Observed spectra normally contain spurious features along with those of interest and it is common practice to employ one of several available algorithms to remove the unwanted components. Low frequency spurious components are often referred to as 'baseline', 'background', and/or 'background noise'. Here we examine a cross-section of non-instrumental methods designed to remove background features from spectra; the particular methods considered here represent approaches with different theoretical underpinnings. We compare and evaluate their relative performance based on synthetic data sets designed to exemplify vibrational spectroscopic signals in realistic contexts and thereby assess their suitability for computer automation. Each method is presented in a modular format with a concise review of the underlying theory, along with a comparison and discussion of their strengths, weaknesses, and amenability to automation, in order to facilitate the selection of methods best suited to particular applications.

Raman microspectroscopy is an attractive approach for chemical imaging of biological specimens, including live cells, without the need for chemi-selective stains. Using a microspectrometer, near-infrared Raman spectra throughout the range 663 cm(-1) to 1220 cm(-1) were obtained from colonies of CA1 human embryonic stem cells (hESCs) and CA1 cells that had been stimulated to differentiate for 3 weeks by 10% fetal bovine serum on gelatin. Distributions and intensities of spectral bands attributed to proteins varied significantly between undifferentiated and differentiated cells. Importantly, compared to proteins and lipids, the band intensities of nucleic acids were dominant in undifferentiated cells with a dominance-reversal in differentiated cells. Thus, we could identify intensity ratios of particular protein-related bands (e.g., 757 cm(-1) tryptophan) to nucleic acid bands (784 cm(-1) DNA/RNA composite) that were effective in discriminating between spectra of undifferentiated and differentiated cells. We observed no discernible negative effects due to the laser exposure in terms of morphology, proliferation, or pluripotency of the stem cells. We conclude that Raman microscopy and complementary data processing procedures provide a rapid, noninvasive approach that can distinguish hESCs from differentiated cells. This is the first report to identify specific Raman markers for the differentiation status of hESCs.

Blood is a bodily fluid that is vital for a number of life functions in animals. To a first approximation, blood is a mildly alkaline aqueous fluid (plasma) in which a large number of free-floating red cells (erythrocytes), white cells (leucocytes), and platelets are suspended. The primary function of blood is to transport oxygen from the lungs to all the cells of the body and move carbon dioxide in the return direction after it is produced by the cells' metabolism. Blood also carries nutrients to the cells and brings waste products to the liver and kidneys. Measured levels of oxygen, nutrients, waste, and electrolytes in blood are often used for clinical assessment of human health. Raman spectroscopy is a nondestructive analytical technique that uses the inelastic scattering of light to provide information on chemical composition, and hence has a potential role in this clinical assessment process. Raman spectroscopic probing of blood components and of whole blood has been on-going for more than four decades and has proven useful in applications ranging from the understanding of hemoglobin oxygenation, to the discrimination of cancerous cells from healthy lymphocytes, and the forensic investigation of crime scenes. In this paper, we review the literature in the field, collate the published Raman spectroscopy studies of erythrocytes, leucocytes, platelets, plasma, and whole blood, and attempt to draw general conclusions on the state of the field.

Ultraviolet resonance Raman spectroscopy (UVRRS), electronic absorption spectroscopy, and X-ray crystallography were used to probe the nature of the binding of 2,3-dihydroxybiphenyl (DHB) to the extradiol ring-cleavage enzyme, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD; EC 1.13.11.39). The lowest lying transitions in the electronic absorption spectrum of DHBD-bound DHB occurred at 299 nm, compared to 305 nm for the monoanionic DHB species in buffer. In contrast, the corresponding transitions in neutral and dianionic DHB occurred at 283 and 348 nm, respectively, indicating that DHBD-bound DHB is monoanionic. These binding-induced spectral changes, and the use of custom-designed optical fiber probes, facilitated UVRR experiments. The strongest feature of the UVRR spectrum of DHB was a Y8a-like mode around 1600 cm(-1), whose position depended strongly on the protonation state of the DHB. In the spectrum of the DHBD-bound species, this feature occurred at 1603 cm(-1), as observed in the spectrum of monoanionic DHB. Raman band shifts were observed in deuterated solvent, ruling out dianionic binding of the substrate. Thus, the electronic absorption and UVRRS data demonstrate that DHBD binds its catecholic substrate as a monoanion, definitively establishing this feature of the proposed mechanism of extradiol dioxygenases. This conclusion is supported by a crystal structure of the DHBD:DHB complex at 2.0 A resolution, which suggests that the substrate's 2-hydroxyl substituent, and not the 3-hydroxyl group, deprotonates upon binding. The structural data also show that the aromatic rings of the enzyme-bound DHB are essentially orthogonal to each other. Thus, the 6 nm blue shift of the transition for bound DHB relative to the monoanion in solution could indicate a conformational change upon binding. Catalytic roles of active site residues are proposed based on the structural data and previously proposed mechanistic schemes.

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In this paper, the effects of solvent flow, dopant flow, and lamp power on proton transfer ionization in dopant-assisted (DA) atmospheric pressure photoionization (APPI) are investigated. A broad theoretical framework is presented, describing the primary photoionization process, the formation of protonated-solvent cluster ions, and the balance between analyte ion creation via proton transfer and loss via recombination. The principal experimental test system utilized methanol as the solvent, toluene as the dopant, and acridine as the analyte. Comparisons are made between acridine and a less basic compound, 9-methylanthracene (9-MA). Experimental determinations of the trends in the analyte MH ϩ signal and the total ion current (TIC) with variations in the subject parameters are provided. Experimental results and theory demonstrate that both the analyte signal and the TIC approach asymptotic limits with increases in dopant flow and/or lamp current (two factors which dictate the rate of photoion generation). The data show that these limits are lowered at higher solvent flow rates. These results are attributed to the recombination loss process, the rate of which increases with the second power of ion concentration. We deduce that the recombination rate constant increases with solvent flow rate, a consequence of the growth of ion-solvent clusters. Cluster growth is also believed to be a factor in the dramatic loss of sensitivity for 9-MA that occurs as the solvent flow is raised, because larger protonated-solvent cluster ions have greater solvation energies and may be unreactive with compounds having low gas-phase basicity and/or low solvation energy. (J Am Soc Mass Spectrom 2005, 16, 1275-1290) © 2005 American Society for Mass Spectrometry P hotoionization (PI) is the latest means of ionization to be incorporated into atmospheric pressure ionization (API) sources for liquid chromatography-mass spectrometry (LC-MS). The original motivation for the development of atmospheric pressure photoionization (APPI) sources was the demand for a method or device capable of expanding the range of compounds amenable to LC-MS to include nonpolar compounds not readily ionized by either electrospray [1,2] or atmospheric pressure chemical ionization (APCI) [3,4]. In recent years, two approaches towards utilizing PI at atmospheric pressure have emerged: dopant-assisted (DA) APPI [5] and direct APPI [6]. Recent review papers provide details of the two APPI-MS methods [7,8]. This paper is concerned mainly with DA-APPI.As is often the case for new technologies, the practical application of DA-APPI has outpaced the development of detailed knowledge regarding the mechanisms responsible for its performance. DA-APPI relies upon gas-phase ion-molecule reactions to place a charge on neutral analytes, so it is especially important that these reactions be well understood. The groups of Kostiainen and Bruins have completed several studies of the reaction chemistry of DA-APPI and the effects of solvent and dopant composition on the ionization effici...

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Atmospheric pressure photoionization (APPI) is capable of ionizing nonpolar compounds in LC/MS, through charge exchange reactions following photoionization of a dopant. Recently, several novel dopants-chlorobenzene, bromobenzene, 2,4-difluoroanisole, and 3-(trifluoromethyl)anisole-have been identified as having properties making them wellsuited to serve as dopants for charge exchange ionization under reversed-phase LC conditions. Here, we report the results of experiments comparing their effectiveness to that of established dopants-toluene, anisole, and a toluene/anisole mixture, for the charge exchange ionization of model nonpolar compounds-the 16 polycyclic aromatic hydrocarbons (PAHs) identified by the US EPA as priority pollutants-when using a conventional reversed-phase LC method. Chloro-and bromobenzene were found to be much more effective than toluene for all the PAHs, due to the relatively low reactivity of their photoions with the solvent. Their overall performance was also better than that of anisole, due to anisole's ineffectiveness toward higher-IE compounds. Further, the experiments revealed that anisole's performance for higher-IE compounds can be dramatically improved by introducing it as a dilute solution in toluene, rather than neat. The two fluoroanisoles provided the highest overall sensitivity, by a slim margin, when introduced as dilute solutions in either chloro-or bromobenzene. With APPI, analyte ionization is mostly due to ionmolecule reactions following photoionization of a primary reagent, typically a dopant [4]. Analyte ionization can occur in positive mode through either proton transfer or charge exchange (electron-transfer) reaction pathways. This article regards the ionization of nonpolar, low proton affinity compounds via charge exchange with dopant radical cations.In APPI, for charge exchange ionization to occur the dopant's ionization energy (IE) must be greater than that of the analyte and to be efficient its radical cations must not be consumed through reactions with the solvent, its own neutrals, or impurities. Toluene was the first dopant to be used for charge exchange ionization in APPI [1], and it has a relatively high IE (8.83 eV), making it suitable for a wide range of analytes (all IE values are from reference [5]). In practice, however, toluene is only an efficient charge exchange dopant under normal-phase [6,7] and/or low-flow conditions[8] because its radical cations are rapidly consumed in reactions with methanol and acetonitrile at conventional LC flow rates [6, 9, 10]. Some other dopant is then required for efficient charge exchange ionization with reversed-phase LC methods. The usual alternative to toluene for promoting charge exchange ionization is anisole, whose photoions are stable in the presence of methanol and acetonitrile [11]. Anisole, however, has a relatively low IE (8.20 eV), restricting its applicability. Mixtures of anisole and toluene have been utilized by Itoh et al. as dopants to promote charge exchange ionization under reversed-phase conditions, i...