Development of 31P Nuclear Magnetic Resonance Methods for the Study of Phosphate Metabolisms in E. coli and B. subtilis

31P NMR experiments were performed on Escherichia coli and Bacillus subtilis at various temperatures under aerobic and anaerobic conditions. The total soluble intracellular phosphate concentration was estimated to be 2 x 10^-17 mole/cell, while intracellular orthophosphate concentration was around 1 x 10^-17 mole/cell. Addition of glucose resulted in a general decrease in intracellular pH and was accompanied by the formation of sugar monophosphates. The concentrations of soluble intracellular phosphates, both inorganic and organic phosphates, were estimated by integration versus methylene diphosphonic acid (MDPA) standard. Although intracellular and extracellular orthophosphate could be observed, these appear to exchange rapidly on the NMR time scale

Application of linear prediction and rapid acquisition to nuclear magnetic resonance

In pulse nuclear magnetic resonance (NMR) spectroscopy, data are obtained by perturbing the nucleus from its equilibrium position and acquiring the transient response. Fourier transformation is the preferred mode used in data processing of the signals due to its ability to compute the NMR spectrum rapidly. To obtain good signal-to-noise ratio, it is common practice to average many transients. To obtain good resolution, lengthier acquisition times are favored. For insensitive nuclei, where thousands of collected transients are necessary, this is a time-consuming procedure; especially if the nuclear relaxation time constant is in the order of seconds or minutes. A faster acquisition method is proposed. Using a modified NMR pulse sequence, the proposed method acquires signals more rapidly than by conventional acquisition methods; however, the signals are truncated. In processing truncated data; the shortcomings of the Fourier transform must be overcome by alternative spectral estimation methods. An alternative processing method - linear prediction (LP) - is used to reconstruct the spectrum from the incomplete time-domain magnetic resonance data. The LP method\u27s application to truncated, fast acquisition of data is discussed in detail. This combination of methods is a novel way of acquiring and processing NMR spectroscopic data

Electrochemistry in an Acoustically Levitated Drop

Levitated drops show potential as microreactors, especially when radicals are present as reactants or products. Solid/liquid interfaces are absent or minimized, avoiding adsorption and interfacial reaction of conventional microfluidics. We report amperometric detection in an acoustically levitated drop with simultaneous ballistic addition of reactant. A gold microelectrode sensor was fabricated with a lithographic process; active electrode area was defined by a photosensitive polyimide mask. The microdisk gold working electrode of radius 19 μm was characterized using ferrocenemethanol in aqueous buffer. Using cyclic voltammetry, the electrochemically active surface area was estimated by combining a recessed microdisk electrode model with the Randles–Sevcik equation. Computer-controlled ballistic introduction of reactant droplets into the levitated drop was developed. Chronoamperometric measurements of ferrocyanide added ballistically demonstrate electrochemical monitoring using the microfabricated electrode in a levitated drop. Although concentration increases with time due to drop evaporation, the extent of concentration is predictable with a linear evaporation model. Comparison of diffusion-limited currents in pendant and levitated drops show that convection arising from acoustic levitation causes an enhancement of diffusion-limited current on the order of 16%

Mixing in Colliding, Ultrasonically Levitated Drops

Lab-in-a-drop, using ultrasonic levitation, has been actively investigated for the last two decades. Benefits include lack of contact between solutions and an apparatus and a lack of sample cross-contamination. Understanding and controlling mixing in the levitated drop is necessary for using an acoustically levitated drop as a microreactor, particularly for studying kinetics. A pulsed electrostatic delivery system enables addition and mixing of a desired-volume droplet with the levitated drop. Measurement of mixing kinetics is obtained by high-speed video monitoring of a titration reaction. Drop heterogeneity is visualized as 370 nl of 0.25 M KOH (pH: 13.4) was added to 3.7 μL of 0.058 M HCl (pH: 1.24). Spontaneous mixing time is about 2 s. Following droplet impact, the mixed drop orbits the levitator axis at about 5 Hz during homogenization. The video’s green channel (maximum response near 540 nm) shows the color change due to phenolphthalein absorption. While mixing is at least an order of magnitude faster in the levitated drop compared with three-dimensional diffusion, modulation of the acoustic waveform near the surface acoustic wave resonance frequency of the levitated drop does not substantially reduce mixing time

Analysis of Monoglycerides, Diglycerides, Sterols, and Free Fatty Acids in Coconut (Cocos nucifera L.) Oil by 31P NMR Spectroscopy

Phosphorus-31 nuclear magnetic resonance spectroscopy (31P NMR) was used to differentiate virgin coconut oil (VCO) from refined, bleached, deodorized coconut oil (RCO). Monoglycerides (MGs), diglycerides (DGs), sterols, and free fatty acids (FFAs) in VCO and RCO were converted into dioxaphospholane derivatives and analyzed by 31P NMR. On the average, 1-MG was found to be higher in VCO (0.027%) than RCO (0.019%). 2-MG was not detected in any of the samples down to a detection limit of 0.014%. On the average, total DGs were lower in VCO (1.55%) than RCO (4.10%). When plotted in terms of the ratio [1,2-DG/total DGs] versus total DGs, VCO and RCO samples grouped separately. Total sterols were higher in VCO (0.096%) compared with RCO (0.032%), and the FFA content was 8 times higher in VCO than RCO (0.127% vs 0.015%). FFA determination by 31P NMR and titration gave comparable results. Principal components analysis shows that the 1,2-DG, 1,3-DG, and FFAs are the most important parameters for differentiating VCO from RCO

Studies on Standards for Commercial Virgin Coconut Oil

A minimum set of analytical methods is recommended for the differentiation of virgin coconut oil (VCO) from refined, bleached and deodorized coconut oil (RBD CNO): % fatty acid composition,% moisture by Karl Fischer (0.10%), % volatile matter at 120°C (0.10-0.20%), % free fatty acids as lauric acid (0.2%), peroxide value (3 meq/kg), and microbial contamination by colony forming units (\u3c10 cfu/mL). The% fatty acid composition was determined using an internal standard and molecular weight correction from the fatty acid methyl ester to the fatty acid. This method yields absolute amounts of fatty acid in the oil. The absolute amount of oleic acid and linoleic acid can be used to replace the iodine value. Principal components analysis of the fatty acid composition indicates that it is not affected by the processing method

Standards for essential composition and quality factors of commercial virgin coconut oil and its differentiation from RBD coconut oil and copra oil

Commercial samples of virgin coconut oil (VCO), refined, bleached and deodorized coconut oil (RBD CNO), and copra oil were analyzed using standard chemical parameters: gas chromatography (GC) of the fatty acid methyl esters (FAME), % moisture by Karl Fischer titration, % volatile matter at 120° C, % free fatty acid, iodine value, peroxide value, and microbial contamination. Principal components analysis (PCA) of the GC-FAME results indicates that the various samples cannot be differentiated by their fatty acid composition, indicating that the fatty acid profile is not affected by the processing method. No trans-fatty acid was detected in all samples down to 0.01% (w/w) detection limit. VCO can be differentiated from RBD CNO and copra oil using the following tests: % moisture by Karl Fischer, % volatile matter volatile at 120° C, and peroxide value

Essential quality parameters of commercial virgin coconut oil

Chemical analyses conducted on commercial samples of virgin coconut oil (VCO) produced by four different methods gave the following ranges of values: % Fatty acid composition: C6: 0.24 to 0.49%; C8: 4.15 to 8.30%; C10: 4.27 to 5.75%; C12: 46.0 to 52.6%; C14: 16.0 to 19.7%; C16: 7.65 to 10.1%; C18: 2.86 to 4.63%; C18:1: 5.93 to 8.53%; C18:2: 1.00 to 2.16%; %moisture by Karl Fischer: 0.05 to 0.12%; %matter volatile at 120 0C: 0.12 to 0.18%; %free fatty acids as lauric acid: 0.042 to 0.329%; and peroxide value: none detected to 1.40. The tests for %moisture by Karl Fischer and %matter volatile at 120 0C can be used to differentiate VCO from and refined, bleached and deodorized coconut oil (RBD CNO). No trans-fatty acid was detected in both VCO and RBD CNO down to 0.01% (w/w) detection limit