Derivative UV-Vis Spectroscopy Guide

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

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

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.

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

MEASUREMENT TECHNIQUES
Measurement of derivative spectra value is achieved out by the following three methods:
1. 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

DERIVATIVE SPECTRA
 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

 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.

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

Zero order derivative spectrum
• Zero order derivative is initial step of giving further derivatives i.e.,
zeroth order spectrum can give nth order derivative. D0 spectrum i.e.
zeroth order is a representative feature of normal absorption spectrum.
• The 1st, 2nd, 3rd and 4th order derivative spectra can be obtained directly
from the zeroth 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

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/dλ=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 λmax of the absorption band.
10

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/dλ2=f’(λ)
• Second derivative has direct relation with concentration i.e. directly
proportional. d2A/dλ2 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/dλ3=f’''(λ)
11

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/dλ4=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

• 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

NOISE-TO-SIGNAL RATIO
• 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 σ0 expresses the noise
of normal spectrum of the absorbance of blank while standard deviation σn expresses nth order
derivative that can be calculated by σ0.
14

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,
𝟏
𝑵
(𝒂𝑵+𝟏-𝒂𝟏),
𝟏
𝑵
(𝒂𝑵+𝟐-𝒂𝟐)….
• A standard deviation of- 𝟐𝟏/𝟐
𝝈𝒐/N
15

INSTRUMENTATION
• Derivative spectra may be generated
by 3 techniques:
1. 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

2. 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.
3. 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

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

APPLICATIONS OF DERIVATIVE SPECTROSCOPY
1. Better qualitative analysis and identification of the number of absorbing species in a sample.
2. Single component analysis.
3. Accurate determination of λ𝑚𝑎𝑥.
4. Obtaining spectra in solutions with high scattering is possible using dual wavelength instruments.
5. Multicomponent analysis- Spectral derivatization can remove the prevalence caused by spectra of
disturbing compounds.
6. Bioanalytical application- Amphotericin and Diazepam has been determined in human plasma
with its order of derivatives.
7. 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.
8. 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

APPLICATIONS OF UV-VISIBLE SPECTROSCOPY
1. Detection of impurities
2. 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
3. Quantitative analysis- Based on Beer’s Law
𝐴 = 𝑙𝑜𝑔
𝐼𝑂
𝐼𝑡
= 𝑙𝑜𝑔
1
𝑇
= −𝑙𝑜𝑔𝑇 = 𝑎𝑏𝑐 = 𝜀𝑏𝑐
4. Chemical Kinetics
5. Qualitative analysis of pharmaceutical substances- Diazepam tablet can
be analysed by 0.5% 𝐻2𝑆𝑂4 in methanol at the wavelength 284nm.
6. As HPLC detector
20

7. Detection of functional groups
8. Molecular weight determination-
• Amine Amine picrate Dissolved in 1ltr of solution
• Optical density measured at λ𝑚𝑎𝑥 380nm
𝑪 =
𝐥𝐨𝐠 𝑰𝒐/𝑰𝒕
𝜺𝒎𝒂𝒙 𝟏
9. Examination of polynuclear hydrocarbons
10. Extent of conjugation- Extent of conjugation in polyene can be
estimated, (RCH=CH)n-R
Naphthalene Diphenyl
21

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

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