Spectrophotometer INDEX

Photoelectric-Colorimetric Method. Although the recording spectrophotometer is, for food work at least, a research tool, another instrument, the Hunter multipurpose reflectometer (4), is available and may prove to be applicable to industrial quality control. (The newer Hunter color and color difference meter which eliminates considerable calculation will probably be even more directly applicable. Another make of reflection meter has recently been made available commercially that uses filters similar to those developed by Hunter and can be used to obtain a similar type of data.) This instrument is not a spectrophotometer, for it does not primarily measure the variation of any property of samples with respect to wave length, but certain colorimetric indexes are calculated from separate readings with amber, blue, and green filters, designated A, B, and G, respectively. The most useful indexes in food color work obtainable with this type of instrument have been G, which gives a... 

Many different detectors are used in RPLC, including ultraviolet-visible spectrophotometers (UV-VIS), refractive index (RI) detectors, electrochemical (EC) detectors, evaporative light-scattering detectors, fluorimeters, and... 

Detection is also frequently a key issue in polymer analysis, so much so that a section below is devoted to detectors. Only two detectors, the ultra-violet-visible spectrophotometer (UV-VIS) and the differential refractive index (DRI), are commonly in use as concentration-sensitive detectors in GPC. Many of the common polymer solvents absorb in the UV, so UV detection is the exception rather than the rule. Refractive index detectors have improved markedly in the last decade, but the limit of detection remains a common problem. Also, it is quite common that one component may have a positive RI response, while a second has a zero or negative response. This can be particularly problematic in co-polymer analysis. Although such problems can often be solved by changing or blending solvents, a third detector, the evaporative light-scattering detector, has found some favor. 

Concentration-sensitive detectors, such as the refractive index detector or UV-VIS spectrophotometer... 

Part—IV has been entirely devoted to various Optical Methods that find their legitimate recognition in the arsenal of pharmaceutical analytical techniques and have been spread over nine chapters. Refractometry (Chapter 18) deals with refractive index, refractivity, critical micelle concentration (CMC) of various important substances. Polarimetry (Chapter 19) describes optical rotation and specific optical rotation of important pharmaceutical substances. Nephelometry and turbidimetry (Chapter 20) have been treated with sufficient detail with typical examples of chloroetracyclin, sulphate and phosphate ions. Ultraviolet and absorption spectrophotometry (Chapter 21) have been discussed with adequate depth and with regard to various vital theoretical considerations, single-beam and double-beam spectrophotometers besides typical examples amoxycillin trihydrate, folic acid, glyceryl trinitrate tablets and stilbosterol. Infrared spectrophotometry (IR) (Chapter 22) essentially deals with a brief introduction of group-frequency... 

Infrared spectra were recorded on a Perkin Elmer Model 567 Spectrophotometer. Ultraviolet spectra were obtained on a Cary 1756 Spectrophotometer. Gas chromatograms were recorded on a Tracor Model 220 with electron capture detector. High pressure liquid chromatography studies were conducted with a Waters Model ALC-200 with ultraviolet and refractive index detectors. 

The main objectives in calibrating the SEC detection system in absolute refractive index and absorption units are the estimation of v and E at the normal flow conditions and the standardization of the measurement errors. The first step in the calibration process is the estimation of the instrument s constants to transform the computer units into absorbances and refractive index units. The Waters AAO UV spectrophotometer displays absorbance units. Therefore, step changes in the instrument s balance and sampling of the signal provide the necessary data for the calibration. The equations obtained are ... 

Once the volume of the injection loop is known, the overall extinction coefficient (e) and refractive index increment (v) for an unknown sample can be calculated directly from equation II6 and the corresponding equations for the uv spectrophotometer at the required wavelengths... 

The spectrophotometer measures the transmission and, if an absorption measurement is carried out, converts the transmission into absorbance using these equations. This conversion works fine for samples where there is no reflection, either specular or diffuse, as is the case for nonturbid solutions. However, for films there is invariably some reflection, which is often quite large, particularly for films of high dielectric constant (or refractive index) materials, such as PbS and PbSe. Additionally, if the films are not completely transparent, then scattering introduces an extra element of reflection. Therefore, to measure the real absorption of a film, a reflection measurement must also be carried out and correction for this reflection made. The correction will be approximate and depends on the nature of the film itself. However, that most commonly used is... 

Electrophoretic mobilities of the quartz particles in cobalt (II) perchlorate solutions were determined with a calibrated Zeta-Meter apparatus. Coagulation sedimentation behavior was followed using a stop-flow type apparatus. The dispersion is pumped in a closed loop from an equilibration vessel through an optical cell located in the sample compartment of a recording spectrophotometer. From the optical densitytime curve obtained from the time the pump is switched off, the turbidity index (in arbitrary units) is obtained as the slope of the curve at zero time. 

X-ray photoelectron spectroscopy (XPS) was used for elemental analysis of plasma-deposited polymer films. The photoelectron spectrometer (Physical Electronics, Model 548) was used with an X-ray source of Mg Ka (1253.6 eV). Fourier transform infrared (FTIR) spectra of plasma polymers deposited on the steel substrate were recorded on a Perkin-Elmer Model 1750 spectrophotometer using the attenuated total reflection (ATR) technique. The silane plasma-deposited steel sample was cut to match precisely the surface of the reflection element, which was a high refractive index KRS-5 crystal. 

Figure 1 is the ultraviolet spectrum of a 10 mcg/ml solution of vitamin D3 in methanol. The spectrum was obtained using a Cary Model 219 recording spectrophotometer (Varian Instrument Co., Palo Alto, CA). Vitamin D3 and related compounds have a characteristic UV absorption maximum at 265 nm and a minimum at 228 nm. The extinction coefficient at 265 nm is about 17,500 and 15,000 at 254 nm. An index of purity of vitamin D3 is a value of 1.8 for the ratio of the absorbance at 265 to that at 228 nm. The high absorbance at 254 nm enables one to use the most common and sensitive spectrophotometric detector used in high performance liquid chromatography (HPLC) for the analysis of vitamin D3 in multivitamin preparations, fortified milk, other food products, animal feed additives etc. 

Activities for cellulase (FPU/mL) and P-glucosidase (IU/mL) were measured by the methods described previously (16,17). ODs (1-cm light path) of cultures were monitored on a Beckman DU-640 Spectrophotometer (Fullerton, CA) at 550 (E. coli) or 660 nm (S. cerevisiae). Concentrations of sugars and ethanol were determined by HPLC using an Aminex HPX-87H column (300 x 7.8 mm Bio-Rad, Richmond, CA) and refractive index detector. Samples were run at 65°C and eluted at 0.6 mL/min with 5 mM H2S04. 

A sample of the suspension can then be taken and titrated separately with a double beam spectrophotometer, the reference being the initial solution. The optical density of the solution at X = 650 mn is actually proportional to the amount of S produced in the experiment. This was checked by a gravimetric titration of the precipitate which served as a standard for the optical method. The experience was repeated at all the injection points indexed on Figures 2 and 3. The amount of precipitate is simply additive from one experiment to the next. These amounts (Cg = 10- to 10 2 moles. m 3 per experiment) are very small as compared to the concentration of reactants. CQ can thence be identified to (Ba +)0 and it is not necessary to readjust the reactant concentration as long as the number of sucessive injections does not exceed about ten. It can be noticed from [4] that precipitation is facilitated by the presence of pre-existing precipitate. 

In an absorbing medium, /cabs / 0, this wave will be attenuated as e" abs rrf. The imaginary part k of the complex index of refraction, nKf + Kabs, is a measure of the attenuation of an electromagnetic wave as seen, for example, in a spectrophotometer. We speak of k k(o>) as the absorption spectrum of light. In the limit of high absorption (k3bS... 

Another approach, which is used in this experiment, is to develop the analytical separation on the high-surface-area packing and increase the amount injected into the column to determine the loading level for preparative work. A common problem in preparative LC is detector saturation. Detector saturation occurs when the concentration of sample eluting from the column is so high that the detection system is electronically overloaded. The result of detector saturation is loss of the ability to observe the peaks. This is demonstrated in this experiment when the spectrophotometer is saturated, and the refractive index detector is not. 

Absolute MWD can be measured using light scattering or viscometry combined with universal calibration. Compositional drift over the MWD of a polymer can be measured using a UV spectrophotometer and a differential refractive index detector. The increase in the available information also expands the complexity of data analysis. We discuss some of the concerns regarding data analysis that arise in multidetector SEC. 

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