Pharmaceutical Chemistry V (Analytical Chemistry), Pabitra Subedi

Unit I: An Introduction to Analytical Methods

Introduction:
-Analytical chemistry is a measurement science consisting of a set of powerful ideas and
methods that are useful in all fields of science and medicines.

-It is most extensively used branch of science and applied throughout industry, medicines, and
all the sciences.

-It is interdisciplinary branch of science.

* Most of the chromatographic methods were invented by biochemists.

* Nuclear magnetic resonance(NMR) & Mass spectrometry by physicists.

-As we know :Analytical chemistry deals with methods for determining the chemical
composition of samples of matter.

-Aspects of modern analytical chemistry:

Qualitative(Identification of substance i.e. information about atomic or molecular species or the
functional groups)

Quantitative(Provides numerical information about the one or more components of the sample)

Elucidation of structure: of organic compounds(especially).

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Examples of application of analytical chemistry:
-The concentration of oxygen and carbon dioxide are determined in millions of blood samples
everyday and used to diagnose and treat illness.
-Quantitative measurements of ionized calcium in blood serum help diagnose parathyroid
diseases in human.
-Quantities of hydrocarbons, nitrogen oxides, and carbon monoxide present in automobile
exhaust gases are measured to assess the effectiveness of smog-control devices.
-Quantitative determination of nitrogen in foods establishes their protein content and thus their
nutritional value.
-Analysis of steel permits adjustment in the Concs. ofsuch elements as carbon, nickel, and
chromium to achieve a desired strength, hardness, corrosion resistance, and ductility.
-Farmers tailor fertilization and irrigation schedules to meetchanging plant needs during the
growing season, gauging these needs from quantitative analyses of the plants and the soil in
which they grow.
-Pharmaceutical analysis may be defined as a process or a sequences of processes to identify
and/or quantify a substance or drug, the components of a pharmaceutical solution or mixture or
the determination of the structures of chemical compounds used in the formulation of
pharmaceutical product.

-Pharmaceutical analysis is heart and brain of quality assurance activities of a pharmaceutical

industry.

-It is used to give shape and to evaluate the quality objectives.

Pharmaceutical analysis is relied upon for:
-Quality control of active pharmaceutical ingredients(API), inactive materials, packaging
materials.
-In-process quality control.
-Quality control of finished dosage forms.
-Stability study of API and finished dosage forms.
-Research and development activities.
-Various validation activities.
-Bioavailability and pharmacokinetics study.
-The mentioned topics are subjects of pharmaceutical analysis(or pharmaceutical analytical
chemistry)
Classification of Analytical Methods:
1.Classical methods:
Gravimetry:
The mass of theanalyte or some compounds produced from the sample is determined.(The
analyteis obtained from the sample by separation, precipitation after derivatisation, ignition).

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Titrimetry:
The volume of the solution is determined after the reactions such as neutralisation, complex
formation, precipitate formation, &oxidation-reduction.
(Classical methods are sufficiently accurate & precise however extent of their general
application is decreasing with the passage of time & with the advent of instrumental
methods to supplant them.)
2.Instrumental methods & types:

-Different properties & phenomena are used other than classical methods.
Separation techniques: Highly efficient chromatography & electrophoresis.

Determination techniques:
Properties relied on for qualitative/qualitative analysis:
Conductivity, electrode potential, light absorption or emission, mass-to-charge ratio etc.

‘Collectively these separation & determination methods are called instrumental method of
analysis.’
Instrumental methods:

a)Electrochemical methods:
Used for the measurement of the current, voltage or resistance.
e.g. Conductometry: measurement of the electrical conductance
Potentiometry: measurement of potential.
Coulometry: measurement of electrical charge.
Voltametry/Polarography/Amperometry: measurement of the current at specified voltage.

b)Optical/Spectroscopic methods:
Based upon the measurement of radiation absorbed or emitted.
e.g. Absorption methods: UV/Vis. Spectroscopy, Infrared(IR), Atomic Absorption
Spectroscopy(AAS).
Emission methods: Plasma/Flame emission spectroscopy, Fluorimetry.
c)Chromatography:
e.g. Paper, High Pressure Liquid Chromatography(HPLC), Gas Chromatography(GC), Ion
exchange, Thin Layer Chromatography(TLC) & column chromatography.

d)Thermal methods:
Differential Thermal Analysis(DTA), Thermogravimetry(TG), & Differential Scanning
Calorimetry(DSC).

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e)Other methods:
X-ray diffraction, radioactive methods, mass spectrometry, refractometry /interferometry &
polarimetry/ORD/CD, Gravimetry (quartz crystal microbalance), kinetic methods for rate of
reaction etc.
Instruments For Analysis
-An instrument can be taken as a communication device between the system under study & the
investigator.
-An instrument for chemical analysis converts information about the physical or chemical
characteristics of the analyte.
-The information is converted such a way that can be manipulated & interpreted by a human.
-To collect desired information from the analyte stimulus should be provided.

-Stimulus:
Electromagnetic,
Electrical,
Mechanical,
Nuclear energy

-The stimulus collects response from the system under study.

-Nature & magnitude of the response are controlled by the fundamental laws of chemistry
& physics.

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-The information is contained in the phenomena(response) resulted by the interaction of
stimulus with the analyte.
Stimulus is a narrow band of wavelength of light.
e.g absorption / transmission of light & its relation to the concentration of the analyte.

Data domains:
-The information is encoded or represented by physical – chemical characteristics &
electrical signals.
-In the measurement process, various devices convert information from one to another form.
Definition

Way of encoding analytical response in electrical or non-electrical signals.

_______________________________________
Non-electrical domains Electrical domains

Physical: (light intensity, color, Analog(EA) :

pressure , density) (Current, voltage, charge)

Chemical: (pH)
Time(EΔt):
Scale Position: (length) (Frequency, Pulse width, Phase)

Digital(ED) :
Number (objects) (Count, serial, parallel, number)

Non-electrical domains:

-Measurement begins & ends in non-electrical domains.

The physical and chemical characteristics that is of interest in a particular Expt. resides in these
data domains i.e, length, density, chemical composition, intensity of light, pressure etc.
e.g.
Determination of the mass of an object with equal & unequal arm mechanical balance,

Measurement of volume with a measuring cylinder, determination of linear dimension with a

ruler.

-Instrument relying exclusively on non-electrical information transfer are becoming obsolete.

-The information sought begins in the properties of the analyte& ends in a number (both are
non-electrical), however,interdomain conversion is frequently practiced:

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electronic devices process information & transform it from one domain to anotherand the final
result is again in non-electrical domain.
Measurement process begins from properties of analyte and ends in number(both are non-
electrical representation.
-Hence, a measurement process can be represented as a series of inter-domain conversion.

Photocell Current meter

Light Intensity → Current → Scale
(Non-electrical) (Electrical) (Non-electrical)

The scale number is generally proportional to the physical and chemical properties.
Electrical domains:

Modes of encoding informations can be divided into 3 categories:

Analog: (Current, Voltage, Charge)

(Continuously variable magnitude)

Time: (Frequency, Pulse width , Phase)

(Vary with time)

Digital: (Count, serial, parallel, number(electrical & non-electrical)

(discrete values)
Analog (Current, Voltage, Charge):
(Continuously variable magnitude)

-Information is encoded as the magnitude of electrical quantities.

Current, Voltage, & charge.

-These quantities: continuous both in amplitude & time.

-Analogue quantities can be measured continuously or sampled at specific points in time.

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-Instead of time any variables :

Wavelength/frequency,

Magnetic field strength or Temp.

may be independent variables as appropriate.

-Analog domain is used in AM radio signal.

Time: (Frequency, Pulse width , Phase)

(Vary with time)

-Information is stored as the time relationship of signal fluctuations but

Not in the change of amplitudes of the signal.

-Three different time-domain signals are recorded as an analog quantity Vs. time.

-The horizontal dashed lines are an arbitrary analog signal threshold .

-These lines are used to decide , a signal=HI, or LO.

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-No. of signals per unit time is frequency.

-The time required for each cycle is period.

-Time between successive LO to successive HI transitions is called the period.

-The time between a LO to HI & a HI to LO is the pulse width.

-The time relationships between transitions of the signals from HI to LO or from LO to HI

contain the information of interest.

In fig.(a), time domain signals represent the data as the frequency of a periodic waveform.

In fig.(b), time domain signals represent the data as the time duration of a pulse.

In fig(c), time domain signals represent the data as the time or rate of pulse.
-Conversion of Time-domain signals to analog-domain signals vice-versa by voltage-to-
frequency&frequency-to-voltage converters.

-Time domain is used in FM radio signal.

-It is also used in Raman spectroscopy and instrumental neutron activation analysis.

In these methods, the frequency of arrival of photons at adetector is directly related to the
intensity of the emissionfrom the analyte, which is proportional to its Conc.

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Digital (Count, serial, parallel, number)
(discrete values)

-By using binary number(binary coding) more efficiently information is encoded to represent
numeric and alphabetical data.

-The count digital data of the signal in fig. (a) represents the number n=5.
-Here the signals are monitored & counted the number of complete oscillations.
-The process requires a period of time that is proportionalto the no. of cycles of the signal.

-In the binary coded serial digital data the time intervals are number consecutively beginning
with zero.

-The numerical value is assigned to each successive interval of time.

The total value represented is :
( 1 X 20)+ ( 0 X 21) + ( 1 X 22) = 5. ( 0= LO)
In binary coding, 101 = 5(decimal numbering) 3 times of counting in (b) is equal to 5 times in
(a) for n=5.

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-Hence , the binary coded serial data is nearly twice as efficient as count serial data.

-In the counting of n=10 as in by count serial data without binary coding, requires 210 = 1024
time intervals.
-Hence, the binary coded serial digital data is 100 folds efficient than count digital(without
binary coding).
-As the result of efficiency of binary coding schemes, most digital information is encoded,
transferred, processed and decoded in binary form.

-The parallel digital data schemes is highly efficient because all of the desired information is
presented simultaneously as just as all of the digits on the face of the digital voltmeter.

-This also adopts binary data coding scheme.

Any measurement process can be represented as a series of inter-domain conversion.

Example:
-Intensity of the fluorescence(at characteristic wavelength) is proportional to concentration of
quinine.

-Extraction of the information from the sample is carried out by applying electromagnetic
energy (from laser) as a stimulus.
-The intensity of the fluorescence emission, nonelectrical information is encoded into electrical
signals.

-The nonelectrical to electrical encoding is done by a special type of device called ‘Input
transducer’ (Phototransducer).

-The mathematical relation between the electricaloutput & the input radiant power is called
thetransfer function of the transducer.
-Devices that serve to convert data from electrical to nonelectrical domains are called
‘output transducer’.

e.g. Voltmeters, alphanumeric displays, electric motors, computer screens etc.

-Voltmeter of the fluorimeter converts the voltage V to a number at LCD.

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FIGURE 1-3 A block diagram of a fluorimeter showing (a) a general diagram of the
instrument,

(b) a diagrammatic representation of the flow of information through various data

domains in the instrument, and

(c) the rules governing the data domain transformations during the measurement process.

Examples:

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i)Detectors
ii)Transducer
iii)Sensors
i)Detectors: It refers the device that identifies ,records, or indicates a change in one of the
variables in its environment.

Devices may be: mechanical, electrical , or chemical.

Variables may be: pressure, temperature,electrical charge, electromagnetic radiation, nuclear

radiation, particulates, or molecules.

e.g.UV detector used in liquid chromatography, pointer of a balance, mercury level in a

thermometer etc.
-Detector is sometimes referred to an entire instrument.
-Hence,
detection system represents entire assemblies that indicates or records physical or chemical
quantities.
e.g.UV detector used in liquid chromatography.
-It is used to indicate & record the presence of eluatedanalytes.

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ii)Transducer

-Represents the devices that convert the information in non- electrical to information in
electrical domains & converse.

e.g. photodiodes, photomultipliers, electronic photodetectors, thermistors, strain gausesetc.

-The radiation falls on the surface of the transducers.
-These produce current or voltage proportional to the radiant power of electromagnetic
radiation.

iii)Sensors

-These are capable of monitoring specific chemical species continuously & reversibly.
e.g. the glass electrode & other ion-selective electrodes, Clark oxygen electrode, fiber-optic
sensors (optrodes)etc.

-Sensors consist of a transducer coupled with a chemicallyselective recognition phase.

Selecting An Analytical Method:

-Array of tools for carrying out chemical analyses are available.

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-The selection is often difficult & requires experience as well as intuition.
Hence,
Prior to selection of analytical method:
A)Nature of the analytical requirements or problem should be defined and their probable
solutions should be proposed.
B)Performance characteristics of instruments should be studied.
C)Final selection of analytical method.

A)Definition of analytical problems & their solution :

1) What level of accuracy is required ?
High accuracy demands more time & care for analysis.
Compromise between accuracy&money & timerequired

2) How much sample is available ?& how expensive the same is ?

3)What is the concentration range of the analyte ?

-Amount of sample &analyte concentration demand method sensitivity & range of concentration
be accommodated

-Less sample amount with low analyte concentration requires more sensitive method.
4) What interferents are/could be present?

- Complexity & the number of components in the sample , compel to select a selective method.
-If the sufficiently selective method is not available, techniques to remove interferents
should be considered. Solvent extraction, chromatography etc.
5) How many samples need to be analyzed ?

- Depends upon, what time and money are available?

-If the No. of sample more, more time & money are to be spent on instrumentation, method
development & calibration.

-Hence, the least operator time consuming method should selected or developed.

-In case of a few samples, more time consuming methods are also acceptable but these should
be simpler & require little or no preliminary work.

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B)Performance Characteristics of Instruments
Performance criteria are useful to decide whether a given Instrumental method can solve an
analytical problem
-These criteria are expressed in numeric terms:

‘figures of merit’
-These help to short-list the choice of instrument for a given analytical problem.

C)Final selection of the instrument is on the basis of desirable characteristics of an

analytical method:
1)Speed.
2)Ease& convenience.
3)Skill required of operator.
4)Cost,service& availability of equipment.
5)Per-sample cost.

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1)Precision
-It is the degree of mutual agreement among data that have been obtained in the same way
or
It is the reproducibility of results.
-It provides a measure of the random, or indeterminate error of an analysis.
-It generally accompanies accuracy.
-But a high degree of precision does not imply accuracy.

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2)Bias:

-It is a measure of the systematic(determinate) error of an analytical method.

Bias Δ is can be expressed as:
Δ = µ- τ

(μ: population mean for the conc. of an analyte in a sample, τ: true value).
-From the analyses, population mean(µ) is calculated.
3)Sensitivity:
Ability to discriminate between small differences in analyte concentration.
-Depends upon: a) The slope of calibration curve.
b) The precision of the measuring device.
-If two methods have equal precision, then the one having the steeper calibration curve will be
more sensitive.
-If two methods have equal slope, then the one exhibiting the better precision, will be more
sensitive.

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Two types of sensitivity:

i)Calibration sensitivity(Slope at the desired Conc.)

S= mc + Sb1.
(S=measured signal, m= slope, c= conc. of the analyte, Sb1= signal for a blank & it is y-
intercept)
(Except in non-linear cal. curve, it is independent of Conc.)
-Hence, fails to take into account the precision of individual measurements.
ii)Analytical sensitivity.(Takes care of precision of individual measurements)

γ = slope(m)/std. dev. of signals(ss).

-It is relatively insensitive to amplification factors.
(as both m &ss increase).

-It is independent of the measurement units for S.

-Often concentration dependent as ss may vary with Conc.

-Since, the precision of measurement decreases at low concentrations, the ability
to distinguish between small concentration difference also decreases.

-Therefore, the sensitivity is a function of the precision.

4)Detection limit(LOD):
It is he minimum concentration or weight of analyte that can be detected at a known
confidence level but not necessarily quantitated as an exact value.
-Depends upon the ratio of the magnitude of the analytical signal to the size of the statistical
fluctuation in the blank signal. (or signal to noise ratio).

-Near to the limit of detection, analytical signal plus its Std. Dev. approach the blank signal plus
its Std. Dev.
-Hence, analytical signal should be larger than the blank by multiple k (e.g 3) of the variation in
blank due to random error(Std.Dev. of blank).So that it becomes distinguishable with less level
of uncertainties.
-Minimum distinguishable analytical signal(Sm) is:

Sm= Sbl + k sb1 (k=3, 95% confidence)

Sum of mean blank signal =Sb1 , Std.dev. ofSbl = sb1

For cm ,we know:

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 S= mc + Sb1or, Sm = mc +Sbl

Hence,

Cm = Sm- Sb1
m

5)Dynamic range

-Extends form the lowest limit of quantitation(LOQ) to the concentration at which the
calibration curve departs from linearity (limit of linearity LOL).

-Deviation of 5% from the linearity is considered the upper limit .

(This is because of non-ideal detector response or chemical effects).

-The lower limit of quantitation should be taken at least 10 times the mean Std. dev. of a
blank(10sb1)
6)Selectivity:

-It refers to the degree to which the method is free from interference by other species in the
sample matrix.
-Absolute freedom from interference is ideal case.
-Remedial action is taken to minimize the effects of these interferences.
Problem:
A least-square analysis of calibration data for the determination of lead based on its flame
emission spectrum yielded the equation:
S=1.12cpb +0.312

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Where cpb is the lead concentration in parts per million & S is a measure of the relative intensity
of the lead emission line. The following replicate data were then obtained:
Conc. ppm(Pb) No of replications Mean value of S s
10.0 10 11.62 0.15
1.00 10 1.12 0.025
0.000 24 0.0296 0.0082

Calculate: a)the calibration sensitivity, b) the analytical

sensitivity at 1& 10ppm of Pb, & c) the detection limit.
Ans.: a)m=1.12, b) At 10ppm, Analyt. Sensitivity=m/ss =7.5, At 1ppm, 45
c)LOD=0.022ppm Pb.
Steps followed for quantitative analytical determinations
-After selecting an analytical method.
-Determination involves the sequence of steps.
-In some instances, one or more the steps can be omitted.

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Errors
-Analyst is to obtain a result as near to the true value.
-It is made possible by the correct application of the analytical procedure employed.
-By the application of the analytical procedure a set of quantitative data is collected.
-The data are subject to errors.
-Error is defined as a difference between a measured value and true value

E= (Measured - true ), where E= absolute error.

- Error α 1/Accuracy.

-Expression of errors: as relative error

Percentage = Measured- True X 100 = % error

True

Per thousand = Measured-True X 1000 = ppt

True
Percentage = Measured value X 100% = % accuracy
True
-The true value of any quantity is never known.

-However, the value of a standard sample certified by institute of standards & regulatory body
may be treated as correct.
e.g,
-National Medicine Laboratory supplies secondary reference substances.
National Bureau of Standards & Metrology calibrates various devices and provides values
Error types:

1)Determinate(Systematic)errors2)Indeterminate(Random)errors
1)Determinate(Systematic)errors.

-These errors are because of definite causes.

-These can be avoided or whose magnitude can be determined.
-These are generally unidirectional W.R.T. the true value
- These are reproducible.
-The sources of the errors can be predicted by a person who thoroughly understands all the
aspects of the measurement.

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-Sources of the errors may be ,e.g.
i) Incorrectly calibrated instrument,
i.e. burette, balances, or pH meter,

ii) An impurity in a reagent,

iii)A side reaction in a titration,
iv)Heating a sample at too high Temp.
v) Reproducible mistakes committed by analyst/operator etc.
Classification

i)Methodic( because of Non-ideal chemical or physical behaviour of analytical system)

ii)Operative(because of Carelessness, inattention, personal limitation of experimenter)

iii)Instrumental(because of Non-ideal instrument behaviour, faulty calibrations, using under

inappropriate conditions)
i)Methodic

-Most serious of analytical errors.

-Inherent in the method applied.
-The sources of the error in the physico- chemical properties of the system.
e.g in the precipitation of aluminium( as aluminium hydroxide) zirconium, titanium & iron are
co-precipitated.
ii)Operative(Carelessness, inattention, personal limitation of experimenter)
-These originate from ineptitude of the experimenter i.e colour blindness, incorrect handling of
instruments or careless weighing.
iii)Instrumental(Non-ideal instrument behaviour, faulty calibrations, using under inappropriate
conditions)

-These are because of failure of measuring devices to perform in accordance with required
standards.

-These can be minimized by calibration or use of proper blanks in case of spectrophotometric

analysis.
2)Indeterminate(Random)errors
-Exist in every measurements.
-Caused by uncontrollable variables.
-Never be totally eliminated & major source of uncertainty in analysis.
-The sources of errors are small & show accumulated effect.

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Calibration of instrumental methods:
-Calibration determines/fix the relationship between the analytical response & the analyte
concentration.
-Usually this is determined by the use of chemical standards.
-However, gravimetric & some coulometric methods do not require chemical standards.
Types of calibration
1)Comparison with Standards
i) Direct comparison ii)Titrations
2) External-Standard Calibration
3) Standard-Addition Methods
4) Internal-Standard Method

1)Comparison with Standards

i) Direct comparison:
-Property of analyte is compared with the standard until the property being tested matches or
nearly matches that of the standard.
e.g in colourimetry, colour produced as the result of a chemical reaction of the analyte is
compared with the coloured produced by the same reaction of the standard.
The concentration of the standard is varied by dilution until its colour exactly or nearly matches
the sample.

-Colour may be the inherent property of analytes& standards.

-Then ,
the concentration of analyte= Conc. of the Std.

-It is also called the null comparison or isomation method.

ii)Titrations
-Analyte reacts with a standardized reagent.
-Reaction should be stoichiometric.
-The amount of titrant is added until chemical equivalent is reached.
-The chemical equivalent is ascertained by the colour change of the chemical indicator.
-The amount of standardized reagent needed to achieve the chemical equivalence is related to
the amount of analyte.

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2)External-Standard Calibration
-It is used to calibrate instruments & procedures.
-It is assumed that the interference effects from the sample matrix are absent.

-Series of standard solutions of the analyte with increasing order of concentrations are prepared.
-At least three solutions should be prepared.
-Instrument response(Absorbance, Peak height/Peak area) is obtained for each Std. solutions.
-The instrument response/signals are taken as a function of the analyte concentration.
-A calibration curve is plotted as signal(intercept) vs. Conc.(abscissa)

or
If data points do not fall exactly on straight line due to random errors ,fitting the data to
mathematical method of linear least square( y=mx+b) or best-fit equation.
-Finally analyte/instrument response is measured for the analyte present in the sample.
-Finally Conc. of analyte in the sample is determined either with the help of the curve prepared
as Std. analyte responses vs Conc. Std.analyte series or by best –fit equation.

-In the calibration curve, because of the indeterminate errors, not all data points fall exactly on
the line.

- In such situation .for the best straight line, regression analysis or method of least square is to
be resorted on.

i)There exists linear relationship between signal(y) & analyte concentration(x)

y=mx+b
where m= slope, b= intercept.
ii)Any deviation of the individual data points from the straight line is because of measurement
errors.

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Measures to prevent Errors in External-Standard Calibration:

-Systematic errors should be avoided as:

*Solvent or reagent blank should be prepared representing the sample matrix,
*Contamination during sampling or sample preparation should be avoided,
*Std. should be prepared correctly,
*Accuracy of the gravimetric, volumetric technique & equipment should be ascertained.
*Chemical form of the std. must be identical to that of the analyte in sample.
*Once preparation, concentration of the Std.solncan change with decomposition, volatilization or
adsorption onto container walls.(necessary measures should be in place to prevent such
phenomena).

-Measurement of the analyte should be made near the center of the calibration curve.

3)Standard-Addition method:
-Useful when: likelihood of matrix effects is substantial , suitable internal std. is unavailable
and analyte present in sample is in the trace amount.
-In this technique identical volumes of samples are spiked with standard (known) solutions
which are in increasing order of concentration.
-Each solution is further diluted to a fixed volume.
-Measurement is made on one original(containing only sample solution)& other std. added
samples.
-In all samples , sample matrix is nearly identical.
After the blank correction, instrument responses are measured for all the samples(Std+Sp).
-Then responses are plotted against conc. of added standards in the sample.
-Extrapolation from “zero” added material determines how much analyte was in the original
sample.(Curve prepared for flame photometry ↓)

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3)Internal standard method:
Requirements for the internal standard:

i)It should provide a signal that is similar to the analyte.

ii)Two signals should be sufficiently distinguishable by the instrument.
iii)It should be chemically similar to but not present in the original sample.
iv)Neither it affects the analyte signal, nor gets affected by the analyte signal.
-Internal standard is added in a constant amount to all samples and calibration standards.
-The ratio of the analyte signal to the internal Std. signal is plotted against as a function of the
analyte concentration of the standards.

-This ratio for the sample is calculated then used to obtain analyte concentration in sample
from the calibration curve drawn.
Benefits:
*This method is useful to eliminate several types of both random & systematic errors. This
happens when the both analyte& internal standard respond proportionately to random
instrumental & method fluctuation.
*The result of ratio of their signals(Analyte/Int.Std) is independent of such fluctuation.
*The matrix effect can also be compensated if two signals are influenced in the same way.
-Finding of the suitable internal standard & its introduction into analyte sample & Std. is a
difficult task.
e.g.
-Lithium for the determination of sodium & potassium in blood serum by flame spectrometry.
-Propan-2-ol for the determination of ethanol in blood sample by GLC.

Model questions:
1)Compare& contrast calibration curve method & standard addition method.
2)What do you understand by calibration?Explain about standard addition method.

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