Femtomole

Titrations conducted with microliter or picoliter sample volumes require a smaller absolute amount of analyte. For example, diffusional titrations have been successfully conducted on as little as 29 femtomoles (10 mol) of nitric acid. Nevertheless, the analyte must still be present in the sample at a major or minor level for the titration to be performed accurately and precisely. 

It was found, that at standard gas-chromatograph sampling of 1 pL of analyte solution the limit of detection for different amines was measured as 0.1-3 ng/ml, or of about 1 femtomole of analyte in the probe. This detection limit is better of published data, obtained by conventional GC-MS technique. Evidently, that both the increasing of the laser spot size and the optimization of GC-capillary position can strongly improve the detection limit. 

Our initial attempts with this design using the NDA/CN" system were encouraging far beyond our expectations. Our enthusiasm was sustained when we found that dipeptide-CBI derivatives could be detected at the femtomole level (Tables IV and V) and that such limits of detection with our purified derivatives under less-than-... 

Reports on the use of fluorescent derivatives abound (5). Some reagents have become widely used. The dansyl group is probably the most thoroughly studied. Dansyl chloride has been widely used as a fluorescent derivatizing reagent for HPLC (6,7). It reacts readily with primary and secondary amino groups (7) and with phenols (8), but forms derivatives of alcohols very slowly (9). The lower detection limit for dansyl derivatives of aliphatic amines is in the range of 300 femtomoles per injection. 

Hancock, D. O. and Synovec, R. E., Refractive index gradient detection of femtomole quantities of polymers by microbore size-exclusion chromatography, Anal. Chem., 60, 1915, 1988. 

Stocchi, V., Palma, F., Piccoli, G., Biagiarelli, B., Magnani, M., Masat, L., and Cucchiarini, L., Analysis of amino acids as DABS-derivatives with a sensitivity to the femtomole using RP-HPLC narrow-bore columns, Amino Acids, 3, 303, 1992. 

Anions of weak acids can be problematic for detection in suppressed IEC because weak ionization results in low conductivity and poor sensitivity. Converting such acids back to the sodium salt form may overcome this limitation. Caliamanis et al. have described the use of a second micromembrane suppressor to do this, and have applied the approach to the boric acid/sodium borate system, using sodium salt solutions of EDTA.88 Varying the pH and EDTA concentration allowed optimal detection. Another approach for analysis of weak acids is indirect suppressed conductivity IEC, which chemically separates high- and low-conductance analytes. This technique has potential for detection of weak mono- and dianions as well as amino acids.89 As an alternative to conductivity detection, ultraviolet and fluorescence derivatization reagents have been explored 90 this approach offers a means of enhancing sensitivity (typically into the low femtomoles range) as well as selectivity. 

ESI and APCI are soft ionisation techniques which usually result in quasi-molecular ions such as [M + H]+ with little or no fragmentation molecular weight information can easily be obtained. However, experimental conditions can also be chosen in such a way that a sufficiently characteristic pattern is obtained, allowing verification [540]. ESI is amenable to thermally labile and nonvolatile molecules. Both ESI and APCI are much more sensitive than PB and very well suited for quantitative analysis, but less so for unknown samples. The choice among the two is usually determined by the application. Recently, nanoscale LC-ESI-MS has been developed [541]. The nano-electrospray ion source offers the highest sensitivity available for LC-MS (atto-to femtomole range) and can also be used as an off-line ion source. 

Bi Oscillating magnetic field vector of the f, fg, fmol Femto (10 15) femtogram, femtomole... 

Wilm, M., Shevchenko, A., Houthaeve, T., Breit, S., Schweigerer, L., Fotsis, T., and Mann, M. (1996). Femtomole sequencing of proteins from polyacrylamide gels by nanoelectrospray mass spectrometry. Nature 379, 466-469. 

Nelson, R. W. Jarvik, J. W. Taillon, B. E. Tubbs, K. A. BIA/MS of epitope-tagged peptides directly from E. coli lysate Multiplex detection and protein identificatin at low-femtomole to subfemtomole levels. Anal. Chem. 1999, 71, 2858-2865. 

Pinkse, M.W., Uitto, P.M., Hilhorst, M.J., Ooms, B., Heck, A.J. (2004).. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 76, 3935-3943. 

The ProteinChip System from Ciphergen Biosystems uses patented SELDI (Surface-Enhanced Laser Desorption/Ionization) ProteinChip technology to rapidly perform the separation, detection, and analysis of proteins at the femtomole level directly from biological samples. ProteinChip Systems use ProteinChip Arrays which contain chemically (cationic, anionic, hydrophobic, hydrophilic, etc.) or biochemically (antibody, receptor, DNA, etc.) treated surfaces for specific interaction with proteins of interest. Selected washes create on-chip, high-resolution protein maps. This protein mass profile, or reten-tate map of the proteins bound to each of the ProteinChip Array surfaces, is quantitatively detected in minutes by the ProteinChip Reader. 

Chemical immobilization procedures of bioluminescent enzymes such as firefly luciferase and bacterial luciferase-NAD(P)H FMN oxidoreductase to glass beads or rods [174, 175], sepharose particles [176], and cellophane films [177] have produced active immobilized enzymes. Picomole-femtomole amounts of ATP or NAD(P)H could be detected using immobilized firefly luciferase or bacterial luciferase-oxidoreductase, respectively. 

Femtomol levels of detection limits were also achieved in the determination of stimulant amines with the benzofurazan derivative 4-(Af,Af-dimethylaminosul-phonyl)-7-fluoro-2,l,3-benzoxadizole (DBD-F) [73], DBD-F was successfully applied to the PO-CL detection of amino acids and epinephrine [74] and a P-blocker, metoprolol [75], 4-(Af,Af-Dimethylaminosulphonyl)-7-hydrazino-2,l,3-benzoxadizole (DBD-H) has also been used for PO-CL determination of a neuronal cell protective compound, propentofylline. The method was applied for the first time to determine propentofylline concentration in the dialysate obtained from the rat hippocampus [85],... 

Examples are given in References 249 and 250 of about 100 ions detected in a single scan. This is about the practical detection limit for image current detection due to thermal noise in the detection system. Bradykinin has been detected from a sample concentration of 3 nM [249] and detection of sub-femtomole levels on a column is readily obtained [251]. 

M. Wilm, A. Shevchenko, T. Houthaeve, S. Breit, L. Schweigerer, T. Fotsis, and M. Mann. Femtomole Sequencing of Proteins from Polyacrylamide Gels by Nano-Electrospray Mass Spectrometry. Nature, 379(1996) 466-469. 

Ultimate temporal and spatial resolution femtoseconds, femtoliters, femtomoles and single-molecule detection... 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

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