Derivative spectroscopy and applications of uv vis spectroscopy

The main obejectives of derivative spectroscopy
Derivative spectra and its measurements
Orders of derivative spectra
Noise to signal ratio
Instrumentation of derivative spectroscopy
Advantages and disadvantages of derivative spectroscopy
Applications of Derivative and UV-Vis spectroscopy

1. Derivative Spectroscopy & Applications of UV-Visible Spectroscopy UNDER THE GUIDANCE OF: Dr. A. Sreedevi M.Pharm., Ph.D. Department of Pharmaceutics SUBMITTED BY: Nayeema Khowser Shaik M.Pharmacy Ist year 2020MPH40A029

2. Contents Introduction Objectives Measurement techniques Derivative Spectra Signal-to-noise ratio Instrumentation Advantages and disadvantages Applications of Derivative Spectroscopy Applications of UV-Visible Spectroscopy References Department of Pharmaceutics, SPMVV 2

3. INTRODUCTION Derivative UV-spectrophotometry an analytical technique of enormous implication commonly in obtaining mutually qualitative and quantitative in order from spectra that are of unresolved bands, with respect to qualitative and quantitative analysis, it uses first or higher derivatives of absorbance in accordance with wavelength . This operation allows to remove spectral interferences and as a consequence leads to increase selectivity of assay. It was originally brought in 1950s with its applicability in a lot of features, but because of its complication in producing derivative spectra via UV-Visible spectroscopy the method found less practice . It is presently available with software's controlling modern spectrophotometers. Department of Pharmaceutics, SPMVV 3

4. OBJECTIVES Department of Pharmaceutics, SPMVV It is a spectroscopic technique that differentiates spectra. The objective with which derivative methods used in analytical chemistry are: Spectral differentiation- As a qualitative method that distinguish small variation between almost similar spectra’s. Spectral resolution enhancement - Overlapping spectral bands gets resolved to simply estimation the number of bands and their wavelengths. Quantitative analysis- It facilitates multicomponent analysis and corrects the irrelevant background absorption. Derivative spectroscopy method forms the beginning of differentiation or resolution of overlapping bands; the vital characteristics of derivative process are that broad bands are suppressed relative to sharp bands. 4

5. MEASUREMENT TECHNIQUES Department of Pharmaceutics, SPMVV Measurement of derivative spectra value is achieved out by the following three methods: Graphic measurement - Graphic measurement is theoretical method to calculate the derivative spectra on paper, its manual method it suffer from disadvantage that it gives inaccurate results because the value can determined numerically can be abolish or diminish beyond restriction . 2. Numeric measurement- The method uses set of points where derivative values is carried out by estimating the derivative value at a given wavelength. It gives derivatives by spectral differentiation using suitable numerical algorithm. 3. Zero crossing technique - The method measures the derivative spectra at a particular wavelength, where the derivative crosses the point at zero line. Interference of one component in determination of other component can be eliminated by zero crossing technique. 5

6. DERIVATIVE SPECTRA Department of Pharmaceutics, SPMVV In quantitative analysis, derivative spectra enlarge difference between spectra to resolve overlapping bands. The digital algorithm method called as Savitzky-Golay is most outstandingly referred for obtaining derivative spectra. The degree of difficulty of derivative spectra increases with presence of satellite peaks. On the basis of solvents polarity, peaks and troughs shifts either to shorter or longer wavelength.  6

7. When spectrum is scanned at a constant rate, real time derivative spectra can be recorded either by achieving the time derivative of the spectrum or by wavelength modulation. Wavelength modulation device is used to record the derivative spectra, where a beam of radiation differs in wavelength by a small change (1-2 nm) and difference between the two readings is recorded, computerized method is widely used to obtain derivative curves. Derivative spectra can obtain by variety of experimental techniques; the differentiation can be done numerically even if the spectrum has been recorded digitally or in computerized readable form. Department of Pharmaceutics, SPMVV 7

8. Department of Pharmaceutics, SPMVV The way of obtaining the derivative orders- Derivative spectroscopy accomplishes conversion of a normal or zero order spectrums to its first, second or higher derivative spectrum. The normal spectra is known as fundamental , zero order or D˚ spectra. Appropriate selection of derivative order gives useful separation of overlapped signals. 8

9. Department of Pharmaceutics, SPMVV Zero order derivative spectrum Zero order derivative is initial step of giving further derivatives i.e., zero th  order spectrum can give n th  order derivative. D  spectrum i.e. zeroth order is a representative feature of normal absorption spectrum. The 1 st , 2 nd , 3 rd  and 4 th  order derivative spectra can be obtained directly from the zero th  order spectrum. An increase in order of derivatives increases the sensitivity of determination. If a spectrum is expressed as absorbance (A) as a function of wavelength (λ), the derivative spectra is given as, A=f( λ) 9

10. Department of Pharmaceutics, SPMVV First order derivative spectrum Spectra obtained by derivatizing zero order spectrum once. It is a plot of change of absorbance with wavelength against wavelength i.e. rate of change of the absorbance with wavelength, dA/d λ = f’( λ ) First order spectra passes through zero as λ max of the absorbance band. Absorbance band of first order derivative shows certain positive and negative band with maxima and minima. By scanning the spectrum with a minimum and constant difference between two wavelengths, dual-wavelength spectrophotometer obtains first-derivative spectra. It is characterized by a maximum, a minimum and cross over point at the λ max of the absorption band. 10

11. Department of Pharmaceutics, SPMVV Second order derivative spectrum Derivatizing the absorbance spectrum twice gives this type of spectra. It is a plot of curvature of absorption spectrum against wavelength, d2A/d λ2= f’( λ) Second derivative has direct relation with concentration i.e. directly proportional. d 2 A/dλ 2  must be large, large the ratio greater is the sensitivity. The method is useful in obtaining atomic and gas molecular spectra. Third order derivative spectrum Unlike second order spectrum third derivative spectrum shows disperse function to that of original curve, d3A/d λ3= f’''( λ) 11

12. Department of Pharmaceutics, SPMVV Fourth order derivative spectrum Fourth order is inverted spectrum of second order and has a sharper central peak than the original band, Narrow bands are selectively determined by fourth derivative (UV-high pressure), d4A/d λ4= f‘’( λ) Quantitatively for second or fourth order derivative curves, peak heights are measured of long-wave peak satellite or for short-wave peak satellite. 12

13. Department of Pharmaceutics, SPMVV In case of multicomponent analysis, the spectral differences of assayed compounds and their selective determination can be increased by the use of different polynomial degrees. Polynomial degree has a great impact on number of polynomial points rather than on shape of derivative. The scope of polynomial is less; differentiation of spectra of half-width is used by low degree polynomials and that for spectra of small half -width by higher degree polynomials. Distorted derivative spectrum is a result of inappropriate polynomial degree. Polynomial Degree 13

14. NOISE-TO-SIGNAL RATIO Department of Pharmaceutics, SPMVV Signal-to-noise ratio  can be defined as the mean value of a  signal  (S) divided by the standard deviation of the background (N). The noise is responsible for sharpest features in the spectrum. Signal is defined as the difference between the largest (most positive) and the smallest (most negative) value of the derivative. S/N can be improved prior to derivatization if spectrophotometer would scan spectra and average multiple spectra. Best signal-to-noise ratio can be obtained by taking the difference between the highest maximum and the lowest minimum, but this leads to enhanced sensitivity to interference from other components . The  signal  becomes impossible to see when S/N drops below 2 or 3. Noise of signal is expressed by standard deviation σ. Standard deviation σ  expresses the noise of normal spectrum of the absorbance of blank while standard deviation σ n  expresses n th  order derivative that can be calculated by σ . 14

15. Department of Pharmaceutics, SPMVV Smoothing of spectra- Increase in signal-to-noise ratio generates many worse conditions, to lessen the condition or to decrease the high-frequency noise, technique is used  viz;  low-pass filtering or smoothing. Smoothing is an operation that is performed on spectra separately on each row of the data and acts on adjacent variables. The smoothing effect depends upon two variables mainly on: (a) Frequency of smoothing and (b) the smoothing ratio i.e. ratio of width of the smoothed peak to the number M of data points . With sufficient smoothing, high order derivatives can be obtained with only modest signal-to-noise ratio degradation compared to zeroth order. A first derivative smoothed by one pass of an N-point sliding average smooth has the general form, ( - ), ( - )…. A standard deviation of- /N   15

16. INSTRUMENTATION Department of Pharmaceutics, SPMVV Derivative spectra may be generated by 3 techniques: Modification of optical system- Spectrophotometers with dual monochromators, set a small wavelength interval ( λ typically 1-3nm) apart or with the facility to oscillate wavelength over a small range are required.   In either case, photodetector generates a signal with an amplitude proportional to slope of spectrum over wavelength interval. 16

17. Department of Pharmaceutics, SPMVV Electronic differentiation of spectrophotometer analogue signal- Resistance capacitance(RC) modules are highly dependent on instrumental parameters, the scan speed and the time constant. Resistance capacitance modules may be incorporated in series between spectrophotometer and recorder to provide differentiation of absorbance. Standard solution of analysis is employed to calibrate the measured value under the instrumental condition selected. Based on microcomputers differentiation- Derivative spectra during or after scan. To measure derivative amplitudes between specified wavelengths. To calculate concentrations and associated statistics from the measured amplitudes. 17

18. Department of Pharmaceutics, SPMVV ADVANTAGES DISADVANTAGES Even in small wavelength range, in presence of two or more overlapped peaks, absorbance bands can be identified. Even though it is sensitive method still it is highly susceptible to various parameters. In presence of strong and sharp absorbance peak, weak and small absorbance peak can be identified. The method is limited to particular system only and has limited applications due to its less reproducibility. Broad absorbance spectrum gives clear idea about the particular wavelength at that maximum spectrum. It is less accurate in measuring zero-crossing spectra. Even in presence of existed background absorption, the quantitative analysis can studied as there is linear relationship between the derivative values and the concentration levels. There is likeness in shape of derivative spectra and zero order spectrum, so small variation in a basic spectrum can strongly modify derivative spectrum. 18

19. Department of Pharmaceutics, SPMVV APPLICATIONS OF DERIVATIVE SPECTROSCOPY Better qualitative analysis and identification of the number of absorbing species in a sample. Single component analysis. Accurate determination of . Obtaining spectra in solutions with high scattering is possible using dual wavelength instruments. Multicomponent analysis- Spectral derivatization can remove the prevalence caused by spectra of disturbing compounds . Bioanalytical application- Amphotericin and Diazepam has been determined in human plasma with its order of derivatives. Forensic toxicology- Derivative spectroscopy has its application in toxicology especially of illicit drugs  viz ; amphetamine, ephedrine, meperidine, diazepam, etc. and can also be used in mixtures. Trace analysis- Derivative signal processing technique is widely used in practical analytical work in measurement of small amounts of substances in the presence of large amounts of potentially interfering substances; in the conditions like non-specific broadband interfering absorption, non-reproducible cuvette positioning, dirt or fingerprints on the cuvette walls, etc.   19

20. Department of Pharmaceutics, SPMVV APPLICATIONS OF UV-VISIBLE SPECTROSCOPY Detection of impurities Elucidation of organic compounds- For example, elucidation of structure of Vitamin K1 & K2 and Vitamin A1&A2. Vitamin K1&K2- dimethyl naphtha-quinone Vitamin A1&A2- additional ethylic bond Quantitative analysis- Based on Beer’s Law Chemical Kinetics Qualitative analysis of pharmaceutical substances- Diazepam tablet can be analysed by 0.5% in methanol at the wavelength 284nm. As HPLC detector   20

21. Department of Pharmaceutics, SPMVV Detection of functional groups Molecular weight determination- Amine Amine picrate Dissolved in 1ltr of solution Optical density measured at 380nm Examination of polynuclear hydrocarbons Extent of conjugation- Extent of conjugation in polyene can be estimated, (RCH=CH)n-R   Naphthalene Diphenyl 21

22. Department of Pharmaceutics, SPMVV REFERENCES J. Chil. Chem. Soc. vol.63 no.3 Concepción  2018, A Review on D erivative UV -S pectrophotometry analysis of drugs in pharmaceutical formulations and biological samples review , Journal of the Chilean Chemical Society, version On-line ISSN 0717-9707. T. C. O'Haver  and  T. Begley , Signal-to-noise ratio in higher order derivative spectrometry, Anal. Chem .  1981, 53, 12, 1876–1878. Thomas C. O'Haver ,    Anthony F. Fell ,    Geoffrey Smith , Derivative spectroscopy and its applications in analysis , Anal. Proc., 1982,19, 22-46 . Dr. S. Ravi Sankar , Textbook of Pharmaceutical Analysis , 5 th edition. Sharma. YR ., Elementary Organic Spectroscopy , 1 st edition, S. Chand publisher; 2010. Chatwal G.R ., Instrumental methods of C hemical Analysis , 1 st edition, Himalaya publisher; 2010. 7. https://www.avantes.com/applications/cases/uv-vis-spectroscopy-applications-in-chemistry/ 23

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