Absorbance Versus Wavelength Curves

TABLE 1.1 Approximate Cutoff Ranges for Solvent Classes 

Alkyl ethers (diethyl ether, methyl /-butyl ether, etc.) 210-220 

Alkyl ketones (acetone, methyl propyl ketone, etc.) 320-340 

One important exception occurs when the solvent is used as a low-level component of the mobile phase, for example, at 10% v/v (volume to volume) or less. In such cases, working at or near the cutoff for the 10% component leads to an equivalent background absorbance of only 0.1 AU (absoibance unit). This is often acceptable despite the concomitant increased noise level (which leads to highs detection limits), decreased linear working range, and lowered sensitivity. 

Nevertheless, the use of the UV cutoff value as the onty criterion for solvent selection leads to potentially missed opportunities for unique and powerful separations, as will be discussed in the following sections. 

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Record absorbance versus wavelength curves for the standard solutions and unknown solutions between 200 to 300 nm. (This step may be deleted if you do not have a recording ultraviolet spectrophotometer.) Does the wavelength of 277 nm appear to be the most suitable wavelength for the determmation of aspirin Do the wavelengths of 250 and 275 nm appear to be the best wavelengths for the measurement of the absorbance for the mixture of phenacetin and caffeine Explain. 

Even though the MPMs are present at low concentrations, consideration of their absorbance versus wavelength curves is also very important. Examples of 3% aqueous solutions of MPMs are presented in Figures 1.5a-c. In these examples, phosphate has little or no absorbance down to 200 nm, whereas acetic and trifluoroacetic acids cut off between 230 and 240 nm. Notice also that aging can also affect the A vs. 2 curve. Compare the fiesh triethylamine curve to the one-year aged material (see Figure 1,5d). Both fresh and old have UV cutoff values between 240 and 250 nm. However, the old material has significant absorbance out to 290 nm (possibly due in part to the extraction of phenolic residues from the bottle cap). 

Set a suitable atomic absorption spectrophotometer to a wavelength of 309.3 nm. Adjust the instmment to zero absorbance against water. Prepare and read the absorbance of four aqueous solutions containing 5, 10, 20, and 50 xg/mL of aluminum, in the form of the chloride, and plot the standard curve as absorbance versus concentration of aluminum. 

Obviously, it is advantageous to make the measurement on an absorption peak whenever possible, in order to minimize this curvature, as well as to obtain maximum sensitivity. Because a band of wavelengths is passed, the absorptivity at a given wavelength may vary somewhat from one instrument to another, depending on the resolution, slit width, and sharpness of the absorption maximum. Therefore, you should check the absorptivity and linearity on your instrument rather than relying on reported absorptivities. It is common practice to prepare calibration curves of absorbance versus concentration rather than to rely on direct calculations of concentration from Beer s law. 

Fig. 4.6. Schematic representation of absorbance/transmittance versus time curves. The symbols give the data points for three wavelengths, measured r tively one after another. Synchronisation is obtained by taking int polated values at equal times (perpendicular lines).

Fig. 4.1.4. Reflexion coefficient at normal incidence versus wavelength for a non-absorbing cholesteric (a) semi-infinite medium, b) film of thickness 25P, where P is the pitch. Curves are derived from the dynamical theory circles represent values computed from the exact theory ( 4.1.3) assuming that the medium external to the cholesteric (e.g., glass) has a refractive index 1.5. The parameters used in the calculations are n = 1.5, Sn = 0.07, X = nP = 0.5 /on. (After reference 21.)...

An infrared spectrum can be a plot of either absorbance A or transmittance T versus wavelength or wave number. The convention in this country is that the ordinate scale is usually set up in such a way that absorption peaks appear as valleys in the curve, regardless of whether absorbance or transmittance is plotted on the ordinate. Foreign laboratories often plot spectra with the scale arranged differently, and these appear upside down compared to ours but both types give equivalent information, and one should be familiar with them both. 

On the A - versus A, curve (not shown), the wavelength at which the absorbance variations upon the shift of equilibrium 7.38 are the greatest is located at 442 nm. A second extremum is found near 522 nm. 

A plot of the optical absorbance at 260 nm (the wavelength of maximum light absorption by nucleic acids) versus temperature is known as a melting curve (Fig. 5-45). The absorbance is lower, by up to 40%, for native than for denatured nucleic acids. This hypochromic effect (Chapter 23) is a result of the interaction between the closely stacked bases in the helices of the native molecules. The melting temperature Tm is taken as the midpoint of the increase in absorbance (Fig. 5-45). As the percentage of G + C increases, the nucleic acid becomes more stable toward denaturation because of the three hydrogen bonds in each GC pair. Tm increases almost linearly with increases in the G + C content. In the "standard" citrate buffer (0.15 M NaCl + 0.015 M sodium citrate, pH 7.0) Eq. 5-22 holds. The exact numerical relationship depends strongly upon the ionic composition and pH of the medium.37 72 552 553... 

Typical standard curves are shown in Figures B1.1.2, B1.1.4, B1.1.6, and B1.1.8 for each of the four assay methods. In each case, the tube protocols were performed in duplicate on diluted BSA or BGG standard. The color in each tube was measured at the appropriate wavelength in a dual-beam spectrophotometer. The net absorbance for each sample was plotted versus its protein concentration. 

Quantification. For accurate quantification, a standard curve of peak area versus concentration should be constructed for each standard using the same chromatographic conditions (e.g., wavelength and solvent) as for the samples under analysis. The concentration range of standard curves should be determined according to both the isoflavone level of soy food samples and dilution factors during sample preparation such that the UV absorbance of the injected sample is within a range of 0 to 1. The appropriate standard curve can then be used to calculate the quantity of isoflavones represented by each HPLC peak in the sample. 

Pig. 7. Relative carrier yield versus excitation wavelength. All data are normalized to the same absorbed photon flux. The electric field was t300 V/cm oriented approximately in the polymer chain direction in a standard surface cell configuration. The peak photocurrent-to-dark current ratio was 300 at 300 °K and >600 at 120 °K. Also shown is the absorption spectrum of PTS. The solid portion of the curve refers to the polymer chain direction. The dashed portion is an extrapolation derived from absorption measurements for a dispersion of PTS in a KBr pellet24 ... 

Figure 24-10 Typical absorption spectra of potassium permanganate at five different concentrations. The numbers adjacent to the curves indicate the concentration of manganese in ppm. The absorbing species is permanganate ion. MnOj the cell path length b is 1.00 cm. A plot of absorbance at the peak wavelength at 525 nm versus concentration of permanganate is linear, and thus the absorber obeys Beer s law.

Sensitive tests for the uniformity of a reaction can be done by global analysis of the complete set of spectra recorded during photolysis. These methods, described in Section 3.7.5, provide the best evaluation of the minimum number of spectral components required to reproduce a sequence of spectra within experimental accuracy and the time-dependent species concentrations thus obtained accurately define the reaction progress. Simpler versions use absorbance differences observed at a few selected wavelengths where the changes are largest. Uniform reactions give linear plots of Aversus AA(/,2, ). For two sequential photoreactions, absorbance difference plots are curved, but plots of absorbance difference quotients, AA(21,7)/AA(/l24) versus AA(21,7)/AA(/l3,7), are linear. Isosbestic points provide the simplest criterion... 

Qualitative iuformatiou is obtaiued from the wavelength of light at which the radiatiou is absorbed (AAS) or fluoresceuce emitted (AFS). Quautitative iuformatiou is derived from the amount of radiatiou absorbed or emitted, i.e. the iuteusity. Couceutratiou measure-meuts are usually determiued from a workiug curve of iuteusity versus couceutratiou after calibratiug the iustrumeut with staudards of kuowu couceutratiou. 

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. 

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