Subtraction chromatography

Pieper R, Su Q, Gatlin CL, Huang ST, Anderson NL, Steiner S. Multi-component immunoaffinity subtraction chromatography An innovative step towards a comprehensive survey of the human plasma proteome. Proteomics 2003 3(4) 422 132. 

Pieper, R. Su, Q. Gatlin, C.L. Huang, S.T. Anderson, N.L. Steiner, S. Multi-Component Immunoaffinity Subtraction Chromatography An Innovative Step Towards a Comprehensive Survey of the Human Plasma Proteome, Proteomics 3(4), 422 32 (2003). 

Albumin, a major constituent in serum (60-80 mg/L), is known to act as a transport carrier for small proteins. Also, many antineoplastic drugs bind to albumin, often at the 80-95% level. Removal or depletion of albumin is a major problem there are problems with ultrafiltration (e.g., membrane binding of small proteins and drugs) and other approaches, e.g., Cibacron dye columns and immunoaffinity-based protein subtraction chromatography. Albumin removal using acetonitrile may be a simple alternative [54] however, there is no truly satisfying method at this time. 

Mixtures can be identified with the help of computer software that subtracts the spectra of pure compounds from that of the sample. For complex mixtures, fractionation may be needed as part of the analysis. Commercial instmments are available that combine ftir, as a detector, with a separation technique such as gas chromatography (gc), high performance Hquid chromatography (hplc), or supercritical fluid chromatography (96,97). Instmments such as gc/ftir are often termed hyphenated instmments (98). Pyrolyzer (99) and thermogravimetric analysis (tga) instmmentation can also be combined with ftir for monitoring pyrolysis and oxidation processes (100) (see Analytical methods, hyphenated instruments). 

Figure 13.10 LC-LC chromatogram of a surface water sample spiked at 2 p.g 1 with ati azine, and its metabolites (registered at 220 nm). Conditions volume of sample injected, 2 ml clean-up time, 2.60 min ti ansfer time, 4.2 min The blank was subtracted. Peak identification is as follows 1, DIA 2, HA 3, DEA 4, atrazine. Reprinted from Journal of Chromatography, A 778, F. Hernandez et al, New method for the rapid detemiination of triazine herbicides and some of thek main metabolites in water by using coupled-column liquid cliromatography and large volume injection , pp. 171-181, copyright 1997, with permission from Elsevier Science.

PBECOLUMN SUBTRACTION REAGENTS USED FOR QUALITATIVE SAMPLE IDENTIFICATION IN GAS CHROMATOGRAPHY... 

The pore volume (Vp) can be evaluated by subtracting 1/ from the elution volume of the smallest PS standard (usually benzene or toluene), which is generally supposed to access all pores, being relevant for chromatography. The corresponding porosity (Sp) is defined by dividing 1/ by the volume of the empty column. 

Figure 11.13 —Schematic am optical path showing the principle and simplified view of a diode array spectrophotometer. The shutter is the only mobile piece in the assembly, allowing subtraction of the background signal (dark current) without any light intensity striking the photodiodes. This inverted optical design allows the sample to be exposed to the exterior light. These instruments are widely used as detectors in liquid chromatography (cf. 3.7).

Internal standard areas and acceptance limits Raw data for each sample, blank, spike, duplicate, and standard (quantitation reports, reconstructed ion chromatograms) Raw and background subtracted mass spectra for each target analyte found Tentatively identified compounds mass spectra with library spectra of 5 best-fit matches Sample preparation bench sheets Gel permeation chromatography clean-up logs S S S S S S S ... 

Although the ionic strength of the eluant may remain the same throughout the run, the background conductivity can decrease due to the changing dielectric constant. These baseline changes can be compensated either by chemical means or by computer baseline subtraction. Often, mobile phase ion chromatography is used to elute ions which are very... 

Fig. 6.12. Chromatography of 3H-labelled extracted products following incubation of homogenates of Hymenolepis diminuta in [3H]tryptophan 5HT, 5-hydroxytryptamine 5HTP, 5-hydroxytryptophan TRY, tryptophan or, origin fr, solvent front. The data were obtained by subtracting the radioactivity in boiled homogenates (blanks) from that in unboiled homogenates. Radiolabelled tryptophan was metabolised in the latter to [3H]5HTP and [3H]5HT. (After Ribeiro Webb, 1983a.)...

Perhaps the best route to evaluate the composition of a lysoPA mixture is through the methods cited earlier, especially FAB-MS. A subtractive type of analysis can done in which the FAB-MS spectrum of the original sample is obtained then, after each treatment (acid, base, etc.) the sample is purified by thin-layer chromatography, the remaining lysoPA recovered and examined again by FAB-MS. These various profiles will yield information of value in determination of the structural characteristics of the sample under study. Thus, if after an acid reaction followed by a base treatment a chloroform-soluble phosphorus compound remains, it would most likely be the lysoalkyl ether GPA. The FAB-MS spectrum would support such a conclusion. 

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