1 Analytical Methods |Introduction -Classification of analytical methods, -Types of instrumental methods, -Instrument for analysis, -Selection of analytical methods,

-Calibration of instrumental methods.

-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 -It is interdisciplinary branch of science. * Most of the chromatographic methods were invented by biochemists. * Nuclear magnetic resonance(NMR) & Mass spectrometry by physicists. Classification of Analytical Methods: 1.Classical methods: Gravimetry: The mass of the analyte or some compounds produced from the sample is determined.(The analyte is obtained from the sample by separation, precipitation after derivatisation, ignition). Titrimetry: The volume of the solution is determined after the reactions such as neutralisation, complex formation, precipitate formation, & oxidation-reduction.

2 (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. 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/Res. 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). 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.

-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, pressure , density) Chemical: (pH ) Scale Position: (length) Time(Et): (Frequency, Pulse width, Phase) Digital(ED) : (Count, serial, parallel, number) Analog(EA) : (Current, voltage, charge)

Number (objects)

Non-electrical domains: -Measurement begins & ends in non-electrical domains.

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: electronic devices process information & transform it from one domain to another. -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 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 electrical output & the input radiant power is called the transfer 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.

Electrical domains: It 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) (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.

-Instead of time any variables : Wavelength, Magnetic field strength or Temp. may be independent variables as appropriate.

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.

Digital (Count, serial, parallel, number) (discrete values) -By using the binary number(binary coding), information is more efficiently encoded.

Relationship between instrument components Vs. flow of information from the characteristics of the analyte An example:
Instrument Energy source (Stimulus) Analytical information Information sorter Input Data domain Signal transducer of transduced processor/ information readout
Photodiode. Electrical current Amplifier, digitizer,

Photometer.

Tungsten lamp

Attenuated

Filter

light beam

LED

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 the 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 eluated analytes. 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 gauses etc. -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 chemically selective recognition phase.

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Selecting An Analytical Method: -Array of tools for carrying out chemical analyses are available. -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. B)Performance characteristics of instruments should be studied. C)Final selection of analytical method.

A)Definition should be able to address the following curiosity: 1) What accuracy is required ? High accuracy demands more time & care for analysis. Compromise between accuracy & money & time required 2) How much sample is available ? 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 interferences 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.

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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.

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 following qualitative performance criteria: 1)Speed. 2)Ease & convenience. 3)Skill required of operator. 4)Cost,service & availability of equipment.

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5)Per-sample cost.

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 accompanies accuracy. -But a high degree of precision does not imply accuracy.

2)Bias: -It is a measure of the systematic(determinate) error of an analytical method. Bias is can be expressed as: = - (: population mean for the conc. of an analyte in a sample, : true value).

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-From the analyses, population mean() 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. 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.) ii)Analytical sensitivity.(Takes care of precision of individual measurements) = slope(m)/std. dev. of signals(ss). 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). -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).

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-Minimum distinguishable analytical signal(Sm) is: Sm = Sbl + k sb1 (k=3, 95% confidence) Sum of mean blank signal =Sb1 , Std.dev. of Sbl = sb1 For cm ,we know: S= mc + Sb1 or, Sm = mc +Sbl Hence, Cm = Sm - Sb1 m

Or, LOD or Cm= k sb l/m where, m is calibration sensitivity, sbl is standard dev. of blank, Value of k=3 for 95% confidence.

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).

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-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: Conc. ppm(Pb) 10.0 1.00 0.000

No of replications 10 0 24

Mean value of S 11.62 1.12 0.0296

s 0.15 0.025 0.0082

Calculate: a)the calibration sensitivity, b) the analytical sensitivity at 1& 10ppm of Pb, & c) the detection limit.

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.