Manual Spectrum Subtraction

Example In the partial TIC obtained by GC-EI-MS of an unknown mixture on a HP-5 column the chromatographic peak at 32.6 min is rather weak (Fig. 12.7). Scan 2045 extracted from its maximum yields a spectrum that is mainly due to background ions from column bleed as demonstrated by comparison to the average of scans 2068 2082. Finally, manual background subtraction ((2035 2052)-(2013 2020)) delivers a spectmm of reasonable quality, although some background signals are not completely erased. 

Methods are available to remove the interferences due to solvents, such as subtraction and compensation. These may be accomplished manually or electronically depending on the instrumentation and how it is configured. However, the preference is always to obtain a spectrum without the interference of a solvent ... 

A scaled saline or water reference spectrum was manually subtracted from each sample spectrum such that the region of the spectrum between 1700 cm and 2000 cm"1 was flat. Each subtracted sample spectrum was baseline corrected between 900 cm "1 and 2000 cm "1, and the peak height of characteristic protein and polysaccharide bands was determined using the baseline correct function and the cursor, respectively, on the Nicolet 660 data station. 

DRS, the method of subtraction of the baseline requires special attention, especially because instructions in various commercial manuals might lead to erroneous results. The procedure adopted by the author involves (i) digital acquisition and storage of the reflectance spectrum of the white standard, Ro(v) over the range of wavenumbers v (cm1) of interest, where v = MX, X is the wavelength in cm and the conversion factor for the photon energy in... 

There are two modes of operation, namely an automatic one started by Subtract and an interactive one launched by Start Interactive Mode. In the automatic mode, the difference spectrum with automatically determined multiplication factors is calculated from a least squares analysis over the displayed wavenumber range. The range of wavenumbers used is the largest range present in all spectra. The interactive mode allows you to set the multiplication factors manually. 

In the absence of the above facilities, the uncorrected baseline can be recorded and subtracted from the difference spectrum manually at, say, 2 nm intervals. The difference spectrum can be assumed to cross the baseline at a known isosbestic point (see below). The method is of course tedious. 

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