Reference cuvet

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. 

In measuring a spectrum, it is routine to first record a baseline with pure solvent or a reagent blank in both the sample and reference cuvets. Cuvets are sold in matched pairs that are as identical as possible to each other. In principle, the baseline absorbance should be 0. However, small mismatches between the two cuvets and instrumental imperfections lead to small positive or negative baseline absorbance. The absorbance of the sample is then recorded and the absorbance of the baseline is subtracted from that of the sample to obtain true absorbance. 

Figure 19-1 Double-beam scanning spectrophotometer. The incident beam is passed alternately through sample and reference cuvets by the rotating beam chopper.

For visible and ultraviolet spectroscopy, a liquid sample is usually contained in a cell called a cuvet that has flat, fused-silica (Si02) faces (Figure 18-5). Glass is suitable for visible, but not for ultraviolet spectroscopy, because it absorbs ultraviolet radiation. The most common cuvets have a 1. OOO-cm pathlength and are sold in matched sets for sample and reference. 

In recording an absorbance spectrum, first record a baseline spectrum with reference solutions (pure solvent or a reagent blank) in both cuvets. If the instrument were perfect, the baseline would be 0 everywhere. In our imperfect world, the baseline usually exhibits small positive and negative absorbance. We subtract the baseline absorbance from the sample absorbance to obtain the true absorbance at each wavelength. 

A 4.37-mg sample of protein was chemically digested to convert its nitrogen into ammonia and then diluted to 100.0 mL. Then 10.0 mL of the solution were placed in a 50-mL volumetric flask and treated with 5 mL of phenol solution plus 2 mL of sodium hypochlorite solution. The sample was diluted to 50.0 mL, and the absorbance at 625 nm was measured in a 1.00-cm cuvet after 30 min. For reference, a standard solution was prepared from... 

Using a 1-ml pipet (0.01 ml subdivision), a suitably-sized aliquot (e.g., 0.3 ml) of the above solution is transferred to a 1-cm path quartz (reference) cuvette (=3-ml capacity) and diluted 10-fold (i.e., to 3.0 ml) with the same solvent used to dissolve the sample. A second aliquot, identical in volume to the first, is similarly transferred to a matching 1 cm path quartz (sample) cuvet and diluted... 

When charge-transfer bonds are obscured by those of the original donors and acceptors, one may find of value a difference method [1] (e.g., Forster s tandem method [70]). Four cuvets of equal path length are used, two containing the charge-transfer complex solutions in series in the indicator beam of a double-beam spectrophotometer, and two cuvets, one with the unreacted donor and the other with unreacted acceptor solution, also in series in the reference beam. A difference spectrum is thus obtained which, however, needs special care in its interpretation. 

Cuvets used for measurements in the UV region should be handled with special care. Invisible scratches, fingerprints, or residual traces of previously measured substances may be present and absorb significantly. A good practice is to fill all such cuvets with distilled water and measure the absorbance for each against a reference blank over the wavelengths to be used. This value should be essentially zero. 

A mechanically robust ECESR cell that is suitable for measurements even at very low temperatures with all kinds of electrolyte solvents and that employs a platinum wire loop as the working electrode located at the bottom of a 4 mm ESR cuvet has been developed by Fiedler et al. [618]. The reference and counter electrodes are placed above the working electrode outside of the sensitive region of the ESR cavity. 

Instrumentation. The spectroelectrochemical cell described by Kobayashi and Ni-shiyama includes a minigrid working electrode in a glass cuvet (approx. 3 mm optical pathlength) attached to the bottom of a cylindrical cell body with counter electrode and reference electrode mounted therein. The cuvet is exposed to the magnetic... 

The following technique for quantification of samples by UV spectroscopy is convenient, uses little sample, and decreases errors associated with unmatched or inadequately cleaned cuvets One milliliter of reference solution (buffer blank for proteins, EtOH for retinoids) is added to a standard 1-mL quartz cuvet. This is placed in the spectrometer and the instrument is zeroed. An appropriate amount of sample is then thoroughly mixed into this cuvet and the absorbance recorded. Examples addition of 100 fiL of a 10- iAf solution of CRABP will yield a final Ajgo of -0.02 addition of 10 pL of a 1-mM stock of typical retinoid will yield a final A280 of 0 4 The measurement should be repeated several times, preferably using several different dilutions... 

The accuracy of the experimental values of the photoluminescence quantum yield can be increased if the standard and the sample (either solid or film) are placed in an integrating sphere, which collects nearly the entire emitted photoluminescence intensity and is therefore less sensitive to the particular shape of the sample or the reference [182,183]. Recent comparative experiments with the circular cuvet geometry and also with an integrating sphere confirmed the quantum yield data presented in Table 30.1 within about the same experimental error [179,183]. 

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