Spectrum subtraction

Derrick studied the interaction of L-tryptophan and ibuprofen with human serum albumin (HSA),74 which is an abundant transport blood protein capable of binding efficiently several species.75 They acquired 1H NMR spectra of L-Tryptophan-HSA system for different ligand protein molar ratios, that is 3 1, 5 1, 7 1 and 10 1. The aromatic resonances of L-Tryptophan are difficult to be observed due to the overlap with HSA signals, even at 10 1 molar ratio, so that the spectral subtraction was performed. D values of L-Tryptophan were calculated by integration of the subtracted spectra and were in good agreement with those predicted by computer simulations. In the case of ibuprofen, only for 140 1 molar ratio, the resonances of ibuprofen are clearly visible also in this case, the... 

Substraction spectra were obtained by setting the aliphatic C-H stretching band at 2969 cm l at about zero. The spectrum comparing AO/60 PHBA/diol polyol and unmodified polyester (Fig. 3) is typical. The aliphatic OH band at about 3535 cm"l has been largely replaced by a phenolic OH band at about 3365 cm"l. Subtraction spectra of the other oligomers (Fig. A) yield similar conclusions. 

FTIR-spectroscopy was performed in a Nicolet 5 SXC instrument using KBr pellets. The spectra were baseline corrected and the 1125 cm-1 band was used as reference peak to compare bleached and unbleached spectra. The 1505 cm-1 peak was used as the reference peak to obtain the subtraction spectra. 

Figure 8. Subtraction spectra of samples bleached with H2O2 under acidic (A) and alkaline (B) conditions. Unbleached HPL control (normal spectrum) is shown in (C). (Subtraction spectra were scaled using the 1505 cm-1 peak as unity.)...

Integrated peak intensity (1055-965 cm 1) of water-subtracted spectra of aqueous methanol solutions... 

The filtered preparation of gum arabic contained 13 pg/mg protein (dry wt). When a 0.01% (w v) aqueous solution, at pH 6.5, was exposed to a Ge IRE, no polysaccharide was observed to adsorb from a flowing solution at the aqueous/solid interface, as the characteristic C-0 stretching bands of gum arabic were not visible in the water-subtracted spectra. The protein (1.3%) associated with gum arabic demonstrated a high affinity for the Ge surface, adsorbing from a flowing solution at a concentration of 1.3 ppm, while polysaccharide, present at a concentration of 100 ppm, did not adsorb on the IRE. Distinct Amide I and Amide II bands of this adsorbed protein are visible in the water subtracted spectra. At the end of 4 hr, the 1549 cm 1 band intensity was 0.9 mAU. Rinsing with Milli-Q water (pH 6.5) did not affect the Amide II band intensity, indicating that the protein was firmly adsorbed to the Ge surface. 

B) HSQC-IP/AP for measurement of Yen couplings with samples at low concentrations. By acquiring spectra with the phase F = x and F = —x, the corresponding added/subtracted spectra can also be used to extract YHh couplings of CH2 groups (see ref. 145 for details). 

FIGURE 38. Scheme of resultant 13C NMR spectmm formation from two subtracted spectra with different selective decoupling 29Si frequencies. Reproduced with permission of Collection of Czechoslovak Chemical Communications from Reference 47... 

Figure 16.3 shows the XAS in the C K-region of various CB samples and references of HOPG, C60, and anthracene. Compared to HOPG, CB shows broad spectral features, especially in the n peak. In order to quantify the broadness of the spectral features, Figure 16.3 also shows the subtracted spectra, (CB)-(HOPG). The subtracted spectra of the CB clearly demonstrate the broad portion of the it peak, which has peak structures at 284.2 and 286.2 eV. The energy... 

SAF, FT) and reference compounds of HOPG, C60, and anthracene. Subtracted spectra, (CB)-(HOPG), are also shown on the absorption spectra. 

FIG, 27. CASS experiments on the carbohydrate derivative [6], (a) Proton-decoupled C spectrum, (b) Proton-coupled C spectrum, (c)-(o) Selectively saturated and subtracted spectra from each of the carbon sites indicated on the right of the traces. From ref. 202. 

The technique of subtracting spectra taken with linearly s- and p-polarized radiation is based on the surface selection rule for reflection-absorption on a metal surface [20] ... 

For spectral subtraction, spectra of true placebos were subtracted, yielding spectra very close to that of the omitted drug. (Since this was a dry blend, a true placebo was possible. Wet blends give different results due to H bonding.)... 

Subtraction spectra for films having been subjected to 2, 4, 6 and 8 minute immersion times using methanol solvent are superimposed in Figure... 

To provide more detail to the end user beyond a purity number and a color code, the UV chromatogram and the background-subtracted spectra that correspond to the integrated peaks are written into a format that can be read by a custom browser that resides within the corporate analytical request-and-tracking system. In this way, the end user can view via the intranet the associated LC-MS data by simply clicking on the numeric value for SI, which is reported via the Web page with the other associated profiling data. There is no requirement to have any vendor software on their personal computer (PC). 

In the DP samples, after two hours deposition-precipitation, there is formation of a clear shoulder with DP time at about 1005 cm, which according to the literature [7], points to the presence of 1 1 nickel phyllosilicate. This is more clearly seen in the subtraction spectra. It appears that in the case of the cationic competitive exchange-prepared sample the formation of hydrosilicate species is only incipient. In fact, these... 

Figure 3. Efficiency of plasma background subtraction. Spectra obtained after on-detector integration for 1.6 s. Key a, spectrum of 10 pg/mL Ti b, detector and spectral background and c. net difference spectrum.

U.V. spectrum of callichiline is a summation of jS-anilino-acrylic-ester and meth-oxyindoline chromophores the exact nature of the indoline is not readily determined from subtraction spectra. 

If cleavage is impossible or only one fission product may be obtained then analysis should be carried out by means of subtraction spectra. Subtraction spectra must be preferred to addition spectra since, in the latter, differences in the model chromophore and the chromophore actually contributing to the spectrum of the dimer are masked, especially when the other contributing chromophore has a high extinction coefficient. 

Fig. 1. Rapid-scanning stoppcd-flow (RSSF) study of the reaction ofN-furylacryloylr-tryptophan methyl ester (FATME) with a-chymotrypsin (a-Ct) at pH 5.0 in the absence and presence of proflavin. (A) RSSF difference spectra for the reaction of 19 pM a-Ctwith 7.5 pM FATME in 0.1 M pH 5.0 sodium acetate buffer at 25°. Spectrum 0 is 7.5 pM free FATME, spectra 1 -5 are difference spectra measured during reaction wherein the spectrum of a-Ct has been subtracted from the set. Spectrum 6 is the spectrum of the hydrolysis product furylacryloyl i-tryptophan with the spectrum of a-Ct removed by subtraction. Spectra were measured at the following time intervals after flow had stopped (1) 8.54, (2) 162.3, (3) 341.6 (4) 1409.1 and (5) 3074.4 ms. Spectrum 6, t = oo.

Fig. 8. The rapid-scanning spectroscopic time courses for the reaction of 0.17 mM Co(II)-T6 with 100 mM phenol at pH 8.0 in the absence (A, C) and presence (B, D) of 100 mM chloride ion are shown. (A) Reaction in the absence of chloride ion. The time interval between scans is 8.54 ms for the first five spectra, followed by spectra at successively longer intervals afterward (see insets in C and D). The total acquisition time was 1.71 s for the 25 spectra collected only spectra numbers 1-5,7,10,12,15,18,21, and 25 are shown. (B) Reaction in the presence of 100 mM chloride ion. The timing sequence of the spectra is the same as that used in (A). For clarity, spectra 6, 8,10,12,14-16, 18-20, and 22-24 have been omitted. (C) The scaled, subtracted spectra, calculated from the second to the sixth spectrum of part A, correspond to the time-course for intermediate formation. The time course plotted in the inset shows the absorbance change at 560 nm for the complete set of scaled, subtracted spectra as a function of time. (D) Scaled, subtraction spectra numbers 2 to 6, as in part C, for the data part B, with chloride ion present. The inset plot also shows the time course at 560 nm obtained from the complete set. (Taken from Gross and Dunn (55) with permission.]...

A HY31 B HY41 and HY4 . a before ethylene adsorption b after ethylene adsorption, b-a Subtracted spectra for the determination of the interacting OH and the shift (v a). 

Figure 6. Effect of H2S adsorption on HTA solids (subtracted spectra).

Subtraction spectra of functionalized supports, recorded at different temperatures, by spectra of bare support recorded at the same temperature evidenced consumption of surface hydroxyls by TMSiOMe grafting. Indeed, as can be seen on Figure 3, bands at 3734 cm" and 3760... 

All spectra were run on a Digilab FTS-10 FTIR system equipped with fast-scan capabilities, a Hycomp 32 data array processor, and a nitrogen-cooled mercury-cad-mium-telluride detector. Transmission spectra were obtained using CaF2 windows with a 6- xm spacer. For each transmission spectrum, 500 scans were co-added at 4-cm-1 resolution. Smoothing was not needed on these spectra. All the spectra in this chapter (both transmission and ATR) are subtracted spectra, that is, they are the resultof subtracting a saline (H20) spectrum from the spectra of the aqueous protein solutions. 

Further addition of C6H5COOCH3 generally increases the intensity of the bands, except for those characterizing the methoxy groups, which tend to disappear. Subtracted spectra (Figure 1B) show that methoxy I species (v(CO) band at 1154 cm-i) are first affected. They also indicate that the spectrum of the second benzoate species formed, hereafter called B, is different from that of species A the vs(C02) band intensity is much weaker whereas that of the 1453 cm" band is enhanced. 

This study was performed either under static or dynamic conditions. In static conditions, H2 (200 Torr) was introduced on Zr02 on which C6H5COOCH3 were chemisorbed. Heating for 2 hours at 300°C does not modify the spectrum of benzoate species much. Subtracted spectra show that only benzoate species B disappear (Figure 2). 

Figure 5.2b, in which the strong infrared spectrum of water has been removed from a relatively weak spectrum of a 1% wt/vol solution of aspirin, illustrates how subtraction can be ve ry useful in FT-IR spectroscopy. You need to take care, however, when subtracting spectra. The concentration of the solvent alone is greater than that of the solvent in the solution and negative peaks may appear in the fegidns of solvent absorption. 

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