Separation Serum, analysis

The column used for blood serum analysis was 100 cm long, 1 mm in diameter and packed with RP 18 reversed phase having a particle size of 10 pm. A concave gradient program was used to develop the separation over a period of 45 min. at a flow rate of 50 pl/min. The initial solvent was 75% methanol 25% water and the final solvent was pure methanol. 

The question often arises whether a sample must be analyzed immediately or can be stored, and if so, under what conditions and for how long (B4a, H5a, W9a). Freshly drawn blood maintained anaerobically (A3) at 38 C decreases in pH at the rate of —0.062 unit per hour and in pCOj, at 4.8 1.3 mg Hg per hour. At 0-4°C, the change is minimal — 0.006 0.004 pH unit and 0.6 0.06 mm Hg. There has been controversy concerning the use of minerol oil to maintain specimens for carbon dioxide analysis (G2). Paulsen found that values of total carbon dioxide in plasma collected in stoppered tubes with and without paraflSn oil were identical if the tubes without oil were completely filled to the stopper (P4). The loss of carbon dioxide in tubes stored at room temperature without oil was about 6 mEq/1 in 2.5-4 hours. The problem for the laboratory is unfilled tubes and the storage of separated serum or plasma before analysis and in plastic cups during continuous-flow procedures. 

Another new technology that offers promise for commercial biotechnology purifications is the use of parametric pumping with cyclic variations of pH and electric field. This has been described by Hollein and cowork-ers.fi26) They worked with human hemoglobin and human serum albumin protein mixtures on a CM-Sepharose cation exchanger. The extensive equations they reported for parametric separations allow analysis of other systems of two or more proteins which may be candidates for this type of separation. 

Cl. Crowle, A. J., A simplified agar-electrophoresis method for use in antigen separation and serum analysis. J, Lab. Clin. Med. 48, 642 (1956). 

Digest dean sample in acid mixture separate serum and plasma by centri fugati on keep stored i n glass tubes at -20°C until analysis thaw to room temprature prior to analysis Ash lyophiUze wet-ash with HNO3 add H2O2 ... 

Some further examples of the application of TLC may be cited a procedure for two dimensional separation [179] analysis of bile from various species [76] fractionation of bile lipids into groups [128] metabolism [73] and analysis of human faecal bile acids [153 a] quantitative determination of free [203] and bile acid conjugates [202] in serum. 

Comprehensive two-dimensional GC has also been employed for the analysis of pesticides from serum, which, although not strictly a forensic analytical problem , provides an example of the promise of this technique to forensic applications, such as the analysis of drugs of abuse (40). Two-dimensional gas chromatograms of a 17-pesticide standard and an extract from human serum are shown in Figure 15.13. The total analysis time of about 5 min, high peak capacity and the separation of all... 

The two examples of sample preparation for the analysis of trace material in liquid matrixes are typical of those met in the analytical laboratory. They are dealt with in two quite different ways one uses the now well established cartridge extraction technique which is the most common the other uses a unique type of stationary phase which separates simultaneously on two different principles. Firstly, due to its design it can exclude large molecules from the interacting surface secondly, small molecules that can penetrate to the retentive surface can be separated by dispersive interactions. The two examples given will be the determination of trimethoprim in blood serum and the determination of herbicides in pond water. 

The analysis demonstrates the elegant use of a very specific type of column packing. As a result, there is no sample preparation, so after the serum has been filtered or centrifuged, which is a precautionary measure to protect the apparatus, 10 p.1 of serum is injected directly on to the column. The separation obtained is shown in figure 13. The stationary phase, as described by Supelco, was a silica based material with a polymeric surface containing dispersive areas surrounded by a polar network. Small molecules can penetrate the polar network and interact with the dispersive areas and be retained, whereas the larger molecules, such as proteins, cannot reach the interactive surface and are thus rapidly eluted from the column. The chemical nature of the material is not clear, but it can be assumed that the dispersive surface where interaction with the small molecules can take place probably contains hydrocarbon chains like a reversed phase. 

GC/MS has been employed by Demeter et al. (1978) to quantitatively detect low-ppb levels of a- and P-endosulfan in human serum, urine, and liver. This technique could not separate a- and P-isomers, and limited sensitivity confined its use to toxicological analysis following exposures to high levels of endosulfan. More recently, Le Bel and Williams (1986) and Williams et al. (1988) employed GC/MS to confirm qualitatively the presence of a-endosulfan in adipose tissue previously analyzed quantitatively by GC/ECD. These studies indicate that GC/MS is not as sensitive as GC/ECD. Mariani et al. (1995) have used GC in conjunction with negative ion chemical ionization mass spectrometry to determine alpha- and beta-endosulfan in plasma and brain samples with limits of detection reported to be 5 ppb in each matrix. Details of commonly used analytical methods for several types of biological media are presented in Table 6-1. 

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

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