of varying phase... 

In RPC systems, the retention is weaker on weakly or moderately polar stationary phases such as on phenyl- or cyanopropyl-bonded phases than on an alkylsilica phase. RPC separations on phenyl or cyanopropyl columns may show selectivities differing from those observed on Cix or Cs phases, but their main advantage is lower concentration of organic solvent required to elute weakly polar samples, which may potentially reduce the separation time. For the great majority of samples, however, the selectivity of separation is generally better on alkylsilica-bonded phases. On the other hand, retention of hydrophilic samples can be increased and their separation improved on columns with a high amount of bonded carbon (polymeric bonded phases) or on hydrophobic organic polymeric materials such as styrene-divinylbenzene copolymers. 

In a later study, Pesek et al. reported the separation of other proteins using a diol stationary phase [61-64]. The use of a diol stationary phase should result in a surface that is more hydrophilic than a typical alkyl-bonded moiety, like Cis or Cg. The overall results showed significant variations in retention times due to differences in solute-bonded phase interactions. Other factors, such as pH, could also influence this interaction, due to its influence on charge and protein conformations. Combining all these factors in the separation of peptides and proteins provides an experimentalist with many decisions to be made in the optimized experimental conditions to be used. Other chemical modifications of etched fused silica need to be studied in order to provide a better understanding of their interactions with proteins and peptides, as well as other classes of biopolymers. 

Bonded phases for HIC consist of a hydrophilic poly- meric layer into whieh hydrophobie ligands are in- 9 serted. The hydrophilie layer totally eovers the silica or ... 

Because HIC supports are designed for macromolecules, they either possess pore diameters of at least 300 A to allow inclusion or are nonporous. Both silica and polymer matrices are used because the hydrophilic polymeric coating minimizes or ehminates most matrix-based effects. The absolute retention and selectivity of an HIC support may be affected by the specific composition of the bonded phase, as well as the ligand. For example, protein mixtures have shown distinct selectivity on different HIC columns which have propyl functional groups [5]. 

The HILIC bonded phases are hydrophilic, consisting of amide and/or polyhydroxy functionalities. Pore diameters are at least 300 A to allow penetration of peptides. Supports can be based on either silica or polymer because the matrix is not exposed to the solutes. 

Hydrophilic interaction chromatography (HILIC) is a variation of normal-phase chromatography in which solutes are retained on a polar bonded phase under high concentrations (80-90%) of organic solvent and released during a gradient to a more aqueous solvent. The organic mobile phase usually causes at least partial denaturation of proteins. 

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