Comparator-densitometer

FIGURE 5-18. Optical arrangement of a comparator-densitometer. [Courtesy Jarrell-Ash Division, Fisher Scientific Co.]... 

To quantitate proteins from staining, a densitometer aided by computer software is used to evaluate band areas of samples compared to band areas of a standard curve. Amido black, Coomassie Brilliant Blue, and silver stains are all appHcable for use in quantification of proteins. 

A representative example of this process is shown in Fig. 2. The spectra of the analyte peaks can be measured at the upslope, the top, and at the downslope, or the whole spectrum of the chromatography peak can be compared. In the latter case, the term totalpeak purity is used, and a purity curve of the peak can also be recorded. These operations can be performed by a HPLC system equipped with a DAD detector [13], or for TLC a densitometer that can measure the UV-Vis spectrum of the analyte spot should be used. If the value of the purity is 0.000 0.8900, it is not pure, and a purity of 0.9000-0.9500 means that the peak is contaminated (Shimadzu Class-VP, Chromatography Data System). 

For determining peak identity, the whole spectral data of the standard and the analyte should be compared, and the value of r or MF or SI can be calculated using the software of the HPLC/densitometer equipment as described above. 

ES—values represent wt %—samples compared with NBS standards by densitometer measurement (averages of 2-4 analyses), semiquantitative... 

To dearly distinguish between these two modes of solvent penetration of the gel, we immersed poly(acrylamide-co-sodium methacrylate) gels swollen with water and equilibrated with either pH 4.0 HQ or pH 9.2 NaOH solution into limited volumes of solutions of 10 wt % deuterium oxide (DzO) in water at the same pHs. By measuring the decline in density of the solution with time using a densitometer, we extracted the diffusion coefficient of D20 into the gel using a least squares curve fit of the exact solution for this diffusion problem to the data [121,149]. The curve fit in each case was excellent, and the diffusion coefficients obtained were 2.3 x 10 5cm2/s into the ionized pH 9.2 gel and 2.4 x 10 5 cm2/s into the nonionized pH 4.0 gel. These compare favorably with the self diffusion coefficient of D20, which is 2.6 x 10 5 cm2/s, since the presence of the polymer can be expected to reduce the diffusion coefficient about 10% in these cases [150], In short, these experiments show that individual solvent molecules can rapidly redistribute between the solution and the gel by a Fickian diffusion process with diffusion coefficients slightly less than in the free solution. 

The densitometer uses a two beam comparator technique of measurement. A single lamp illuminates, through a variable neutral density filter, the emitter of an eleven stage photomultiplier tube. Because the frequency of measurement is five times the AC line frequency and the amplifiers are extremely high gain, the lamp must be operated from a regulated DC source, otherwise, 120 Hz brightness ripple of the lamp is recorded in the data. 

Test to determine the proper exposure and development to give the first discernible or meaningful density above film base + fog. This can be done with a densitometer or by developing a single sheet of unexposed film with which to compare against. I tested using ND filters under the lens to achieve this density at 4 seconds. Once you have this benchmark, then make approximately nine exposures, doubling the time... 

The correct measurement of the Rp values is very important because it affects the calculation of the Rm values and the further correlations. The manual measurement of the Rp values will include a error of 1 mm, comparing with the precision of a densitometer, which is 0.01 mm. The start and the front lines should be also exactly established, in order to reduce the measurement error. The front line in TLC is approximately equiva-... 

Often, the Rf value of the separated substances is compared with the Rf values of references. Not a long time ago, this information, together with the similar color, was usually enough to identify a substance. Today, modem densitometers are capable of measuring, more accurately, migration distances (therefore the Rf value) and to scan the in situ UV-Vis spectmm of the spot. 

For quality checks on colour or graphics, controlled lighting conditions are virtually obligatory. Eyeball colour comparison, by trained inspectors, is still a valid quality tool, although colour comparators or densitometers will provide recordable measurements. 

A densitometer is an instrument that can be used to determine the density of a spectral line on a photographic plate or film. A comparator is a device that allows easy comparison of two or more spectra recorded on separate photographic plates or films. In modern instruments these two functions usually are combined in one instrument. 

Comparators are commonly available as an integral part of a densitometer-comparator combination. Comparators are constructed so a reference spectrum from one photographic plate and another spectrum from a second plate may be projected onto the same viewing screen. Alignment of the two spectra provides a convenient method for comparing them. The technique is especially useful for qualitative analysis of an unknown, and in some cases semiquantitative information can be obtained. It also can be used... 

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