Nanogram quantities

The available sensitivity depends strongly on the equipment as well as the sample. H is the nucleus of choice for most experiments. 1 mg of a sample of a medium-sized molecule is adequate for almost all types of H-only spectra, and with specialized equipment one can work with nanogram quantities. At this lower level, the... 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Another analysis handled effectively by use of gc/ir/ms is essential oil characterization which is of interest to the foods, flavors, and fragrances industries (see Oils essential). Even very minor components in these complex mixtures can affect taste and aroma. Figure 4 shows the TRC and TIC for Russian corriander oil which is used extensively in seasonings and perfumes (15). The ir and ms are serially configured. Spectra can be obtained from even the very minor gc peaks representing nanogram quantities in the it flow cell. 

Other methods of sensitive detection of radiotracers have been developed more recently. Eourier transform nmr can be used to detect (nuclear spin 1/2), which has an efficiency of detection - 20% greater than that of H. This technique is useful for ascertaining the position and distribution of tritium in the labeled compound (14). Eield-desorption mass spectrometry (fdms) and other mass spectral techniques can be appHed to detection of nanogram quantities of radiolabeled tracers, and are weU suited for determining the specific activity of these compounds (15). 

Gram-negative baeteria eontain lipopolysaeeharides (endotoxins) in their outer membranes that ean remain in an active condition in products even after eell death and some ean survive moist heat sterilizatioa Although inactive by the oral route, endotoxins can induce aeute and often fatal febrile shock if they enter the bloodstream via contamirrated irrfirsion fluids, even in nanogram quantities, or via diffusion aeross membranes ftxm eontarrrinated haemodialysis solutions. 

The partition coefficient of a substance between several Immiscible solvent pairs can be combined with retention time data to confirm the identity of a substance when a pure standard is available [706]. Devised by Bowman and Beroza, the substance specific partition coefficient ("p-value") was defined as the fractional amount of substance partitioning into the less polar phase of an equal-volume, two-phase system. Only nanogram quantities of sample are required for the measurement and p-values are often sufficiently characteristic to distinguish between closely related substances. 

Andreae [324,325] has described a gas chromatographic method for the determination of nanogram quantities of dimethyl sulfoxide in natural waters, seawater, and phytoplankton culture waters. The method uses chemical reduction with sodium borohydride to dimethyl sulfide, which is then determined gas-chromatographically using a flame photometric detector. 

Turner et al. [328] determined nanogram quantities of dimethyl sulfide and dimethyl sulfoniopropionate in Antarctic waters. 

For the very heavy elements which are not available in micro-or nanogram quantities or which are synthesized one atom at a time , partition methods are the only practical way of determining complexing constants. When the half-life of an element is short, dynamic rather than static procedures are used since they give the most rapid experimental results (12). 

If the product is an antibody, then it is essential to distinguish the immunoglobulin product, e.g., mouse IgG, from any media immunoglobulin components, e.g., bovine IgG. Lucas et al.16 developed an immunoassay to measure nanogram quantities of bovine IgG in the presence of a large excess of a structurally homologous protein, mouse MAb. The bovine IgG was a contaminant that copurified with the product from a protein A column. For the bovine IgG assay, whole IgG and protein A-purified IgG reacted differently in the assay. It is important to evaluate these types of assays for cross-reactivity. For other media components, such as chemicals or antibiotics, ELISA is probably not the most appropriate method due to the low immunogenicity of chemicals. Techniques such as HPLC would be better to detect these chemical components. 

In vitro studies indicate that cotton mill dust extracts do activate complement by the alternative pathway. Kutz et al. (36) proposed that endotoxin is not the only agent responsible for complement activation, as microgram rather than nanogram quantities of purified endotoxin are required to induce the degree of complement activation observed with crude cotton dust extract. Several investigators (36, 37) have thus proposed endotoxin... 

Three primary tests are incorporated in the health effects area. The microbial mutagenesis assay is based on the property of selected Salmonella typhimurium mutants to revert from a histidine requiring state to prototrophy due to exposre to various classes of mutagens. The test can detect nanogram quantities of mutagens and has been adapted to mimic some mammalian metabolic processes by the addition of a mammalian liver microsomal fraction. The test is used as a primary screen to determine the mutagenic activity of complex mixtures or component fractions. 

In summary, we have isolated three toxins from lipid extracts of tqxicus. We have demonstrated that the guinea pig ileum preparation is an effective assay for these toxins in nanogram quantities. All three fractions effectively inhibited the guinea pig ileum preparation in its response to histamine at nanogram concentrations. The GT-3 toxic fraction is quite different from the other two (GT-1, GT-2) in being very slow acting and nonreversible in nature. 

Cl 5. Curry, S. H., Determination of nanogram quantities of chlorpromazine and some of its metabolites in plasma using ga.s-liquid chromatography with an electron capture detector. Anal. Chetn. 41, 1251-1256 (1968). 

Dias Neto, E., Harrop, R., Correa-Oliveira, R., Wilson, R.A., Pena, S.D. and Simpson, A.J. (1997) Minilibraries constructed from cDNA generated by arbitrarily primed rt-PCR an alternative to normalized libraries for the generation of ESTs from nanogram quantities of mRNA. Gene 186, 135-142. 

Cassidy and Frei [23] designed a microflow cell for the Turner Assoc. Model III fluorimeter for use with HPLC. Nanogram quantities of fluorescent materials could be detected. The volume of the flow cell was only 7.5 jul. The detector was unaffected by the flow-rate or composition of the solvent. This gives this detector a decided advantage over refractive-index or UV detectors. The peak shapes were symmetrical and the linear range of response was 2-3 orders of magnitude. 

The separation of the 2-naphthacyl esters of several fatty acids is shown in Fig.4.16. Corasil-Cig (3 ft. X 0.07 in. I.D. column) was used as the stationary phase and the fatty acid esters were eluted with methanol—water (17 3) at a flow-rate of 0.2 ml/min. Nanogram quantities of the fatty acids may be detected by this procedure. 

The reaction of anisoyl chloride (p-methoxybenzoyl chloride) with hexachlorophene (HCP) produces a derivative which can be detected in nanogram quantities by HPLC with UV detection [51]. The reaction product is the dianisate ester 2,2 -methylenebis(3,4,6-trichlorophenylp-methoxybenzoate). 

Most of the older methods of fluorimetric analysis of pesticides involved hydrolysis to form fluorescent anions. Co-ral (coumaphos) [147] was hydrolyzed in alkali to the hydroxybenzopyran, which was subsequently determined by means of its fluorescence. Guthion (azinphosmethyl) was hydrolyzed to anthranilic acid for fluorimetric analysis [148,149]. A method was developed [150] for Maretin (N-hydroxynaphthalimide diethyl phosphate) in fat and meat which involved hydrolysis in 0.5 M methanolic sodium hydroxide followed by determination of the fluorescence of the liberated naphthalimide moiety. Carbaryl (1-naphthyl N-methylcarbamate) and its metabolites have been determined by a number of workers using base hydrolysis and the fluorescence of the resulting naphtholate anion [151-153]. Nanogram quantities of the naphtholate anion could be detected. Zectran (4-dimethylamino-3,5-xylyl N-methylcarbamate) has been determined by the fluorescence of its hydrolysis product [154]. The fluorescence behaviour of other carbamate insecticides in neutral and basic media has been reported [155]. Gibberellin spray used on cherries has been determined fluorimetrically after treatment with strong acid [156]. Benomyl (methyl N-[l-(butylcarbamoyl)-2-benzimidazolyl]carbamate) has been analyzed by fluorimetry after hydrolysis to 2-aminobenzimidazole [157]. 

J.T. Colston, P. Kumar, E.D. Rael, A.T.C. Tsin, J.J. Valdes and J.P. Chambers, Detection of sub-nanogram quantities of Mojave toxin via enzyme immunoassay with light addressable potentiometric detector, Biosens. Bioelectron., 8(2) (1993) 117-121. 

It is very important to remember that HPLC is both an analytical and a preparative tool. Often the preparative capabilities of the HPLC are overlooked. While normal analytical injections contain picogram to nanogram quantities, HPLCs have been used to separate as much as 1 lb in a single injection (obviously by a candidate for the Guinness Book of World Records). Typical preparative runs inject 1-3 g to purify standard samples. 

These separations are run at 1.0-2.0 mL/min on the same 4.5-mm x 25-cm column used for our analytical runs. Normally, in analysis we shoot from picogram to nanogram quantities. Most separations maintain their resolution until we reach an injection quantity of about 1 fig. The valleys between peaks begin to rise indicating some overlapping. 

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