RAM column

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). 

FIGURE 9.3 Typical SCX-RAM column separation profile peaks (a) represent physical exclusion by pore size. Trapped retained biomolecules are separated by a gradient in the second step (b). Conditions column—LiChrospher 60 XDS (S03/Diol), 25 x 4 mm I.D., flow rate— 0.5 mL/min, gradient from 0 to 1M NaCl in 20 mM KH2P04 pH 2.5, containing 5% ACN in 30 min. Sample 100 pL Human Hemofiltrate (3.7mg/mL), UV detection at 214 nm. 

After sample loading, the cation-exchange RAM-column was placed in-line with the analytical cation-exchange column and analytes were eluted with a salt gradient. A total of 24 fractions of 4 min duration (2 mL of eluent) were transferred to the... 

Direct injection of pretreated biological samples (also called online sample cleanup) greatly simplified sample preparation for LC/MS/MS analysis. The normal process involves sample aliquot steps, internal standard addition, and centrifugation. Compared to traditional off-line LLE and SPE sample preparation procedures, online methods are easier and faster. Two types of online SPE columns are commercially available. One is the restricted access media (RAM) column. The other is the turbulent flow chromatography (TFC) column. 

A RAM column functions through a size exclusion mechanism. Large biomolecules such as proteins are restricted from the adsorptive surfaces inside silica particles. Small analyte molecules are able to penetrate into the inner surfaces of the particles. As a result, protein molecules pass through the column rapidly and analytes of interest are retained on the adsorptive sites. Depending on the application, the analyte molecules are directed to MS for detection or transferred onto an analytical column for separation prior to MS detection. Detailed applications are discussed in a recent review.8... 

Mass loadability of SPE and RAM columns play a key role in executing the sample clean-up. It is advisable to work below the overload regime of the column. Otherwise, displacement effects and other phenomena such as secondary interaction by adsorbed species might take place, which will lead to nonrepro-ducible results. This last statement is particularly important when the task is to monitor medium-to-low-abundance proteins large sample volumes in the milliliter range are therefore usually applied. 

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]. 

J. Martinez Fernandez, J.L. Martinez Vidal, P. Parrilla Vazquez, A. Garrido Frenich, Application of RAM column in coupled-column RPLC with UV detection and ESI-MS for determination of azole pesticides in urine, Chromatographia, 53 (2001) 503. 

Another technique that allows for the direct injection of plasma or serum online with the chromatographic system uses an analytical coliunn containing particles referred to as restricted-access media (RAM) traditional laminar flow liquid chromatography is employed. These RAM particles are designed to prevent or restrict large macromolecules from accessing the inner adsorption sites of the bonded phase. Commercially available RAM columns, all... 

The dual-phase nature of these RAM materials allows the direct injection of the biological sample matrix onto the column without pretreatment. Some disadvantages with the use of RAM columns are that retention times can be long (> 10 minutes) the column must be washed between injections and the mobile phases are not always compatible with some ionization techniques used in LC-MS/MS. Dual-column RAM techniques are also used. These methods use an analytical separation column placed in series downstream from the RAM column. A general overview of the use of RAMs in LC has been published in two parts [117,118]. 

Oliveira and Cass described a method for the analysis of cephalosporin antibiotics in bovine milk using RAM columns for on-line sample clean-up. The system was composed of a RAM bovine serum albumin (BSA) phenyl column coupled to a C18 analytical column. Milk samples were directly injected after addition of 0.8 mM solution of tetrabutylammonium phosphate. The standard curve was linear over the range 0.100-2.50 tig/ml for five cephalosporin antibiotics (cefoperazone, cephacetril, cephalexin, cephapirin, and ceftiofur). The limits of quantification and detection reported were 0.100 and 0.050 ttg/ml, respectively. The method showed high intermediate precision [coefficient of variation percent (CV%) 2.37-2.63] and recovery (CV% 90.7-94.3) with adequate sensitivity for drug monitoring in bovine milk samples. 

Pressurized columns with cr)ratal or melt transport by external pumps or ram, columns with mechanical forced conveyors, i.e., mixer, screw, spiral, etc. 

Hollow fibers have proved to be useful to separate labeled hapten from the complex labeled hapten-Ab after a competitive on-line HPLC-immunochromato-graphic detection method [121]. First, hapten eluting from the HPLC column was made to react with Ab. Then, labeled hapten was added to react with the excess of Ab. Separation of free labeled hapten from labeled hapten-Ab was performed using a hollow-fiber module. The cross-flow membrane of this module allowed separation based on size, as opposed to a restricted-access media (RAM) column in which size and hydrophobicity account for separation [122]. No restriction on polarity and size of the label and no need for regeneration of the separation device arc characteristic of the use of hollow fibers. Although the method has been used for analysis of haptens, it could be used to analyze proteins as it has been shown that proteins of 40kDa can be separated from the corresponding protein-Ab complex if the appropriate hollow fiber is chosen. 

Competitive flow immunoassay using RAM columns has been used to separate immunocomplexes from free haptens [123]. The system used was valid for antigens of molecular mass lower than 3000 Da. 

Besides the RAM column for sample preparation, the multidimensional separation platform comprised one analytical ion-exchange column and four parallel reversed-phase columns. At any given time, two reversed-phase columns were being gradient eluted simultaneously, one column was being loaded with an analyte fraction from the ion-exchange dimension, and the fourth column... 

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