Photometric linearity

Checking for photometric linearity by the use of a set of transmittance or reflectance standards (e.g. Spectralon, carbon black mixtures). 

Different operators, using the same instrument, may obtain different results due to variations in technique which, for example, markedly affects the precision of some automatic pipettes (B19). Manufacturers instructions may give little or no information on how to obtain the best results. The optimal absorbance required to obtain maximum precision varies for different types of spectrophotometer from 0.43-0.88 (H26) the user may not know this if it is not stated in the instructions. Inadequate maintenance is undoubtedly a major source of error, and includes such simple faults as greasy spectrophotometer cuvettes and pipettes and dirty tubing in continuous-flow systems, resulting in excessive sample interaction. Errors of spectrophotometers, arising from poor technique and faults in wavelength accuracy, photometric linearity, and photometric accuracy, are discussed in Section 4.5. 

Photometric linearity is usually tested with colored solutions of varying concentration (B20, Rl), but apparent deviations from Beer s law do not distinguish between the properties of the solution (S6) and true... 

Linearity of signal with integration time to check photometric linearity of the detector. 

If desired, or required for a particular application, repeat step 4 for a range of integration times, from near the detection limit to near detector saturation. The detector response should be linear with integration time. For an FT-Raman system, a similar test may be performed by varying the laser power to check photometric linearity. 

Table 1 displays recommended specifications for wavelength accuracy, photometric linearity, and spectrophotomet-ric noise levels for pharmaceutical applications. The first step... 

Photometric linearity is tested using a set of standards with known relative transmittance or reflectance, depending on the application. Repeated measurements on stable standards give information about the long-term stability of an instrument. For analyte systems with absorbance levels below 1.0, at least four reference standards are to be used in the range of 10 to 90%. When analytes provide absorbance values that exceed 1.0, the proposed USP chapter recommends adding a 2 or 5% standard, or both. 

The wavelength uncertainty test verifies the accuracy and precision of the spectrophotometer x-axis. Typically, the x-axis will be in nanometers for a dispersion instrument and cm for a FT instrument. The use of cm for the spectral axis of an FT instrument is due to the mathematics of the interference term (Atkins 1996). The wavelength standards have stable isolated peaks usually based on a mixture of rare-earth oxides. The center of mass of the peaks is compared to standard values established on master instruments at National Institute of Standards and Technology (NIST). The typical tolerance values for the peak accuracy are 1 nm [19]. The observed precision values are usually much smaller than 1 nm due to the high reproducibility of modern spectrophotometers. The photometric linearity verifies that the y-axis of the spectrophotometer is linear over a typical refiectance range. The linearity is verified by scanning a series of standards of known reflectance (absorbance) values. The measured absorbance is plotted versus the standard values. The USP chapter specifies that the slope of this curve is equal to 1.0 0.05 with an intercept of 0.0 0.05. Photometric standards are available from instrument vendors and third party suppliers. 

Table 6.1 displays recommended specifications for wavelength accuracy, photometric linearity, and spectrophotometric noise levels for pharmaceutical applications. The first step in validating any NIR method is to test the suitability of these specifications for a given application. Wavelength accuracy tests conducted using appropriate external standards will prevent potential problems that could occur with proprietary internal calibration protocols. The exact nature of any calibration standard must be noted in a validation protocol. Rare earth oxides and glass standards are candidates for such calibration. 

Photometric linearity is tested using a set of standards with known relative transmittance or reflectance, depending on the application. Repeated measurements on stable standards give information about the... 

An absolute method of checking a spectrophotometer s photometric linearity exists. It combines the use of Bouguer s law and the superposition of optical fields (17). A simple example of this technique from Hawes paper explains the method clearly. Two neutral density filters are measured separately and then together. If the transmittance of one is 57.19 and the other is 48.66, the transmittance of the two together, separated by an air space, would be 57.19-0.4866 = 27.83. Other filters can be measured separately and combined in multiple stacks to check various photometric levels. 

Photometric linearity is an important area as well. Photometric qualification is based on a set of transmission or reflectance standards with known values. In transmission, filters with known transmittance values are used. In reflectance, Labsphere Spectralon gray standards with reflectance values from 0.99 to 0.02 are used. 

An important issue in the restoration process is photometric linearity—the ability of the restoration technique to maintain a linear relationship between the brightness of a star and the response. Unfortunately, the biases in maximum entropy methods make photometric linearity a difficult proposition. 

The flame-photometric detector (FPD) is selective for organic compounds containing phosphoms and sulfur, detecting chemiluminescent species formed ia a flame from these materials. The chemiluminescence is detected through a filter by a photomultipher. The photometric response is linear ia concentration for phosphoms, but it is second order ia concentration for sulfur. The minimum detectable level for phosphoms is about 10 g/s for sulfur it is about 5 x 10 g/s. 

The worked out soi ption-photometric method of NIS determination calls preliminary sorption concentration of NIS microamounts from aqueous solutions on silica L5/40. The concentrate obtained is put in a solution with precise concentration of bromthymol-blue (BTB) anionic dye and BaCl, excess. As a result the ionic associate 1 1 is formed and is kept comparatively strongly on a surface. The BTB excess remains in an aqueous phase and it is easy to determinate it photometrically. The linear dependence of optical density of BTB solutions after soi ption on NIS concentration in an interval ITO - 2,5T0 M is observed. The indirect way of the given method is caused by the fact the calibration plot does not come from a zero point of coordinates, and NIS zero concentration corresponds to initial BTB concentration in a solution. 

Figure 3 Least squares calibration line for photometric detector. (From Dorschel, C. A., Ekmanis, J. L., Oberholtzer, J. E., Warren, Jr., F. V., and Bidlingmeyer, B. A., LC detectors evaluations and practical implications of linearity, Anal. Chem., 61, 951 A, 1989. Copyright American Chemical Society Publishers. With permission.)...

GC is coupled with many detectors for the analysis of pesticides in wastewater. At the present time the most popular is GC-MS, which will be discussed in more detail later in this section. The flame ionization detector (FID) is another nonselective detector that identifies compounds containing carbon but does not give specific information on chemical structure (but is often used for quantification because of the linear response and sensitivity). Other detectors are specific and only detect certain species or groups of pesticides. They include electron capture,nitrogen-phosphorus, thermionic specific, and flame photometric detectors. The electron capture detector (ECD) is very sensitive to chlorinated organic pesticides, such as the organochlorine compounds (OCs, DDT, dieldrin, etc.). It has a long history of use in many environmental methods,... 

In many instruments the meter read-out is calibrated in absorbance units using a logarithmic scale while other instruments retain the convenience of a linear scale but convert the signal from the detector to a logarithmic one by electronic or mechanical means. It is essential when using a photometric instrument to know if it is calibrated in absorbance or transmittance units. 

The amount transformation process is illustrated with data for chlorpyrifos in the flame photometric detector, phosphorus mode, and shown in Table VI. Level 1 transformations were calculated where the amount power was increased by 0.03 units for each step. At an amount power of 0.20 the F statistic of 32.7 showed a minimum but at a confidence level of 95% did not satisfy the F test for linearity. Power steps changed by only 0.01 and 0.001 units in the vicinity of the minimum were then calculated as shown in levels 2 and 3. The best linearity was found in this case at a power transformation of 0.182 although the F statistic of 8.33 did not indicate linearity when compared with the critical F of 2.99 at P=.95. Calculations at these second and third levels were not always necessary and even when performed did not always lead to a satisfactory condition of linearity. 

Table VI. Convergence of the Optimal Amount Transformation for the Determination of Data Linearity. Chlorpyrifos Data with Flame Photometric Detection.

---