Instrumental Methods of Analysis 2022-2023

UNIT I
INTRODUCTION TO INSTRUMENTAL METHODS

ANALYSIS

Analysis refers to the detailed examination of the elements and their structure.
Chemical analysis uses instruments and methods used to separate, identify, and quantify
matter. Analytical chemistry is the science of obtaining, processing, and communicating
information about the composition and structure of matter.

Analysis

Qualitative Quantitative
Analysis Analysis

Qualitative analysis
Qualitative analysis deals with the identification of elements or functional groups
present in a sample. In short, quantitative analysis answers, “What is it?”
Example: inorganic salt analysis (identification of cations and anions), organic elemental
analysis, organic functional group analysis.

Quantitative analysis
Quantitative analysis deals with the quantification of the substance. It tells you
how much substance is present. In short, quantitative analysis answers, “How much is
it?” Example: Volumetric analysis (titrations)

Methods of
Analysis

Classical Instrumental
Methods Methods

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 Classical methods are the techniques, which forms the fundamentals of laboratory
practices. These are the traditional method of chemical analysis, which is still being used
by scientists. Classical method is cheaper and easily available for schools and industries.
It is sometimes called as "wet chemistry" where there are too many chemical reactions
are used to identify certain compounds. The classical method consumes more time than
the instrumental analysis. These methods are often labor intensive. The results are often
accurate and precise. Examples: Quantitative analysis performed by gravimetric and
volumetric (titrimetric) methods.

Instrumental Analysis
Instrumental methods are often called modern method of analysis. The reason is
that these analytical techniques use modern equipment’s such as computers and other
electronic equipment. Instrumental method is expensive because the machines are highly
specialized for a particular chemical analysis. Quantitative results are often more accurate
and precise than the classical methods. Precision is dependent less on the operator and
more on the instrument and sources of noise. Not all schools and colleges can afford
those machines. Machines used are quite sophisticated that experts are needed to operate
the machines to avoid system malfunctioning.
Examples: Qualitative analysis using Gas Chromatography, High Performance Liquid
Chromatography, NMR (Nuclear Magnetic Resonance) spectroscopy etc.

Advantages
• Less labor intensive
• Easy to automate
• Simultaneous multi component analysis
• Fast analysis
• Lower detection limits

Disadvantages
• Higher expense
• Harder to trouble shoot problems, more technical expertise

Lambert’s Law
Lambert’s law explains the relationship between absorption of light by molecules and
thickness of the medium. Lambert’s law can be stated as “When a beam of monochromatic radiation
is allowed to pass through a homogeneous absorbing medium, the rate of decrease of intensity of
radiation dI, with the thickness of absorbing medium dx is proportional to intensity of incident
radiation, I”.
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Mathematically Lambert’s may be stated as,

Where I denotes the intensity of incident radiation of wavelength λ, t denotes the thickness
of the medium and kdenotes the proportionality factor. On integrating the above equation and putting
I=I0 and t=0, we get

Where I0 is the intensity of incident radiation, It is the intensity of transmitted radiation and
kis the constant which depends upon the wavelength and absorbing medium used.

In the above equation K is the absorption coefficient defined as, reciprocal of thickess
which is require to reduce the light to 1/10 of its intensity

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 The ratio I0/It is termed as transmittance, T and the ratio It / I0 is termed as absorbance, A
of the medium. Formerly, absorbance was termed as optical density, D or extinction coefficient, E.
the ratio is termed as opacity.

Beer’s Law
Lambert’s law show that there exist a logarithmic relationship between transmittance and
the length of the optical path through the sample. Beer observed that a similar relationship holds
between transmittance and concentration of solution. The intensity of monochromatic light decreases
exponentially with the increase in concentration of the absorbing substance arithmetically.

Where k and k; are constant ad c is the concentration of the absorbing substance.

The above equation is termed as the mathematical statement of Beer-Lambert’s law. This
is also the fundamental equation of spectrophotometry.

Where ε is a new constant called as molar absorptivity or molar extinction coefficient. Its
unit depends on the unit of concentration and thickness. The concentration is measured in terms of
mol dm-3 and x in centimeters. When c=1mol dm-3, x = 1 cm, then

A=ε
Molar absorption coefficient is the absorbance of the solution when concentration is 1mol
-3
dm and path length is 1 cm.

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Application of Beer-Lambert’s law
Beer-Lambert’s law is useful for qualitative and quantitative analysis and also for
determination of percentage purity of an analyte sample.
1. Determination of purity of sample
The value of ε is calculated by Beer-Lambert’s law from the measured absorbance value of
a standard solution of the absorbing species. By comparing the ε value of the test sample with that of
the standard substance, the percentage of purity of sample can be determined.
2. Determination of unknown concentration
By knowing the absorbance of the standard solution the absorbance of the unknown
solution can be found.
As = ε C s x
Au= ε Cu x
Where As and Au are the absorbane of standard and unknown solution respectively, Cs and
Cu are concentrations of standard and unknown solution respectively and x is the path length.

Experimental procedure
A series of concentrations of standard solution (10, 20, 30, 40, 50 ppm) are prepared.
Corresponding absorbance is measured using colorimeter or spectrophotometer. A blank solution with
reagents alone is also prepared and the absorbance of this blank is set as zero. Absorbance increases
linearly with concentration. After measuring absorbance of standard solutions, the absorbance of the
unknown sample is measured. Concentration corresponding to that particular absorbance is the
concentration of unknown sample.

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3. Identification of sample
ε, the molar extinction coefficient is a physical constant. Therefore, by knowing or
comparing ε values, chemical identity of a sample can be found out.
4. Determining rate of a reaction
The change in concentration can be followed by measuring absorbance (A) as a function
of time. This is useful in chemical kinetics for determining rate of chemical reactions.
5. Colorimetric estimation of metal ions
Lambert-Beer’s law is the principle of colorimetric estimation. Metals like Fe, Co, Ni, and
Cu etc. can be estimated even in low concentrations using colorimetry. Basic principle of
colorimetry is that absorbance of a solution varies with concentration. Color of the solution is
measured using a spectrophotometer. Ferric ion reacts with thiocyanate ion, forming a deep blood
red colored complex which can be estimated by colorimetry.
Fe3+ + 6KSCN → [Fe (SCN) 6]3– + 6K+
Colorless colorless Red

Limitations of Lambert-Beer’s law

1. The law is obeyed when radiation is monochromatic
2. It is applicable only for dilute solutions.
3. The temperature of the system should not vary significantly.
4. It is applicable only for true solutions (non colloidal solution)
5. Impurities present in solution can cause deviation.
6. The law is not obeyed, if there is association or dissociation of sample in solution
Deviations of Lambert-Beer’s law
In general three types of deviations from the law occur under experimental conditions
limiting the application of law. The deviation includes

• Real deviations
• Chemical deviations
• Instrumental deviations

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 Real deviations
It occurs at higher concentration of the absorbing species. The absorptivity ε of the
solution changes with the concentration depending on the refractive index η of the solution
according to the equation,
ε = ε true [η / (η2+22)]
Since refractive index varies considerably at concentration higher than 10-3 M, absorptivity also
varies. In addition refractive index also depends on the wavelength of the incident radiation and is
a constant for a particular wavelength. Beer – lambert’s law was derived on the assumption of an
incident beam of monochromatic radiation and even the best monochromator system of an
instrument provides only polychromatic beam of radiation spread over a few wavelength.
Chemical deviations
These are absorbed due to the presence of more than one absorbing species in the solution.
The measured absorbance in such case is actually the sum of the absorbance of individual species
each having its own absorptivity value. For example the aqueous solution of potassium dichromate
containing dichromate ions (Cr2O72-) is orange in color and shows absorption maximum at 350 nm
and 450 nm in the visible region. Diluting this solution with water hydrolyses the dichromate ion to
chromate ion (CrO42- λ max = 375nm) which is yellow in color. Hence the diluted solution contains
two different absorbing species and results in no-conformity with Beer-Lambert’s law attributed to
the chemical deviation within the absorbing medium. The presence of two species may be
represented by the equilibrium

Conformity with Beer-Lambert’s law can be maintained by ensuring the presence of only
one absorbing species by maintaining appropriate experimental conditions. For recording the
absorbance of potassium dichromate solution at its absorption maximum at 450 nm a small amount
of dilute sulphuric acid is added to ensure that the above equilibrium is shifted to the left and only
dichromate ion remain in the solution. The absorbance of potassium dichromate solution can be
measured at 375 nm by adding a few ml of 0.05M potassium hydroxide to ensure the presence of
chromate ions only.
Instrumental deviations
This deviation arises depending upon the bandwidth of the instrument. The bandwidth of
the instrument depends on the wavelength resolving capacity of the optical system consisting of the
monochromator and slits and varies from instruments to instruments. More sophisticated instruments
with better resolving power have narrow bandwidth and allows incident radiation of wavelength with

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narrow wavelength range. The effect of instrument deviation is more with instrument having wide or
broad bandwidth and chemical system exhibiting narrow absorption bands. Instrument deviation can
be minimized by using the instrument with narrow bandwidth and proper selection of wavelength for
quantitative assays. The below figure indicates the effect of wavelength of selected for measuring the
absorbance and bandwidth of the instrument on the calibration plot. Measurement made at λ1, at or
close to the λ max , with narrow bandwidth gives a better conformity with Beer-Lambert’s law
compared to the other measurement with wide bandwidth.

Effect of selection of wavelength

Estimation of inorganic ions using Beer -Lambert's law

1. Estimation of iron
Iron content in the sample can be determined by complexing with 1, 10-phenanthroline.
Iron (II) forms an orange red color complex [Fe (C12H8N2)3]2+ while iron (III) forms yellow color
complex with the same reagent [Fe (C12H8N2)3]3+. Both iron (II) and (III) can be determined from
the absorbance of the complexes. The test sample containing both iron (II) and (III) is made
slightly acidic with sulphuric acid and is treated with 1, 10-phenanthroline reagent and buffered to
pH 3.9 with potassium hydrogen phthalate. The absorbance at 396 nm gives the total amount
while absorbance at 515 nm is due to iron (II) complex alone.
The iron (II) complex is quite stable and the intensity of orange-red color is independent of
pH in the range 2-9, and hence is used for spectrometric measurements. Any iron (III) that may be
present is reduced to iron (II) with hydroxylammonium chloride. A calibration chart is prepared
by using the series of standard solution of ferrous ammonium sulfate, buffering the solution with
0.2 M sodium acetate, reacting with 0.25% solution of 1, 10-phenanthroline monohydrate in water
and measuring the absorbance at 515 nm. The test sample is treated in a similar manner to form a
comlex. And its absorbance is measured under identical conditions. The calibration chart of
measured absorbance as a function of iron concentration is linear and the concentration of iron in
the test sample is determined graphically.

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2. Estimation of nickel
Nickel content of the alloy can be determined by forming a soluble red colored nickel-
DMG complex after completely removing the copper by precipitating as sulphide. The intensity of
color is directly proportional to the amount of nickel. The procedure involves dissolving a
weighed quantity of alloy in dilute hydrochloric acid and making up to a known volume. An
aliquot of the made up solution is pipetted out into a beaker and copper is precipitated as cupric
sulphide by passing hydrogen sulphide. The precipitate is filtered off and the filtrate is boiled with
a few mL of nitric acid to completely oxidize and expel any hydrogen sulphide. Nickel (II) in the
solution is oxidized to nickel (III) with bromine water (nickel (II) forms a precipitate with DMG
while nickel (III) forms a soluble complex) and the excess bromine water is destroyed by adding
ammonia. An alcoholic solution of DMG (1%) is added to the colorless solution nickel (III) and
excess aqueous ammonia is added to render the solution ammoniacal. The red color developed is
stable and its intensity is directly proportional to the amount of nickel present. The amount of
nickel present in the analyte solution is determined by comparing the intensity of the color with
that of a standard nickel-DMG solution prepared in a similar manner.

3. Estimation of nitrite and nitrate

Nitrite in environmental sample is determined spectrophotometrically by measuring the
absorbance of the dye formed by diazotizing sulphanilamide and coupling with N-(1-naphthyl)-
ethylenediamine dihydrohloride. The nitrite sample is treated with 2 mL of sulphanilamide
solution (prepared by dissolving 0.5 g of the amide in 100 mL of 20% hydrochloric acid) is added.
After about 5 minutes, 2 mL of N-(1-naphthyl)-ethylenediamine dihydrohloride solution (prepared
by dissolving 0.3 g of reagent in 100 mL of 1% hydrochloric acid) is added. The pH of the
reaction mixture at this point should be 1.5. After about 10 minutes, the absorbance of the dye
solution a 550nm against blank. The amount of nitrite in the test sample is determined from the
calibration chart prepared by using standard nitrite solution and treated in a similar manner.
Nitrate content in drinking water is determined by reacting with phenoldisulphonic acid to
give yellow colored solution which has absorption maxima at 410 nm. Chloride and nitrite at
concentration less than 2 ppm interfere in the determination. Chloride is precipitated by adding
silver sulphate solution (0.44%) and centrifuged. The clear centrifuge is used for nitrate
determination. The sample water is adjusted to pH 7 by adding dilute sodium hydroxide solution
and evaporated to dryness in china dish. The dry residue is dissolved in 2 ml of with
phenoldisulphonic acid reagent (prepared by dissolving 2.5 g phenol in 15 mL concentrated
sulphuric acid and adding 7.5 mL of fuming sulphuric acid followed by heating for 2 hrs in hot
water tub). The solution is diluted with about 20 mL of distilled water and ammonia (6mL) is
added till maximum color is developed. If any turbidity is formed dissolve it by adding EDTA
solution. The clear solution is transferred quantitatively to 50 mL volumetric flask and made up to

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the mark. Absorbance of the solution is read at 410 nm using a blank and the concentration of
nitrate in the sample is determined from the calibration chart prepared by using standard nitrate
solution treated in a similar manner.

Photometric titrations
Photometric titrations also known as spectrophotometric titrations involve monitoring the
changes in the absorbance of the reaction mixture at specified wavelength during titration in order
to locate the end point. During the titration the change in the absorbance is recorded for each
incremental addition of the titrant well beyond the end point. The plot of absorbance versus the
volume of the titrant consists of two intersecting straight lines of different slopes and the end point
is located graphically by dropping a perpendicular from the intersection point to the x-axis. A
curve may be obtained instead of sharp intersecting lines near the end point due to incomplete
reaction or dilution. End point location will be better by plotting the absorbance corrected for
volume changes or by using a concentrated solution of the titrant to minimize the volume changes.
Photometric titrations involving variety of reactions may be carried out if the absorbing system
obeys Beer-Lambert’s law. Different types of graphical plot can be obtained based on whether the
analyte alone absorbs or product alone absorbs, or titrant alone absorbs, etc., at the chosen
wavelength as shown in below figures.

Different types of photometric titrations curves (ε A, ε p and ε T refer to the absorptivities of

analyte, product and titrant respectively)

Examples for curve a is iron (III) – salicyclic acid complex versus EDTA at 525 nm or p-
toulidine versus perchloric acid in butanol at 290 nm. Cure b example is copper (II) versus EDTA
at 745 nm, curve c – arsenic (III) versus bromate bromide mixture at 326 nm and curve f-
formation of colored products of different compositions.

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 Even a mixture of metal ions can be quantitatively analyzed by photometric titrations
without involving any preliminary separations step as in the case of the titration of a mixture of
copper (II) and bismuth (III) with EDTA as shown below.

Advantage is taken of bismuth (III) with EDTA compared to that of copper (II) with EDTA. At a
chosen wavelength of 745 nm the cations and EDTA don’t have any absorbance and the Bi-EDTA
complex also don’t absorb. Hence, the absorbance remains close to zero until all the bismuth have
reacted with EDTA. Copper reacts with EDTA only after all bismuth has completely reacted and
absorbance shows an increase as more Cu-EDTA complex is formed. When all the Cu has reacted
absorbance level off as the excess EDTA added does not absorb. Thus the two end point can be
detected in a single titration curve.
A similar photometric titration can be carried out with the mixture of arsenic (III) and
antimony (III) with potassium bromate-bromide mixture at 326 nm. Arsenic (III) is oxidized first
by bromate-bromide mixture, the absorbance remaining unchanged. After all arsenic is oxidized
antimony is oxidized, the absorbance decreasing to a minimum till the end point and increasing
with the excess titrant.
Advantages of photometric titrations
• Better precision and accuracy compared to direct absorbance measurement
• Very dilute solutions can be handled
• Greater choice of experimental conditions as it is sufficient that any one of species, namely
analyte, product or titrant only need to absorb at the chosen wavelength
• Even slightly turbid solutions can be handled
• Relative changes in absorbance alone measure and not the absolute values
• The end point can be easily detected by graphical as long as straight lines segment can be
obtained and extrapolated.
Multicomponent Analysis
Analysis of samples with numerous components presents a major challenge in modern
analysis. Multi- component analysis has become one of the most appealing topics for analytical
chemists in the last few years, in fields as clinical chemistry, drug analysis, pollution control,…. etc.
Different analytical techniques can be applied for multicomponent analysis including;
spectrophotometry, chromatography, and electrophoresis. UV spectrophotometric techniques are

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mainly used for multicomponent analysis thus minimizing the cumbersome task of separating
interferents and allowing the determination of an increasing number of analyte, consequently reducing
analysis time and cost. Multicomponent UV spectrophotometric methods are based on recording and
mathematically processing absorption spectra
Combination drug products occupy a time-honored and important role in therapeutics.
When rationally formulated, fixed-combination drugs may produce greater convenience, lower cost,
and sometimes greater efficacy and safety. Because most analytes of interest are accompanied in their
dosage forms by other compounds absorbing in the same spectral region, classical UV spectral
measurements could not be used for their determination. The use of traditional methods like extraction
is quite difficult because extraction techniques require large solvent consumption, with accompanying
risks of analyte loss or contamination, and possibility of incomplete separation. The procedure may be
expensive and time consuming.

Advantages
• It avoids prior separation techniques e.g. extraction, concentration of constituents, and cleanup
steps that might be required
• spectral data are readily acquired with ease
• process is fast, accurate, and simple
• wide applicability to both organic and inorganic systems
• typical detection limits of 10-4 to 10-5 M
• Moderate to high selectivity.
• If a sample contains two absorbing drugs

Different UV spectrophotometric multicomponent analysis methods

1. Simultaneous equation method

If a sample contains two absorbing drugs (x and y) each of which absorbs at the λ max of the
other, it may be possible to determine both drugs by the technique of simultaneous equation
(Vierordt’s method) provided that certain criteria apply.
The information required is
• The absorptivities of x at λ1 and λ2, ax1 and ax2 respectively
• The absorptivities of y at λ1 andλ2, ay1 and ay2 respectively
• The absorbance of the diluted samples at λ1 and λ2, A1 and A2 respectively.
Let Cx and Cy be the concentration of x and y respectively in the diluted samples. Two
equations are constructed based upon the fact that at λ1, the absorbance of the mixture is the sum of
the individual absorbance of x and y.

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For measurements in 1 cm cells, b =1cm. Rearrange Eq. (2)

Substituting for C y in eq. (1) and rearranging gives

Criteria for obtaining maximum precision, based upon absorbance ratios, have been suggested
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 reasonably
dissimilar and if the two components do not interact chemically, thereby negating the initial
assumption that the total absorbance is the sum of the individual absorbances. Simultaneous equation
method was developed for simultaneous determination of several mixtures, e.g. atenolol and
indapamide, and dexibuprofen and paracetamol.

2. Difference spectrophotometry
The selectivity and accuracy of spectrophotometric analysis of samples containing absorbing
interferents may be markedly improved by the technique of difference spectrophotometry. The
essential feature of this method is that the measured value is the absorbance difference between two
equimolar solutions of the analyte in different chemical forms which exhibit different spectral
characteristics. The criteria for applying difference spectrophotometry to the assay of the substance in
the presence of other absorbing substances are that Reproducible changes may be introduced in the
spectrum of the analyte by the addition of one or more reagents. 1- The absorbance of the interfering
substances is not altered by that reagent. The simplest and the most commonly employed techniques
for altering the spectral properties of the analyte is the adjustment of the pH by means of aqueous
solutions of acids, alkalies or buffers.

3. Derivative spectrophotometry (DS)

DS involves the conversion of a normal spectrum (fundamental, zero-order spectrum) to its
first, second or higher derivative spectra by differentiating absorbance of the sample with respect to

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wavelength. The differentiation of zero-order spectrum can lead to separation of overlapped signals,
elimination of background caused by presence of other compounds in a sample, improvement of
resolution of mixtures as it enhances the detectability of minor spectral features, and enhancement of
sensitivity and specificity. Derivative spectra yield a more characteristic profile in comparison to the
parent one; new maxima and minima appeared and points where derivative spectra cross the X- axis.
DS keeps all laws of classical spectrophotometry, e.g. dependence of derivative value on analyte
concentration and additivity law. These features allow the determination of several components in a
mixture by measuring the amplitude of derivative spectrum of mixture at several wavelengths. If the
measured height of derivative peak of analyte is performed at those wavelengths at which spectra of
other components undergo zeroing, the measured amplitude is proportional only to concentration of
this analyte. This approach of quantitative determination is called “zero-crossing technique”.DS has
been used for simultaneous determination of different mixtures in pharmaceutical formulation, e.g.
loratadine and pseudoephedrine sulfate, aceclofenac and tramadol with paracetamol,tramadol and
ibuprofenor dexketoprofen, paracetamol and tapentadol, naproxen and acetaminophen or
diphenhydramine,phenylephrine and ketorolac, and amiloride and hydrochlorothiazide with timolol.

4. Absorbance ratio spectra method

Consider a mixture of two compounds x and y. The absorption spectrum of the mixture
"measured in 1 cm cell" is defined by the equation

Where; AM is the absorbance of the mixture, are the molar absorptivities, C x and C y are the
concentrations of x and y, respectively. If the absorbance of the mixture is divided by the absorbance
of a standard solution of x (its absorbance ), the following equation results

The ratio is a constant value (Fig. 1) which can be eliminated by taking the difference in absorbance ratio
amplitudes between two wavelengths λ 1 and λ 2 (peak to peak measurement)

Eq. (8) illustrates that the amplitude difference in the mixture absorbance ratio between two
wavelengths (termed; peak to peak, peak to trough, maximum to minimum measurement, or ratio
difference spectrophotometric method) is equal to the same amplitude difference for compound y after
canceling the constant interference due to compound x. The concentration of compound y (C y) is
proportional to the peak to peak amplitudes of its absorbance spectra. A calibration graph is obtained
by recording and storing the spectra of solutions of different concentrations of pure y, and the
spectrum of a solution of pure x (the divisor x0). The stored spectra of the solutions of pure y are

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divided by the standard spectrum of the divisor (x0). In the generated ratio spectra, the peak to peak
amplitudes between the selected wavelengths are measured and plotted against C y to obtain the
calibration graph. By using the calibration graph, the concentration of compound y in the mixture is
determined after similar treatment for the mixture solution. The concentration of x in the mixture is
determined by an analogous procedure. Ratio spectra method was developed for the simultaneous
determination of several binary mixtures e.g., emtricitabine and tenofovir, diclofenac and pantoprazole
and ternary mixtures, e.g., omeprazole, tinidazole and clarithromycin.

5. Derivative ratio spectra method

This simple spectrophotometric method, developed by Salinas et al., is based on the derivation
of the ratio spectra for resolving binary mixtures. It permits the use of the wavelength of highest value
of analytical signals with several maxima and minima, which give an opportunity for the
determination of active compounds in the presence of other compounds and excipients which could
possibly interfere in the assay. Calculation of the first derivative will remove the constant value due to
C x / C x0 in Eq. 9, so concentration of y can be easily determined without any interferences from the
drug x.

The difference between the two spectra and (Fig. 1) is due to the constant interference value
due to compound x (C x / C x0 ). Elimination of such interference can be done by measurement of ratio
spectra difference between two wavelengths or calculating derivative of the ratio spectra. Second
derivative of the ratio spectra may be also used to improve linearity, mean % recoveries and decrease
relative standard deviation. A derivative ratio spectrum was modified for the determination of ternary
mixtures using the derivative ratio spectra zero-crossing method. This method is realized by
measurement of amplitudes at the zero- crossing points in the derivative ratio spectra. Elimination of
such interference can be done by measurement of ratio spectra difference between two wavelengths or
calculating derivative of the ratio spectra. Second derivative of the ratio spectra may be also used to
improve linearity, mean % recoveries and decrease relative standard deviation. Derivative ratio
spectra was modified for the determination of ternary mixtures using the derivative ratio spectra zero-
crossing method. This method is realized by measurement of amplitudes at the zero- crossing points in
the derivative ratio spectra.

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 6. Double divisor ratio spectra derivative method
This method is based on the use of the derivative of the ratio spectrum obtained by
dividing the absorption spectrum of the ternary mixture by a standard spectrum of a mixture of two of
the three compounds in the mixture, and the measuring at either the maximum or minimum
wavelengths. It can only be used for the mixtures that the ratio of the concentrations of two interfering
compounds (used as double divisor) is known. In other words, the ratio of the concentrations of two
interfering compounds should be the same in calibration, prediction and unknown samples. It is
obvious that the ratio of the concentration of the analytes in real samples is always unknown.
Of a standard solution of x (its absorbance ), the following equation results

The ratioC x/ C 0 is a constant value which can be eliminated by taking the difference in
absorbance ratio amplitudes between two wavelengths λ 1 and λ 2 (peak to peak measurement)

Eq. (8) illustrates that the amplitude difference in the mixture absorbance ratio between two wavelengths
(termed; peak to peak, peak to trough, maximum to minimum measurement, or ratio difference
spectrophotometric method) is equal to the same amplitude difference for compound y after canceling the
constant interference due to compound x. The concentration of compound y (Cy) is proportional to the
peak to peak amplitudes of its absorbance spectra. A calibration graph is obtained by recording and
storing the spectra of solutions of different concentrations of pure y, and the spectrum of a solution of
pure x (the divisor x0). The stored spectra of the solutions of pure y are divided by the standard spectrum
of the divisor (x0). In the generated ratio spectra, the peak to peak amplitudes between the selected
wavelengths are measured and plotted against C y to obtain the calibration graph. By using the
calibration graph, the concentration of compound y in the mixture is determined after similar treatment
for the mixture solution. The concentration of x in the mixture is determined by an analogous procedure.
Ratio spectra method was developed for the simultaneous determination of several binary mixtures e.g.,
emtricitabine and tenofovir, diclofenac and pantoprazole and ternary mixtures.

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