Restricted access material columns

The sample homogenization with an MeCN THF mixture was used for the simultaneous determination of SMM, miloxacin, and oxolinic acid. The supernatant was filtered and injected directly into the ion-pair chromatographic system using a shielded hydrophobic phase. This method did not require time-consuming and complex extraction procedures moreover, the use of a restricted-access-material column prevented both column clogging and peak broadening throughout the analysis. On the other hand, no preconcentration of the sample affected the LOD... 

The first attempts employing two Cjg columns showed that the selectivity was not high enough, although this improved when the first column was substituted by a 5 p.m GFF n internal surface rcversed-phase material. This is known as a restricted-access-material (RAM) column which, since it restricts some compounds because of their size and includes rcversed-phase interaction and ionic exchange, is very useful for analysing herbicides in samples with high contents of humic and fulvic acids (54). 

Another solution to this particular problem is to use a restricted-access-material (RAM) column prior to the precolumn (54). 

Petrovic and coworkers [6] used PLE applying MeOH acetone (1 1) for the extraction of APEOs, APECs, APs and their halogenated derivatives from sediment samples. In combination with column-switching LC, using a restricted access material precolumn, hours of sample treatment were saved. 

High flow rate is generally the basis for the high-throughput efficiency of several Turbulent Flow Chromatography (TFC), Restricted Access Material (RAM), and monolithic columns methodologies. 

The simultaneous HPLC-UV determination of sulfamonomethoxine (SMM), miloxacin (MLX), and OXO in serum and muscle of cultured fish was developed (153). A sample of muscle was extracted with MeCN-THF (95 5) after centrifugation, the supernatant was injected into the HPLC system. A Hisep column, used in this study, is packed with restricted-access materials (RAMs) consisting of the polymeric hydrophilic/hydrophobic phase bound to silica gel. This column did not require time-consuming and complex extraction procedures. The RAM sorbent could also be applied in short precolumns, which are combined directly on-line with the HPLC equipment. This approach is much more convenient than that applied in the present paper. The guard column had to be changed very often in order to protect the analytical column in a sufficient way. The extraction recovery was 79.5%, RSD of 6.0%. 

Body fluids, such as serum, contain several different carotenoids in low amounts. The crucial point in the isolation and analysis of these samples is the enrichment factor. Serum samples can be directly analysed with hyphenated extraction-sample enrichment-separation systems, such as on-line SPE-HPLC employing tailored stationary phases [29]. By using special restricted access materials (RAMs) for sample enrichment, the carotenoids are retarded on the pre-column while the protein binding is broken and the macromolecules are eluted. The preparation of artifacts is hindered, as the whole analysis steps take place under conditions of light- and oxygen-exclusion. The scheme of on-line SPE-HPLC is presented in Figure 5.2.2. 

Restricted-access material (RAM) columns combine the size-exclusion of proteins by the hydrophilic outer surface of the packing and the simultaneous enrichment by SPE of analytes that interact with hydrophobic groups at the inner surface of the packing. These columns allow the direct injection of plasma samples without protein precipitation. Often, on-line RAM-LC-MS is described, following a procedure identical to on-line SPE-LC-MS (Ch. 1.5.4). The use of RAM columns has been reviewed by Souverain et al. [29]. 

The use of on-line immrmoaffinity extraction in combination with coupled-column LC-MS-MS was demonstrated for the characterization of benzodiazepine libraries [34]. The benzodiazepine library was injected onto a Protein G colurtm loaded with benzodiazepine antibodies. The benzodiazepine-antibody complexes are stripped of the column at low pH and eluted to a restricted-access material in order to separate the benzodiazepines from the antibodies. In the last step, the compotmds interacting with the benzodiazepine antibodies are separated on a Cg-teversed-phase colunm and identified by MS-MS. 

Solid-phase extraction (SPE), which can be done off-hne or on-line by means of cartridges, cartridge columns, or extraction disks (Ch. 11.6.4), and eventually by using special packing materials, such as restricted access materials (RAM) or turbulent-flow chromatography (TFC) (Ch. 11.6.5). 

SadiTek P, Satinsky D, Solich P, Using restricted-access materials and column switching in high-performance liquid chromatography for direct analysis of biologically-active compounds in complex matrices. Trends Anal. Chem. 2007 26 375-384. 

Special sorbents possessing restricted-access properties have been developed to allow the direct injection of biological matrices into the online SPE-LC systems. These sorbents, the so-called restricted-access materials, combine the size exclusion of proteins and other high-molecular-mass matrix components that are prohibited from entering the pores of the packing, and they are not well retained by the column. Therapeutic drugs and other small molecules permeate the pores of the column packing material where they partition and are retained by... 

Restricted access phases are another approach to exploiting the differences in characteristics of analytes. Large analytes are excluded from an internal surface on which an adsorptive stationary phase is present. A herbicide analysis for Metsulfuron methyl, Bentazone, Bromoxynil, methylchlorophenoxy acid, and Mecoprop in the presence of humic acid was performed on restricted access reversed phase media.52 The cytostatic compound epirubicin and its metabolites were separated from plasma using a Pinkerton GFF II column.53 Gradient separations of polymers on reversed phase and on normal phase represent an alternative to gel permeation chromatography. Polyesters of noncrystalline materials were separated on a variety of such phases.54... 

Viehauer S. et al., 1995. Evaluation and routine application of the novel restricted-access precolumn packing material Alkyl-Diol Silica Coupled-column high-performance liquid chromatographic analysis of the photoreactive drug 8-methoxypsoralen in plasma. J Chromatogr B 666 315. 

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... 

On the basis of the separation mechanism, restricted-access media can be classified into physical or chemical diffusion barrier types. The limited accessibility of the former type is due to the pore structure of the support that represents physical diffusion barriers for macromolecular compounds. The restricted access of the latter type is due to covalently or adsorptively bonded synthetic or natural polymers that cover the support surface, preventing macromolecules from being adsorbed on or denatured by the column packing material. 

Bayenes, W.R.G., Van der Weken, G., Haustraete, J., Aboul-Enein, H.Y., Corveleyn, S., Remon, J.P., Carcia-Campana, A.M., Deprez, P. Application of the restricted-access precolumn packing material alkyl-diol silica in a column-switching system for the determination of ketoprofen enantiomers in horse plasma. J. Chromatogr. A 871, 153-161 (2000)... 

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