External reference compounds

An external reference is a compound placed in a separate container from the sample. For liquid samples, an external reference compound is often placed as a neat (undiluted) liquid either in a small sealed capillary tube inside the sample tube or in the thin annulus formed by two precision coaxial tubes. In either case, the usual rapid sample rotation (Section 3.2) makes the reference signal appear as a sharp line superimposed on the spectrum of the sample. An external reference is advantageous in eliminating the possibility of intermolecular interactions or chemical reaction with the sample. Also, there are no problems with solubility of the reference in the sample solution. There is, however, a serious difficulty raised by the difference in bulk magnetic susceptibility between sample and reference. 

Figure 19.8 Distribution of fIuvoxamine at steady-state concentration in the brain of a volunteer measured by 19F MR CSI at 3 T. (a) Spatially localized, 9F MR CSI spectra overlaid on the axial proton image of the brain and including the external reference sample., 9F spectra of a voxel containing (b) the background (e.g., no structure and no signal), (c) posterior brain tissue, and (d) external reference compound (trifluoroethanol). The density of the compounds is reflected by peak amplitude and area under the curve (AUC).

Stored in a table where columns are descriptors, and rows are compounds (or conformers), QSAR data sets contain separate columns for the measured target property (Y), attributed to the training set, as well as computed descriptors for (external) reference compounds on which the QSAR model is tested—the test set. Statistical procedures, e.g., multiple linear regression (MLR), projection to latent structures (PLS), or neural networks (NN) [38], are then used to establish a mathematical soft model relating the observed measurement(s) in the Y column(s) with some combination of the properties represented in the subsequent columns. PLS, NN, and AI (artificial intelligence) techniques have been explored by Green and Marshall in the context of 3D-QSAR models [39], and were shown to extract similar information. A problem that may lead to spurious (chance) correlations when using MLR techniques, the colinearity between various descriptors, or cross-correlation, is usually dealt with in PLS [40],... 

It is technically possible, but very difficult, to measure the exact frequency of a radio signal, and in practice the frequency of the energy absorbed by a test compound (usually called the resonance frequency) is measured relative to that of a reference compound. This reference may be mixed with the sample (direct referencing), or if contamination of the sample is undesirable it may be placed in a separate container within the sample tube (external referencing). In proton and 13C NMR, the reference compound usually used is TMS (tetra-methyl silane) or its water-soluble derivative DSS (2,2-dimethylsilapentane 5-sulphonic acid). These compounds give a sharp proton peak at the right-hand side of a typical NMR spectrum (Figure 2.39). 

Any mass spectrometer requires mass calibration before use. However, the procedures to perform it properly and the number of calibration points needed may largely differ between different types of mass analyzers. Typically, several peaks of well-known m/z values evenly distributed over the mass range of interest are necessary. These are supplied from a well-known mass calibration compound or mass reference compound. Calibration is then performed by recording a mass spectrum of the calibration compound and subsequent correlation of experimental m/z values to the mass reference list. Usually, this conversion of the mass reference list to a calibration is accomplished by the mass spectrometer s data system. Thereby, the mass spectrum is recalibrated by interpolation of the m/z scale between the assigned calibration peaks to obtain the best match. The mass calibration obtained may then be stored in a calibration file and used for future measurements without the presence of a calibration compound. This procedure is termed external mass calibration. 

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

To obtain absolute concentrations of metabolites, calibration techniques are necessary. For this purpose an external calibration compound of known concentration can be measured to which the metabolite signals are referenced. Another possibility is the use of spectral signals from a tissue compound with known concentration serving as internal reference. 

For calibration with an external reference, a sample with known concentration of the compound of interest is positioned outside the examined object, but inside the receiver coil. The desired metabolite concentration in the tissue can be calculated from the known concentration in the external reference by... 

The chemical shift is measured relative to a reference compound, for example LiBr in THF or H2O. Due to the common dynamic processes observed between organohthium complexes, an external reference is often used. One problem is that there is not a single reference compound in general use, but different external references are employed. Thus, chemical shifts reported in different investigations must be compared with caution. 

The H NMR shifts (ppm relative to TMS as internal standard) shown in Figure 1 were calculated from the reported shifts (neat compounds ) relative to benzene as external reference under the assumption that internal TMS is shifted 7.26 ppm highfield from external benzene <64HCA942). The shifts for phenyl are the centers of complex signals. 

As derived from Fig. 4, we plot next in Fig. 5 the augmentation in the /" ground-state energy (relative to the external reference) which arises between successive elements. This is what in Fig. 1 was made to serve as one-electron-like energy for the electron of configuration/ , to describe processes involving /-electron loss such as photoionization, or intracation / - d transitions. This seems feasible because both the p- and d-bands and the work function are all relatively invariant in energy across any particular series of lanthanide compounds. 

Curve d -asc, but corrected to most stable level with Smax, giving energy of ground-state of / set below external reference. Latter, within compound series, approximately fixed at appropriate level w.r.t. p- and d-bands. 

An unknown aqueous solution of 9-aminoacridine in water leads to an intensity of fluorescence which, measured at 456 nm, is of 60% with respect to an external reference. A standard solution of this compound, the concentration of which is 0.1 ppm in the same solvent, leads to a fluorescence of 40%, under the same conditions. Water alone, equally under the conditions of the experiment, presents a negligible fluorescence. 

The reference compound can be added to the sample solution (internal reference) or kept separate from the sample in a sealed capillary (external reference, Fig. 2.36). If an external reference is necessary, a correction term accounting for the difference between the bulk susceptibilities of reference ( R) and sample solution ( s) must be added to the observed shift, dob5 ... 

Neither CS2 nor TMS are ideal standards. The 13C signals of CS2 and carbonyl carbons overlap, as do the 13C signals of cyclopropane and some methyl carbons with TMS (Fig. 3.3). Furthermore, the 13C resonance of TMS has been shown to suffer from solvent shifts of the order of + 0.1 to 1.5 ppm in common NMR solvents, even at infinite dilution [74]. This must be considered if 13C shifts relative to TMS of one compound in different solvents are to be compared. There are two alternative methods to overcome this problem one is to use cyclohexane as the internal reference cyclohexane was shown to have 13C solvent shifts lower than + 0.5 ppm [74], The other alternative is to use TMS as an external reference (Sections 1.9.3 and 2.8.5) and to make bulk susceptibility shift corrections according to eq. (1.44). 

The other group comprises silver-silver halide electrodes, mercury pools, metal-metal-ion electrodes, and others normally prepared In the solvent used for the compound being studied (and often, indeed, employed as internal "reference" electrodes). For such an electrode, the abbreviation alone signifies that the solvent was the same throughout the cell, while the symbol "(w)" for ("water") following the abbreviation signifies that the reference electrode was prepared with water and used as an external reference electrode. 

The qualitative and quantitative analyses of monosaccharide, glycine and ARP were performed on a Waters HPLC-system equipped with a Nucleosil 5-NH2 (aminopropylsilica) HPLC-column using acetonitrile/phosphate-buffer (pH-3 75/25 (v/v)) as eluent. An external standard method was used to determine the mono- saccharide-, glycine-, and the ARP-intermediate concefttrations (reference compounds were available). 

Depending upon whether or not the reference compound is present in the same solution as the measured substance, the method of referencing is denoted as internal or external. Internal referencing, when the reference is in the same solution as the measured substrate, eliminates the effect of magnetic susceptibility, xvi both reference and substrate are in the same magnetic field. It does not eliminate, however, any of the many possible specific... 

In some cases, notably 3lP NMR, the chosen reference compound is not inert toward most samples. One way around this problem is to put the reference compound in a small capillary tube, which itself is carefully centered within the sample tube. Such a reference compound is referred to as external, that is, not dissolved in the sample solution. 

Two types of reference are used in NMR—Internal and external. An internal reference is a compound giving a sharp NMR line that is dissolved directly in the sample solution under study. The reference substance is then dispersed uniformly at a molecular level through the sample. The magnetic field acts equally on the sample and reference molecules, so that Eq. 4.6 and the other relations derived before are completely valid. Provided the reference compound does not react chemically with the sample, the only serious drawback of an internal reference is the possibility that intermolecular interactions might influence the resonance frequency of the reference. Usually, by careful choice of relatively inert... 

An NMR method had been utilized as part of a patent claim by Stimac et al. [11], in a publication dealing with the synthesis of ezetimibe. Here, and NMR assignments were applied to characterize ezetimibe anhydrate in Form-S. In the NMR assay, the methyl group of hexam-ethylbenzene 5 = 17.3 ppm) was used as the external reference, and the compound resonance bands were observed over 28.4—170.2 ppm. 

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