Photometric Techniques Are Important To The Pharmaceutical Analyst Biology Essay

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Photometric techniques are the most important instrumental techniques which are available to the pharmaceutical analyst. For example: flame photometers are used to determine the concentration of certain metallic elements. UV visible and nuclear magnetic resonance spectrophotometers are used to determine the structural and quantitative analysis of molecules. The basics of all these instrumental techniques measure the interaction of electromagnetic radiation with matter in quantized specific energy levels.

The quantitative measurement of such multi component formulations were very important and the quantitative estimation method is to determine the how much of each constituent present in the sample

For these quantitative measurements spectrophotometer has become a useful instrument for drug analysis, and simultaneous estimation methods are suitable methods for the quantitation of such multi component formulations .And such various methods are employed in the quatitation of multi component formulations, method validation which ensures the selective method for reproducible and reliable results used for intended purpose were explained in this review.

Introduction: Analytical methods in a broad sense classified in to two methods one is chemical method and the second is instrumental method. Chemical operations depends upon combination with simple instruments in general the measurement of mass and volume i.e. of gravimetric and volumetric .And instrumental methods which depends upon the use of complicated instrumentation based on analytical methods.

Although spectrophotometric methods are extensively used in recent years but it is wrong to conclude that chemical methods are totally replaced by instrumental methods. Various chemical steps are involved as a part of analytical methods like sampling dissolution change in oxidation state addition of complexing agent removal of excess reagent pH adjustment. The HPLC is used is extensively used in the recent years because of good ability in the separation of macromolecules and ionic species, liable natural products, polymeric materials and also wide variety of polyfunctional groups of highly molecular weight are actively performed because of high pressure created in HPLC

In spite of more advantages of HPLC we often select spectrophotometric methods for the analysis of drug .because high cost of the instrument and the costly analytical process, small scale industries cannot afford to procure the HPLC. Hence spectrophotometric methods are often used for the quantification of the analytes.

8.1.1 Theory of Spectrophotometry and Colorimetry: Wavelength and Energy:

Absorption and emission of radiant energy by molecules and atoms is the source for the optical spectroscopy. By based up on the data of interpretation both qualitative and quantitative estimations can be determined. We can quantitatively identify the presence of a specific substance based on positions of absorption and emission bands are lines obtains on electromagnetic spectrum. Quantitatively, we can identify the intensities of the absorption and emission lines or bands for the unknown and standards. The concentrations and intensities of the of the unknown is determined from the data,

In the electromagnetic spectrum the absorbance's and emission of energy occurs as discrete packets of photons. The relation between the energy of a photon frequency is described of its propagation as


Where E is represented as Energy in ergs

V is represented as frequency as cycles per second

And h is represented as planks constant (6.6256x10-27erg/sec)

In spectroscopic measurement the plotting of data based on radiant energy absorbed are emitted as a function of position in the electromagnetic spectrum. And this is known as spectrum and the absorbance or emission is measured in the units of energy wavelength and frequency.

Beer-Lambert's law:

Colorimetry is based on the determination of light absorbing capacity of a system and the quantitative determination is therefore carried by subjecting to those wavelengths which are absorbed by the visible energy. The absorption bands of UV and Visible are due to the electronic transitions in the region of 200-800nm.and there are four types of absorption bands that occur due to the electronic transitions of a molecule,

R - Bands: Due to C=O or NO2 group

K-Bands: in conjugated systems

B -Bands (Benzonoid bands): Due to aromatic and heteroaromatic systems

E -Bands (etylinic bands): In aromatic systems

When the monochromatic light falls on the homogeneous medium, some of the portion of incident light is reflected and a portion of light is absorbed with in the medium and the remaining is transmitted.

                                              I = Ia + It + Ir   .......................... (1)

Incident light is expressed as I

Absorbed light is expressed by Ia

Transmitted light is expressed by It

Reflected light is expressed as Ir

Credit for investigating the change of absorption of light with the thickness of the medium is frequently given to Lambert; Beer later applied similar experiments to solutions of different concentrations and published his results. The two separate laws governing absorption are usually known as Lambert's law and Beer's law. In the form they are referred to as the

Beer-Lambert law. Mathematically, the radiation-concentration and radiation-path-length relation can be expressed by [40]

                      Image...................... (2)

The more familiar equation used in spectrometry

log (Io/It) = Î cl .........................(3)

Where                          'Io'    is the intensity of the incident energy

                                     'It'      is the intensity of the emergent energy

                                     'c'      is the concentration

                                     'l'        is the thickness of the absorber (in cm)

And Π     is the molar absorbtivity for concentration in moles/L

  Image, which is encountered less frequently in the literature, represents absorbance of a solution of concentration 1% w/v and sample thickness of 1 cm cell and is used primarily in the investigation of those substances of unknown or undetermined molecular weight. A typical UV absorption spectrum, shown in fig no 7.1, is the result of plotting wavelength v/s absorbtivity, Îmax is denoted by lmax.


Fig. 7.1: A representative Beer-Lambert law plot

Principal of Quantitatative spectrometric Assay :

Assay of the absorbing substance can be carried out by preparing the solution by using transparent solvent then the solution absorbance can be checked at a suitable wave length.

Principle procedures to measure the absorbance:

By using standard absorbitivity value

Calibration graph method

Single or Double point standardization method

Different Spectrometrometric simultaneous estimation methods for multi component samples:

It mainly involves measurement of absorbance of analyte containing more than one absorbing component.

Principle procedures to measure the absorbance in multi component formulations:

Assay as a single component sample:

By using the spectrophotomeric measurements the concentration of sample contain other absorbing substances can determined.

Assay using absorbance corrected for interferences:

The concentration and absorbitivity of the absorbing of the interferences are knows from that we can calculate their calculation from the total absorption of the mixture.

Simultaneous equation method: If a sample contains two absorbing drugs (X and Y) each of which absorbs at the lmax of the other, it may be possible to determine both drugs by the technique of simultaneous equations (Vierodt's method).



a) The absorptivity of X at λ 1 and λ 2, ax1 and ax2 respectively.

b) The absorptivity of Y at λ 1 and λ 2, ay1 and ay2 respectively.

c)  The absorbance of the diluted sample at λ 1 and λ 2 are A1 and A2 respectively.

Criteria for obtaining maximum precision, based upon absorbance ratios that place limits on the relative concentrations of the components of the mixture.

The criteria are that the ratios               


Should lie outside the range 0.1- 2.0 for the precise determination of Y and X respectively. These criteria are satisfied only when the λ max of the two components are reasonably dissimilar. An additional criterion is that the two components do not interact chemically.

To reduce the random errors during measurements, sometimes instead of carrying out analysis of two components at two wavelengths, it is carried out at 3 or 4 wavelengths. The equations will no longer have a unique solution but the best solution can be finding out by the least square criterion.

Absorbance Ratio method [43]: The absorbance ratio method is a modification of the simultaneous equations procedure. It depends on the property that, for a substance, which obeys Beer's law at all wavelengths. Q-analysis is based on the relationship between absorbance ratio value of a binary mixture and relative concentrations of such a mixture. The ratio of two absorbance determined on the same solution at two different wavelengths is constant. This constant was termed as "Hufner's Quotient' or Q-value which is independent of concentration and solution thickness e.g. two different dilutions of the same substances give the same absorbance ratio A1/ A2. In the USP this ratio is referred to as a Q value. In the quantitative assay of two components in admixture by the absorbance ratio method, absorbance are measured at two wavelengths, one being the λ max of one of the components (λ 2) and the other being a wavelength of equal absorptivity of the two components (λ 1), an iso-absorptive point.                                   


A1 =Absorbance of the mixture at isoabsorptive point i.e 270 nm

A2 = Absorbance of the mixture at 245 nm

ax1and ay1 = A (1%1cm) values of both drugs at iso absorptive point

ax2 and ay2 = A (1%1cm) values of both drugs at 245 nm





 Equation gives the concentration of X in terms of absorbance ratios, the absorbance of the mixture and the absorptivity of the compounds at the iso-absorptive wavelengths. Accurate dilutions of the sample solution and of the standard solutions of X and Y are necessary for the accurate measurement of A1 and A2 respectively.

Geometric correction method:

To reduce or eliminate the irrevelent absorption of the sample of the biological origin, a no. of mathematical correction procedures have been developed. In those the simplest procedures have been developed. In those the simplest procedure is the three point geometric procedure.

Three point geometric procedure: This is applied only if the irrevelent absorption is linear at the three wave lengths λ1 λ2and λ3 to that back ground absorbances B1B2andB3 are linear than the corrected absorbance D of the drug may be calculated from the three absorbances A1,A2, A3of the sample solution at λ1 λ2and λ3

Let vD and wD be the absorbance of the drug alone in the sample solution at λ 1 and λ 3 respectively i.e.,v and w are the absorbance ratios λ 1 and λ 3 Respectively.

B1 = A1 - vD, B2 = A2 -D and B3 = A3 -wD

Let y and z be the wave lengths intervals (λ 2 - λ 1) and (λ 3- λ 2) respectively.

D= y(A2 -A3) + z(A2 - A1) / y (1-w) + z(1-v)

This general equation which may be applied in any situation where is A1, A2 and A3 of the sample , the wavelength intervals y and z and the absorbance ratio V and W are known

Orthogonal polynomial method:

Another mathematical correction procedure .it is more complex than three point correction procedure. It is more complex than three point correction procedure

The basic of the method that an absorption spectrum is represented in terms of orthogonal functions as follows.

A(λ ) = p P (λ ) + p1 P1 (λ ) + p2 P2 (λ ) ….. pn Pn (λ )

A-Absorbance at wavelengths λ belonging to a set of n+1 equally spaced wavelengths at which the orthogonal polynomials P (λ ) ,  P1 (λ ), P2 (λ ) …..  Pn (λ ) are defined.

In orthogonal functions procedures The accuracy depends on the correct choice of the set wave lengths and polynomial order

The selection of Quadratic or cubic polynomials depends on the shape of absorption spectra of the drug and irrevelent absorption .

The set of wavelengths is defines to the number of wavelengths , interval and the mean wavelengths of the(λ m)..apparent linear irrevelent absorptions normally eliminated using 6-8 wavelengths if the irrevelent absorption contains high frequency components.than more than 20 wave lengths required The wave lengths interval and (λ m) are obtained from the convulated absorption curve.. The absorptivity coefficient were plotted for a specified order of polynomial, a specified No.of wavelengths and specified wavelengths interval against the ; (λ m) of the set of wave lengths . The maximum or minimum wavelengths corresponds to the convoluted curve of the analyte and with the coefficient Zero for irrevelent absorption. in this connvuluted curve In the concentration of an absorbing drug in ad mixture with another may be caluculated

Difference spectrophotometry:

It is a sensitive method for detecting small changes in the environment of a chromophore or it can be used to demonstrate ionization of a chromophore leading to identification and quatitation of various components in a mixture. difference spectrometry is improved technique because the selectivity and accuracy of spectrophotometric samples can be analysed containing the absorbance interferents markedly improved. The essential feature for this different spectrometric assay is that it shows the difference absorbance (Δ A) between two equimolar solutions of the analyte in different forms which exhibit different spectral characterstic s.

In the presence of other absorbing substances we can apply the difference spectrophotometry for the assay of the method

A; Reproducible by addition of one or more reagents. Reproducible changes may be included in the spectrum of the analyte

B. The absorbance of interfering substances is not altered by the reagents.

The adjustment of the pH by means of aqueous solutions of acids, alkali or buffers is most common and simplest technique employed for altering the spectral properties of the analyte .The UV-Vis absorptions spectra of many ionisable functional groups. Eg: phenols, aromatic carboxylic acids and its eminent depend on the state of ionization of the functional groups and pH of the solution.

If the individual absorbances Aalk and Aacid which proportional to the concentration of the analyte and path length the Δ A also obeys the Beer-Lambert law and a followed equation may be derived

  Δ A = Δ abc

When Δ a is the difference absorptivity of substance of the wavelength of measurement.

when one or more other substances are present in the sample at analyte absorbance.;;;;;;;;;;;;in the alkaline and acidic solutions the interferences of spectrophotometric measurement were eliminated

Δ A = (Aalk + Ax) - (Aacid + Ax)

The selectivity of the Δ A procedure depends on the choice of pH values to induce the spectral change of the analyte without altering of the interfering components absorbance of the sample. For inducing the Δ A of an analyte is convenient The use of 0.1M NaoH &0.1.M Hcl to induce the pKa of the analyte is convenient and satisfactory.

Derivative spectrophotometer: For the determination multi component formulation in the direct spectrophotometric determination is often complicated by interference from formulation matrix and spectral overlapping such interferences .by the derivative spectrophotometry the useful means of resolving two overlapping spectra and eliminating interferences which caused by the matrix which is imprecise on side of an absorption band. Derivative spectrophotometry involves in the conversion of a normal spectrum to its first, second or higher derivative spectrum . Here the normal absorption is referred to as fundamental,Zeroth order or D spectrum. The absorbance of a sample is differentiated with respect to wavelength; λ to generate first second or higher order derivative.

[A]     = f (λ ): zero order

[dA/dλ ]   = f (λ ): first order

[d2A/d λ 2]  = f (λ ): second order

is characterized by a and a cross over point at the λ max of the absorption band maximum a minimum can be characterized by The first derivative spectrum of an absorption band The by two satellite maxima and an inverted band of which the minimum corresponds to the λ max of the fundamental band is characterized by second derivative spectrum

The important features of derivative technique in quantitative analysis it enhances the enhanced information content, discrimination against back ground noise and greater selectivity derivative technique is useful for detection and determination of determination of impurities in drugs chemicals and also in food additives and industrial wastes.

Analytical method validation:

Regulatory perspective: in us there was no word of validation in cGMP's 1971 but precision and accuracy were stated in the laboratory samples. The need for the validation was implied in the cGMP's guide lines of march 28, 1979

It is done in the two sections:

Section 211.165, word validation was used

Section 211.194 proof for suitability

Accuracy and reliability are the two parameters which made compulsory for the regulatory submission. The guide line published WHO expert for the examination of pharmaceutical materials.

WHO reported in the 32nd report the expert committee (titled validation of analytical procedure

Published in the year of 1992

ICH harmonized tripartite guidelines developed ICH titled on text on validation of analytical procedures (Q2A)' (Q2B)' on the 1st march 1999

And the FDA is also published a final guideline on analytical procedures Selection of parameters in the analytical method:

When the problem is obtained the following factors should be considered in the analytical method: these are of concentration range and sensitivity selectivity time requirements required accuracy and cost of analysis.

Concentration range:

Concentration range is the parameter that which ability to match the method to optimum size this awareness is usually gained through experience on different methods

Sensitivity which is applied to the analytical method which corresponds to the minimum or lowest concentration of substance which is detected by specific reliability. This is numerically expressed as detection of limit and sensitivity. Accuracy is parameter which refers to the correctness of the result achieved by the analytical method


The ability of the method ensures the analytical concentration and its response in the presence of other potential components or interferences.

Time and cost:

For the completion of determination time and cost goes to hand in hand personnel and space.

Analytical method validation:

Regulatory perspective: in us there was no word of validation in CGMP's 1971 but precision and accuracy were stated in the laboratory samples. The need for the validation was implied in the CGMP"S guide lines of march 28, 1979

It is done in the two sections:

Section 211.165, word validation was used

Section 211.194 proof for suitability

Accuracy and reliability are the two parameters which made compulsory for the regulatory submission. The guide line published WHO expert for the examination of pharmaceutical materials.

WHO reported in the 32nd report the expert committee (titled validation of analytical procedure

Published in the year of 1992

ICH harmonized tripartite guidelines developed ICH titled on text on validation of analytical procedures (Q2A) (Q2B) on the 1st march 1999

And the FDA is also published a final guidelines on analytical procedures

Method validation:

Method validation ensures that, the analytical method is suitable for its reliable and its intended use.

The acceptability range of all validation parameters for a particular method based according to the guidelines (USP, ICH, and FDA)

Depending on the Drug study, drug development stage and regulatory reviews, the validation requirements can change. At the 1 st stage of the drug development performance of various validation studies is not necessary Linearity, accuracy, specificity and precision are the validation parameters during preclinical phase II stages. And the remaining validation parameters can be proved on drug research at phase III. Based on the results of validation data we can assume any changes in the method procedure if necessary. The method validation parameters proved during the validation play key and helpful for subsequent validation steps.

Method validation parameters:







Detection limit

Quantification limit


Establish minimum criteria:

The first step in the method development and validation cycle should be to set minimum requirements, which are essentially acceptance specifications for the method. A complete list of criteria should be agreed on by the developer and the end users before the method is developed so that expectations are clear. For example, is it critical that method precision (RSD) be 2%? Does the method need to be accurate to within 2% of the target concentration? During the actual studies and in the final validation report, these criteria will allow clear judgment about the acceptability of the analytical method. The statistics generated for making comparisons are similar to what analysts will generate later in the routine use of the method and therefore can serve as a tool for evaluating later questionable data. More rigorous statistical evaluation techniques are available and should be used in some instances, but these may not allow as direct a comparison for method troubleshooting during routine use.


The specificity parameter ensures that, the intended method can able to measure the analyte response in the presences of other potential sample components. In this experiment the response of the analyte test sample which contains analyte and other (placebo formulation, synthesis intermediates, process impurities, degradation product and excipients) is compared against the analytical response of the solution of only analyte sample. If the method is able to give the analytical responses in the presence then the method is aid to specific.


Accuracy defines the closeness of the experimental values with that of true value.

The accuracy always expressed in terms of % Mean accuracy.

Determination range:

For the determination of an active substance or a finished product normally from 80-120% of the test concentration.

For the determination of an impurity; from reporting level o an impurity to 120% of the Specification.

For content uniformity; covering minimum range is from 70-130% of the test concentration, unless a wider more range based on the nature of dosage form.

For the dissolution testing ±20% over the specified range.


Precision is defined as the "Degree of the reproducibility or repeatability while doing the (experiment) measurements or calculations shows the similar the results.

Precision method is represented by % CV (coefficient of variation.

%CV= (SD/Mean)*100

SD=Standard deviation

Both accuracy and precision determined by two ways:

Within batch precision/ accuracy

Between batch precision/ accuracy

Detection limit:

It is one of the validation parameter used to ensure that the intended analytical method can able detect the responses of lowest analyte concentration from the noise level of the is very importent parameter done during the early stages of method- development and validation process.

Quantification limit:

It is the parameter which gives the lowest level of analyte concentrations accurate and precisely.


This parameter mainly proved to ensure the ability of method that means it remains unaffected by the small changes in the method parameters .(e.g: temperature, change in buffer concentration, PH of the solvent)


Stability experiment procedure ensures the stability of analyte during sample analysis.

It can be performed by analysis of the Stability samples against comparative (freshly prepared) samples.