Spectrophotometer errors

Figure 18-6 shows measured spectrophotometer errors. Electrical noise was only modestly dependent on sample absorbance. The largest source of imprecision for A < 0.6 was irreproducible positioning of the cuvet in the sample holder, despite care in placing the cuvet. The resulting error curve reaches a minimum near A = 0.6. In Section 5-2, we learned that the detection limit for an analytical procedure is determined by the reproducibility of the measurement. The less noise, the lower the concentration of analyte that can be detected. 

Finally, values of sx are directly proportional to transmittance for indeterminate errors due to fluctuations in source intensity and for uncertainty in positioning the sample cell within the spectrometer. The latter is of particular importance since the optical properties of any sample cell are not uniform. As a result, repositioning the sample cell may lead to a change in the intensity of transmitted radiation. As shown by curve C in Figure 10.35, the effect of this source of indeterminate error is only important at low absorbances. This source of indeterminate errors is usually the limiting factor for high-quality UV/Vis spectrophotometers when the absorbance is relatively small. 

When using a spectrophotometer for which the precision of absorbance measurements is limited by the uncertainty of reading %T, the analysis of highly absorbing solutions can lead to an unacceptable level of indeterminate errors. Consider the analysis of a sample for which the molar absorptivity is... 

The comparison of more than two means is a situation that often arises in analytical chemistry. It may be useful, for example, to compare (a) the mean results obtained from different spectrophotometers all using the same analytical sample (b) the performance of a number of analysts using the same titration method. In the latter example assume that three analysts, using the same solutions, each perform four replicate titrations. In this case there are two possible sources of error (a) the random error associated with replicate measurements and (b) the variation that may arise between the individual analysts. These variations may be calculated and their effects estimated by a statistical method known as the Analysis of Variance (ANOVA), where the... 

Visual methods have been virtually displaced for most determinations by methods depending upon the use of photoelectric cells (filter photometers or absorptiometers, and spectrophotometers), thus leading to reduction of the experimental errors of colorimetric determinations. The so-called photoelectric colorimeter is a comparatively inexpensive instrument, and should be available in every laboratory. The use of spectrophotometers has enabled determinations to be extended into the ultraviolet region of the spectrum, whilst the use of chart recorders means that the analyst is not limited to working at a single fixed wavelength. 

The sampling of solution for activity measurement is carried out by filtration with 0.22 pm Millex filter (Millipore Co.) which is encapsuled and attached to a syringe for handy operation. The randomly selected filtrates are further passed through Amicon Centriflo membrane filter (CF-25) of 2 nm pore size. The activities measured for the filtrates from the two different pore sizes are observed to be identical within experimental error. Activities are measured by a liquid scintillation counter. For each sample solution, triplicate samplings and activity measurements are undertaken and the average of three values is used for calculation. Absorption spectra of experimental solutions are measured using a Beckman UV 5260 spectrophotometer for the analysis of oxidation states of dissolved Pu ions. 

Heller and Tabibian (13) noted that errors, due to laterally scattered light and the corona effect, as large as to cause a 30 reduction in measured turbidity, may result if instruments which are perfectly suitable for ordinary absorption measurements are used for turbidity measurements without proper modifications. To evaluate the performance of our turbidity detector, particle suspensions of various concentrations of several polystyrene latex standards were prepared. Their extinction coefficients were measured using both a bench-top UV spectrophotometer (Beckman, Model 25) and the online detector (Pharmacia). 

Most of the instruments, commonly used in an analytical laboratory, such as UV-Spectrophoto-meter, IR-Spectrophotometer, single—pan electric balance, pH-meter, turbidimeter and nephelometer, polarimeter, refractometer and the like must be calibrated duly, before use so as to eliminate any possible errors. In the same manner all apparatus, namely pipettes, burettes, volumetric flasks, thermometers, weights etc., must be calibrated duly, and the necessary corrections incorporated to the original measurements. 

Plots of Residuals. Residuals can be plotted in many ways overall against a linear scale versus time that the observations were made versus fitted values versus any independent variable (3 ). In every case, an adequate fit provides a uniform, random scatter of points. The appearance of any stematic trend warns of error in the fitting method. Figures 4 and 5 shows a plot of area versus concentration and the associated plot of residuals. Also, the lower part of Figure 2 shows a plot of residuals (as a continuous line because of the large number of points) for the fit of the Gaussian shape to the front half of the experimental peak. In addition to these examples, plots of residuals have been used in SBC to examine shape changes in consecutive uv spectra from a diode array uv/vis spectrophotometer attached to an SBC euid the adequacy of linear calibration curve fits (1). 

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 ... 

Student 2 has obtained a set of results which are closely clustered but give a mean which is less than the correct answer. Thus although this assay is precise it is not completely accurate. Such a set of results indicates that the analyst has not produced random errors which would produce a large scatter in the results but has produced an analysis containing a systematic error. Such errors might include repeated inaccuracy in the measurement of a volume or failure to zero the spectrophotometer correctly prior to taking the set of readings. The analysis has been mainly well controlled except for probably one step which has caused the inaccuracy and thus the assay is precisely inaccurate. 

The more the precision of the instrument, and the more the points for the time unit in the acquired profile, the better the result of the fitting of experimental data. For this reason instruments with a low measure error and connectable to a computer for the automatic and continous aquisition of data are very much prefered. The UV-Vis spectrophotometer is by far the most used instrument in chemical kinetics. It has a good sensitivity and a good control of the temperature. It is connected or easily connectable to a computer and is available nearly everywhere. The absorbance has a very low dependence on the temperature so that, in the used temperature range, its variation can be neglected during the VTK experiments. 

Wavelength accuracy is defined as the deviation of the wavelength reading at an absorption band or emission band from the known wavelength of the band. The wavelength deviation can cause significant errors in the qualitative and quantitative results of the UV-Vis measurement. It is quite obvious that if the spectrophotometer is not able to maintain an accurate wavelength scale, the UV absorption profile of the sample measured by the instrument will be inaccurate. The true Amax and A.min of the analyte cannot be characterized accurately. 

The identification of a compound using only its retention time is vulnerable to error. It is essential that a standard compound is injected in order to verify the retention time. As is the case in gas chromatography, more sophisticated detectors can be used. These detectors provide complementary information and can be installed at the end of the column. These can be other types of spectrophotometers or a mass spectrometer and they are used simultaneously as classical detectors (to obtain the chromatogram) or for identification purposes of the analytes (cf. Chapter 16). For example, the coupling of HPLC to NMR, which has long been considered impossible, has now been realised through the miniaturisation of the probes and the increased sensitivity of the NMR instruments (cf. Chapter 9). 

Some spectrophotometers allow the measurement of absorbance over a dynamic range of 4 to 6 decades. However, elevated values of absorbance are less reliable because they correspond to very weak transmitted intensities (///0 = 10-6 for A = 6). For most instruments, there are three independent causes of error that can affect transmittance (Fig. 11.23) ... 

Slight mismatch between sample and reference cuvets, over which you have little control, leads to systematic errors in spectrophotometry. For best precision, you should place cuvets in the spectrophotometer as reproducibly as possible. Random variation in absorbance arises from slight misplacement of the cuvet in its holder, or turning a flat cuvet around by 180°, or rotation of a circular cuvet. 

Basic components of a spectrophotometer include a radiation source, a monochromator, a sample cell, and a detector. To minimize errors in spectrophotometty, samples should be free of particles, cuvets must be clean, and they must be positioned reproducibly in the sample holder. Measurements should be made at a wavelength of maximum absorbance. Instrument errors tend to be minimized if the absorbance falls in the range A — 0.4—0.9. 

Figure 20-9 Absorbance error introduced by different levels of stray light. Stray light is expressed as a percentage of the irradiance incident on the sample. [M. R. Sharp."Stray Light In UV-VIS Spectrophotometers,"Anal. Chem. 1984,...

Samples are usually placed in 1mm thick quartz spectrophotometer cells sealed with Parafilm or similar. Samples in which the aqueous phase has a very high D to H ratio are sometimes thicker, as the level of incoherent scatter due to H will be low. Samples may be in the scattering apparatus for several hours, and so H20/D20 exchange due to faulty sealing can cause errors. For gel-like samples, it is very important that there are no air bubbles trapped in the sample. Gel or viscous samples can be centrifuged to the bottom of cells, and air bubbles removed, using a Helma Roto-Vette or similar. 

---