Separation and Quantitation

Separation and Quantitation.—The specific radioactivity of [y-32P]ATP of very high activity (up to 240 Ci per millimole) may be measured by using it to phosphorylate [dT(pT)10] quantitatively, using polynucleotide kinase, isolating the labelled undecanucleotide, and measuring its activity.170 Isotope dilution is used to confirm the values. An alternative method of measuring specific radioactivities of ribo-nucleoside triphosphates utilizes a 3H-labelled nucleoside triphosphate (e.g. UTP) of unknown specific activity, a 14C-labelled nucleoside (e.g. ATP), a suitable primer in 

The determination of the molecular weight of animal cell RNA, using electrophoresis on exponential polyacrylamide gels under fully denaturing conditions, has been described. Effects due to RNA secondary structure are fully suppressed if dry formamide and high temperatures are used.177 

In an elegant method for the isolation of translating ribosomes, the dihydrazide of dithioglycollic acid is first attached to cellulose, and treated with poly(rU) which has been oxidized at the 3 -terminus with periodate, to form (83). After passage of a 

---

High Pressure Liquid Chromatography. This modem version of the classical column chromatography technique is also used successfully for separation and quantitative analysis of dyes. It is generally faster than thin-layer or paper chromatography however, it requires considerably more expensive equipment. Visible and uv photometers or spectrophotometers are used to quantify the amounts of substances present. 

Heavy isotopes endow the compounds in which they appear with slightly greater masses than their unlabeled counterparts. These compounds can be separated and quantitated by mass spectrometry (or density gradient centrifugation, if they are macromolecules). For example, O was used in separate experiments as a tracer of the fate of the oxygen atoms in water and carbon dioxide to determine whether the atmospheric oxygen produced in photosynthesis arose from HgO, COg, or both ... 

High performance liquid chromatography is used for the separation and quantitative analysis of a wide variety of mixtures, especially those in which the components are insufficiently volatile and/or thermally stable to be separated by gas chromatography. This is illustrated by the following method which may be used for the quantitative determination of aspirin and caffeine in the common analgesic tablets, using phenacetin as internal standard where APC tablets are available the phenacetin can also be determined by this procedure. 

One purpose of this work was to develop a gas chromatographic procedure based on separation and quantitation of each of the com-... 

One can use an LFER to facilitate the separation and quantitation of the effects of different variables on the reaction. Thus, steric and electronic effects can be explored by selecting a subseries in which only one of them varies. 

The above considerations apply to samples where all the components are of interest and all need to be separated and quantitatively assessed. In practice, for many samples, only specific components of the mixture are important and only those need to be separated from the matrix and be analyzed. The components of the matrix need not be resolved and they are of no interest. It follows, that under these circumstances the critical pair will be comprised of the component of interest that has the closest neighbor and the neighbor itself. Such a situation usually greatly simplifies the separation problem but it should be noted that the last peak must still be eluted before the next analysis can be carried out and so the analysis time may not be significantly reduced. 

Note Under the conditions employed emetine and cephaeline were not well separated but there was good resolution of the subsidiary alkaloids of the ipecacuanha tincture (Fig. 1). The separation and quantitative determination of the main alkaloids (Fig. 2) can be carried out under the following conditions Ascending, one-dimensional development in a trough chamber with chamber saturation layer HPTLC plates Silica gel 60 (Merck) mobile phase dichloromethane — methanol — ammonia solution (25%) (34+6+1) migration distance 6 cm running time 13 min h/ f cephaeline 65-70 emetine 75-80. 

In a study of the metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes, a high performance liquid chromatography (HPLC) method has been developed that separates and quantitates methyl parathion and six of its hepatic biotransformation products (Anderson et al. 1992). The six biotransformation products identified are methyl paraoxon, desmethyl parathion, desmethyl paraoxon, 4-nitrophenol, />nitrophenyl glucuronide, and /wiitrophenyl sulfate. This method is not an EPA or other standardized method, and thus it has not been included in Table 7-1. 

Saito, K., A new enzymatic method for extraction of precarthamin from dyer s saffron (Carthamus tinctorius) florets, Z. Lebensmitt. Untersuch. Forsch., 197, 34, 1993. Cserhati, T. et ah. Separation and quantitation of colour pigments of chili powder (Capsicum frutescens) by high-performance liquid chromatography-diode array detection, J. Chromatogr. A, 896, 69, 2000. 

Tpnnesen, H.H. and Karlsen, J., Studies on curcumin and curcuminoids Vll. Chromatographic separation and quantitative analysis of curcumin and related compounds, Z. filr Lebensm. und Forsch. A, 182, 215, 1986. 

Water-soluble polymeric dyes have been prepared from water-insoluble chromophores, viz., anthraquinone derivatives. Unreacted chromophore and its simple derivatives, which are all water-insoluble, remain in solution due to solubilization by the polymeric dye. A method has been developed to separate and quantitate the polymeric dye and these hydrophobic impurities using Sephadex column packing. The solvent developed has the property of debinding the impiirities from the polymer, and further allows a separation of the imp irities into discrete species. This latter separation is based on the functional groups on the impurity molecules, having a different interaction with the Sephadex surface in the presence of this solvent. The polymer elutes at the void volume... 

Residues of alachlor and acetochlor are determined by similar methods involving extraction, hydrolysis to the common aniline moieties, and separation and quantitation by reversed-phase FIPLC with electrochemical detection. The analytical method for acetochlor is included as a representative method for residue determination of alachlor and acetochlor in plant and animal commodities. Propachlor and butachlor residues, both parent and metabolite, are determined by similar analytical methods involving extraction, hydrolysis to common aniline moieties, and separation and quantitation by capillary GC. The analytical method for propachlor is included as a representative method. The details of the analytical methods for acetochlor and propachlor are presented in Sections 4 and 5, respectively. Confirmation of the residue in a crop or... 

The analytes, EMA and MEMA, are separated and quantitated by reversed phase HPLC/OECD. Details of the operating conditions are as follows ... 

Adjust the volume collected to 5 mL, if necessary, and mix the contents thoroughly. The sample is now ready for separation and quantitation by GC/NPD. 

LC/MS/MS. LC/MS/MS is used for separation and quantitation of the metabolites. Using multiple reaction monitoring (MRM) in the negative ion electrospray ionization (ESI) mode, LC/MS/MS gives superior specificity and sensitivity to conventional liquid chromatography/mass spectrometry (LC/MS) techniques. The improved specificity eliminates interferences typically found in LC/MS or liquid chro-matography/ultraviolet (LC/UV) analyses. Data acquisition is accomplished with a data system that provides complete instmment control of the mass spectrometer. 

Beecher, G. R., Design and assembly of an inexpensive, automated, microbore amino acid analyzer separation and quantitation of amino acids in physiological fluids, Adv. Exp. Med. Biol., 105, 827, 1978. 

Nuryono, Huber, C. G., and Kleboth, K., Ion-exchange chromatography with an oxalic acid-alpha-hydroxyisobutyric acid eluent for the separation and quantitation of rare-earth elements in monazite and xenotime, Chromatograph-ia, 48, 407, 1998. 

For the analysis of nonvolatile compounds, on-line coupled microcolumn SEC-PyGC has been described [979]. Alternatively, on-line p,SEC coupled to a conventional-size LC system can be used for separation and quantitative determination of compounds, in which volatility may not allow analysis via capillary GC [976]. An automated SEC-gradient HPLC flow system for polymer analysis has been developed [980]. The high sample loading capacity available in SEC makes it an attractive technique for intermediate sample cleanup [981] prior to a more sensitive RPLC technique. Hence, this intermediate step is especially interesting for experimental purposes whenever polymer matrix interference cannot be separated from the peak of interest. Coupling of SEC to RPLC is expected to benefit from the miniaturised approach in the first dimension (no broadening). Development of the first separation step in SEC-HPLC is usually quite short, unless problems are encountered with sample/column compatibility. 

XIV. The Separation and Quantitative Estimation of Glucosamine and Galactosamine in Blood Group Substances. J. Amer. chem. Soc. 76, 4887 (1954). 

Schleyer, E., Reinhardt, J., Unterhalt, M., Hiddemann, W. (1995). Highly sensitive coupled-column high-performance liquid chromatographic method for the separation and quantitation of the diastereomers of leucovorin and 5-methyltetrahydrofolate in serum and urine. J. Chromatogr. B 669, 319-330. 

Ricardo-da-Silva JM, Rosec JP, Bourzeix M and Heredia N. 1990. Separation and quantitative determination of grape and wine procyanidins by HPLC. J Sci Food Agric 53 85-92. 

Montedoro G, Servili M, Baldioli M and Miniati E. 1992a. Simple and hydrolyzable phenolic compounds in virgin olive oil. 1. Their extraction, separation, and quantitative and semiquantitative evaluation by HPLC. J Agric Food Chem 40 1571-1576. 

High performance liquid chromatography is used to determine the purity of calcitriol, and to separate it from related compounds. Using a 10 micron silica column of 25 cm length, and a mobile phase of spectroquality heptane ethyl acetate. methanol (50 50 1) at a flow rate of 1.7 ml/ minute, separation and quantitation are achieved. p-Dimethyl-aminobenzaldehyde may be used as an internal standard to compensate for variations in injection technique and instrumental conditions. With a 254 nm ultraviolet absorbance detector, 0.01 ug of calcitriol may be detected (3). 

Zagotto, G. et al. 2001. Anthracyclines Recent developments in their separation and quantitation. J Chromatogr B. 764 161. 

The choice of the leading electrolyte for the second stage, in which the micro-constituents were finally separated and quantitatively evaluated, was straightforward, involving a low concentration of the leading constituent (low detection limit) and a low pH of the leading electrolyte (separation according to pK values). 

The rise in popularity of HPLC is due in large part to its high-performance nature and the advantages offered over the older, noninstrumental open-column method described in Chapter 11. Separation and quantitation procedures that require hours and sometimes days with the open-column method can be completed in a matter of minutes, or even seconds, with HPLC. Modern column technology and gradient solvent elution systems, which will be described, have contributed significantly to this advantage in that extremely complex samples can be resolved with ease in a very short time. 

If interference is a major problem the sample must be partially purified before analysis. This breaks the analysis into preparatory and quantitative stages. In order to reduce the technical difficulties resulting from such two-stage methods much work has gone into the development of analytical techniques such as gas and liquid chromatography in which separation and quantitation are effected sequentially. 

---