Second derivative spectroscopy

D.T. Williams et al, Evaluation of Second Derivative Spectroscopy For Monitoring Toxic Air Pollutants , SAM TR-74-19, Proj AF-7164, Contract F 41609-73-C-0011 (1974)... 

The extent of homogeneous mixing of pharmaceutical components such as active drug and excipients has been studied by near-IR spectroscopy. In an application note from NIRSystems, Inc. [47], principal component analysis and spectral matching techniques were used to develop a near-IR technique/algorithm for determination of an optimal mixture based upon spectral comparison with a standard mixture. One advantage of this technique is the use of second-derivative spectroscopy techniques to remove any slight baseline differences due to particle size variations. 

Wahbi reported on the use of second-derivative spectroscopy for the determination of canrenone in spironolactone, and found that the method is stability-indicating for spironolactone [21]. First and second-derivative spectroscopy were also utilized for the simultaneous analysis of spironolactone in combination with either hydrochlorothiazide or frusemide [22], The drugs are extracted with ethanol and analyzed through the use of the zero-crossing method. Nowakowska [23] analyzed mixtures of spironolactone and hydrochlorothiazide in tablets by measuring the absorbance of all species at 239 nm, and that of hydrochlorothiazide at 318 nm. The recoveries for spironolactone were found to be 97.9 to 101.8%, with a coefficient of variation of less than 0.1%. 

Second derivative spectroscopy is a means to extract information from the spectrum. In contrast to the relatively featureless UV spectra, the second derivatives of UV spectra are characterized by 2 sharp peaks and troughs [6,7]. Figure 1 shows the ultraviolet spectra of yeast enolase at 0.28 and 200 MPa and Figure 2 shows the second derivative of those spectra. 

The use of second derivative spectroscopy allows us to observe the effects of pressure on this enzyme, to quantitate the changes occuring (we can use the values of r to calculate Keq as a function of pressure and hence AG and AV for dissociation) and to interpret those changes in terms of the known structure of the enzyme. Second derivative spectroscopy has both advantages and disadvantages vis-a-vis fourth derivative spectroscopy. With second derivatives, one uses the ratio of the amplitudes, which is independent of the absolute absorbance. Therefore, one does not have to correct the spectra for compression effects and can easily compare samples studied at different times. The changes, however, are dominated by the contributions of tyrosines and provide much less information about the tryptophan residues. 

The selective resolution enhancement in derivative spectroscopy is pushed even further in the fourth derivative mode. As in the case of second derivative spectroscopy, the amplitude and the position of the derivative spectral bands of the aromatic amino acids are related to the polarity of the medium. We have undertaken a systematic investigation of these spectral features of the N-acetyl O-ethyl esters of tyrosine and tryptophan in various solvents of different polarity (from cyclohexane to water). Astonishingly, a simple relationship between the spectral parameters of the fourth derivatives and the dielectric constant was found [11]. As shown in Figure 5, for tyrosine it is the position of >.max, and for tryptophan it is the derivative amplitude which depends linearly on the dielectric constant er. Since in addition the fourth derivative spectra of these model compounds do not depend significantly on pressure (at least up to 500 MPa), these spectral features may be used as an intrinsic probe to sense the dielectric constant in the vicinity of tyrosine and tryptophan. 

Levine, R.L., Federici, M.M. (1982) Quantitation of Aromatic Residues in Proteins Model Compounds for Second-Derivative Spectroscopy, Biochemistry 21, 2600-2606. 

Crumpton W., Isehart G. M., and Mithell P. L. (1992) Nitrate and organic N analyses with second-derivative spectroscopy. Limnol. Oceanogr. 37, 907-913. 

Modern UV spectrometers may offer the option of first and second-derivative UV spectroscopy and measurements in the 240-320 nm range using these techniques can be interpreted to determine the ratios of the various a romatic residues in peptides. Thus, tyrosine can be differentiated from tryptophan through first-derivative spectroscopy and phenylalanine can be differentiated from tyrosine and tryptophan through second-derivative spectroscopy (Miclo et al., 1995). 

Figures 8B and 8C illustrate the use of second-derivative spectroscopy and FSD, respectively, to resolve the overlapping component bands of the amide I band profile in the FTIR spectrum of p-lactoglobuhn (Figure 8A). Both of these techniques should only be applied to spectra that exhibit a high signal-to-noise ratio (>1000 1), or otherwise artefacts may be introduced by resolution enhancement. The presence of water vapour in the region of the spectrum of interest will also produce features in the processed spectrum that may be misinterpreted as absorption bands of the sample (Figure 9).

Ragone, R., Colonna, G., Balestrieri, C., Servillo, L. and Irace, G, 1984, Determination of tyrosine exposure in proteins by second-derivative spectroscopy. Biochemistry 23, 1871-1875. 

Mahrous, M.S. et al (25). Derivative uv spectroscopy was used to determine the cited drugs in capsules and tablets. First derivative spectroscopy was used to determine indomethacin in O.IN-H2SO4 solution, and second derivative spectroscopy was used to determine naproxen and iboprofen in O.IN-NaOH solution. The method is rapid and accurate. 

Balestieri C, Colonna G, Giovane A, Irace G, and Servillo L (1978) Second derivative spectroscopy of proteins. A method for the quantitative determination in proteins. European Journal of Biochemistry 90 433—440. 

A more recent publication demonstrating the power of PDA analysis for conformational studies focused on the stability of closely related proteins [35]. Using lysozyme as a model protein for showing the influence of protein conformation on the experimentally determined r value (Fig. 6), the second-derivative spectroscopy then provided a straightforward means of comparing the... 

Botton, D, Honkawa, T., Tohyama, S., Perkin-Elmer Corp. Norwalk, C.T., US.A. Application Data Sheet Second Derivative Spectroscopy. Perkin-Elmer Corp. Norwalk, C.T., US.A., 1977 22 p. 

Mach, H. Middaugh, C.R. Simultaneous monitoring of the environment of tryptophan, tyrosine and phenylalanine residues in proteins by near-ultraviolet second-derivative spectroscopy. Anal. Biochem. 1994, 222, 323-331. 

The estimation of the concentration of polyenes from their absorption spectra requires prior empirical calculation of the absorption coefficients [512]. A very sensitive method for the registration of polyene structures in degraded poly(vinyl chloride) is second derivative spectroscopy (cf. section 10.16) [1792, 1888]. 

As can be seen from these figures, the polyene chromophores are much more easily detectable by second derivative spectroscopy, from the commencement of irradiation, than by normal absorption spectra. 

Second derivative spectroscopy also allows the determination of very low concentrations of polyene structures so formed (Fig. 10.61). 

Vg P04 band to its underlying components through either second derivative spectroscopy or deconvolution methods, and quantification of their contribution through curve fitting [7, 9]. 

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