UNIT 18 ELECTROANALYTICAL METHODS
Structure
18.1 Introduction Objectives 18.2 pH Metry Definition of pH Measurement of pH Colourimetirc Measurement of pH 18.3 Electrometric Measurement of pH Principle of Potentiometry Electrodes Meas urement of pH using pH Meter pH of Water and Waste Water Acid Rains and pH pH of Soils 18.4 Ion Selective Electrodes 18.5 Counductometry Some Basic Concepts of Conductometry 18.6 The Measurement of Conductance The Wheatstone Bridge Principle Measurement of Conductance of a Solution Experimental Measurement 18.7 Application of Conductometry 18.8 Summary 18.9 Terminal Questions 18.10 Answers
18.1 INTRODUCTION
Electroanalytical methods find applications in all branches of Chemistry, industries, engineering and a number of other technologies. The possibility of the determination of low level of pollutants has prompted the use of these methods in environmental studies.
An electroanalytical method can be defined as one, in which the electrical response of a chemical system or sample is measured. These methods can be classified into a number of types characterized by measuring the electrical response in terms of different electrical quantities such as: potential, current, quantity of current, resistance and voltage etc. and bear the corresponding names as potentiometry, amperometry, coulometry, conductometry and voltammetry etc. During the past few years, there has been sudden increase in interest in electroanalytical techniques. This is partially attributed to the development in instrumentation and partially due to the heavy demands by environmental scientists for the determination of a large number of heavy metal, organic and inorganic substances present in water and soil samples.
In this unit we will study how to measure pH of water and soil samples using pH metry. We will also discuss the potentiometric measurement of concentration of ions selectively with the help of ion selective electrodes. Then we will discuss conductometry.
Objectives
After studying this unit, you will be able to:
• define pH, • define electrode potential, • describe the use of some electrodes,
5 Instrumental Methods • measure the pH of a solution, of Analysis • define conductivity, • measure the conductivity of a solution, • apply the concept of pH metry, ion selective potentionmetry and conductivity for water and soil analyses
18.2 pH METRY
There is a widespread usage of electrochemical methods in general and of potentiometric determination of pH and concentration of several ions in particular. Measurement of pH is one of the most important and widely used test in water analysis. For natural water treatment as well as for waste water treatment a large number of reactions e.g. coagulation, disinfection, water softening, acid base neutralisation etc. are all pH dependent. Most of chemical laboratories are equipped with pH meters. Modernization of potentiometry by the development of ion selective electrodes has increased the interest in the study of environmental samples.
The principle of potentiometry is applied to measure the potential difference in terms of pH unit on pH scale by suitably modifying the common voltmeter to high input impedance mV meter and such pH measurement can be termed as pH metry instead of potentiometry. In pH metry, pH meter is used to measure the pH. Before going in further details of potentiometric measurement of pH, let us know the basic concept of pH.
18.2.1 Definition of pH
The hydrogen ion concentration plays an important role in many areas of chemistry A rigorous definition of and its determination and control is of great practical value in the study of pH would obviously environment. involve activities, accordingly: The shorthand notation of hydrogen ion concentration is given in terms of pH for p H = log a + a − H 'puissance de hydrogen'.
The pH value, originally formulated in 1909 by S.P. Sorensen, is defined as the negative logarithm of hydrogen ion concentration:
+ pH = −log10 [H ] ..… (18.1)
where [ ] represents equilibrium concentration and logarithm is taken to the base 10. In practice 'p' preceding a variable is used to express the negative logarithm of that variable. Likewise, pOH is to designate the negative logarithm of hydroxyl ion concentration.
In aqueous solutions the product of [H+] and [OH− ] is always a constant at a particular temperature. Thus,
+ − KW =[H ] [OH ] ..... (18.2)
−14 0 where Kw is the ionic product constant of water, its value is 1 × 10 at 25 C.
Taking logarithm of both sides of equilibrium of equation 18.2 and substituting 'p' for negative logarithm we get
0 pH + pOH = pKw = 14, at 25 C . .… (18.3)
For pure water [H+] = [OH−] = 1×10−7 (at 250C), which gives the pH value of pure water equal to 7 at this temperature.
6 For an acidic solution [H+] > [OH−] and pH is below 7, whereas for a basic solution Electroanalytical [OH−] > [H+] and pH is above 7. Methods
Neutral Acidic Range Basic Range
0 7 14
pH Scale
SAQ 1
Find the concentration of H+ ions of a solution for which pH value is 4.5. ………………………………………………………………………………………… ………………………………………………………………………………………… ………………………………………………………………………………………...
18.2.2 Measurement of pH
The pH of a solution is commonly found by the use of either an indicator or a pH meter. Because of their accuracy and speed, pH meters have superseded the older indicator method in many applications. However, the indicator method remain in use because it is simple and convenient specially for field work in pollution analysis. In next part of this section we are taking a brief description of ind icators method which also known as colorimetric method for the measurement of the pH.
18 .2.3 Colo rimetric Measurement of pH
For the approximate and rapid estimation of pH and in studies in non-aqueous media, it is convenient to make use of coloured indicators. Colorimetric measurement can be carried out visually or photometrically.
Visual Measurement of pH
The use of coloured indicators for the visual measurement of pH is well known. The approximate pH of a solution can be determined by comparing its reaction with different indicators or on papers impregnated with the indicator solution. In this method the colour change is observed in a particular pH range. The chief advantage is the low cost and also the method is suitable for routine pH measurement. A very common example is litmus which is red below pH 5 and blue above pH 8. The colour changes from red to blue when pH changes from 5 to 8. To find colour changes in a wide range of pH, the mixtures of indicators, the so called universal indicators are to be used. For example, the Kolthoff universal indicator is a mixture of five indicators and gives a conspicuous colour change within unit pH values. The colours at different pH values are given in Table 18.1.
Table 18.1: Variation of colour of Kolthoff Universal Indicator with change in pH .
pH 1 2 3 4 5 Colour R R-P R-O O Y-O pH 6 7 8 9 10 Colour L-Y Y-G G G-B V
Abbreviations: R=Red, P=Pink, O=Orange, Y=Yellow, LY=Light Yellow, G=Green, B=Blue & V=Violet 7 Instrumental Methods Photometric Measu rement of pH of Analysis The visual method for pH measurement using indicators has low accuracy due to difficulties of light intensity estimation. The accuracy can be increased by instrumental means using a colorimeter or a spectrophotometer to measure the absorbanc e at a particular wavelength. Indicators are considered to behave as weak acids or weak bases and the degree of dissociation of indicator substance depends on hydrogen ion concentration in solution. Consider, e.g. an indicator acid, HIn, which dissociates as
HIn ƒ H+ + In− ..… (18.4) Colour A Colour B Our eyes can generally detect only one colour The dissociation constant K of indicator HIn is if the ratio of the concentration of the + - two colour forms is [H] [In] K = ...... (18.5).....18.6 10:1. Only the colour [HIn] of the more concentrated form is − + [In] seen. logK =+log[H]log...... (18.6) [HIn] [In]− orpH=+pK log...... (18.7) [HIn]
Indicator colours are indicated by the In− and HIn concentration ratio which depends on degree of dissociation and hence the pH can be indicated by the intensity of either colour A or colour B with the assumption that the Beer's law is obeyed. To get satisfactory results by photometric measurement, it is necessary to keep the indicator concentration as small as possible. The principle of photometric measurement is discussed in detail in Unit 19 of this course. In next section, we will take up the principle of pH metry.
18.3 ELECTROMETRIC MEASUREMENT OF pH
The electrometric method of pH determination is based on the measurement of potential of a pH cell, whereby the potential of a hydrogen sensitive electrode is directly proportional to pH, and pH is defined in an operational manner on a potentionmetric scale.
The pH meter is calibrated potentiometrically with an indicator electrode (glass) and a reference electrode using a standard buffer. The operational pH is defined as:
EE(cell)− (cell) (pH) = (pH) ± ( )us( ) ..… (18.8) u s 0.0591
where
(pH)u = potentiometrically measured pH of the sample (unknown solution) (pH)s = assigned pH of the standard buffer used for calibration (E cell)u = cell potential of glass electrode and reference electrode system with unknown solution (Ecell)s = cell potential of glass electrode and reference electrode system with standard buffer
In order to understand this operational definition of pH, we will take up general principles of potentiometry.
8 18.3.1 Principle of Potentiometry Electroanalytical Methods Potentiometry deals with the measurement of difference in potential between two electrodes which have been combined to form an electrochemical cell. The difference in potential between the two electrodes in electrochemical cell is known as cell potential. The cell potential depends on the composition of the electrodes, concentration of the solution or more correctly activity of a species in solution (or pressure of gases) and the temperature. Relationship connecting the cell potential with the activity of the species involved in the concerned chemical reaction, known as Nernst equation, can be derived using thermodynamic principles. A detailed discussion is given regarding this in Sec.17.6 of Unit 17 of Physical Chemistry (CHE- 04) course. Based on the dependence of cell potential on the activity or concentration of the species in the electrochemical cell, we use this concept to obtain the activity or concentration, and pH of a species in solution from potential difference measurements using potentiometer or pH meter.
In order to understand the above concept, we shall study the galvanic cell in some detail. For example, consider the simple galvanic cell illustrated in Fig.18.1. In this cell there are two half cells. Where a half cell is the combination of an electrode and the solution with which it is in contact. In one half cell zinc gets oxidized to Zn 2+ ions.
2+ − Zn(s) Zn (a1) + 2e (Oxidation)
This electrode is negatively charged relative to the solution and is referred to as anode i.e. oxidation always occurs at the anode. On the other hand, in second half cell Copper (II) ions get reduced to copper.
`
V Zinc rod Copper rod Porous division Solution of Solution of 2+ 2+ Zn Cu
Fig. 18.1: Simple galvanic cell having Cu 2+/Cu and Zn 2+ /Zn half cells.
2+ − Cu (a2) + 2e Cu(s) (Reduction)
This electrode is, therefore positively charged relative to the solution and, this is referred to as cathode . Note that reduction always occur at the cathode.
As shown in the Fig.18.1 both electrodes of half cells are connected externally via an electric circuit and the circuit is completed by ionic conduction through the solution and KCl salt bridge. The voltmeter will then measure the difference in potential between the two electrodes.
Note that the one half cell involves an oxidation process and the other half cell a reduction process. These are then combined in the cell to give a redox reaction represented below by Equation,
2+ 2+ Zn(s) + Cu (a2) Zn (a1) + Cu(s) ….. (18.9)
Using IUPAC convention this can be represented by
9 2+ 2+ Instrumental Methods Zn(s) | Zn (a1) ¦ Cu (a2) | Cu(s) of Analysis This notation starts with the left hand electrode and move to the right through the solution to the right hand electrode. The simple vertical bars signify phase boundary, whilst the double vertical bar the salt bridge. a1 & a2 are the activities of two ions.
Measurement of Potential Difference
The potential difference between the electrode and solution in a half cell is referred to as the electrode potential. It is impossible to determine the potential of a single electrode and rather it is the potential difference that is measured. A number of conventions have been established in order to compare the potential of different half reactions. The half-reaction is written as a reduction process i.e. for the metal, M and its own ion Mn+, it is written as
Mn+ + ne-= ƒ M(s)
If the constituents in the half -cell are present at unit activity, the potential difference is measured at 25ºC with respect to a standard hydrogen electrode (SHE), which has been arbitrarily assigned the zero potential. Under these conditions the potential difference or electrode potential is known as the standard electrode potential, Eº. We will take up standard hydrogen electrode in detail in the next section.
In general, the potential difference of a cell is given by the difference between the two electrode reduction potentials E1 and E2, of the cathode and anode, respectively
Ecell = E1 − E2
The junction potential developed at the junction between the two half cells are also contributing to the cell potential, the Ecell calculation can be rewritten as:
E(cell) = [E1 − E2] + Ej
Where Ej is the liquid junction potential and can be a positive or negative quantity. By using salt bridge between two half cell the liquid junction potential can be minimized.
Using Nernst equation, the relationship between cell potential and activity of species involved can be developed.
The Nernst equation is:
RTa(reduced) E = Eº − ln ..… (18.10) nFa(oxidised)
Where R is the universal gas constant, T is the absolute temperature, n is the numbers of electron involved in the transfer, F is Faraday’s constant and a(reduced) and a(oxidized) are the activities of the reduced and oxidized species of each half cell.
For the Copper (Copper II) half cell of the galvanic cell mentioned earlier it is written as RTa(Cu) EE0 ln 2 + =− 2 + Cu Cu2 + Cu Cu 2Fa(Cu)
But we can take the activity of a pure substance to be unity, so
0 RT 1 EE2 + =−ln Cu 2+ Cu 2+ ()Cu Cu 2F a(Cu) or
10 Electroanalytical 02RT + E2+ =+Ealn(Cu) Methods Cu 2 + Cu ()Cu Cu 2F A similar expression could be written for the left-hand half-cell:
02RT + E2 + =+Ealn(Zn) Zn 2 + Zn ()Zn Zn 2F
If the liquid junction potential is negligible, the potential of the cell is then given by:
Ecell =−EE2 + Cu2+ Zn Cu ()Zn
Clearly the potential of the cell will depend upon the activity of both the copper(II) and zinc(II) ions. In such situation it is not possible to determine the activity or concentration of the two ions from the cell potential. In practice, we determine the activity or concentration of a single substance, rather than a combined value for two or more substances. For this reason if we keep the activity of zinc(II) ions at a fixed value so that its potential also remain constant. Then,
02RT + Ecell =E+−lnaE(Cu) 2 + Cu2 + Zn Cu 2F Zn ()
0 As EEand 2 + are both constant, they can be combined into one value E’. Cu2 + Zn Cu ()Zn Then, RT E=+E'aln(Cu)2+ cell 2F
Now, the measurement of cell potential is clearly proportional to the natural logarithm of the activity of the Copper (II) ion. This equation relates the activity of the oxidized form of the metal to potential. However, a general equation can be written for both oxidized and reduced forms of electrodes.
RT E=±E'aln ..… (18.11) cellinF
Where n is the charge on the ion, I, and E' is a constant incorporating the potential of first half cell which is kept constant and the standard potential of the second half cell containing the solution under investigation and the first half cell. Note the ± sign in the equation is used to signify that it will be positive if I is a cation and negative if I is an anion. This equation can be applied to a typical cell used in potentiometric analysis and shown in Fig. 18.2
Potentiometer (Voltmeter)
Indicator electrode Reference electrode Salt bridge Solution under test Stirrer 11 Instrumental Methods of Analysis Fig. 18.2: Diagrammatic representation of a cell used for potentiometric analysis.
This is the common practice in potentiometric measurements. The first half cell which is having constant potential is referred to as a reference electrode . The second electrode in combination with the reference electrode is called an indicator electrode and its response should be dependent upon changes in activity or concentration of the species of interest.
Before considering electrodes in detail, let us now discuss the measurement of cell potential. If a current is drawn from a cell as discussed above, in the course of measurement of cell potential, the cell reaction proceeds and the concentration of the solutions change in the two half-cells. Hence, it is important to measure the cell potential without allowing current to flow. The cell potential measured nearly under zero or negligible current flow is called electromotive force (e.m.f) of the cell. The instruments which used for accurate measurement of potential are potentiometer and pH meter. These instruments draw only negligible current.
18.3.2 Electrodes
In previous part we saw that a electrochemical cell were composed of two electrodes: indicator and reference electrodes. A reference electrode must be easy to construct, and must maintain a constant, reproducible potential even if small currents are passed. An indicator electrode respond to change in activity of the species to be measured. Now we are going to consider some systems which can be used as reference electrodes.
Reference Electrode
There are three common reference electrodes used for potentiometric analysis:
• Standard Hydrogen Electrode • Calomel Electrode • Silver -Silver Chloride Electrode
Standard Hydrogen Electrode (SHE)
The standard hydrogen electrode (Fig. 18.3) is constructed from a platinum foil plate, which has been platinized, that is coated with platinum black (finely divided platinum) by chemical or electrochemical reduction of chloroplatinic acid. The finely divided layer of platinum black helps in achieving the largest possible surface area. Its + composition can be formulated as Pt, H2 (1 atm) 1 H (a =1)
Glass hood
H2 (1 atm)
Pt electrode H2
Fig.18.3: Standard hydrogen electrode.
12 The half cell reaction, may occur in either direction depending upon the type of Electroanalytical electrode which is coupled with it. Methods
+ 2H + 2e ƒ H2 (g) The potential of a hydrogen electrode is given by the Nernst equation:
2 0 2.303RT (a + ) E=E + log H ..… (18.12) 2F p H 2 where E0, the standard electrode potential for hydrogen electrode is zero. For the + standard hydrogen electrode, having H activity, aH + equal to one and pressure exerted by hydrogen gas p also equal to one atmosphere, the logarithmic term on H 2 the right of Eq. (18.12) becomes equal to zero and E = E0 = 0. By convention, the potential of SHE is assigned the value of exactly zero volt at all temperatures.
The SHE was selected as a primary reference electrode for several reasons as given below:
(i) It can be used for the entire pH range. (ii) The accuracy in measurement is high. (iii) The reagents used in its preparation can easily be purified. (iv) The potential of the electrode is not affected by mechanical stress of the electrode.
Besides these advantages, the use of SHE has certain limitations as:
(i) It can not be used in solutions containing strong oxidizing and reducing substances. (ii) It is not handy and hence transfer from one place to another is not easy. − (iii) The platinum surface is poisoned in presence of species like H2S, CN and Hg.
However, for experimental measurement of the electrode potential the use of SHE requires many precautions, therefore, other electrodes of simpler design whose potentials are well known with respect to the SHE e.g. Saturated Colomel Electrode (SCE) and Ag/AgCl electrodes. Such electrodes are known as secondary reference electrodes and are generally preferred.
Saturated Calomel Electrode (SCE)
A saturated calomel electrode is the most frequently used reference electrode. Many designs of saturated calomel electrodes have been reported in literature. One of those is shown in Fig. 18.4.
Insulated wire lead
KCl filing hole
Saturated KCl solution
Connector Glass Platinum connector wire Hg, HgCl2 2KCl paste Opening to inner tube KCl
Fine capillary plugged with asbestos fiber or a porous ceramic junction
13 Instrumental Methods of Analysis Fig.18.4: A commercial calomel electrode.
It consists of an outer glass tube with a crack in the end of the tube. A crack is made by an asbestos filament or fitted porcelain plug or quartz fiber. A mercury and mercurous chloride paste is filled in the inner tube, which is connected to the saturated potassium chloride solution in the outer tube through a small opening. The saturated potassium chloride solution in the outer tube can be easily renewed through a lateral hole. Calomel, i.e., mercurous chloride is a sparingly soluble salt. Its solubility product (Ksp) is given as:
−18 Ksp = 2 + × aa − = 1.1 × 10 ..… (18.13) Hg 2 Cl
A calomel electrode may be symbolised as:
Hg2Cl2 (satd.), KCl (x M) | Hg
Where x represents the concentration of potassium chloride and must be specified in describing the electrode. Commonly, 0.1 M, 1 M, and saturated solutions of potassium chloride have been employed and the calomel electrodes are named as decimolar, molar and saturated calomel electrodes, respectively.
The half cell reaction is
− − Hg2Cl2 (s) + 2e ƒ 2Hg (l) + 2Cl
and the potential at 250 C is given by the equation: 0 0.0591 EE=+ log a Hg 2+ Hg 2 2
Substituting for 2+ from Eq.18.13 gives: a Hg 2 K 0 0.0591 sp ..… (18.14) or EE=+Hg log 2 a Cl --
The activity of chloride ions (by excess of chloride ions obtained from potassium chloride) remains constant for a particular electrode. The experimental value of the potential of the saturated calomel electrode is 0.244 V at 25 0 C.
Silver-Silver Chloride Electrode
The silver -silver chloride electrode is another frequently used reference electrode. It is prepared by plating a layer of silver chloride onto a metallic silver wire and immersing in a solution containing chloride ions (usually KCl) of known concentration which is also saturated with silver chloride (see Fig. 18.5).
Ag wire
Rubber policeman
KCI solution Frit (filled with Agar-KCI mixture)
14 Electroanalytical Fig. 18.5: Silver-silver chloride electrode. Methods
Silver chloride is a sparingly soluble salt. Its, solubility product, Ksp, is:
Kspaa1.7710−100atC25 ..… (18.15) =Ag+−Cl =×
The silver -silver chloride electrode may be symbolised as:
AgCl (satd.), KCl (x M) | Ag
Where x represents the molarity of potassium chloride solution. Normally, a saturated potassium chloride solution is taken.
The half cell reaction is:
- AgCl (s) + es→+Ag ()Cl and the potential at 25 0C is given by the equation
EEa0 0.0591log =+Ag Ag + Substituting for a from Eq.18.13 gives: Ag +
0 Ksp EE=+Ag 0.0591log...... (18.16) a Cl - When a large excess of chloride ion (potassium chloride) is present, the contribution of chloride ions obtained from dissolution of silver chloride can be (in the half cell) controlled by the concentration of potassium chloride (which remains constant). The experimental value of potential for an electrode prepared by saturated solution of potassium chloride is 0.199 V.
SAQ 3
What is a ref erence electrode? Give some examples. ………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………………
Indicator Electrodes
As described earlier, electrodes that respond to a specific ion are referred to as indicator electrodes and the selection and use of such electrodes is the key to modern potentiometry. Several metals can be served as indicator electrodes. But they are not selective as these electrodes can respond to its own ions and can also respond to a number of other metal ions. There are several other indicator electrodes which are selective to particular ions. Many of them are very valuable in potentiometric analysis and are collectively referred to as ion selective electrodes (ICE). The most common of these is the glass electrode, which is selective to H+ ions and, consequently, pH. In next part we will discuss glass electrodes in detail.
Glass Electrode
The most convenient way for determining pH has been by the use of the glass electrode. It is an ion-selective electrode. Glass electrode is a membrane type electrode whose membrane is made by a special type of glass. A potential develops across a thin glass membrane separating two solutions of different acidities. The 15 Instrumental Methods measurement of potential difference can thus be related to hydrogen ion concentration. of Analysis This phenomenon was first recognized by Cremer in 1906 and systematically explored by Haber in 1909 with the construction of a glass bulb electrodes. After the work of Sorenson (1909) to determine hydrogen ion concentration in terms of pH and the use of vacuum tube voltmeter by A. Beckmann (1930) made the use of glass pH electrodes more practical. Development of transistor based pH meters and the special purpose glass for the measurement of high pH values has further improved the technology.
The glass electrodes of various sizes, shapes and for different pH ranges are commercially available. Fig. 18.6 illustrates the construction of a common type of glass electrode. It consists of a thin, H+ sensitive glass membrane bulb at the end of a heavy-walled glass tubing. A buffer solution (or 0.1M HCl) is filled in the glass membrane bulb. A reference electrode (usually silver-silver chloride electrode) is placed in contact with the inner solution. It is connected to one terminal of the pH meter. The bottom portion (bulb) of the glass electrode is immersed in the external solution whose pH is to be measured. An external reference electrode (usually an SCE) is immersed in the external solution and is connected to the other terminal of the pH meter.
Schematic diagram of cell containing a pH glass electrode can be represented as below:
Internal Internal H+ sensitive External External Reference buffer glass solution reference + + Electrode (aH )1 membrane (aH )2 electrode
Saturated potassium chloride solution
Silver electrode coated with silver chloride
Thin walled glass bulb
Fig.18.6: A typical glass electrode for pH measurements.
The potential for this cell response is related to the logarithm of hydrogen ion activities on the two sides of the glass membrane and is given by Nernst equation:
(aH + )1 Ecell = k + 0.0591 log ….. (18.17) (aH + )2
Where k is a constant. The constant includes the difference in juntion potential between the reference electrode and solution and the asymmetry potential for the glass membrane. The asymmetry potential is due to the difference in surface of the inner and outer layers of the glass membrane.
16 Since the pH on the internal side of the glass membrane is held constant using a Electroanalytical buffer, the potential of the glass membrane electrode will depend upon the pH of the Methods external solution.
Since pH = − log aH+
This substituting into Eq.18.17 gives,
Ecell = k1 + 0.0591 pH ..… (18.18)
+ Where k1 now includes the constant factor related to aH . Thus, the emf produced in the glass electrode system varies linearly with pH. From the Eq.18.18 the constant k1 can be eliminated by measuring potential, first with standard buffer solution whose pH is precisely known and then with unknown sample solution. Thus for the standard buffer
(Ecell)s = k1 + 0.0591 (pH)s
For unknown sample solution
(Ecell)u = k1 + 0.0591 (pH)u
Then the difference in both the cell potentials
(Ecell)u − (Ecell)s = 0.0591 (pH)u − 0.0591 (pH)s or (EE ) - (cells) (pH)u = (pH)s + cellu 0.0591
This is the operational definition of the pH.
As said above, the constant k 1 includes the asymmetry potential which exists across the glass membrane even if the two sides of the cell are of identical composition. For this reason, a pH meter is to be calibrated from time to time (preferably every time when pH measurements are done) with standard buffers.
In pH meter, voltmeter is used to measure the potential of the cell. The voltage scale is calibrated in pH units so that 0.0591V correspond to 1pH Unit at 25°C. This value will change with temperature and modern pH meters have a temperature compensation device. This may be set before taking pH readings.
Combination Electrodes
For the purpose of convenient both an indicator and a reference electrodes (with salt bridge) can be combined into a single probe to make a complete cell or combination electrodes. For such electrodes only small volume is needed for potentiometric measurements. A typical assembly of combination electrodes is shown in Fig.18.7. It consists of a tube within a tube, the inner one having the pH indicator electrode and the outer one having the reference electrode and its salt bridge.
SAQ 4
Why is it necessary to calibrate glass electrode before determin ing pH of a solution? ………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………….….
17 Instrumental Methods of Analysis
Electrical leads
Refil hole Protective sleeve
Plastic or glass body
Saturated KCI
Ag/AgCI wires
Porous plug
Glass membrane
Fig.18.7: Combination pH reference electrode.
18.3.3 Measurement of pH Using pH Meter
A pH meter is an electronic voltmeter of requisite sensitivity having a scale calibrated directly in pH units. The scale is normally taken as extending from 0 to 14 pH units. A pointing needle moves across the graduated scale and the pH of the solution can be read directly on the scale.
Numerous pH meters of various designs are marketed by several instrument manufactures. General purpose pH meters are either line operated instruments that are readable to 0.05 pH unit or battery operated instruments suitable for field job. These days digital pH meters readable to 0.01 pH unit are more popular as compared to scale-needle instruments.
Measurement of pH of a solution with the instrument (analog meter) shown in Fig.18.8 can be made following the procedure given below in a step-wise manner.
1. Keep the selector switch on ‘zero’ position and adjust the zero position by a screwdriver if the pointer does not indicate zero. 2. Mount the electrodes (a glass electrode and a saturated calomel electrode or a combined glass-calomel electrodes) in the clip on the stand. Wash the electrode(s) well with distilled water. 3. Connect the power cable to a 220V AC supply. Switch on the instrument and wait for a few minutes till the instrument warms up. 4. Adjust the temperature/solution temperature value. 5. Take the standard buffer solution of desired range (e.g. buffer of pH 4 for acidic solutions) in a beaker. The electrode assembly is immersed in the pH reference buffer and the solution is agitated gently by swirling the solution in the region of the glass electrode surface so as to bring it into pH equilibrium. It should be ascertained that the glass electrode membrane is completely immersed in the 18 solution. The electrodes should not touch each other or the side or the bottom of Electroanalytical the beaker. Methods
II III
VII IV
I VI V
Fig. 18.8: A direct reading pH meter (front view) legend: I. On/off switch II. Set zero III. Selector IV. Electrode support V. Temperature compensation VI. Set buffer VII. Meter
6. Put the selector switch to suitable pH range (0-7 for acidic or 7-14 for basic solutions) and adjust set buffer knob in manner that the pointer reads the pH of the standard buffer solution (placed in the beaker). 7. Put the selector switch back to zero position. Remove the electrodes from the buffer solution, wash the electrodes with distilled water and wipe them gently with tissue paper. 8. Transfer the standard buffer back to the storage bottle and wash the beaker well with distilled water. 9. Take the sample solution in the beaker. Introduce the electrodes in the solution and swirl it gently. 10. Set the selector switch in the suitable range position and read the pH on the scale. 11. Put the selector switch back to zero position. Remove the electrodes from the solution, wash them with distilled water and keep the electrodes in distilled water, when not in use.
Precautions
(i) Never touch the membrane of the glass electrode with anything else except soft tissue paper since it is fragile and is easily ruined if scratched or bumped. (ii) The electrode(s) must not be removed from the solution unless the selector switch is at zero. (iii) Never dip the glass electrode in a solution with a dehydrating action. (iv) If used for measuring pH of albuminous substances, the glass electrode must be cleaned with suitable solvents and th en the electrode is placed in distilled water for a few hours before it is used to measure the pH of the other solution. (v) For basic solutions with pH more than 11, glass electrodes of special composition are required to avoid interference due to sodium ion. (vi) The glass electrode may be covered with a sleeve to save it from jerks. (vii) The standard buffer of pH value as close as possible to the sample pH value must be taken for the calibration of the system. Commercially available standard buffers of pH values 4, 7 and 9.2 are commonly used.
18 .3.4 pH of Water and Waste Water
The pH of a water sample tells, whether the water is acidic or basic. The pH is a measure of an aggregate property of natural water and waste water and can be
19 Instrumental Methods interpreted in terms of specific substances only when the chemical composition of the of Analysis sample is known.
Acidity of water may be caused due to the presence of strong mineral acids, weak acids, such as, carbonic acid and acetic acid, and hydrolysing salts of polyvalent metals like iron and alumin ium. Acids contribute to corrosiveness and influence chemical reaction rates and biological process in water.
Alkalinity of water is primarily due to carbonate, bicarbonate and hydroxide content. Other bases such as phosphates, borates, silicates may also be present to cause alkalinity.
The waste waters discharged from various industries are sometimes seriously acidic, for example acid mine drainage water, steel industry pickling and tin plating rinse water. The acid contents of such waste waters are quite high and can be checked by measuring pH. The acid of the waste must be neutralized to pH 6-8 before it is drained to a river.
Measuring pH of water and industrial waste water has attained significant importance and for this purpose pH measurements have been used more and more in chemical industries and in natural waters. The electrometric pH measurement is applied in most parts of the industrial plants. A commercial pH meter can be used for this purpose. Nearly always (except in some specific cases) the glass electrode is used as a pH measuring electrode. A special attention to temperature compensation and electrode care must be paid. For field work, the battery operated instruments are used. Glass electrodes can be used for measurement in continuous plant control. However, before use, the electrodes must be standardized with buffer solutions and the electric recorder must be adjusted in such a way, that the measured value is in agreement with the recorder value.
When measurements of pH in waste water, containing hydrofluoric acid, are to be made, antimony electrodes have been suggested, since a glass electrode can not be used because of the reaction of fluoride with silicon of the glass.
18 .3.5 Acid Rains and pH
One of the most serious environmenta l problems facing many regions of the world today is ACID RAIN. A rain that is appreciably more acidic (pH < 5.0) than the unpolluted natural rain is considered to be “acid rain”. In a more general way, it also includes a variety of other phenomena such as acid fog and acid snow. Acid rain has a variety of ecological damaging consequences, and also the presence of acidic oxides in air probably has the direct effects on human health.
The natural (i.e. unpolluted) rain itself is mildly acidic due to the atmospheric carbon dioxide dissolved in it. Carbon dioxide in water forms carbonic acid which is a weak acid and partially ionizes to produce a proton causing the lowering of pH of the system:
CO2 + H2O ƒ H2 CO3 − + H2CO3 ƒ H + H CO3
and due to the produced proton even the unpolluted natural rain is having a pH of about 5.6, which is lower than 7 and lies in the acidic region. Hence, a truly acid rain must be more acidic than the natural rain, that is, the pH of acid rain is less than 5.
The primary pollutants of acid rain are: Sulphur dioxide, SO2; and Nitrogen oxides, NOx. Sulphur dioxide is mostly produced by volcanoes and by combustion of coal. 20 SO2 is also emitted into air directly or indirectly by oil refineries and thermal power plants. Nitrogen oxide pollutant gases are produced by burning of fuels in air with a Electroanalytical hot flame. First nitric oxide, NO, is formed which is gradually oxidized to nitrogen Methods dioxide, NO2. Collectively, NO and NO2 in air is referred to as NOx .
These primary pollutants, over a period of time (by air oxidation and water vapours) are converted into the secondary pollutants: sulphuric acid, H2SO4, and nirtic acid, HNO3, both of which are highly soluble in water, are strong acids, and are responsible for making rains acidic.
The quality of acid rain is determined by its pH. In general pH values of rains have been falling with passing years, that is the rain is becoming more acidic. However, the pH of the rains vary from place to place. So far, the lowest pH was recorded as 2.4 for a rainfall in Scotland in April 1974.
Decreasing pH of rains has serious ecological consequences. Because of acid rainwater falling on the ground drains into rivers and lakes. Acidified lakes have high concentration of dissolved aluminium, Al3+. Both the acidity (i.e. low pH) itself and the high concentration of aluminium are responsible for the devasting decreases in fish population that have been observed in many acidified water systems.
SAQ 5
List the substances that cause acid rain. ………………………………………………………………………………………… ………………………………………………………………………………………… ……………………………………………………………………………..………….
18 .3.6 pH of Soils
Soil is an important environmental segment because of its basic importance in food production. Therefore, an attention toward the study of physical characterization of soil in relation to crop needs is essentially required. The environmental happenings, such as: certain physical and chemical processes taking place due to climat e and vegetation etc. affect the soil properties. The difference in various physical and chemical properties of soils depends on its colour, alkalinity, acidity, pH, presence of exchangeable cations etc. pH of the soil plays an important role to determine the nature of the soil, that is, the acidic and basic nature of the soil is ascertained by measuring pH of the soil.
The pH of a soil is influenced by the presence of electrolytes, sulphides, carbonates, ammonia and ammonium salts. Variation in pH is also observed with the proportion of water present in the soil. The pH of the soils containing carbonate or bicarbonate in the soil solutions are closely associated with the carbon dioxide pressure. Water percolating through soil causes leaching of bases and increases acidity of soils in humid regions and decreases the productivity. The pH of the soils also depend on the amount and nature of the exchangeable cations. Thus for calcium saturated soils the pH usually varies from 7 to 8.5, whereas for sodium saturated soils it may be as high as 10.
Determination of pH of the soil does not have the same meaning as the pH of a true solution. A suspension of soil in water is not homogenous and does not have a uniform distribution of ions. If the supernatent solution of the soil suspension is taken for pH measurement, it may be different from the suspension. Some persons favour leaching soil samples to remove electrolytes before pH determinations, but for the leached soil parts the pH values are higher than the unleached soils by 0.2 to 0.5 pH units. Measuring pH of the water saturated paste of the soild may also be taken in the routine procedure. It gives the lowest moisture level at which the determination can be 21 Instrumental Methods conveniently made. The pH values obtained by paste method are very near to field of Analysis conditions.
18.4 ION SELECTIVE POTENTIOMETRY
In previous section you have learnt the use of glass electrode to determine selectively the hydrogen ion concentration of a solution in terms of pH. The potential difference across the glass membrane separating a reference solution of fixed H+ ion concentration from the test solution depends on the pH of the test solution.
Similar as the glass electrode in principle, the new classes of ion selective electrodes have been introduced for more or less selective determination of other ions such as: + + + ¯ Na , NH4 , Ag , F, CN etc. The ion selective electrodes respond logarithmically to the activity of an ion of interest, thus they can be used to determine the concentrations of the particular ions selectively.
The need for the new (specific) ion electrodes had existed from a long time and the technology to use them (i.e. ion meters just as pH meters) had also been available. The development in this field has surprisingly become very rapid, after its conceptual discoveries in nineteen sixties. An ion selective electrode consists of an ion selective membrane with essential properties of having workable electrical conductivity, capability of selective binding the analyte ion, and practically its zero solubility in analyte solutions.
Ion selective electrodes can be constructed with various designs. In one common type it consists of an ion selective membrane sealed at the lower end of a glass or plastic tube. An internal reference solution of fixed concentration of ion of interest (I) is filled in the tube and an internal reference electrode is immersed in this solution. A cell with an ion selective electrode is represented in Fig. 18.9. The ion selective electrode is dipped in the test solu tion of ion I, and is coupled with an external reference electrode.
A famous procedure in potential measurements is based upon the use of colomel reference electrodes, assuming that the electrode potentials of the two saturated calomel electrodes to be equal. Under these conditions the emf of the cell is equal to the membrane potential.
Electronic voltmeter
Internal External reference electrode reference electrode
Internal standard solution of I
Test solution of I Ion selective membrance
22 Electroanalytical Methods Fig.18.9: A cell with selective ion electrode.
Using two saturated calomel electrodes the cell can be represented as:
Hg Hg 2Cl2 KCl Solution Membrane Solution KCl Hg 2Cl2 Hg Satd. 1 2 Satd. under these conditions, potential of the cell is equal to the membrane potential
0 0.05910.0591 (Em), at 25 C is, Ecell =Em = −+logaa'iilog"...... (18.19) ZZii
Where Zi is the charge of ion I, a’i & a” i are the activities of internal and external solutions of ion I. The activity of internal solution being constant, equation (18.19) changes to
0.0591 Ecell = constt+pI ….. (18.20) Zi
Where pI is the negative logarithm of activity (conc. in dilute solutions) of ion I. The above equation, which relates the potential of cell to the concentration of ion I is analogous to that for the glass pH electrode.
Fluoride Ion Sele ctive Electrode
It is an ion selective electrode prepared by placing a piece of a membrane disc nearly 1 mm thick and a few mm in diameter consisting [a single crystal of Europium fluoride (EuF 2) doped lanthanum fluoride (LaF3) into the end] of a plastic tube (as shown in Figure 18.10). Doping with europium fluoride improves electrical conductivity of the LaF3 membrane. A silver-silver chloride electrode is placed in the tube (as an internal standard, and the tube is filled with equimolar solution of KCl (1M) and NaF(1M). To measure the potential difference of the cell, this electrode is coupled with a saturated calomel electrode placed in the fluoride containing test solution
Ag/AgCl electrode Teflon KCl/NaF
LaF (Eu F doped) 3 2 membrane
Fig.18.10: A fluoride selective electrode.
The potential of the cell, will be equal to,
Ecell = constt − 0.0591 log aF¯ ..… (18.21) = constt + 0.0591 pF – ..… (18.22)
23 Instrumental Methods Measurements as low as 10–5 M of F¯ ion concentrations are possible with this of Analysis electrode.
Caution: Since F−¯ attacks silicon of glass, measurements are made in plastic or Teflon containers.
Besides, the flouride ion electrode, various solid and liquid membrane electrodes are available from commercial sources. The electrodes can be used to determine the ions causing pollution in the environment, for example, Cd2+ CN¯, Pb2+, SCN¯ etc.
18.5 CONDUCTOMETERY
In conductometry we examine the transport of electricity in solution and application of this phenomenon to chemical analysis. The principle advantage of this technique is its simplicity and relatively good sensitivity. It is, one of the earliest techniques to study the behaviour of electrolytic solutions. Since the conductance (which is reciprocal of resistance) of an electrolytic solution depends on the number of ions present, their charge, their mobility, and temperature; analytical applications of conductometry are thus possible.
Conductance measurement is one of the best way for determining the purity of water. A continuous recording of conductance is helpful in monitoring the purity of water purified by ion exchange or obtained from large distillation units. Such measurements are also used in power plants to check the purity of steam distillate, demineralized water, raw water and solid contents of boiler water. Moisture content of soils can also be determined in the field with portable instruments.
Conductance is an additive property of a solution. This principle is the essential basis for condutometric titrations, in which the change in conductance is related to concentration changes of the ionic species involved in the titration reaction. This can be applied for acid-base, precipitation, and complex forming reactions. It is possible to get the equivalence point in titrations of weak acid vs weak base and for mixtures of acids or bases in a better way as compared to other methods of analysis, such as potentiometry. In addition conductomety can be applied for the determination of ionization contents, solubilities and complex formation.
18.6.1 Some Basic Concepts
Conductance
The ease of flow of electric current through a body is called its conductance. In metallic conductors it is caused by the movement of electrons, while in electrolytic solutions it is caused by applied electrical field. The electrolytic conductance , G, of a medium is equal to the reciprocal of its electrical resistance R in ohms:
1 G= ….. (18.23) R
The flow of current through an electrolytic solution is due to the charge carried out by ions and causes electrolytic conduction. We thus expect movement of ions in aqueous solutions of electrolytes and also in molten electrolytes.
Specific Conductance
Since a solution is a three dimensional conductor, the exact resistance will depend on the spacing (l) and area (A) of the electrodes.
24 Electroanalytical Methods
B
2 2 A m A m
The resistance of a solutions is proportional to the distance between the electrodes and inversely proportional to the electrode surface area. If two electrodes having a cross- sectional area of A m2 and separated by l m are dipped in a solution of some electrolyte, the resistance (R) of the solution present between the two electrodes is:
R 8 l R 8 1 A l and R = ρ ..… (18.24) A Conductivity increases with: Where ρ (Rho) is proportionality constant and is called as specific resistance or (i) concentration of resistivity. the solution and (ii) speed of the ions Reciprocal of specific resistance is known as specific conductance or conductivity concerned. designated by κ (kappa):
? = 1/ρ ..… (18.25)
Hence Eq. 18.24 can be written as
11 R = × κ A
1 l Or ? = × ..… (18.26) RA
l The ratio ( ) is known as cell constant, Kcell and (1/R) = G (from Eq. 18.23). A
Now we can summarise as, κ = 1/ρ = (1/R) (l/A) = G Kcell ..… (18.27)
Conductivity = observed conductance × cell constant
Since the resistance is expressed in ohm, Ω (omega) the reciprocal ohm (Ω−1) was earlier used as the unit for conductance. However, in SI system, the unit for conductance is ‘Siemens’ and, given the symbol ‘S’. Hence, the unit for conductivity will be S m−1 (1S = 1Ω−1) or S cm−1. It may be remembered that S m−1 = 1/100 S cm−1. However specific conductance is customarily reported in smaller units as milli Siemens per meter (mS m−1) and micro Siemens per cm (µS cm−1).
25 Instrumental Methods Some conductivity values of typical samples are given in Table 18.2. of Analysis
Table 18.2: Some Conductivity Values of Typical Samples
Sample at 25°C Conductivity µS cm-1
Ultra pure water 0.055 Power plant boiler water 1.0 – 5.0 Drinking water 50 – 1100 Ocean water 53, 000 5% NaOH 2,23,000 50% NaOH 1,50,000 10% of HCl 6,30,000 32% HCl 7,62,000
31% HNO3 8,00,000
Molar Conductivity
In order to compare quantitatively the conductivities of electrolytes, a quantity called
molar conductivity is frequently used. The molar conductivity, Λm , (capital lambda) is the conductivity per unit molar concentration of dissolved electrolyte. It is related to conductivity, κ by the relation:
κ Λm = ..… (18.28) c
where c is the concentration mol m −3. The molar conductivity is usually expressed in S m2 mol−1 or S cm2 mol−1. It may be remembered that S m 2 mol−1 = 10,000 S cm 2 mol−1.
It is to be remembered that c in equation 18.28 is to be expressed in mol m−3 unit. If the concentration is given in terms of Molarity (mol dm −3), then the following conversion is to be carried out
c (mol m−3) = Molarity × 1000 ..… (18.29)
Earlier equivalent conductivity (Λeq), which is given by the following expression, was in use
1000 κ Λeq = ..… (18.30) c
Where c is the concentration expressed in terms of normality of the solution. However, IUPAC recommends the use of molar conductivity only.
In next part we will see how conductance is measured.
18.6 MEASUREMENT OF CONDUCTANCE
26 Principle of Wheatstone Bridge is used to measure the conductance of solution. Electroanalytical Therefore, before taking up the measurement of conductance of solution, let us study Methods the principle of Wheatstone Bridge.
18.6.1 The Wheatstone Bridge Principle
A wheatstone bridge (Fig. 18.11) be employed to measure the resistance of an electronic conductor. It works on the principle of obtaining balance between two arms with the help of a balance indicator (e.g. a galvanometer) at the condition of potential being equal.
Let Rx be an unknown resistor, R1 and R2 two standard resistors, R3 an adjustable resistor and G a galvanometer. The bridge is connected to a source of power S, a battery, and a tapping key K is placed in the path to control the connections.
A
i i 1 2 R1 R2
+
S B D G K _ i 1
R i R 3 2 X
C
Fig.18.11: A DC W heatstone bridge circuit.
To measure the resistance Rx, the tapping key K is held down momentarily and the bridge is balanced by adjusting R3 to get no deflection in galvanometer under these conditions.
In the bridge the total current is divided into two paths: i1 through R1 and R3, and i2 through R2 and Rx. Under the balancing conditions, the potential at points B and D must be the same, i.e. the ohmic voltage drop through the resistors R1 and R2 must be the same. Hence, the potential at B (EB) must be equal to potential at D (ED).
EB = ED ..… (18.34)
Or i1 R1 = i2R2 ..… (18.35)
Similarly i1 R3 = i2 Rx ..… (18.36)
Dividing (18.35) by (18.36) we get,
RR 12= RR3 X
RR23 and Rx = ..… (18.37) R1
27 Instrumental Methods Thus, we can calculate Rx as R1, R2 and R3 are all known. Conductance G, being the of Analysis reciprocal of resistance will be,
R G = 1 ..… (18.38) RR23 Alternatively, the conductometric cell can be incorporated into operational amplifier control circuit, as shown in Fig.18.12. The amplifier balances the potential of two inputs. The current from the input potential, Ei, is balanced by the current from amplifier output which passes through a feedback resistor (Rf). The output potential, E0 is in terms of resistance:
E0 = Ei (Rf/Rx + 1)
Where Rx is the resistance of conductometric cell.
With respect to the solution conductance, G, above equation becomes
E0 = Ei(RfG + 1)
Rf
_
D E + 0
Rx E i
Fig.18.12: An operational amplifier control circuit for conductometric measurement. Rx is the solution resistance and Rf is the feedback resistance.
18.6.2 Measurement of Conductance of a Solution
The Principle of the Wheatstone bridge can be used to measure the conductance of solutions. However, the following considerations must also be kept in mind:
(i) Since a direct current would polarize the electrodes in the conductivity cell by electrolyzing the solution to avoid polarization an alternating current (ac) source of power must be used in place of a dc source (battery) usually ac voltages of 3-6 volts with frequency of 50 Hz or 1000 Hz used across points A and C of Fig. 18.13. (ii) A suitable conductivity cell (with electrodes dipped in the solution) is located between points C and D. Thus Rx represents the resistance of the conductivity cell. (iii) Since, the cell also acts like a small capacitor (Cx), and to balance its capacitive resistance a variable capacitor, CB, must be inserted into the bridge.
A
R R 1 2
ac B BI D
Conductivity cell R 3
RX CB
CX 28 C
Electroanalytical Methods
Fig.18.13: A conductivity bridge circuit.
(iv) The balance indictor (BI) may be an ac galvanometer, but some other devices are also be used: • An earphone can act as a balance indicator if the frequency of the ac source is in audio-range. • A magic eye, which gives a green fluorescence as a result of electrons striking a phosphor coating inside the glass tube, is used in several commercial instruments. • For much precise conductance measurements a cathode ray oscilloscope is used as the balance indicator. (v) Conductance (G=1/Rx) can be read directly on commercially available instruments as a panel mounted meter. Now several digital instruments are also available, such instruments give the conductance directly as the numerical value.
18.6.3 Experimental Measurement
Conductance is reciprocal of resistance and the resistance of a cell can be measured by placing it in an arm of a Wheatstone bridge. The inverse of the resistance gives the conductance and can be directly read on a conductivity measuring instrument, known as “Conductometer”.
A typical conductometer, consists of an ac source, a wheatstone bridge circuit, a null detector or direct reading display and a conductivity cell.
To avoid the effects of polarization, i.e. the change is composition of the measuring cell, alternating current (ac) is used. The instrument has an arrangement to convert the supply of 50 Hz to higher frequency, say 1000 Hz. For measuring low conductance solutions, the lower frequency is preferable and for high conductive solutions higher frequencies are preferably used.
Several inexpensive conductometers are commercially available. The instruments come as a line-operated unit with and without digital readout. For spot checking on a process stream or tank, a dip-type of conductivity cell is used. For titration work conductivity cells of varied designs are available. In some titrations on open beaker with fixed electrodes is sufficient. However, for fairly dilute solutions an open beaker would not be satisfactory because atmospheric CO2 may alter the conductance.
Fig. 18.14 gives the view of a typical conductometer, which can be operated as with given instructions.
Read .4 .8 1.2 Cal. 0 1.6 2.0 Sensitivity
mS range selector Conductometer 29
Instrumental Methods of Analysis
Fig.18.14: Conductometer.
1. Plug the instrument to an ac supply.
2. Put the frequency selector switch to required frequency (say 1000 Hz).
3. Set the mode selector on CAL and set the range selector on the desired setting e.g., 2, 20 or 200. These figures refer to the full scale meter value in milli Siemens (mS). With the help of sensitivity knob keep the pointer roughly midway between the lowest and highest sensitivity say at 1 position.
4. Connect the conductivity cell electrodes to the appropriate terminals of the instrument. Clean the conductivity cell with distilled water (Conductivity water).
5. Take the standard KCl solution (say 0.1M) in a clean beaker. Introduce a stirring rod (to be used for magnetic stirring) in the solution and put the solution beaker on a magnetic stirrer plate.
6. Insert the conductivity cell in the solution. Ensure that the platinum plate electrodes are completely immersed in the solution and they do not touch the stirring rod or the sides or the bottom of the beaker.
7. Switch on the instrument and allow it to warm up for 2-5 minutes.
8. Measure the conductance, Gs, of the standard KCl solution by putting meter switch to READ position.
9. Remove the KCl solution from the beaker, wash the conductivity cell properly with distilled water. Take the unknown solution in the beaker and measure its conductance, Gu, in the manner as for standard KCl solution.
10. Calculate the cell constant, from the conductance and conductivity values of the standard,
Conductivity (specific conductance) K = cell Observed conductance of the standard κ 11 = s cm−− G s 11. Calculate the conductivity (specific conductance) of the unknown solution = cell constant × observed conductance
κuu= KGcell .
12. For titration work, the value of cell constant, Kcell is not required to be calculated, since the cell constant will remain unchanged during the course of any given titration.
Notes:
(i) When the range selector is switched to a new position, it is essential to check the calibration again. Set the meter again to read one with the sensitivity control, if any deviation is observed. (ii) The conductivity cell, when not in use, should be kept in distilled water to 30 prevent drying the platinum electrodes. (iii) In case of fouling the conductivity cell electrode plates, clean them by keeping Electroanalytical in dilute K2Cr 2O7 containing H2SO4 solution (i.e. dilute chromic acid) for 24 Methods hours and then washing with running water followed by rinsing with distilled water.
SAQ 6
At 298 K, the resistance of 2.00 × 10−2 M KCl is 195.96 Ω and that of 2.50 × 10-3 M −2 K2SO4 is 775.19 Ω. The conductivity (κ) of 2.00 × 10 M KCl at 298 K is 0.2768 −1 S m . Calculate molar conductivity of K2SO4 solution. ………………………………………………………………………………………… ………………………………………………………………………………………… ………………………………………………………………………………………...
18.7 APPLICATIONS OF CONDUCTOMETRY
The high sensitivity of the conductometric measurements makes it an important analytical tool for environmental analysis and certain other applications. A continuous or spot check measurement of conductance is employed, usually, with a dip electrode cell and meter. In certain cases continuous recording of conductance is also employed. Since conductance depends on ionic concentrations, the purity of steam distillate, demineralized water, and the ionic contents of raw water can be checked with measuring conductance directly. Metal industries, electroplating baths and rinse baths are monitored by conductance methods.
Perhaps the most common application of direct condutometry has been for estimating the purity of distilled water. Kohlrausch with a painstaking work after 42 successive distillations of water in vocuo obtained a conductivity water with specific conductance, κ = 4.3 × 10?8 S cm?1 at 180C. Traces of an ionic impurity will increase the conductance appreciably. Ordinary distilled water in equilibrium with the carbon dioxide of the air has a conductivity of about 7.0 ×10–7 S cm–1. The sea water has much higher value of conductivity and the conductometric measurements are widely used to check the salinity of water in oceanography.
Measuring conductance of soil helps in finding the moisture content of soils at various places with portable instruments. All soils contain varying amount of water soluble salts upto 0.1% or even more. These salts are usually present as sulphate, chloride, carbonate or bicarbonate of sodium, potassium, calcium and magnesium and contribute to the conductance of the soil. The soil may be classed as saline and non- saline depending on the nature and quantity of the salts present. Conductivity of a saturated extract with water of saline soil at 250C has a conductivity greater than 4 mS cm−1.
18.8 SUMMARY
You have learnt that pH tells the acidic or basic nature of an aqueous solution and is defined as negative logarithm of hydrogen ion concentration. pH can be readily measured electrometrically in an accurate manner with the help of a pH meter. The quality of an acid rain is determined by its pH. The use of ion selective electrode can be made to determine certain ions in a similar way as H+ by glass electrode.
Conductance is the ease of flow of current through a solution. Conductance can be determined following the Wheatstone Bridge principle. The instrument used for its
31 Instrumental Methods determination is conductivity bridge. Direct conductometry has been used for of Analysis estimating the purity of water and the nature of the soil.
18.9 TERMINAL QUESTIONS
1) Define pH. 2) Calculate the pH of 2 × 10?3 M HCl solution. 3) Draw the diagram of a glass electrode. 4) What is asymmetry potential? 5) Define molar conductivity. 6) The conductivity of 0.20 M solution of KCl at 250C is 2.5 × 10−1 S cm−1. Calculate its molar conductivity.
18.10 ANSWERS
Self-Assessment Questions
1) Using the equation, pH = − log [H+]
We get, 4.5 = − log [H+]
log [H+] = − 4.5
[H+] = 10−4.5 M or 10(−5 + 0.5) M = 100.5 × 10−5 M
= 3.2 × 10−5 M
2) The electrode whose potential is to be measured, is coupled with a standard hydrogen electrode and the voltage of the resulting cell is the electrode potential of the electrode being studied, the conditions being such that the liquid-liquid junction potential is negligible.
3) A standard electrode with reference to which the potential of an indicator electrode is determined is called “reference electrode”. Some examples are Standard Hydrogen Electrode (SHE), Saturated Calomel Electrode (SCE).
4) Because of asymmetry potential, a glass pH electrode should be calibrated before determining pH of a solution.
5) H2SO4, HNO3 etc.
6) From Eq.18.27 for KCl solution:
Kcell = κ × R
= 0.2768 S m -1 × 195.96 Ω = 54.24 m−1.
Conductivity of K2SO4 solution can be given by,
32 K 54.24 m−1 Electroanalytical κ = cell = Methods R 775.19Ω
= 0.06997 S m−1.
−3 Concentration of K2SO4 in mol m unit:
c = 1000 × 2.50 × 10−3 mol m−3
= 2.50 mol m−3
Molar conductivity of K2SO4 can be calculated using Eq.18.28
? 0.06997 21− Λm = = Smmol c 2.50
= 0.028 S m2 mol−1
Terminal Questions
1) pH is defined as the negative logarithm of hydrogen ion concentration: pH = −log [H+]
2) [H+] = 2 × 10?3 M pH = −log 2 × 10?3 = −log 2 + 3 log 10 = −0.30 + 3.0 = 2.7
3) Please see Figure 18.4.
4) The glass electrode has an asymmetry potential and is the potential difference across the glass membrane when the two sides of the cell are of identical composition.
5) The molar conductivity is the conductance per unit molar concentration of the electrolyte. It is related to the specific conductance κ and molarity of the solution.
6) Conductivity of KCl solution at 25°C = 2.5 × 10−1 S cm−1 or 25 S m −1
Concentration of KCl in mol m−3 unit:
c = 1000 × 0.2 mol m−3
= 200 mol m −3
Molar conductivity of KCl can be calculated using Eq. 18.28.
k 25 ? == S m2 mol−1 m c 200
= 0.125 S m2 mol−1
33 Instrumental Methods of Analysis
FURTHER READINGS
1. Electroanalytical chemistry, edited by H.W. Nurnberg, (Inter science), 1974, John Willy & Sons Ltd.
2. Physical chemistry by W.J. Moore, Longmans Green and Co.
34 UNIT 19 OPTICAL METHODS
Structure 19.1 Introduction Objectives 19.2 Basics of Spectroscopy The Nature of Electromagnetic Radiation Spectral Regions Classification of Spectroscopic Methods 19.3 Absorption Methods Fundamental Laws of Absorption Methods Absorbing Species 19.4 Ultraviolet-visible Spectrophotometry Instrumentation Some Typical Instruments Analytical Techniques Determination of Substances in Waters, Soil and Air 19.5 Emission Methods Flame Photometry Atomic Absorption Spectrophotometry 19.6 Summary 19.7 Terminal Questions 19.8 Answers
19.1 INTRODUCTION
In the previous unit, we have studied the electroanalytical methods of analysis. Another widely used instrumental methods of analysis in the field of environmental chemistry are optical methods. These methods measure the results of interaction between electromagnetic radiation and matter. The range of electromagnetic radiation may vary from X-rays, through visible, to radio waves.
In optical methods of analysis we consider emission, absorption, scattering, or change in some property of radiation (such as: direction and state of polarization). Measurement of these effects gives different types of optical methods, which may be utilized, to identify and determine one or more constituent of the sample.
In modern usage the word spectroscopy is used to characterize important optical methods where, in general, the study is made of the emission, absorption or scattering of electromagnetic radiation involving energy changes in nuclei, atoms or molecules. The absorption of electromagnetic radiation is used in absorption spectrophotometry and emission used in emission spectophtometry. Whereas scattering results: Raman spectroscopy, nephelometry and turbidimetry.
Spectroscopy has been found to be an important tool that can quickly solve certain difficult tasks in chemistry as well as other branches of science. Spectroscopic methods are used in widely diverse fields, such as, structure elucidation, identification of compounds and functional groups, qualitative and quantitative analyses, determination of thermodynamic properties, and pollution analysis. The recent proliferation of government regulations with respect to atmospheric contaminants has demanded the development of sensit ive, rapid and specific methods for a variety of chemical compounds. Absorption Spectrophotometry solves the problem better than any other single tool. Spectrophotometry involves measuring the extent to which radiation energy is absorbed by a chemical system as a function of wavelength, as well as, the measurements at a fixed predetermined wavelength of the radiation.
All spectroscopic methods have in common the interaction of electromagnetic radiation with the quantized energy states of matter. The emission or absorption of 35 Instrumental Methods radiation of a particular frequency results the transitions between different states of of Analysis energy. The aim of spectroscopist is to measure the relative amounts of radiant energy absorbed or emitted at such frequency and to relate these changes with the nature and amount of various substances.
Especially ultraviolet-visible spectroscopy is still probably the single most frequently used analytical method for quantitative analysis of trace components in routine work. Therefore, our main concern here is to deal with ultraviolet-visible absorption methods and to apply them to pollution studies.
The discussion will also include flame photometry and atomic absorption spectrophotometry. Flamephotometry although applicable to the determination of a few elements but is simple.
Objectives
After studying this unit, you will be able to:
• describe electromagnetic radiation, • classify the spectroscopy methods, • define and relate various parameters such as wavelength, frequency, wave number etc. associated with the electromagnetic radiation, • define Beer-Lambert’s Law, • describe the different components of a spectrophotometer, • define absorbing species such as chromophores, auxochromes, etc. • determine substances in water, waste waters, soil and air samples using ultraviolet-visible spectrophotometry, and • describe flame photometry and atomic absorption spectrophotometry.
19.2 BASICS OF SPECTROSCOPY
Before taking up absorption and emission spectrophotometry in detail, let us review some of the concepts, whic h you may have studied in CHE 1 (Atoms and Molecules), CHE 5 (Organic Chemistry) and CHE 10 (Spectroscopy) courses of our B.Sc. Programme.
19.2.1 T he N ature of Electromagnetic Radiation
All optical methods involve the interaction of electromagnetic radiation with matter following several mechanisms. A brief description of electromagnetic radiation and their characteristics should be considered first.
The electromagnetic radiation (emr) has dual nature: (i) a stream of discrete particles, called photons (or quanta), and (ii) a type of energy that is transmitted through space with enormous velocities, called waves. Various optical phenomena are best interpreted with the wave nature of emr.
The electromagnetic radiation, in general, may be described as wave motion characterized by electric and magnetic displacement at right angles to each other and to the direction of propagation of radiation, as shown in Fig. 19.1
36 Optical Methods
Electric field y
Magnetic field x z
Direction of propagation
Fig. 19.1: The electric and magnetic Fields of the electromagnetic radiation.
For optical phenomenon only the electric displacement needs to be considered. In order to characterize many of the properties of emr it is convenient to portray these waves by such parameters as velocity, frequency, wavelength and wave number.
• Velocity: The velocity of an electromagnetic wave is the rate at which the wave front moves through a medium. The velocity of all electromagnetic waves in vacuum is the same (equal to 2.998 x 108 m s-1 (meter per second) and is denoted by c. However, velocity is dependent upon both the composition of the medium and the frequency. But in vacuum the velocity of radiation becomes Angstorm unit, A° after independent of frequency and is at its maximum. A.J. Angstrom, a • Wavelength: It refers to the distance between (two) adjacent crests or troughs. Swedish Physicist o (See Fig. 19.2). It is designated by λ (lambda). The units of wavelength depend A =10 −10 m o upon the region of the spectrum. In ultraviolet and visible region Angstrom (A ) nm = 10 −9 m and nanometer (nm) are widely used.
+ Wavelength
Amplitude, A
0
Electric field
- Time of distance
Fig. 19.2: Wavelength (λ) and amplitude (A) associated with electromagnetic radiation.
• Wave number: The number of waves in unit length is referred to as the wave Wave number is often used by chemist (particularly in number. The wave number is reciprocal of wavelength. The symbol for wave infrared region as a number is ν (nu bar). The common unit of wave number is reciprocal frequency unit since centimetre (cm-1). ν = cν • Frequency: Frequency is the number of waves (or cycles) passing a point of space in unit time. Unit of frequency is Hertz, 1Hz = 1 cycle per sec. Frequency is denoted by ν (nu).
Frequency is determined by the source and remains invariant regardless of the medium through which the radiation travels. 37 Instrumental Methods of Analysis These parameters are related among themselves as follows:
1c ν =, ?==cν ,c=?? ??
Interaction of Radiation with Matter
When electromagnetic radiation comes in contact with atoms or molecules of matter, there may be an exchange of energy between them. The system may absorb energy and go from the lower energy state(ground state), E1, to higher energy state(excited state), E2. Alternatively, a system initially in the higher energy state E2 can lose energy and go to the lower energy state E1. This transfer of energy is quantized and energy difference, ∆E, between these two states is given by the following equation.
∆E = E2 – E1 = hν ..… (19.1)
This equation can be related to λ and ν by following equation
∆E = hc/λ = hc ν
where h is the Plank’s constant and has the value 6.626 × 10 −34 J s (Jules second) ν is the frequency of electromagnetic radiation which is causing energy changes. The energy changes are shown in Fig. 19.3.
E2
E
E 1
Fig. 19.3: Energy level diagram; E1, lower energy level, E2 high energy level.
By determining the energy absorbed or emitted, we can know about the energy levels or transitions present in an atom or a molecule. In other words, these transitions can be related to the structure of the atoms or the molecules. The energy absorbed or emitted by matter can be detected by an instrument called spectrophotometer. These instruments are designed to measure the frequencies or wave number or wavelength of radiations which are absorbed or emitted by a particular sample on irradiation.
SAQ 1
Give the wave number in cm-1 and the frequency in Hz for radiation of the wavelength o 4000 A .
………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………………
19.2.2 Spectral Regions
38 The spectrum of electromagnetic radiation is broken down in several regions Optical Methods (Fig. 19.4). The limits of these regions are determined on the basis of different mechanisms involved and different kinds of information achieved from the interaction of electromagnetic radiation. Table 19.1 provides the different spectral regions with their approximate wavelength ranges and the types of transition involved.
Ultraviolet Infrared
Far IR - rays X- rays Visible
Near IR Near UV Middle IR Microwave Vacuum UV Radio waves
10 nm 100 nm 1000 nm 10 m 100 m 1000 m (1 m)
Fig 19.4: Representation of electromagnetic spectrum.
Table 19.1: Regions of the electromagnetic spectrum and types of transition involved.
Region of Electromagnetic Wavelength Type of Transition Radiation Range (consequential effect)
o Nuclear transition γ- Rays (Moss Bauer 0.005 – 1.4 A spectroscopy) (change of nuclear configuration)
X-rays (Diffraction, 0.1 – 100 Å Inner electron transition Absorption, Emission, (change of electron distribution) Fluorescence)
Vacuum UV (Absorption) 10 – 180 nm Bonding electrons transition (change of electron distribution)
Ultraviolet- visible 180 – 780 nm Bonding electrons transition (Absorption, Emission, (change of electron distribution) Fluorescence)
Infrared (Absorption) and 0.78 – 300 µm Vibration/Rotation of molecules Raman (Scattering) (change of configuration)
Microwaves (Absorption) 0.75 – 3.75 nm Rotation of molecules (change of orientation)
Electron Spin Resonance 3 cm Spin of electrons in a magnetic (ESR) field (change of electron spin)
Nuclear Magnetic Resonance 0.6 – 10 m Spin of nuclei in a magnetic field (NMR) (change of nuclear spin)
39 Instrumental Methods You may note that the visible portion of the spectrum to which the human eye is of Analysis sensitive is a very small part of the emr spectrum.
19.2.3 Classification of Spectroscopic Methods
In the preceding sub-section you have studied that the spectroscopic methods are based on the energy changes occurred due to the interaction of emr with matter. Thus, they can be classified on the basis of the energy changes (i.e. nuclear, electronic, vibration, or rotational etc.) involved in the transition. Another way of classification is on the basis of the type of the process (i.e., emission, absorption, or scattering) involved in the transition. Yet, another way to characterize spectroscopic methods is according to the spectral region of electromagnetic radiation involved. They include: γ-ray, X-ray, ultraviolet, visible, infrared, microwave, electron spin resonance and nuclear magnetic resonance methods.
We can choose a suitable spectroscopic method in order to solve the problems of structure elucidation, quantitative estimation, or measurement of such properties as the value of dipole moment, equilibrium constant etc.
X-rays usually cause transitions of inner shell (K and L shell) electrons. The most widespread use of X-rays has been in the field of metallurgy, but X-rays may also be used to analyse metals, minerals, liquids, glasses and ceramics. They can be used to determine the crystal structure and to measure the thickness of a very thin layer of tin plating.
Ultraviolet-visible spectroscopy involves the electronic transitions in atoms and molecules. It is mainly used for quantitative analysis of substances of different categories, such as, inorganic, organic and bio-chemicals. The use of this technique is of wide importance in clinical laboratory and to perform chemical analysis of environmental samples.
Infrared spectroscopy can be applied to determine the molecular structure. Identification of compounds can be done by identifying the functional groups. Quantitative determination is also possible. Raman spectroscopy is another technique, which utilizes this range of frequency and is used for the same kinds of applications as infrared (IR) and is complementary to IR spectroscopy.
Nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spec troscopic methods are rather recent techniques but are very useful in structure elucidation particularly of organic compounds. In these methods the splitting of energy levels takes place in the influence of an external strong magnetic field.
With these fundamental backgrounds, now we will concentrate our discussion on absorption method with special emphasis to methods involving ultaviole-visible region of electromagnetic radiations.
19.3 ABSORPTION METHODS
Absorption Methods in general and using ultraviolet-visible region in particular is probably the most frequently used analytical technique for the quantitative estimation of substances in traces. May be a clinical laboratory to analyse the samples of blood or urine, or an environmental laboratory to know toxic metals in natural waters or waste waters, or a routine analysis in industries, science laboratories and so forth, this technique is widely used. In absorption methods involving ultraviolet-visible region, the sample solution is ideally irradia ted with electromagnetic radiation of single wavelength (monochromatic radiation), and the amount of absorption at each wavelength is measured as the wavelength is varied. By plotting the absorbance or 40 percentage transmittance, against the wavelength, an absorption spectrum is obtained. Optical Methods An instrument called spectrophotometer is used to make the measurement. The plot of absorbance (A) vs. wavelength (λ) is illustrated in Fig. 19.5. In this figure you can see that two maxims occur, corresponding to point (i) and (ii), denoting intense absorption at the corresponding wavelength. We will discuss terms, absorbance and transmittance in detail in next sub-section. Spectrum shown in Fig. 19.5 is usually characterized by two parameters.
2.0 100
max=279 nm (a) O
CH-33 C- CH Acetone 0 210 230 250 270 290 310 (ii) =255 nm max (c) Benzene vapour A
Absorbance 1.0 % T 50 (i) 0 230 240 250 260 270
max =255 nm (d) Benzene in hexane 0 230 240 250 260 270 (i) (ii) A few typical absorption curves 0 0 /nm 300 340 380 420 440 300 340 380 420 440
Fig. 19.5: Presentation of absorption spectral data.
? max value: The value of the wavelength at which maximum absorption occurs is called wavelength maxima, ? max. Values of ? max for different molecules are different. For example, ? max for acetone is 279 nm, whereas for benzene, it is 255 nm. Compounds may have more than one maxima, Fig. 19.5 is showing two ? max values (i) and (ii).
∈ Value: The extent of absorption for a given concentration of a compound at any given wavelength is defined by molar absorptivity, which is indicated by ∈ (epsilon) There is a useful term value. It is related to the height of the absorption band. We shall define this precisely Transmittance , and is defined in next sub-section. The parameters, ? max (the position) and ∈ (the extent of as the ratio of radiant power absorption) are characteristic property of a molecule. These parameters depend on the transmitted through sample to structure and concentration of the molecules in solution. Therefore, absorption the incident radiant power spectrum especially in ultraviolet-visible region is extensively used in characterisation T = P/Po and also in quantitative estimations. Transmittance is often In this section we will take up fundamental law s of quantitative analysis relating the expressed in percentage, amount of radiation absorbed to the concentration of an absorbing substance and % T = P/P0 × 100 structural requirement of a substance to absorb the Ultraviolet visible region. while working with solution a 19.3.1 Fundament Laws of Absorption Methods comparison can be made with a blank, where the ratio of There are two fundamental laws used in absorption methods. One is Bouguer’s law or transmitted powers through Lambert’s law, which expresses the relationship between the light absorptive capacity solution and that through blank (or solvent) is called as and the thickness of the absorbing medium; and the other is Beer’s law, which transmittance. For this 100% T expresses the relationship between the light absorptive capacity and the concentration adjustment is made by putting of a solution. The two laws are combined together to give Beer-Lambert’s law. You the blank in the light path. will know about these laws in the following discussion. Note: Transmittance being a ratio has no unit. Beer-Lambert’s Law
Reciprocal of transmittance is known as Opacity. 41
Opacity = 1/T = P0/P Instrumental Methods Lambert’s Law: Earlier Bouguer and later Lambert gave a mathematical relation based of Analysis on the transmission of monochromatic light by homogeneous absorbing medium and stated that, “each unit length of absorbing material through which light passes absorbs the same fraction of entering light”.
Px P-Px d x P P 0
dPx b
Fig. 19.6: Illustration of Lambert’s Law.
In Fig. 19.6, if Po represents the radiant power of incident light and P represents the radiant power of transmitted light after passing through a slab of thickness b, consider a small slab of thickness dx, then the change in power, dPx, is proportional to the power of incident light (Px) multiplied by the change in thickness dx of the slab through which the light is passed. That is,
dPx ∝ Pxdx or dPx = - k Pxdx ..… (19.2)
where k is the proportionality constant and the negative sign indicates that radiant power decreases with absorption Eq. (19.2) can be rearranged to,
dP x =−kxd ..… (19.3) Px
Integrating Eq. 19.3 we get,
PbdP x =−kxd ∫∫P Poo x ..... (19.4) P orln =−kb Po
Eq. (19.4) is the mathematical expression for Bouguer-Lambert law or Lambert’s law.
Changing Eq. (19.4) to base 10 logarithms and rearranging we get,
P k log'o ==bkb ..… (19.5) P 2.303
Note that the ratio P/Po has been inverted to remove the negative sign. Lambert’s law applies to any homogeneous non-scattering medium, regardless of whether it is gas, liquid, solid, or solution.
42 Beer’s Law: Beer (1852) modified the Lambert’s law from the point of view of the Optical Methods effect of concentration of the absorbing species under a constant path length b. He indicated that, “the decrease in power of a radiant beam of monochromatic radiation is proportional to the power of the beam multiplied by the change in concentration of absorbing substance in the path”. The mathematical expression of Beer’s law is derived in the same manner as the Lambert’s law. Consider a parallel monochromatic radiation beam traversing any thickness of solution of a single absorbing substance of concentration c if c is changed by a small amount dc to c + dc, the change in transmitted power is:
d ∝ PP dc x x " dPx −= k Px dc where k″ is the proportionality constant and the negative sign indicates that radiant power decreases with absorption. This equation can be rearranged to:
dP " x −= dck Px
On integration, we get
P d P c ∫ x −= " ∫dck Px Po o P or ln −= " ck Po P or log o = " ck P ..… (19.6)
It can be shown mathematically that the two Eqs. (19.5) and (19.6) may be combined to yield:
P log o = abc ..… (19.7) P
Where a is a constant (combining two constants k′ and k″) known as absorptivity for concentration c given in grams per dm 3. It has unit cm−1g−1 dm3.
The term log Po/P is given a special symbol, A, known as “absorbance”. Eq. (19.7) then is,
A = abc ..… (19.8)
When concentration c is expressed in moles per dm3 (mol dm−3)
A = ∈bc ..… (19.9)
Where∈ (Epsilon) is called molar absorptivity (formerly called the molar extinction coefficient) and it has unit cm−1 mol-1 dm 3
Eqs. (19.8) and (19.9) are expressions of Beer-Lambert’s law or often referred to as Beer’s law.
43 Instrumental Methods The Beer -Lambert’s law provides a quantitative relationship to determine the of Analysis concentration of a solution by measuring the amount of light absorbed by that solution in a known path length cell.
Deviations from Beer’s Law
The Beer -Lambert’s law (or, as in common practices, simply Beer’s law) given by equations A = abc, states that a plot of absorbance versus concentration should give a straight line passing through origin, Fig. 19.7a. However, deviations from this linear relationship between absorbance A and concentration c may sometimes be encountered and instead of a straight line a curvature in the plot may be observed. The upward curvature, curve (b), is known as positive deviation and the downward curvature, curve(c), as negative deviation.
a b
c
A
0 c
Fig. 19.7: Deviations from Beer’s law.
From Eq. (19.8), it is evident that the slope of the absorbance versus concentration plots will be equal to a b. When both a and b are constant the slope is constant and the relationship between A and c is linear. However, when any of the two i.e. either a or b is not constant, there is departure from linearity in the Beer’s law plot. Generally, cell length is a constant factor and is not involved in deviation. That is, deviations to Aαb, the Lambert’s law, are not known (if the instrument factors are not changed).
The deviation is then caused due to the variation in absorptivity. Absorptivity, a, is a function of wavelength and the nature of the substance whose absorbance is being measured. The nature of the substance depends on a number of variables. The variation in absorptivity may thus be caused due to several factors, such as: non- monochromatic radiation, dissociation, association, complex formation, polymerisation, solvolysis, stability of the absorbing species, pH, photochemical reaction, reaction time and temperature. Failure of Beer’s law due to these factors is grouped in apparent deviations since they reflect experimental difficulties rather than the limitation of Beer’s law itself. These deviations are called apparent since they are caused by the deviation from the conditions for which the law was derived, and disappear if the actual conditions are used. The effects of some of these factors are being discussed in the following paragraphs.
Non-monochromatic Radiation : Beer’s law requires that the radiation be monochromatic. In usual practice, however, one works with a narrow band of wavelength and not with a single wavelength. Since absorptivity is a function of wavelength, the absorptivity, a, at one wavelength λ may not be identical with absorptivity, a’, at the other wavelength λ’ in the band. Therefore, the relationship between absorbance and concentration may be non-linear. The deviation will be greater as the difference between a and a’ becomes greater. 44 Optical Methods Association and dissociation : Deviations from Beer’s law arise when an analyte undergoes dissociation, association, or polymerisation to produce a species with a different λmax than that of analyte. An example of this behaviour is found with dichromate and chromate equilibrium, given in equation below:
22−+− Cr2O7+H24O→+2H2CrO
Obviously the two species dichromate and chromate have different colours and 2− 2 − different spectra with different λmax. The concentrations of CrO27and CrO4 be 2− A wavelength at which two affected by pH. CrO27 which is the dominating species at a lower pH will change to or more species in chromate on dilution with water and the deviation from Beer’s law will be observed. equilibrium with one another have the same absorptivity value is called However, the measurement of absorbance at isobestic point (or is absorptive an isobestic point. wavelength) at which the two absorbing species in equilibrium have a common value of absorptivity will not show deviation. Beer’s law then holds, though the measurements have low sensitivity, even when there is a shift in equilibrium.
Another example of this behaviour is observed with the equilibrium of an acid-base indicator, where the
HIn ƒ H+ + In−
Two species HIn and the dissociated ion In− have different colours. The colour changes with change in pH. Therefore, to avoid the deviation effect, we should buffer the solution before the measurements are made.
Temperature: Although temperature is not considered as an important factor since ordinarily the measurements are made at a constant temperature. However, changes in temperature, sometimes, may shift ionic equilibrium and the absorptivity. For example, the colour of acidic ferric chloride solution changes from yellow to reddish brown on heating and therefore λmax and absorptivity are changed.
Photochemical reactions: If analyte undergoes a photochemical reaction due to the effect of radiation the product will be different than the analyte and deviation is obser ved.
Real Limitations: The other class of deviations, which may be considered real, rather that apparent, may be encountered due to the following factors:
Concentration: Beer’s law is applicable to dilute solutions only (that is for concentration lower than 0.01M). For higher concentrations deviations are caused due to diminishing the average distance between the species responsible for absorbance in the solution (i.e., area for capture of photon by the absorbing particles in the solution is decreased).
Refractive Index: Absorptivity is changed when wavelength is changed. Since wavelength changes when the medium is changed, that is, the refractive index (µ) of the medium is changed, absorptivity, therefore, depends on the refractive index of the solution. Changes in concentration can change the refractive index and therefore changes in absorptivity resulting deviation from Beer’s law. However, this effect is very small and is generally well within the experimental errors in spectrophotometry.
Additivity of Absorbance
45 Instrumental Methods According to Beer’s law equation (Eq. 19.9), the absorbance at a given wavelength is of Analysis proportional to the number of radiant particles, which are effective in absorbing radiation power. When it is applied for more than one absorbing species, we have,
A = ∈x bcx + ∈y bcy ..… (19.10)
In general,
A = ∑ Ai = b ∑ ∈i ci ….. (19.11)
That is, the total absorbance of a solution at a given wavelength is equal to the sum of the absorbance of the individual components present. This means that the absorbance is an additive property. This property can be useful in the following ways:
i) To find the contribution of solvent in absorbance measurements, that is the familiar use of blank. ii) To find the absorption spectra of unknown chromophore in presence of a known chromophore by subtraction. iii) In multiple component analysis, that is, the simultaneous determination of the concentration of two or more components in a mixture.
SAQ 2
In a photometer at the λmax of a sample using a 2.00 cm cuvettes the value of Po was 85.4 with the solvent and with 1 × 104 M solution of sample, P was 20.3. What is the molar absorptivity of the sample? ………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………………
Analysis of Binary Mixtures
The property of additivity of absorbance may be applied to determine the concentration of two absorbing constituents present in a single solution, provided that
the two constituents have separate wavelength maxima (λmax) and these constituents do not interact with each other. Consider a solution containing two absorbing
constituents X and Y with wavelength maxima at λ1 and λ2 respectively. In order to calculate the concentration cx and cy in the mixture, we need to take measurement of
absorbance at two different wavelengths (say λ1 and λ2) then,
A1 = (Ax)1 + (Ay)1
= (∈x)1bc x + (∈y)1bcy ..… (19.12)
and A2 = (Ax)2 + (Ay)2
= (∈x)2bc x + (∈y)2bcy ..… (19.13)
Where (Ax)1 is the absorbance contribution due to component X at λ1; (Ay)1 is the absorbance contribution due to component Y at λ1; (Ax)2 is the absorbance
contribution of X at λ2; and (Ay)2 is the absorbance contribution of Y at λ2. Molar absorptivities (∈) are the respective values with the given suffixes in a manner similar to that for absorbance. cx and cy are the concentrations of components X and Y in the mixture being analysed.
Eqs. 19.12 and 19.13 can be solved for cx and cy provided we have the value of the four constants (molar absorpitivities), (∈x)1, (∈y)1, (∈x)2, (∈y)2. The molar
46 absorptivities are determined by making absorbance measurements on pure molar Optical Methods solution of X and Y at wavelength λ1 and λ2.
Since, absorptivity is the function of the wavelength and the nature of the substance, its value will remain constant at a given wavelength for the given component and, therefore, the values of molar absorptivities calculated for known concentrations from the absorption spectra can be substituted in Eqs. (19. 12) and (19.13) for unknown concentrations. These equations can be solved algebraically to find the values of cx and cy in the unknown solution, with knowledge of the components spectra, which may occur with any of the three possible situations shown in Fig. 19.8.
(a) (b) (c)
A A A x x y x y y
1 2 1 2 1 2
Fig. 19.8: Absorption spectra of X and Y with different possibilities. a) No overlapping b) One -sided overlap c) Double-sided overlap
Fig. 19.8a shows the two spectra with two separate absorption peaks and with no overlapping. Absorbance of component X is maximum at λ1 where component Y does not absorb and absorbance of component Y is maximum at λ2 where component X does not absorb. Thus, from Eq. 19.12, A1 is equal to (Ax)1 and from Eq. 19.13, A2 is equal to (Ay)2, since (Ay)1 and (Ax)2 are zero. cx and cy can then be calculated directly from Beer’s law with the molar absorptivities already calculated for known concentrations. That is, the concentrations of the constituents X and Y are measured directly at λ1 and λ2 respectively, without interference.
Fig. 19.8b shows one-sided overlap, that is, at λ1 there is an overlap of spectrum of Y on the spectrum of X; but the spectrum of X does not overlap on the spectrum of Y at
λ2. From Eq. 19.12, A2 is equal to (Ay)2 because (Ax)2 is zero and then (Ay)2 is equal to
(∈y)2bcy gives the direct calculation of cy. Now this value of cy is substituted in Eq. 19.12 to give the value of (Ax)1 and cx is then calculated in simple way.
In Fig. 19.8 c, there is a double -sided overlap that is the spectra of X and Y overlaps each other. From Eqs. 19.12 and 19.13, we observe that the absorbance of the mixture of λ1 is A1 which is the sum of (Ax)1 and (Ay)1; and the absorbance of the mixture at λ2 is A2 which is the sum of (Ax)2 and (Ay)2. The two equations are then solved algebraically to calculate cx and cy.
19 .3.2 Absorbing Species
47 Instrumental Methods Absorption of electromagnetic radiation in the near ultraviolet region (175 – 375 nm) of Analysis and visible region (375 – 750) results in electronic transition for both organic and inorganic substances.
Absorption by Organic Species
The absorption of emr by organic compounds is based on the difference in energy between the ground state and the various excited states (electronic) of the molecule. Most molecules show only one or two electronic transitions in the visible and near ultraviolet region and for this only the outermost electrons need to be considered. Organic molecules have electrons in three kinds of orbital viz: σ (sigma) - bonding, p (pie) -bonding and n (non)-bonding.
The electronic transitions of these electrons are characterized by their elevation to excited state antibonding molecular orbitals (π* and σ *). The quantum energies required for these transitions will be different and will also vary with structure of organic molecules. The relative energies of the probable transitions are qualitatively illustrated in Figure 19.9.
Antibonding
Antibonding
Energy n Nonbonding Bonding
Bonding
Fig. 19.9: Order of energy for various types of molecular orbital and electronic transitions.
As shown in Fig. 19.9 four types of transition are possible σ → σ*, n → σ*, π → π* and n → π*. The probable regions of electronic spectrum is regarded as follows:
* ∆E1 σ → σ vacuum ultraviolet * ∆E2 n → σ far ultraviolet * ∆E3 π → π ultraviolet * ∆E4 n → π near ultraviolet and visible
Let us discuss these transitions in some detail.
σ → σ * transition: It is obvious from the preceding discussion that relative to other possible transitions the excitation energy required to induce a σ → σ* transition is large and corresponds to the absorption peaks in vacuum ultraviolet region (λ < 175 nm). Thus, to excite C – H or C – C single bond electrons in alkanes, radiation of wavelength lower than 160 nm is required. For example methane shows a peak at 122 nm corresponding to σ → σ* transition.
n → σ * transition: The energies required for n → σ* transitions are lower than σ → σ* transitions but larger than π → π* and n → π* type transitions and can be brought about by the radiation of wavelength range 150 to 250 nm with most absorption peaks appearing below 200 nm (far ultraviolet range)
48 π → π* transition: The energies required to these transitions are lower and result in Optical Methods longer wavelength absorption than σ → σ* and n → σ* transitions. The absorption in hydrocarbons containing double bonds and triple bonds is observed at wavelengths approaching near ultraviolet regions. Conjugation further increases λmax and is appreciable in aromatic molecules. For example single ring aromatics absorb in the vicinity of 250 nm, naphthalene in the vicinity of 300 nm and anthracene in the vicinity of 360 nm. n → π* transition: In such transitions one of the non-bonding electrons (lone pairs) may be excited into an empty π* orbital. The energies required to these transitions are lower than π → π* transitions and result in ultraviolet and visible region. Presence of atoms or groups containing n - electrons can cause remarkable changes in the spectrum. Thus, nitrogen, sulphur and halogens tend to move absorption to higher wavelengths. Chromophores
Among the most obvious characteristics of a chemical compound is its colour. In 1876 Witt related the appearance of colour in molecules due to the existence of certain chemical groups, termed “chromophores”. Chromophore in Greek means, “colour bringer”. However, the term chromphore is now not limited to colours only but is used in a general way for groups which are responsible for causing an absorption of electromagnetic radiation between 175 and 1000 nm, which is convenient to use experimentally.
The absorption of emr of a particular frequency results the transitions between states of different energy. Therefore, the wavelength at which a chromophore shows its maximum absorbance depends on the type of electronic transition, that is the energy required to carry out the excitation of a particular kind of electron from the lower energy state to the possible higher energy state. In the ultraviolet-visible region most of the absorption bands are due to the excit ation of π-electrons and n-electrons. Energies required for the transition of π electrons and n-electrons to the π* excited state bring the absorption peaks into an experimentally convenient spectral region (175
– 1000 nm). Such as carbonyl in aldehydes and ketones give λmax in ultraviolet region due to the absorption of photons by π or n-electrons. Acetaldehyde has a λmax at 293 * * nm due to n → π transition; acetone has λmax at 186 and 280 nm due to n → σ and n → π* transitions respectively. Some other examples of chromophores in this region are: >C=C<, − C ≡ C, − Ν = Ο & −Ν=Ν − and so on.
A listing of common chromophores, along with the types of transitions and approximate location of their absorption maxima, is given in Table 19.2 although the peaks are ordinarily broad because of vibration effects, these peaks, however, can serve as rough guides for the identification of functional groups in organic compounds.
When two or more chromophores are present in a molecule, absorption depends on their relative positions of λmax and intensity of absorption varies with the nature of the solvent. Usually polar solvents tend to shift π to π* transitions to a longer wavelength (RED SHIFT); an n to π* transitions to a shorter wavelength (BLUE SHIFT).
Table 19.2: Absorption characteristics of some common chromophores.
Chromophore Example λmax Type of Transition * >C = C< C6H13 CH = CH2 177 π → π
* C C C5H11 C ≡ C − CH3 178 π → π − ≡ − 49 * Instrumental Methods >C = Ο CH3CO CH3 186 n → σ of Analysis 280 n → π *
CH CHO 180 * 3 n → σ 293 * O n → π
−C−ΟH CH3COOH 204 * O n → π
CH3CONH2 214 * −C−NH2 n → π
CH N = NCH 339 * −N = N− 3 3 n → π
CH3NO2 280 * −NO 2 n → π
C H NO 300 −N = Ο 4 9 - 665 n → π * In conjugated molecules (i.e. containing alternating double bonds) the absorption is shifted to a longer wavelength due to the fact that the resonance structure results delocalisation of electrons that is in a conjugated system the electron is less tightly bound than once in a non-conjugated system.
“Auxochromes” though do not themselves absorb emr but when attached to a chromophore, alter both the wavelength and the intensity of absorption of the chromophore. An auxochrome is a saturated (functional) group with non.. -bonding.. electrons,.. which can be donated to the conjugated system. Examples are – OR,.. – NH2, – NR2 ect.
Absorption by Inorganic Compounds
The absorption of emr by inorganic compounds, involving d electrons, f electrons and the electronic transitions are briefly mentioned below.
The spectra of transition metal ions are due to the involvement of d orbitals in the co- ordination bonding of these ions with solvent molecules or forming complexes with specific ligands. The degeneracy of the five d orbitals of the transition metal ions is removed in complex formation and electronic transitions from the lower energy d orbitals to higher energy d* orbitals are observed when the emr of proper frequency (which covers the range from ultraviolet to the near IR region) is absorbed.
The ions of lanthanides and actinides absorb ultaviolet and visible radiation due to the f to f* transitions. Here, the f electrons being shielded from external influences by occupied orbitals of higher principal quantum number absorb the radiation of ultraviolet and visible region in narrow bands.
The absorption of emr involving the charge transfer process may be responsible for the absorption in many transition metal complexes. This type of transition involves the electron transfer (via an internal oxidation reduction process) between the two components of a complex upon the absorption of a photon, where one component of the complex acts as an electron donor while the other component of the complex acts as an electron acceptor. Before going further let us try following SAQs.
SAQ 3
Identify the type of absorption π → π*, n → π* or n → σ* among the following compounds:
O λmax
50 a) CH3CCH3 293 Optical Methods b) CH3 COOH 294 c) CH3NO2 280 ………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………………
SAQ 4
Arrange following transitions in increasing order
n → π*, π → π*, σ → σ*, n → σ* ………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………………
19.4 ULTRAVIOLET-VISIBLE SPECTROPHOTOMETRY
Ultraviolet-visible spectrophotometry is a discipline in which the absorption of ultraviolet (UV) or visible light is used to detect one or more components in a solution and measure the concentration of these species. The prime advantage of using this discipline as an instrumental technique is that traces of substances can be determined in a simple way, which is not possible with classical methods. This technique is one of the oldest instrumental methods of analysis. In the early stage of development of this physico-chemical method of analysis, the natural or the artificial white light was used as light source. The measurements were made with simple instruments and in the earlier development of the technique the naked eye was used for comparing the colour intensity of the solution. However, naked eye was replaced by photometers for the measurements of colour intensity and the instrument thus developed were known as photometers or colorimeters. Filters were introduced in colorimeters to choose a spectral band for colour measurement. Later on instrument, which could select a definite wavelength, were introduced and such instruments are known as spectrophotometers. Ordinary colorimetric methods do not give accuracy greater than about 1 percent. For greater accuracy spectrophotometric methods have to be used.
However, both these instrumental techniques have the great advantage of being more simple and economic.
19.4.1 Components of Instruments for Absorption Measurements
An instrument to be used for measuring intensities of emr requires five basic components shown in the block diagram in Fig. 19.10.
Radiant Wavelength Sample Detector Signal Source Selector Indicator
Fig. 19.10: Block diagram showing basic components of an instrument used for measuring absorption of radiation.
A suitab le radiation source must provide sufficient radiant power over the wavelength region where absorption is to be measured. The proper wavelength is achieved by a wavelength selector either by filtering the radiation of the source or by using a monochromator. Filters are applied in filter photometry and are used mainly in the visible range whereas the use of a monochromator is applied in spectrophotometry in the ultraviolet, visible and infrared ranges. The selected wavelength is allowed to pass 51 Instrumental Methods through the solvent or sample placed in the cuvette. The detector measures the of Analysis intensity of the transmitted radiation and gives a signal, which can be read by a signal indicator. Now we describe these components in greater detail.
Sources: The usual source of radiation in the ultraviolet region (180-350nm) is hydrogen or a deuterium discharge lamp operated under low pressure. The important feature of these lamps is to maintain the discharge to a narrow path with the help of a mechanical aperture between the cathode and the anode. The use of deuterium in place of hydrogen enhances brightness of the lamp.
The source of radiation in visible region as well as in near infrared region (325 nm-3 µm) is usually an incandescent lamp with a tungsten wire filament. The coiled wire filament is enclosed in a sealed bulb of glass filled with an inert gas or vacuum. On heating the filament by an electric current, radiation is emitted. Incandescent lamps are rugged and low cost sources, which are adequate to work in the visible and near ultraviolet region.
Wavelength Selector: The purpose of wavelength selector is to isolate a narrow range of wavelength from the source. Radiation sources such as tungsten lamps, hydrogen discharge tubes emit almost continuous radiation over relatively wide ranges of frequencies. Narrow spectral regions of selected wavelength ranges may be isolated from a continuous spectrum by the use of filters. However, a monochromator is required to produce a monochromatic radiation of the desired frequency. A narrow range can be isolated by either filters or still narrower (monochromatic) can be achieved by a monochromator equipped with a prism or grating as the dispersing device.
Filters: An absorption filter is a coloured piece of glass, which absorbs light of some wavelength to a greater extent than others. We know that white light is made up of seven different colours (VIBGYOR). An object of a particular colour looks of that colour because this colour is transmitted and its complementary colour is absorbed. As a first approximation, the filter should transmit a colour nearly complementary to that of the sample. Or in other words, the filter should absorb the light of the colour, which is transmitted by the sample. For example, a blue cobalt glass transmits blue violet light, but absorbs yellow light. The use of such a filter is illustrated in Figure 19.11.
Yellow light Yellow light absorbed
Violet light Violet light transmitted
Filter
Fig. 19.11: Use of blue cobalt glass filter.
To find the colour of the filter we can take the help of colour wheel (Fig. 19.12).
O 520 nm Y R 580 nm 700 nm
52 G V 530 nm 420 nm B 470 nm Optical Methods
Fig. 19.12: Colour wheel.
In Fig. 19.12 the colours are shown with their approximate wavelengths. The colours, which face one another, are said to be complementary to each other. A filter of complementary colour is most suitable for the measurement. For example, for a red coloured solution, its complementary, that is, a green coloured filter should be used which indicates that the λmax of a red coloured solution should lie between 490-525 nm.
The performance of characteristic of a filter is judged by its effective bandwidth, which is expressed as the wavelength interval at one half of the maximum transmittance value when the response of a filter is plotted with variation of wavelength. See Fig.19.13.
A max %A
A max 2 = Effective band width
Fig. 19.13: Response of a filter.
The narrower the effective bandwidth (∆λ) the better the filter is. Generally ∆λ is of the order of 30 to 50 nm.
Absorption filters are simple and are totally adequate for many applications in visible range of emr spectra. However, for extended ranges we need interference filters. The interference filters cover a wider range than the absorption filters. Interference filters are essentially composed of two transparent parallel films of silver, which are so close as to produce interference effects. Such interference filters are available for ultraviolet, visible and near infrared region. The performance characteristics of interference filters are significantly superior to those of absorption (coloured) filters. The effective bandwidths of these filters are narrower than absorption filters.
Monochromators: In order to get approximately monochromatic radiation a dispersing device is to be used. A monochromator, in general consists of an entrance slit for the heterochromatic radiation from the source, a collimating lens or mirror (to make the radiation parallel), a prism or grating to disperse the radiation into its component wavelengths, a lens or mirror to focus the dispersed radiation (more or less monochromatic), and an exit slit through which the monochromatic radiation is allowed to pass. 53 Instrumental Methods Grating monochromators are now more widely used than the prism monochromators. of Analysis A grating is a small piece of metal or glass with numerous parallel and identical grooves (as many as 10,000 grooves/cm) ruled on it. The grating is ruled with a diamond knife on the metal piece with numerous precautions. Ruling a high quality grating is a tedious task. Replica gratings, which are casted by pouring molten plastics on the original grating, are now used. The replica grating are less expensive and are not much inferior to the original grating. Advancement in the grating was the development of concave gratings where the use of lens to focus the radiation is not required.
Fig. 19.14 represents the optical design of a typical monochromator with grating as the dispersing device. Light striking the grating is diffracted so that different wavelengths come off at different angles. Rotating the grating allows radiation of the desired wavelength to be selected.
Incident Diffracted beams Selected beams wavelength
Slit Grooves
Grating
Fig. 19.14: A typical monochromator-employing grating.
Sample: In ultraviolet-visible spectrophotometery sample is introduced in a cell called cuvette. The cuvettes must be constructed of a material that does not absorb radiation in the region of interest. Quartz cells can be used in the range 190nm-84µm. Silicate glass cells are adequate in the whole visible range (375-950nm), and also a part of UV region. Cylindrical cuvettes are often used in the interests of economy; but care should be taken that each cuvette is marked so that its insertion in the cuvette holder always provides the same incident and emergent surface. The cuvettes are usually one centimetre in path length.
Detectors : The detectors of most instruments generate a signal, which is linear in transmittance that is they respond linearly to radiant power falling on them. The transmittance values can be changed logarithmically into absorbance units by an electrical or mechanical arrangement in the signal to read out.
A detector is, of course, a transducer, which converts one type of signal to another. Early instruments used eye or photographic plate as the detector. Most modern detectors are the photoelectric detector where the intensity of emr (i.e. energy of photon) is converted into electrical energy causing an electron flow and subsequently, into a current flow or voltage in the read out circuit.
Human Eye as Detector: In older days identification of colours through naked eye was one of the major analytical tools. The eye of a common person is quite sensitive to notice differences in radiant power transmitted through two coloured solutions. It is a natural photosensitive detector in the visible range. Fig. 19.15 shows average sensitivity characteristics for the human eye. 54 Optical Methods
100
50 Response
0 400 500 600 700 nm
Fig. 19.15: Response of an average human eye as a function of wavelength. The optical nerves carry the signal from retina to the brain through rods and cones. Comparison of colours and their intensities can be made, approximately, by matching with a reference.
Photoelectric Detectors: In these detectors the radiant energy is converted into electrical energy. They are classified as photovoltaic cells and photo emissive detectors. You will know about them in the following discussion.
Photovoltaic Cell: The photovoltaic cell is used primarily to detect and measure radiation in the visible region. A typical photovoltaic cell or photo cell (schematically shown in Fig.19.16) consists of a flat iron or copper electrode (anode) upon which is deposited a layer of a semi conducting material, such as selenium (or cuprous oxide). The selenium layer is coated with a transparent film of silver, gold, or some other metal, which is protected by a transparent plate of glass. A metal ring, which works as the other terminal (cathode) of the cell, is pressed on the transparent metallic film. The two terminals are connected to a galvanometer. The whole arrangement is placed in a plastic case.
Glass Thin layer of silver
Selenium Plastic Iron case
Fig. 19.16: Schematic of a typical photovoltaic cell.
When the radiation fall upon the cell a current flows through the galvanometer. The current depends upon the intensity of the photons impinging on the cell. Under proper conditions the current through photovoltaic cells is proportional to the energy absorbed per unit of time. Photovoltaic cells require simpler circuitry and no amplification. In general, they are used in filter photometers.
Photo emissive Detectors: The photo emissive detectors are very sensitive and are employed to detect even very small variations in the light intensity that is not possible with a photovoltaic cell. Therefore, in spectrophotometers where the wavelength resolution using monochromators is the essential requirement, such detectors are made use of. Two kinds of photo emissive detectors are in use: (i) vacuum phototubes and (ii) photo multiplier tubes.
55 Instrumental Methods (i) Phototubes: A phototube (Fig. 19.17) consists of two electrodes, a semi of Analysis cylindrical cathode and a wire anode sealed inside in an evacuated transparent vessel. The cathode is coated with a photo emissive material, such as potassium or ceasium. Phototubes with a potassium coated cathode are employed in the range 200-600nm, and ceasium coat ed cathode are utilized mainly in the 600-1000nm range. The most sensitive cathodes are bi-alkali types, for example, one is made of potassium, ceasium and antimony.
Electrons Wire anode
Photon beam Cathode
DC amplifier and readout
R
90 V DC Power supply
Fig. 19.17: Schematic diagram of a phototube.
Fig. 19.17 is a diagram of a phototube and its accessory circuit. When photons of sufficiently high energy hit the cathode, the electrons are dislodged from the photo emissive material by photoelectric effect and are collected at anode. The number of electrons ejected from the photosensitive cathode surface is directly proportional to the radiant power of the beam striking the cathode surface. When a potential is applied across the electrodes through a dc source, the emitted electrons flow to the wire anode generate a photocurrent, which depends on the radiant power of the beam and the applied potential through the dc source. At the saturation potential (about 90 V) across the two electrodes, essentially all of the electrons emitted by the cathode then become independent of applied potential and directly proportional to the radiant power of the beam. Thus, the current flow in the system is related to photon flux coming to the concave surface of the photosensitive cathode.
(ii) Photo multiplier Tubes: Photo multiplier tubes, which do not require external amplification proved to be more sensitive and accurate than phototubes. Fig. 19.18 shows the cross-section and electrical circuit of a phtomultiplier tube, which of course, may be considered as a combination of several phototubes arrang ed in a special manner. The intermediately dynodes of photo multiplier tubes are covered with a material which emits several (2 to 5) electrons for each electron being collected on its surface. The dynodes behave in a manner that the anode of the first stage is the cathode of the second.
The primary electrons ejected from the first cathode (e1) strike a small area on the first dynode (e2), which is about 90V more positive than the first cathode (e3). The dynode (e2) covered with photo emissive material ejects 2 to 5 secondary electrons for each electron collected on its surface. These secondary electrons strike the second dynode (e3) and the process multiplies on in each stage. Thus electron amplification occurs on the dynodes, and for a tube of 9 dynodes the overall amplification factor is between 29 and 59.
900 V dc Several electrons + - for each incident electron 90 V Quartz Numerous envelope electrons Quartz 3 5 for each envelope photon 4 1 9 8 7 6 5 4 3 2 1 6 2 Anode Cathode 8 Grill R Numbered dynodes 7 shown in (a) Radiation, hv 9 - 56 7 Photoemissive To readout Anode, -10 + electrons for Cathode each Photon
(a) (b) Optical Methods
Fig. 19.18: A Photo multiplier tube: a) Cross-section; b) Electrical Circuit.
19.4.2 Some Typical Instruments
The instrumental components discussed in the preceding section have been combined in various ways to produce a variety of commercial instruments for measurement of absorption of radiation. In all absorption measurements, the intensity of radiation transmitted through sample is compared with that of the reference (solvent/blank); hence relative rather than absolute measurements are made.
In a single beam instrument, the reference and the sample are placed successively in the path of the monochromatic beam. In a double beam instrument the monochromatic beam is divided by optical means into two equal intensities. One beam passes through the reference and the other through the sample. Some advance type of spectrophotometers are capable of giving direct results of analysis in concentration units and other information with the help of microprocessors used in automation. For example, one digital spectrophotometer with programmable statistical calculator can provide storage of data, calculation of first and second derivative spectra, peak location, peak area etc. Selection of the instrument is governed by the type of the work and the cost of the instrument.
A few simple instruments that are typical of ones the student at this stage is likely to encounter will be described in this section.
Filter Photometers
A relatively inexpensiv e and simple type of instrument is a filter photometer that works around a set of filters in the visible range. Such instruments are adequate for many methods especially for absorbing systems with broad absorption bands. There are two types of filter photometers, namely, single beam and double beam instruments. The single beam filter photometer will be discussed first.
Single Beam Filter Photometer
It consists of a source of light, S, which is simply a light bulb; a condenser lens, L, to produce parallel radiation beam; a filter, F, to give the appropriate wavelength band; a cuvette C; a photovoltaic cell, D, as detector; and a galvanometer as a signal indicator (See Fig. 19.19).
Variable Solvent diaphragm cell to set 100% T D S L F % T 50 (a) Single- 0 100 beam C photometer Tungsten Microammeter lamp Filter Shutter Sample Photocell Cell
Fig. 19.19: Schematic diagram of a single beam filter photometer. 57 Instrumental Methods of Analysis The radiation from the light bulb passes through a convex lens fitted in such a way that its distance from the light bulb is equal to the focal length of the lens. This arrangement gives the parallel beam of radiation, the parallel beam falls on the filter, which permits to pass a narrow band of wavelength. The filtered radiation is passed to the sample cell (cuvette), and the transmitted radiation power is evaluated by a photovoltaic cell. The function of the photovoltaic cell is to convert the energy of photons into electrical energy, which moves the galvanometer scale that is calibrated for percent transmittance or for absorbance.
Working
The following steps are involved in measuring absorbance and transmittance of the sample solution.
1) Turn the instrument “on”, using the power switch, and wait for about 15 min to warm up. 2) Insert the proper filter in its place in the instrument. 3) Fill the cuvette with blank (or solvent) and place it in the light path at proper position. 4) Adjust the pointer of the galvanometer scale at 100% T (or zero A) mark. 5) Remove the solvent/blank from the cuvette, fill it with sample solution and place in the path at proper position. 6) Read the meter and note the percent transmittance/absorbance.
The single beam filter photometers have the advantages of simple construction, low cost and simple operation; but have limitations of low accuracy and low sensitivity because of small galvanometer scale (about 10 cm).
Double Beam Filter Photometers
In a double beam filter photometer the light beam is divided into two parts and two photocells are used as detectors. The schematic diagram of a double beam filter photometer is given in Fig. 19.20. S is the Source, which is a tungsten filament lamp. L is the lens, which is placed at a distance equal to its focal length from the source. The parallel beam after the lens then falls on the filter F that permits only a narrow band of wavelength to pass through it. The filtered radiation is allowed to fall on half - silvered mirror. This divides the original beam into two portions. One beam passes through the cuvette C1 to fall on photovoltaic cell D1 and the other beam through cuvette C2 and then falls on identical photovoltaic Cell D2. P1 and P2 are the two potentiometers and G is the galvanometer, which is used as the null detector. P1 is calibrated in percent transmittance units. K1 and K2 are the two keys.
Filter Shutter Sample cell Photocell Half-silvered mirror D S 2 Double- beam photometer
Tungsten L F C 2 lamp Solvent C 1 cell 100 Null detector Reference D photocell 1 C %T 50 K2 K1 G P P 0 1 2
Fig. 19.20: Schematic of typical double beam filter photometer.
58 Working Optical Methods
1. Turn the instrument “on” and allow warming up for about 15 min. 2. Place the appropriate filter in its place in the instrument. 3. Fill the cuvettes C1 and C2 with blank and solvent, respectively and place it in the light path 4. Put K1 at 100% T value on the scale of potentiometer P1 and move K2 on P2 so that the galvanometer G reads zero. 5. Fill the sample solution in the cuvette C2 and place in the light path. 6. Move K1 on the potentiometer P1 scale so that the galvanometer reads again zero (without disturbing the position of K2). Note the %T on P1. The double beam filter photometer has the following advantages over a single beam instrument:
i) Since the beam of light is divided into two parts and is allowed to fall on two identical detectors, the fluctuations (if any) of the source will not disturb the reading, which is noted after balancing the two potentiometers. ii) Since the galvanometer is used as a null detector, no current is used in moving the galvanometer needle and the results are more accurate than that with a single beam instrument. iii) Since the reading is noted on a large potentiometer scale, the sensitivity is high.
Spectrophotometers
Spectrophotometers are more sensitive instruments than filter photometers. A spectrophotometer is usually a combination of a monochromatic and a photometer. The light is monochromated by a diffraction grating and slit device. Nevertheless several designs of spectrophotometers are available, we shall consider here the Bausch and Lomb spectronic – 20 Spectrophotometer, which is simple to use and is satisfactorily precise.
The operating features of Bausch and Lomb spectronic – 20 are shown in (Fig. 19.21a) and the schematic optical arrangement in Fig. 19.21b. It is a single beam spectrophotometer operating between 340 – 950 nm. With standard phototube the basic range is 340 to 600 nm, which is extended to 950 nm by adding a red filter and replacing the standard phototube with the red phototube. The scale of the instrument is colour-coded to correspond to the operating range of the phototube: black gradations for the basic 340-600 nm range and red gradation for the 600-950 nm range of the optional red phototube/filter combinations. Readings are taken directly from the meter in either absorbance or transmittance mode.
Absorbance %T scale scale
Wavelength Cell selection compartment
On-off %T Light control calibration (100%T calibration)
59 Instrumental Methods of Analysis
Fig. 19.21: a) Bausch and Lomb spectronic – 20 Spectrophotometer; b) Schematic optical lay out for the Bausch and Lomb Spectronic –20 spectrophotometer.
Working
1. Plug the instrument into a grounded outlet to oper ate on a 230 V AC line. 2. Turn the instrument “on” and allow warming up for about 15 min. 3. Select the wavelength with the wavelength selector knob. 4. Choose matched cuvettes of the appropriate path length (usually 1 cm) for the analytical method. The cuvettes of the same path length must be used for all blanks, standards, and samples. 5. Fill one cuvette with blank (or solvent) having sufficient solution to align with the mark on the cuvette. The solution volume should be enough to cover the light beam passing through the sample compartment. 6. Open the sample compartment cover and insert the cuvette containing blank into the sample compartment. 7. Close the sample compartment cover. 8. Set zero absorbance or 100% transmittance on the scale for the blank using the control knob located on the left side. 9. Remove the blank from the sample compartment. 10. Fill the matched cuvettes with standard solutions and insert in the sample compartment one after the other. Read and record the absorbance values for each standard solution. 11. Construct a calibration curve by plotting the absorbance on the y-axis vs the concentration of each standard solution on the x-axis. 12. Fill the matched cuvette with the sample to be measured and insert in the sample compartment. Read and record the absorbance value. From the calibration curve read the concentration corresponding to the absorbance value of the sample.
19.4.3 Analytical Technique
In this part we will take up general procedures in ultraviolet-visible spectrphtometeric analysis. In quantitative det ection of a substance, the Beer-Lambert’s Law forms the basis of the measurement procedure. The amount of light radiation absorbed by a compound is directly related to the concentration of the compound. Following steps are involved in general measurement procedure:
1) Preparation of sample to make absorbing species 2) Selection of wavelength 3) Preparation of the calibration plot
Preparation of sample to make absorbing species
60 Samples absorbing in the wavelength range 200 to 800 nm are generally analysed by Optical Methods ultraviolet-visible method. But it is a common practice; measurements are carried out in visible range. Sometimes the substance being analysed has its own characteristically strong absorption in the visible range but more often it may require the addition of a reagent that form derivative or complex with the necessary high absorptivity.
While choosing the reagent, following points should be considered. i) The reagent should react selectively with the substance to be determined. ii) Conditions must be chosen to obtain optimum colour formation if we wish to detect substance colorimetrically. iii) Product or complex formed should have high molar absorptivity and should be stable.
Selection of Wavelength
Quantitative analysis is generally made at λmax. This is because this give rises to maximum sensitivity in the analysis. λmax is determined by plotting absorption spectrum. Spectrum: A plot of absorbance versus wavelength is known as absorption spectrum. Usually the wavelength is as abscissa and absorbance as the ordinate. All ultraviolet- visible spectrophotometers have a wavelength scale properly graduated in nanometer or in Angstrom units.
Proper Wavelength: An absorption spectrum (a plot of A vs λ discussed above) for a single absorbing species will normally yield a curve having a maximum value of absorbance at a particular wavelength. This wavelength is designated as λmax. At this wavelength, the sensitivity is maximum, that is, the change in absorbance per unit change in the concentration of the absorbing species is a maximum. λmax is the proper wavelength for determination. However, it should not be located in that portion of the spectrum where a small change in wavelength causes excessively high change in absorbance. Under such conditions, λopt, optimum wavelength, must be used. In Fig.19.22a the spectrum appears with only one maximum with a suitable curvature and λmax is also λopt.
A A
1 2
max = opt max opt
(a) (b)
Fig. 19.22: Absorption spectra to locate proper wavelength for determination.
61 Instrumental Methods In Fig. 19.22b λmax (λ1) is in the form of a sharp peak where a small change in of Analysis wavelength will cause an excessively high change in absorbance, therefore it is not the
proper wavelength for spectrophotometeric determination. Here λ2 with a suitable
curvature, though not λmax, should be selected as proper wavelength (λopt) for determination.
Preparation of the Calibration Plot
After setting the instrument, with the monochromator providing the optimum wavelength, a plot is prepared for absorbance values obtained by inserting solutions of known concentrations in successively increasing order. A plot (See Fig. 19.23a) obtained for absorbance versus concentration is known as the calibration plot. Basically, where Beer’s law is obeyed, a plot of absorbance versus concentration will yield a straight line. A plot of absorbance versus concentration may deviate from a straight line after certain range of concentration. Such a departure yields a zone of non-conformity to Beer’s law.
Absorbance Absorbance
Concentration Concentration
Fig. 19.23: a) Calibration plot; b) Finding the concentration of unknown sample.
For measuring concentration of the unknown solution, it should be adjusted in a manner to yield concentration ranges where Beer’s law is obeyed. After measuring the absorbance of the unknown solution, its concentration may directly be read from the calibration plot Fig. 19.23b.
Concentration of unknown may also be calculated using a ratio method by a comparison of its absorbance with the absorbance of a solution of known concentration as:
cCAunknownknown × unknown cunknown = A known
19.4.4 Determination of Substances in Water, Soil and Air
Ultraviolet-visible spectrophotometer is most widely used instrumental method, particularly for routine work at moderately low concentration environmental detection. 62 In metal ions detection in water and soil, ions are converted into coloured species by Optical Methods treating them with chromophoric reagent such as dithizone or diethyldithiocarbamate. In table 19.3 we have summarised few metal reagent pairs used in metal ion detection in water and soil. Inorganic anions and ammonia can also detected by forming coloured derivative with organic compounds, such as nitrate with xylenol (See Table 19.4). For the detection of gases in air, they must first be absorbed in a selective reagent and then reacted to give a dyes stuff, which can be measured using spectrophotometer (Table 19.5), such as a diazo dye for the determination of oxides of nitrogen.
Organic impurities such as anionic surfactants, cationic surfactants, phenols etc. are detected by derivatizing or pairing with a coloured dye molecules.
Anionic surfactants: Methylene Blue Cationic surfactants: Bromophenol Blue Phenols : 4-Aminoantipyrine
Table 19.3: Reagents for Spectrophotometric determinations of metals.
Metal Reagent Wavelength (nm)
Aluminium Chrome azurol S 545 8-hydroxyquinoline 390 Antimony Iodine 425 Arsenic Diethyldithiocarbamate 515 Bismuth Dithizone 490 Iodide 465 Xylenol Orange 450 Cadmium Cadion 480 Dithizone 520 Chromium Diphenylcarbazide 545 Cobalt Nitrosonaphthol 415 PAR 510 Thiocyanate 620 Copper Diethyldithiocarbamate 436 Dithizone 550 Iron 1,10-phenanthroline 512 Thiocyanate 495 Lead Dithizone 520 PAR 520 Manganese PAN 564 Mercury Dithizone 485 Molybdenum Thiocynate 470 Nickel Dimethylglyoxime 400 Selenium Diaminobenzidine 420 Tellurium Bismuthiol II 330 Vanadium 8-Hydroxyquinoline 550 Zinc Dithizone 538 PAN 560
Table 19.4: Reagents for spectrophotometric determinations of anions and ammonium.
Anion Reagent Wavelength (nm) 63 Instrumental Methods of Analysis Ammonium Hypochlorite/phenol 625 Bromide Hypochlorite/Phenol Red 580 Chloride Mercury thiocyanate/Fe(III) 480 Chlorine Orthotolidine 625 Cyanide Chloramine-T/pyridine/barbituric 580 acid Iodide Bromine/starch 590 Nitrate 3,4-Xylenol phenoldisulphonic acid 410 Nitrite Sulphanilic acid with naphthylamine 520 sulphonic acid Phosphate Molybdate/vanadate (yellow) 400 Molybdenum Blue 780 Sulphate Barium Chloranilate 530 Sulphide Dimethylaminoaniline/Fe(III) 662
Table 19.5: Reagents for spectrophotometric determ inations of gases.
Gas Reagent Wavelength (nm) Ozone Iodide/starch 590 Hydrogen sulphide Zinc acetate, then 662 Dimethylaminoaniline/Fe(III) Oxides of nitrogen Sulphanilic acid with naphthylamine 520 sulphonic acid Sulphur dioxide Tetrachloromercurate, then 545 Pararosaniline/formaldehyde Formaldehyde Phenylhydrazine/ferricyanide 515
SAQ 5
Write down the steps of a typical procedure involving a spectrophotometric method of analysis of the substance. ………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………………
19.5 EMISSION METHODS
You may be familiar with flame test for sodium, which emits a yellow light, and for other alkali and alkaline earth metals. Beside this, many other metallic elements, when subjected to suitable excitation, also emit radiation of characteristic wavelengths. Under proper control conditions, the intensity of the emitted radiation at some particular wavelength can also be correlated with the quantity of the element present. Thus both a quantitative and a qualitative determination can be made using emission methods. The various analytical methods, which make use of emission spectra, are characterized by the excitation method used, the nature of the sample (whether solid or liquid) and the method of detecting and recording the spectra produced. Methods in this category are Flame emission spectrphotoometry (Flame photometry), inductively coupled plasma, atomic emission spectrophotometry etc. Out of this flame photometry is more wid ely used method. This method is used in water and soil analysis for determining the concentration of alkali and alkaline earth metals such as sodium, potassium and calcium. 64 Optical Methods 19.5.1 Flame Photometry
In this technique a flame is used to excite the atoms. When a solution containing an ion is nebulized through a flame, a series of process occur: i) the solvent is vaporized leaving particles of salt(s) ii) the salt is subsequently vaporized and dissociated into atoms iii) some of the atoms are excited by the flame iv) the excited atoms emit radiation characteristic of their species
Because of the relatively low energy of the flame, only few elements can be excited. Therefore, the main application of flame photometry in the quantitative determination of the alkali and alkaline earth elements at concentrations as low as 0.1 µg/cm3 solution (0.1 ppm). A diagram showing the basic elements of a flame photometer is given in Fig. 19.24. The basic components are the flame, monochromator, and detector readout system. The flame is produced with a burner -nebulizer assembly as shown in Fig19.25. The fuel and oxidant are fed into two separate chambers within the burner and mix outside the exit orifices. Thus, a turbulent flame is produced. As the oxidant flows through the sample capillar y a vacuum is produced which draws the solution into the flame.
A number of fuel gases, such as acetylene, hydrogen or the liquid petroleum gas used for heating in most laboratories can be used for the flame. The oxidant used is usually oxygen rather than air. Flames yielding high temperature are capable of exciting more elements and so are more versatile. List of some common flame gas mixtures are given in Table 19.5
Table 19.5: Some common flame gas mixtures.
Fuel Oxidant Temperature Hydrogen Air 2000 Hydrogen Oxygen 2200 Acetylene Air 2000 Acetylene Oxygen 2800 Natural Gas (LPG) Air 1900 Acetylene N2O 2800
The emitted radiation is passed through a monochromator or prism, which separates the various wavelengths so that the desired regio n can be isolated. A photocell as detector and some type of amplifier are than used to measure the intensity of the isolated radiation.
Slits Lens
Burner Monochromator Readout nebulizer Photomultiplier
Fuel Oxidant supply supply
65 Instrumental Methods Fig.19.24: Basic elements of flame photometer. of Analysis The emission spectrum of each metal is different and its intensity depends upon the concentration of atoms in the flame, the method of excitation used, and the after - history of the excited atoms. Sodium produces a characteristic yellow emission at 589 nm, lithium a red emission at 671 nm, and calcium a blue emission at 423 nm. Each also gives a less intense emission at shorter wavelengths. Concentration of these elements can be measured down to 0.1 µg/cm3 (or µg/dm 3) or less with some degree of accuracy, depending upon the sensitivity of the instrument used.
Application of Flame emission spectrophotometry (FES)
In Table19.6 we have listed typical detection limits for the determination of selected pollutant elements by flame emission spectrometry in nitrous oxide-acetylene flame. In the present context, FES should be regarded as an inexpensive complementary techniques to AAS (Atomic Absorption Spectrophotometry).
Table 19.6: Elements that can be determined by Flame emission spectromotometry.
Element Wavelength Detection Element Wavelength Detection nm limit nm limit µg/cm3 µg/cm3 Ag 328.1 20 In 451.1 2 Al 396.2 5 Mg 285.2 5 Ba 553.6 2 Mn 403.1 5 Ca 422.7 0.1 Mo 390.3 5 Co 345.4 50 Ni 341.5 300 Cr 425.4 5 Pb 405.8 200 Cu 324.7 10 Sr 460.7 0.2 Ga 417.2 10 Tl 377.6 20
19.5 .2 Atomic Absorption Spectrophotometry
Atomic absorption although is an absorption method but it is very similar to flame emission spectrophotometry. In this technique radiation is absorbed by non excited atoms in the vapour state. This method has following advantages over flame emission because:
i) More elements can be quantitatively determined ii) The spectral interference are decreased iii) The sensitivity is higher for most elements
Like the flame photometer, an atomic absorption instrument consists of a light source, flame unit, a prism or grating to disperse and isolate the emission lines, and a detector with appropriate amplifiers. The light source (Hollow Cathode Lamp) emits a stable and intense light of a particular wavelength. Each element has characteristic wavelengths that it will absorb. A light source with wavelength readily absorbed by the element to be determined is directed through the flame and a measure of its intensity is made without the sample, and then with the sample introduced into the flame. The decrease in intensity observed with the sample is a measure of the concentration of the element.
The amount of radiation absorbed follows Beer -Lambert’s law i.e. it is proportional to the concentration of the element in the sample. A disadvantage of this method is that a different light source/hollow cathode lamp has to be used for each element. The 66 advantage of AAS is that it is quite specific for most of the elements. Absorption depend upon the presence of free unexcited atoms in the flame which ar e generally Optical Methods available in much greater abundance than the excited atoms.
Flame
Light source Atomizer burner Photo Prism or Grating Slit tube
Sample
Fig. 19.26: Schematic diagram of an atomic absorption spectrophotometer.
Application of AAS in element determinations
Atomic absorption spectrophotometry has been used for the determination of approximately 70 elements. Application include clinical and biological samples, forensic materials, foods and beverages, water and effluents, soils, plants, and fertilizers, iron, steel and various other alloys; minerals, petroleum products, pharmaceuticals and cosmetics.
A modification to the flame AAS is termed electrothermal atomic absorption spectrophotometry, which employs a small graphite furnace, which allows analysis for many heavy metals in further lower ranges i.e. in m icrogram per dm3 range. Here, the atomizer burner is replaced by a small cylindrical graphite furnace/tube that can be programmed through a series of different temperatures. The radiation from the cathode lamp source passes through the open ends of the horizontal cylinder through a hole in the side and the temperature programme is initiated. The temperature first rises to just over 100°C to allow the sample water to evaporate, leaving the metal containing salts behind. The temperature then increases to several hundred degrees Celsius (upto 3000°C), which volatilizes the cations so they fill the cylindrical space, and the particular cation to be determined absorbs the characteristics radiation form the cathode tube. The graphite furnace, as it is called, allows the development of a greater density of atoms and thus affects greater sensitivity to the atomic absorption procedure.
Inductively Coupled Plasma (ICP)
ICP method (emission spectroscopy) is a relatively new technique developed in the 1970’s for analys is of trace metals. ICP method uses Plasma Sources for higher- energy excitation source. The advantage of these more energetic automization sources is there is lower inter element interference, good spectra can be obtained for many elements under the same excitation conditions and hence spectra for many elements can be recorded simultaneously. The disadvantages are high instrument and operating costs, the need for more skilled operators and often less precision than with atomic absorption.
In the ICP method, a stream of argon gas flows through three concentric quartz tubes, which are surrounded by a water -cooled induction coil that is powered by a radio- frequency generator to form a strong magnetic field. When a spark initiates ionisation of the argon, the ions with their associated electrons are caused to follow a spiral flow pattern within the tubes as a result of the magnetic field and heating is the result of their collisions and resistance to this movement. The result ing temperature is 4000 to 67 Instrumental Methods 8000°C which is two to three times hotter than obtained with the hottest of the of Analysis combustion flame temperatures. This temperature is sufficient to almost completely dissociate molecules there by making atomic emission highly efficient. The sample to be analysed is introduced at the head of the argon flow and into the central tube. The emissions produced by the elements are focussed through an entrance slit for either a monochromator or polychrometor, and a portion of the spectrum is isolated for intensity measurements. The instrument can make measurements in the entire ultraviolet-visible spectrum form 180 to 900 nm.
SAQ 6
Describe the basic components of Flame emission spectrophotometer. ………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………………
19.6 SUMMARY
In this Unit, you learnt about the nature and characteristics of electromagnetic radiation. Various parameters such as frequency, wavelength etc. associated with electromagnetic radiations were defined and their interrelationship was discussed. Then we discussed the fundamental law of qualitative analysis, Beer -Lambert’s Law. Absorbing species responsible for ultraviolet-visible electromagnetic radiations in organic and inorganic substances were described. This was followed by the functions of the components of an ultraviolet-visible spectrophotometer. Then wide range of the application of ultraviolet-visible spectrophotometry in environmental analysis was highlight. Finally flamephotometry and atomic absorption spectrophotometry were discussed in brief.
19.7 TERMINAL QUESTIONS
1. Define following terms:
a) absorbance; b) percent transmittance; c) absorptivity; d) molar absorptivity.
2. List four different cases for deviation from Beer-Lambert’s Law.
3. What are the most frequent electronic transitions in organic molecules observed during absorption of electromagnetic radiation in ultraviolet-visible region?
4. Define chromophore and auxochrome.
5. What is the main advantage of the double beam photometer over single -beam photometer?
6. The optimum wavelength used for the analysis of the mixture of o-xylene and p-xylene are 271 nm and 275 nm. The absorbance of the single components and the mixtures at these two wavelengths are given below.
Solution 271 nm 275 nm
o-xylene 0.90 0.10 p-xylene 0.34 1.02 68 mixture 0.47 0.54 Optical Methods
Calculate the various absorptivities and the concentration of o- xylene and p-xylene. Assume b = 1 cm.
7. Compare flame emission and atomic absorption Spectrophotometry with respect to instrumentation.
19.8 ANSWERS
Self -Assessment Questions
11 1.ν ===2.5×106m−−1=×2.51010−414 cm λ 4000×10 −10 c 2.998× 108 ν ===×7.4951014 Hz λ∈ 4000× 10−10 P 2.A=log o =∈∈ bc P
85.4 −4 =log=∈∈ ×××2110 20.3 =log4.207=∈∈ ××210−4 0.62396 ∈∈ =×104=×3.121033dm/mol.cmmol cm 2 3.a)nn→→ππ*;b)**,c) n→σ *
4. σ → σ* > n → σ* > π → π* > n → π*
5. i) Preparation of sample to make absorbing species, ii) Selection of wavelength, iii) Measurement of absorbance for the reagent blank, standards and sample at
λmax , iv) Preparation of calibration curve, and v) Read the conc. of sample from calibration curve.
6. Flame, monochromator and detector readout system
Terminal Questions
1. Absorbance (A): The logarithm (base 10) of the ratio of incident radiant power to the radiant power transmitted through sample. Po ?? A=logorlog P ? 69 Instrumental Methods of Analysis Percent Transmittance (T)
The ratio of radiant power transmitted through sample to the incident radiant power is known as transmittance. P T= Po Where transmittance is expressed in percentage, it is called percent transmittance P o T= × 100 o P o
Absorptivity (a):
It is defined as the absorbance of the solution having unit concentration (g dm3) and unit path length (1 cm). That is
a = A/bc
Molar Absorptivity (ε ):
When the concentration of absorbing substance is taken in mol dm 3, the ratio A/bc is called as molar absorptivity. Molar absorptivity has Unit dm3 mol-1cm-1.
2. Use of non-monochromatic radiation, Association and Dissociation of analyte, change in temperature during experiment, change in analyte due to photochemical reaction, high concentration of sample etc.
3. σ → σ* > π → π* > n → σ* > n → π* etc.
4. Chromophore: Chromophore has groups, which are responsible for causing absorption of electromagnetic radiation between 175 and 1000 nm. These groups have characteristic molar absorptivity and absorb at fairly well defined wavelengths. Example >C=C<, − C ≡ C, − Ν = Ο & −Ν=Ν − etc.
Auxochromes: They do not themselves absorb emr but when attached to a chromophore, alter both the wavelength and the intensity of absorption of the
chromophore. Example − OR, − ΝH2, − ΝR2 etc.
5. i) Since the beam of light is divided into two parts and is allowed to fall on two identical detectors, the fluctuations (if any) of the source will not disturb the reading, which is noted after balancing the two potentiometers. ii) Since the galvanometer is used as a nu ll detector, no current is used in moving the galvanometer needle and the results are more accurate than that with a single beam instrument. iii) Since the reading is noted on a large potentiometer scale, the sensitivity is high.
6. For o-xylene (compound X)
70 0.90 3−−−111−1 Optical Methods 271nm()(λ1ax1)==2.25ddmmg g cmcm 0.40 0.10 275nm()(λ a )==0.25dmdmg3 g−1−− 11cmcm−1 2x2 0.40 Forp − xylene(compoundY)
0.34 3−1−−11−1 271nm()(λ1ay1)==2.0dmdmg g cmcm 0.17
1.02 33−1−−11−1 275nm()(λ2ayz)==6.0dmdmg g cmcm 2 0.17
Therefore,λ10.47=+2.25ccxy2(1)
forλ20.54=+0.25ccxy6.0(2)
First eliminate cy by multiplying the first equation by 3 and subtracting (2) from −3 the result. Thus give cx = 0.13 g dm
−3 cy can be calculated by putting the value of cx, cy = 0.084 g dm
7. See Fig .19.24 and Fig. 19.26. AAS has a light source.
FURTHER READINGS
1. Analytical Chemistry, Gary D. Christin, John Wiley & Sons, Inc. 2. Principal of Instrumental Analysis, Skoog, Holler and Nieman, Saunders Golden Sunburst Series .
71 UNIT 20 MICROBIOLOGICAL EXAMINATION OF WATER, SOIL AND AIR
Structure
20.1 Introduction Objectives 20.2 Microbiological Examination of Water Hydrologic Cycle Bacteriology of water Water Quality Assays 20.3 Microbiological Examination of Soil Microbial Populations Method for Studying Soil Microorganisms 20.4 Microbiological Examination of Air 20.5 Summary 20.6 Terminal Questions 20.7 Answers 20.8 Glossary
20.1 INTRODUCTION
In the previous units of this Block and Block 5 you have studied instrumental and non insturmental methods of analysis. These methods indicate whether water, soil and air are polluted and provides other useful informations as well. However, these methods are not sensitive or specific enough to detect microbiological contamination. On the other hand, microbilogical tests have been designed which are extermely sensitive and specific in revealing evidence of pollution caused by microbes.
In this unit you will cover the some microbiological techniques for the examination of water, soil and air.
What is Microbiology
The planet earth came into existence about 4.5 billion years ago when primeval atmosphere consisted primarily of nitrogen, hydrogen and carbon monoxide and was almost devoid of free oxygen. Between the sun and the surface of the earth, the ozone shield as we have in the present day atmosphere, was lacking in the primeval atmosphere and hence lethal UV rays of the sun bathed the barren rocks and left them sterile. The primitive sea had plenty of dissolved ammonia, carbon dioxide and abundant deposits of sulphates and iron. This was earth's hostile environment, prior to the origin of life. It is believed that life arose in the primitive sea and migrated to the sheltered crevices on land which were beyond the reach of the UV rays of the sun.Since organic matter was virtually absent, it has been conjectured that chemosynthetic microorganisms were the pioneer colonizers of the primitive earth.
The discoverer of the microbial world : the world of "animalcules", or little animals, was a Dutch merchant Antony van Leeuwenhoek. All the main kinds of unicellular microorganisms that we know today- protozoa, algae, yeast and bacteria- were first described by Leeuwenhoek. In addition to the diversity of the microbial world, Leeuwenhoek emphasized its incredible abundance.
As knowledge of living organisms accumulated it gradually became evident that these microorganisms have a major role to play in illness, industry and ecology and that they have a power to change peoples lives. The study about microorganism is called Microbiology.
71 Instrumental Methods Presence of microorganism in soil, water and air affect the chemical and physical of Analysis properties of environment. Therefore, their microbiological examination allows us to assess the quality of environment in which we are living.
Objectives
After studying this unit, you should be able to:
• explain the importance of microbes in water, soil and air, • describe methods of evaluating the potability of water, • identify the coliforms in water, and • describe various microbial techniques for enumerating soil microbes.
20.2 MICROBIOLOGICAL EXAMINATION OF WATER
Before going into the detail of microbiological examination of water, let us first take the hydrological cycle and bacteriology of water.
20 .2.1 Hydrologic Cycle
Water is the dominant compound on earth. It occupies nearly 3/4 of the earth's surface, and 97% of all water is in the ocean. The earth's supply of water is continuously cycled between the hydrosphere, atmosphere, and lithosphere. The "hydrologic cycle" begins when surface water (lakes, oceans, and rivers) exposed to the sun and wind evaporates and enters the vapor phase of the atmosphere. Plants contribute to this reservoir through transpiration (evaporation through leaves) and respiration (see Fig. 20.1). Water is returned to earth through condensation or precipitation (rain, snow, hail). While a drop of water may appear simple, it is quite complex, often containing chemicals and microorganisms of many kinds.
Sun
Snow Vapour Surface runoff Transport Precipitation Evaporation Precipitation Evaporation Infiltration Lake River
Oceans Ground water River flow flow
Fig. 20.1: Hydrologic cylce.
In this cycle, water is classified into three major categories based on their location. Microorganisms of various kinds are present at different stages of this cyclic process.
a) Atmospheric Water
Moisture contained in clouds, and precipitated as snow, hail and rain constitutes atmospheric water. The microbial flora of this water is contributed by the air. Air contains dust particles to which microorganisms are attached. These are actually washed away by precipitation.
b) Surface Water
Surface water is represented by bodies of water such as lakes, streams, rivers and oceans. These waters are susceptible to contamination with microorganisms from 72 atmospheric water, the surface run off from soil, and many wastes dumped into them. Number and type of the microbial community varies with the source of water, with the Optical Methods composition of the water in terms of microbial nutrients and with geographical, biological and climatic conditions. c) Ground Water
Ground water is subterranean water that occurs where pores in the soil or rock- containing materials are saturated. This process forms a deep ground water source called as an aquifer. It's an important source for surface water. The permeability of the soil and depth to which the water has penetrated, helps through filtration, removal of bacteria and suspended matter. Springs consists of groundwater that reaches the surface through a rock fissure or exposed pores in soil. Wells are made by sinking a shaft into the ground to penetrate the groundwater level. Wells and Springs, properly located, produce water of very good quality, and have negligible microbial content.
The aquatic environments being so different, it is natural that different species of microbes are considered to be indigenous to specific habitats.
The total amount of water in the hydrologic cycle has changed over millions of years. However, its distribution and quality have been greatly altered by human activities. Some nutrients / organic matter are added naturally through seasonal upwelling and disasters (floods or typhoons), but the most significant alteration of natural waters comes from effluents from sewage, agriculture, and industry that contain heavy loads of organic debris or nitrate and phosphate fertilizers. The addition of such large quantities of these nutrients to aquatic ecosystems, called "eutrophication" wreaks havoc on the communities involved. It is to be understood that the proper management of water resources is one of the greatest challenges of the century.
SAQ 1 a. What are three categories of natural water? Which one is of good quality and negligible microbial content? …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… b. How much of earths surface is occupied by water? …………………………………………………………………………………… …………………………………………………………………………………… ……………………………………………………………………………………
SAQ 2
Explain the following terms a) Aquifer …………………………………………………………………………………… …………………………………………………………………………………… …………………………………………………………………………………… b) Eutrophication …………………………………………………………………………………… 73 ……………………………………………………………………………………
Instrumental Methods 20.2.2 Bacteriology of Water of Analysis Water is a site of tremendous microbiological activity. Water meant for human consumption must be free from chemical substances and microorganisms which could endanger the health of the community. It is to be kept in mind that all waters are liable to pollution. The situation, the construction and maintenance of water supply, its storage and distribution system must exclude any possibility of pollution.
The objective of Disease producing micro-organisms are also known as ‘pathogens’. Among the most microbiological prominent water-borne pathogens of recent times are the protozoans: Giardia and examination of water Entamoeba; the bacteria: Campylobacter, Vibro cholerae, Salmonella, Shigella , is to detect whether Vibrio and Mycobacterium; and viruses: the polio and hepatitis A (See Table 20.1). pollution of water by pathogenic organisms Some of these agents (especially encysted protozoans) can survive in natural waters has occurred or not. for long periods without a human host, whereas others are present only for a short time and will be rapidly lost. Water -borne pathogens are commonly carried in feces, and this is usually how they get into drinking water supplies like wells or reservoirs.
Table 20.1: Classification of water born pathogens.
Type Examples Diseases
Protozoans Giardia lambia Giardiasis (Diarrhoeal disease) Entamoeba histolytica Amoebic dysentery Bacteria Campylobacter jejuni Diarrhea Vibro cholera Cholera Salmonella species Typhoid Shigella species Dysentery Viruses Polio viruses Polio Hepatitis A Hepatitis
It would be best to look for the pathogen responsible for the contamination of water, however it is not practicable to do so. This is because pathogens are usually few and far outnumbered by nonpathogenic organisms. Also, methods to detect them are costly in time and money. Therefore, we generally look for indicators of human/animal pollution i.e. intestinal organisms. The organisms most commonly used as indicators E.coli is one of the of pollution are indicator microorganisms: Escherichia coli (E.coli) and the bacteria of coliform bacteria coliform group as a whole. These orgainsms survive in natural waters but do not population and is more multiply there, so finding them in high numbers indicates levels of fecal representative of fecal contamination. Water sanitation standards of the Environmental Protection Agencies sources than other coliform group. are based primarily on the levels of indicator microorganisms. Microorganisms of fecal streptococci group are also searched for:
1. When the nature of pollution is doubtful, 2. When water is examined at infrequent intervals and 3. When a new source of water supply is being considered.
Like coliform bacteria, enteric viruses can be carried by human wastes into water. Analysis of water sample for presence of viruses requires more elaborate procedures than those used for isolation of bacteria. No 'standard method" for the detection of viruses in water has been adopted yet. Now, let us know more about indicator mciroorgansims.
Indicator Microorganisms
The indicator microorganism serves as an "alarm" system. The term refers to a kind of 74 microorganism whose presence in water is evidence that the water is polluted with fecal material from humans or other warm-blooded animals. This kind of pollution means that any pathogenic microorganisms that occur in the intestinal tract of these Optical Methods animals may also be present. Some of the important characteristics of an indicator organism are:
1. It is present in polluted water and absent from potable (unpolluted) water. 2. It is present in water when pathogens are present. 3. It survives better and longer than the pathogens. 4. The quantity of indicator organism correlates with the amount of pollution. 5. It has uniform and stable properties. 6. It is present in greater numbers than those of pathogens (making detection easy). 7. It is generally harmless to humans and other animals. 8. It is easily detected by standard laboratory techniques.
Two groups of microorganisms normally are used as indicators of microorganisms contamination of water supplies: the coliform group and the fecal streptococci group. The coliform group consists of all aerobic and facultative an aerobic gram negative, asporogenous bacilli that ferment lactose with the production of gas within 48 hours at 35° C. In this group, Escherichia coli (E coli) and Enterobacter aerogenes are commonly found coliforms.
E. coli is a normal inhabitant of human intestine and other warm-blooded animals and is regarded as a fecal type of coliform. Other members of the coliform group for e.g. Enterobacter aerogenes are widely distributed in nature in soil, water, grain and also in the intestinal tract of humans and other animals and are regarded as nonfecal coliforms. These species are very similar in many of their characteristics. These can be differentiated by the following biochemical tests:
Table 20.2: IMViC reaction of Escherichia coli and Enterobacter aerogenes. ______The coliforms have several TEST characteristics in common with species of the genera ______Salmonella and Shigella all Organism Indole Methyl Red Voges-Proskauer Citrate of which are pathogenic. However, a major Escherichia coli + + - - distinctive biochemical difference is that the coliforms ferment lactose Enterobacter aerogenes - - + + with the production of acid and gas. Salmonella and Shigella donot ferment lactose. Hence, 1. E.coli produces indole from tryptophan, Enterobacter does not. fermentation of lactose is 2. Amount of acidity produced in a special glucose-broth medium is detected by the the key reaction in the methyl red pH indicator. Both organisms produce acid from glucose. However, laboratory procedure for E.coli produces a lower pH which turns the indicator red, whereas Enterobacter determining the coliforms.
aerogenes do not produce as large an amount of acid and thus donot produce the colour change. 3. Ability to produce the compound acetylmethylcarbinol (acetoin) in a glucose- peptone medium: This chemical is detected by Voges -Proskauer test procedure.
E.coli does not produce acetoin, Enterobacter areogenes does. 4. Utilization of sodium citrate: Enterobacter areogenes is capable of growing in a chemically defined medium with sodium citrate as the sole carbon source. E.coli does not grow in such medium. For convenience, these tests are collectively designated as the IMViC reactions
(I=indole, M=methyl red, Vi=Voges-Proskauer reaction, and C-citrate).
The fecal streptococci are present in the form of Streptococci faecalis, S. faecium, S. S. bovis, and S. equines are bovis, and S. equinus in human and animal intestine. They also serve as indicators of bacteria that are indicators fecal water pollution. In the next section we will see how the fecal streptococci are of pollution from catle and horses. used together with coliforms to identify the source of pollution. 75 Instrumental Methods There are additional coliform species in the genera Klebsiella and Citrobacter for of Analysis which more detailed biochemical, genetic, and immunologic data are needed for identification.
SAQ 3
a) What is an indicator organism? …………………………………………………………………………………… ……………………………………………………………………………………
b) Why coliforms and not pathogens are tested for potability of water? …………………………………………………………………………………… ……………………………………………………………………………………
c) What are coliforms? …………………………………………………………………………………… ……………………………………………………………………………………
d) If coliforms are present in water then……………… might also be present, and the water is potentially dangerous to drink.
e) Why are coliforms grown at 350C? …………………………………………………………………………………… ……………………………………………………………………………………
20.2.3 Water Quality Assays
Most potable water supplies come from rivers and underground wells and springs. Water from under -ground sources is partially purified by filtration as it passes through the soil column, removing particulate matter and microorganisms. Water used for drinking purposes is boiled or treated with antimicrobial chemicals (called disinfectants, such as chlorine and its compounds, bromine, iodine, ozone, etc. ) to ensure its safety. UV radiations are also used as disinfectants.
The following points should be considered when water samples are to be collected and submitted for bacteriological analysis:
1. Sterile bottle/container should be used for collecting the water sample. 2. The sample must be representative of the supply from which it is taken. 3. Contamination of the sample should be avoided during and after sampling. 4. Sample should be tested as soon as possible after collection. 5. The sample should be stored at a temperature between 0 C and 10 C if there is Plate counts are useful in ° ° determining the efficiency a delay in testing. of water treatment operations for removing or The usual bacteriological procedures consists of: destroying organisms, e.g. sedimentation, filtration, a) A plate count to determine the number of bacteria present, and and chlorination. A count b) Tests to reveal the presence of coliform bacteria. can be made before and after the specific treatment. The result indicat es extent (a) Total count of microorganisms to which the microbial population has been A determination of the total numbers of viable bacteria in a water sample, although of reduced. limited value by itself, gives an indication of the amount and type of organic matter present in the supply. Standard plate count method is generally used to determine the 76 total numbers of microorganisms present in water sample. This method is based on the assumption that each cell in a solid medium can multiply repeatedly and eventually 3 form a distinct colony. The test is carried out by plating 1 cm of the water sample, Optical Methods using a culture medium such a plate count agar or standard methods agar. The plates are incubated at 35°C for 48 to 72 hours and then colonies in the medium are counted, using a colony counter. This device may be a magnifying glass or a sophisticated electronic colony counter. Water of good quality is expected to give a low count, less than 100 colonies per cm3.
(b) Test for coliforms
Ther e are two standard test for the detection of the presence of coliform group: i) the multiple-tube fermentation test (MTF) ii) the membrane filter test (MF) i) Multiple Tube Fermentation Test
The test involves a most probable number (MPN) procedure and use of selective and Concentration of total differential media for examining water for coliform organisms. This test has been coliform bacteria are successfully used in many countries for the anlysis of drinking and other waters. The generally reported as the coliform results are reported in terms of the most probable number (MPN) of most probable number “per 3 3 organism. Therefore, this test is also known as MPN method. The test gives the most 100 cm (MPN/100 cm ). The MPN is based on the likely number of coliforms bacteria rather than the actual number. It consists of three application of the Poisson steps (i) presumptive, (ii) confirmatory and (iii) completed tests (Fig.20.2). distribution for extreme values to the analysis of • Presumptive Test the number of positive and negative results obtained Typically, this involves a series of three subsets of fermentation tubes. Each subset when testing multiple contains 5 fermentation tubes, and each tube has suitable lactose culture medium portions of equal volume and in portions constituting (lactose broth) and an inverted Durham's tube (for collection of gas). The three subsets a geometric series. It is 3 3 3 are inoculated with different amounts of water sample e.g. 10 cm ,1 cm , and 0.1 cm emphasized that the MPN respectively (See Fig. 20.3). After 24 ± 2 hours of incubation at 35°C, the tubes are is not the absolute concentration of organism evaluated for gas production. Tubes showing positive gas production give presumptive that are present but only a evidence of coliforms, and negative gas production means no coliforms. The number statistical estimate of the of such positive and negative tubes are counted from each subset and this value is Poisson distribution applied to statistical tables to get most probable number (MPN). One such MPN table directly. is given in Appendix 1. From MPN table the most likely, or probable number of coliforms can be estimated.
Water sample
Presumptive Test: Lactose broth inoculated; incubated for 24 to 48 hours
Gas not produced = Gas produced = Negative presumptive test: Positive presumptive test Coliform group absent OR Confirmed test : Brilliant green lactose bile broth Confirmed test : Sample from lactose broth streaked (BGLB) inoculated; incubated for 24 hours onto EMB lactose plates; incubated for 24 hours
Typical coliform colonies : Dark centers, Colonies not coliform = Metallic sheen = Negative confirmed test Positive confirmed test
Gas not produced = Gas produced = Negative Coliforms confirmed test present Completed test : Typical coliform colonies selected; lactose broth and agar slant inoculated; incubated for 24 hours
Gas not produced = Lactose broth Agar slant Negative completed test : Original isolates not coliform
Acid and Gas Gram-negative rods present; Produced no endospores present
Coliform group present = Positive completed test 77 Fig. 20.2: Analysis of drinking water for coliforms by the multiple tube fermentation test.
Instrumental Methods • Confirmatory Test of Analysis The second step is the confirmatory test, in which a fresh fermentation tube that contain brilliant green, bile, and lactose (BGBL) broth with Durham tube is inoculated with the sample of positive tube of the presumptive test. The ingredient in BGBL broth are selective for coliform. After 24 hours of incubation this tube is observed for gas production. This gives confirmation for the presence of coliforms.
Alternatively, presence of coliforms can also be confirmed by growing cultures of coliform bacteria of the samples from tube showing positive presumptive test on the selective media that suppresses the growth of other organism such as Eosin-methylene blue agar (EMB). After, incubation at 35°C for 24 hours this is examined for typical colonies of coliform. If a typical colonies are seen, the ‘completed test’ is carried out.
• Completed Test
Several colonies from the EMB plate are subcultured into lactose broth fermentation tube and on a nutrient agar slant. Both are incubated at 35°C for 24 hours. Gas in the broth and a Gram negative non-sporing rod on the slant is evidence of coliforms.
For most routine water analysis, only the preseumptive test is performed. Important steps of presumptive test are summarized in Fig. 20.3.
Water sample
3 3 3 10 cm 1 cm 0.1 cm
Presence of gas taken as positive test Inner fermentation tube
Fig. 20.3: Illustration of presumptive test.
For non-portable and polluted waters, small volumes are used i.e., 1 cm3, 0.1 cm3 and 0.01 cm3 sample. Transferring small sample amounts is difficult, so first a series of dilution is made as shown below
3 33 3 3 1cm 1cm 1cm 1cm
Sample inoculation 3 3 3 and dilution should be 9cm 9cm 9cm carried out in sterile surroundings i.e. either in inoculation chamber -1 -2 -3 Dilution 10 10 10 having UV-light or in the surroundin g of the flame. 1 cm3 sample from the first tube will contain 1/10 the number of cells present in 1 cm3 78 of the original sample and so on.
(ii) Membrane Filter Test Optical Methods
This test is extensively used nowadays for rapid examination of water. It involves the following steps:
1. A membrane filter is placed in a sterile filtration unit. The membrane filter has a very small size pores (diameter of 0.45 µm). The bacteria are retained on the filter when water sample has been passed through because they are larger than the pores (See Fig. 20.4). 2. A known volume of sample (usually 100 cm3) is drawn through this unit with the help of a vacuum pump. The bacteria are retained on the surface of the membrane filter. 3. The membrane filter is removed and placed on an absorbent pad that has previously bee n impregnated with the appropriate medium. Or else, the filter can be placed aseptically on the surface of an agar medium (EMB or Endo agar) in a Petri dish. 4. After incubating for 24 hours at 350C observe for the development of colonies on the membrane filter. On ENB, typical coliform colonies are dark purple and have a green metallic sheen and on Endo, colonies are dark red and have a pink or red metallic sheen on the surfaces. Coliform colonies are counted and the results are expressed as total coliform per 100 cm3. Verification of these colonies needs to be done by the earlier procedure (i).
Sterile forceps Filter Sample Filter Filter holder funnel Remove filter Filter Filter support Transfer filter
Filter base Vacuum source
0 Incubate at 35C for 24 hours Nutrient pad or Agar medium Receiving flask Bacterial colonies (a) on filter surface Count lactose positive colonies (b)
Fig.20.4: Membrance -filter set up a) Filtration unit; b) After the sample has been filtered, the membrane filter is placed in a petri dish containing a cuture medium and then incubated. But due to the high This technique has several advantages over the earlier techniques : cost of equipment and consumables evolved i) Any amount of water sample can be examined. in membrane filter technique, in India, ii) The same membrane filter can be used or transferred from one medium to multiple tube another for purpose of selection or differentiation of organisms. fermentation method iii) Results are obtained rapidly than standard MPN method. is more used. iv) Qualitative estimations of some bacterial types, e.g. coliforms can be made on appropriate media.
This technique has some disadvantages such as: i) Membrane filtration is unsuitable for waters with high turbidity and low count 79 because the filter will become blocked before sufficient water can pass through. Instrumental Methods ii) Large number of non-coliform organism capable of growing on the medium may of Analysis interfere with coliform group.
A total count of coliform bacteria obtained from above tests determines the portability Use of FC/FS ratio can of the water sources. Water can be classified in four categories on the basis of total be very helpful in coliform count per 100 cm3 (see Table 20.3) establishing the source of pollution in rainfall run- off studies and in Table 20.3: Classification of drinking water according to bacteriological tests. pollution studies conducted in rural areas, Presumptive Coliform E. coli(FC) especially where septic count MPN per 100 cm3 MPN per 100 cm3 tanks are used. I. Excellent 0 0
Determination of Fecal II. Satisfactory 1-3 0 coliform bacteria group is III. Suspicious 4-10 0 based on the ability to IV. Unsatisfactory >10 0-10 produce gas (or colonies) at a elevated incubation The type of fecal pollution, if any, are established by means of a fecal coliform (FC) temperature (44.5°C for 24±2 hours) count, indicative of human pollution. There is another group of indicator bacteria, fecal streptococci group (FS) and a fecal streptococcal count, indicative of pollution from other animal origins.
The ratio of fecal coliforms to fecal streptococci per cm3 (FC/FS) of sample is interpreted as follows:
Between 2 and 4 - human & animal pollution > 4 - human pollution < 0.7 - poultry and livestock pollution.
Standards for tolerable levels of indicator bacteria groups
The standards vary according to the intended use of water (See Table 20.4).The most stringent standards are imposed on the municipal water supplies to be used by many people. A minimum infectious dose of several hundred to several thousand bacteria is usually necessary for an actual infection to be established. Drinking water supplies meeting the 1/100 cm3 coliform standard have been demonstrated to be safe for use.
Table 20.4: Some Indian and International water quality standards for indicator bacteria groups.
Use Total Coliform Faecal Coliform Agency/ MPN per 100 cm 3 MPN per 100 cm3 Country Public Water 0 0 WHO Supply Drinking water < 50 No value India source, without conventional treatment, but after disinfection Drinking water < 5000 No value India source, without conventional treatment and disinfection Bathing, 5000 guide 100 guide Europe recreation water 10,000 mandatory 2000 mandatory Outdoor bathing < 500 No value India (organized) Shellfishing 70 No value US No value 14 Venezuela 80 Mexico
SAQ 4 Optical Methods a) The presumptive test is used for which organism. …………………………………………………………………………………… …………………………………………………………………………………… b) What does MPN stand for? …………………………………………………………………………………… …………………………………………………………………………………… c) What colour of colonies does E. coli and Enterobacter make on EMB agar. …………………………………………………………………………………… …………………………………………………………………………………… d. In the confirmed test, transfer is made from positive presumptive test tubes to either……….. broth or………… agar? e. A water analysis gives following results. Determine the colifrom concentration using MPN table in Appendix 1.
Water sample in cm3 No. of positive tubes No. of negative tubes 10 4 1 1 4 1 0.1 1 4
20.3 MICROBIOLOGICAL EXAMINATION OF SOIL
Soil is often thought of as an inert substance by the average lay person. However, contrary to this belief, it serves as a repository for many life form, including a huge and diverse microbial population. The beneficial activity of these inhabitants far outweigh their detrimental effects.
Life on this planet could not be sustained in the absence of microorganisms that inhabit the soil. This flora is essential for degradation of organic matter deposited in the soil such as dead plant and animal tissue and animal wastes. Hydrolysis of these macromolecules by microbial enzymes supplies and replenishes the soil with basic elemental nutrients. By means of enzymatic transformations, plants assimilates these nutrients into organic compounds essential for their growth and reproduction. In turn, these plants serve as a source of nutrition for animals. Thus, many soil organisms play a vital role in a number of elemental cycles. Most important of them are: Nitrogen cycle, carbon cycle and sulfur cycle. Some microorganisms also play a role in the enzymatic transformation of other elements, such as phosphorus, iron, potassium, zinc, manganese and selenium. These biochemical changes make these minerals available to plants in a soluble form.
20.3.1 Microbial Populations
It is essential to bear in mind that the soil environment differs from one location to another and from one period of time to another. Therefore, factors such as moisture, pH,temperature, gaseous oxygen content,and organic and inorganic composition of soil are crucial in determining the specific microbial flora of a particular sample. Soil contains myriad of microorganisms, including bacteria, fungi, actinomycetes, protozoa, algae and viruses. They may reach a total of billions of organisms (Table 20.5). Despite this diversity,bacterial population of the soil exceeds the population of 81 all other groups of microorganisms in both number and variety.
Instrumental Methods Table 20.5: Micorbial population in a fertile soil. of Analysis Type Number per gram Bacteria Direct count 2,500,000,000 Dilution plate 15,000,000 Actinomycetes 700,000 Fungi 400,000 Algae 50,000 Protozoa 30,000
Macroscopic forms of life also abound such as earthworms, nematodes, mites, and insects and also the root systems of plants. Thus, soil is a very complex living environment.
Soils are characterized by horizons, parallel layers of various thickness and structures. Each horizon differs from that above or below it in such properties as the organic and mineral content, colour, texture, structure, porosity and pH. These properties inturn influence the moisture content, gaseous content and biological content of the horizon. For example, a horizon that is rich in organic matter has great biological activity and is quite different from one that is made up of bedrock and has little organic matter and low biological activity.
SAQ 5
a) How are soils characterized? …………………………………………………………………………………… ……………………………………………………………………………………
b) What factors affect the numbers and kinds of microorganisms in soil? …………………………………………………………………………………… ……………………………………………………………………………………
c) How do the following compare in numbers in soil: viruses, protozoa, algae, fungi, bacteria? …………………………………………………………………………………… ……………………………………………………………………………………
d) What are the three important elemental cycles? …………………………………………………………………………………… ……………………………………………………………………………………
e) What is the role of organic matter in soil? …………………………………………………………………………………… ……………………………………………………………………………………
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20.3.2 Methods for Studying Soil Microorganisms Optical Methods
A soil sample taken for microbiological study is a composite of a large number of micro-habitats occurring in nature. Approaches for estimating the kinds, numbers and metabolic activities of the form and arrangement of microorganisms in soil, include, determination of the form and arrangement of microorganisms in soil, isolation and characterization of subgroup and species and detection and measurement of metabolic processes.
Obtaining Soil Samples
1. Soil samples are collected normally at a depth of 6 inches and transferred to clean containers. 2. Three to five samples are taken for each replicate and mixed thoroughly. 3. Atleast 10-25 gms of soil is taken as a representative sample of a particular replicate.
Just as the soil differs, microbiological methods used to analyse soil also vary. Following methods are commonly used for studying soil microorganisms:
1 Direct Microscopy
Direct examination is useful for determining the form and arrangement of microorganisms in soil. Fluorescent stains such as, the magnesium sulfonic acid, acridine orange and fluorescein isothiocyanate (FITC) make it possible to observe microorganisms on soil particles with normal light microscopy. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) give better resolution and show both shape and structure of the microbial habitat and the microorganisms themselves. SEM gives a three dimensional view.
For direct microscopic examination a dilution of a soil sample is spread in a thin film on a clean glass slide. After fixing and staining the film, the microorganisms can be counted under a microscope. Although this method is used to estimate the total microbial population, special staining techniques are necessary to distinguish living from the dead microorganisms. There are a number of staining techniques. Acridine orange the oldest and widely used, stains the DNA inside the cells. Living cells appear green and dead cells lacking a functional cytoplasmic membrane absorbs large quantities of the dye and appear red. Fluoresein isothiocyanate ( FITC) adsorbs to the disulphide groups in protein and is excellent for bacteria but does not adsorbs to all fungi. For fungi water -soluble fluorescent dye aniline blue which is preferentially adsorbed to the β(1-3) glycan linkages in fungal cells is suitable.
2 Agar - Plate Technique
This is one of the most commonly used methods for isolation and enumeration of soil microorganisms. Dilutions of th e soil sample (1 cm3, or 0.1 cm3 aliquots) are added to tubes containing melted and cooled agar medium. After proper mixing these contents are poured in sterile petriplates. Alternatively, the dilutions may be spread over the surface of plates of the solidified agar medium to permit colonies to develop. From the number of colonies that develop on the plates, one can calculate the number of living organisms per gram of soil.
Appropriate inhibiting agents should be added to encourage the desired group and suppress the other types of microorganisms. For example, to suppress bacteria and encourage fungal growth rose bengal and streptomycin is added in the media. To inhibit fungi nystatin/cyclohexamide is added.
It should be kept in mind that only a portion of the total microbial population will be able to grow under any given set of cultural conditions. For example, if you use nutrient agar medium and incubate the plates at 350C in air, in the dark for 48 hours, 83 you will not be able to detect the following kinds of microbes:
Instrumental Methods 1. Anaerobes and microaerophiles, since they are poisoned by air atmosphere. of Analysis 2. Strict thermophiles, mesophiles and psychrophiles because they donot grow at 350C. 3. Photoautotroph since they require light as an energy source. 4. Chemoautotrophs because they prefer inorganic nutrients. 5. Slow growers because they take a week or more for making visible colonies.
Hence, it should be noted that a wide variety of media and incubation conditions can be used depending upon the requirement.
3 Most Probable Number (MPN)
Where plate counts are not appropriate, dilution counts can be made. The dilution count, in essence, is a determination of the highest soil dilution that will still provide growth in a suitable medium. Inocula from three successive serial dilutions are used to inoculate three sets of tubes (10 usually, 5 minimum) respectively (Fig. 20.5). Visual growth in each tube is counted and taken positive, no growth as negative. These are then converted into numbers with the help of standard table as done in case of water sample. This is called the most probable number (MPN) method of enumeration of microorganisms.
3 1 cm 3 3 3 3 3 1 cm 1 cm 1 cm 1 cm 1 cm
10g Soil + 3 90 cm 3 3 3 3 3 Water 9 cm 9 cm 9 cm 9 cm 9 cm -2 -3 -4 -5 -6 Dilution 10 10 10 10 10 -1 Dilution : 10 Taken for inoculating 5 sets of tubes for each dilution
5 Tubes 5 Tubes 5 Tubes
Fig. 20.5: Most Probable Number Technique.
4 Enrichment Culture Technique
Another method is the culture technique. It does not provide quantitative information of the microbial flora present in any given sample. Instead, it helps to isolate microorganisms that are able to metabolize particular substrate that may be present in very small numbers in th e original sample - for example assume that you wish to isolate soil microorganisms that can degrade a complex compound such as lignin (Fig. 20.6). You would first have to prepare medium in which lignin is the only source of carbon (the energy source). A sample of soil would then be inoculated into the first of a series of flasks and that flask is then placed in an incubator. After several days of incubation some material is transferred aseptically from the first flask into a second flask. This process, is repeated, a number of times. After incubation of the last inoculated flask in this series, material from this flask is transferred to an agar medium containing lignin as the sole carbon source. After incubation colonies will develop on the agar that may consist of lignin utilizing microbes. To confirm this, a small amount of each colony to be tested is inoculated into liquid medium that either contains the lignin substrate or not. After incubation, growth will occur only in the medium containing the substrate, if the enrichment process has selected for and promoted the 84 growth of lignin utilizing microbes.
Optical Methods
Soil sample inoculated into liquid enrichment medium containing lignin as substrate Incubation Incubation
Transfer to solid medium containing lignin
Incubation Colonies
Medium Medium + lignin - lignin
Growth No growth
Fig. 20.6: Enrichment Culture Technique.
Even though this method is desired for isolating specific organisms, its not foolproof. That is, failure to isolate the desired metabolic type of microorganism may be due to the lack of a required component e.g. vitamin; alternatively, degradation of the complex substrate may result by more than one kind of the microorganisms.
5 The Burial Slide Technique
This method was originally devised by Rossi and Cholodny. It has been variously modified to suit the needs of individual workers. This technique is useful to study the qualitative changes in soil microflora under the influence of soil amendments. It requires a clean glass slide to be introduced into a slit in soil. This is to be left in position for three weeks. The slide is then removed carefully without disturbing its one side. This side called 'top side' is gently washed in water, air dried and heat fixed under low flame. The top side is then stained with erythrosin or rose bengal and observed under the microscope to study the microflora.
6 Soil Respiration Technique
When carbon containing substrates are oxidized in soil, CO2 is evolved. CO2 estimation is one of the parameter or index of microbial activity in soil. Many simple methods of determining CO2 are made use of viz. gravimetric, volumetric and 85 manometric.
Instrumental Methods i) Manometric of Analysis Warburg manometers are used. One can take 1-10 g of soil in a manometric flask,. Water or buffer solution is added to practices 60% saturation. In the center well 0.2 cm3 of 3 % potassium hydroxide solution is placed . A small roll of filter paper is also kept there to increase the surface area for the absorption of CO2. Flasks are now attached to Warburg manometer (having Brodies solution as an indicator) and suspended in the water bath at 30 0C. The changes in the readings on the manometer are noted at regular intervals. The rate of oxygen uptake is expressed as l/g/hr.
ii) Volumetric
The technique involves the absorption of CO2 evolved during a given period of time in NaOH solution in an airtight flask. 100g soil is taken in a one dm3 flask. A test tube containing 15 cm3 of std. M/2 NaOH is to be suspended in this flask and stoppered immediately. After 24 hr. incubation the contents of the test tube is to be transferred to a new flask, and the test tube again filled with 15 cm3 of M/2 NaOH solution. This is again kept in the flask and incubated. The removed NaOH is to be treated with saturated solution of barium chloride with the addition of 2-3 drops of phenolpthalin solution. This is now back titrated with standard HCl (M/2).
7 Soil Enzymes
Life is composed of a series of enzyme reactions, and enzymes carry out most of the reactions in nutrient cycling. A great variety of enzymes are produced by soil microorganisms during their metabolism. The total activity of soil microflora is the sum total of a number of activities of different microorganism. Dehydrogenase activity is often increased because they are constitutive and found only in living cells, and is a convenient laboratory technique. 10-20 g of soil at 90 % water holding capacity is taken and mixed with 200 mg of CaCO3 and 2 ml of 1 % solution of triphenyl tetrazolium chloride (TTC). The mixture is incubated for 24 hrs. In the absence of O2, TTC serves as the terminal acceptor for the hydrogen evolved through the microbial dehydrogenases. TTC which is colourless in oxidized state gets reduced to triphenyl formazon (TPF) which is red in colour. TPF is extracted into methanol, at 485 nm, its optical density is proportional to concentration, and hence can be read with spectrophotometer.
8 Molecular Technique
The development of a number of molecular techniques in recent times holds a great promise for the identification and examination of soil flora, without the requirement of artificial media. This involves either the direct extraction of DNA or RNA from soil sediments, or water, or the extraction of nuclear material from the microorganisms. An intermediate approach such as the analysis of 16S RNA often separates groups of microorganisms. The most specific approach involves gene probes which separate at the species or often at the enzyme level. One of the most rapid and exciting technique developed is that of polymerase chain reaction or PCR. This is based on the activity of the enzyme DNA polymerase, specifically Taq polymerase which is heat stable. In a series of heat and cool cycles of few minutes each, an exponential reproduction of desired strand takes place. This can then be analysed by gene probes. This technique allows one to measure very small amounts of complex genetic material that one finds in soil or sediments.
SAQ 6
a) Why do we enumerate microorganisms in soil ? …………………………………………………………………………………… …………………………………………………………………………………… 86 b) What kind of bacteria from a soil sample would not be recovered by inoculating a Optical Methods nutrient agar plate that is incubated at 300C? …………………………………………………………………………………… …………………………………………………………………………………… c) Which method is best suited for isolating organism metabolizing p- hydroxybenzoic acid? …………………………………………………………………………………… …………………………………………………………………………………… d) Would count be the same in plate count and microscopy methods? …………………………………………………………………………………… ……………………………………………………………………………………
SAQ 7 a) Which gas is measured in Warburg method? …………………………………………………………………………………… …………………………………………………………………………………… b) Which enzymes give the measure of soil microbial activity. …………………………………………………………………………………… …………………………………………………………………………………… c) What is PCR? …………………………………………………………………………………… …………………………………………………………………………………… d) Taq polymerase is heat ……………………………
20.4 MICROBIOLOGICAL EXAMINATION OF AIR
The earth’s surface – both land and water – is the source of micro organisms in the atmosphere. Dust is created by wind from soil, and the dust particles carry soil microorganisms in to the air. Microbial population of the atmosphere is transient and variable. Air is not a medium in which microorganism grow. Organisms introduced into the air may be transported a few feet or many miles. Some air borne microorganisms die in a matter of seconds, others survive for weeks, months or longer. The ultimate fate of airborne microorganisms is governed by a complex set of circumstances that include humidity, temperature, the amount of sun light and the size of the particles bearing the microorganisms. The nature of the micro-organisms is also important. For example , organisms that form spores or cysts are likely to survive in the atmosphere for long periods.
Algae, protoza, yeasts, molds and bacteria have been isolated from the air near the earth’s surface. One method for the microbiological sampling of air is to expose a Petri dish containing an agar medium to the air for a certain periods of time. Incubate the dish and find that microorganisms that have settled on the agar surface grow into colonies. This method gives a rough count of the numbers and kinds of airborne 87 organisms. Instrumental Methods SAQ 8 of Analysis a) How are microorganisms carried in the atmosphere? …………………………………………………………………………………… ……………………………………………………………………………………
b) How can the number of microorganisims in a sample of air be determined? …………………………………………………………………………………… ……………………………………………………………………………………
20.5 SUMMARY
In this unit you have studied the microbiological examination of water, soil and air. We have discussed that water, air and soil abounds in microbial life. The earths supply of water undergoes continued recycling; the stages constitute the hydrologic cycle. For water potability it is essential that it be checked for contaminants. Different microlobiological techniques are used e.g. membrane filter technique, plate count etc. However, the three step test to reveal the presence of coliform bacteria is the most used and important one. In practice, it is the presence of coliforms directly and not the pathogens which determines the quality of water.
Microorganisms inhabiting soil and air can also be examined by various methods - depending on the need of the worker. However, no technique is foolproof . Each has own advantages and disadvantages.
20.6 TERMINAL QUESTIONS
1. What is hydrologic cycle? 2. The definitive ingredient of the medium used in performing the presumptive water test is a) lactose, b) glucose, c) methylene blue, d) bile salts , e) nitrate salt. 3. Which of the following elements is most widely used in large scale purification of water? a) fluorine, b) chlorine, c) iodine, d) bromine, e) mercury. 4. Why is the MPN three step procedure qualitative rather than quantitative? 5. Why is the term 'presumptive' used to describe the first in series of such tests? 6. What microorganisms other than coliforms are liable to give a positive presumptive test? 7. What are the advantages of membrane-filter technique in analysis of water? 8. Which three of the following genera belong to the coliform group of bacteria? a) Klebsiella, b) Shigella, c) Salmonella, d) Escherichia, e) Enterobacter 9. Which one of the following characteristics can be used to distinguish between fecal coliforms and Salmonella? a) Gram reaction b) fermentation of glucose c) sporulation d) fermentation of lactose e) temperature requirement for growth 10. What are the important characters of an indicator organism. 11. What other elements have cyclic processes similar to the N, C and S cycles? 12. Which are true with reference to procedures for the enumeration of microorganisms in soil? a) All physiological types can be grown by one plate culture procedure. b) Microscopic examination of a soil specimen does not ordinarily distinguish dead from living microbial cells. c) The method of choice for isolating a bacterial species from soil with unique 88 biochemical characteristics is the enrichment culture technique. d) Agar plate cultures made from samples of soil reveal only a portion of the Optical Methods total microbial flora. 13. What techniques are used to estimate the numbers and kinds of soil microorganisms. 14. What is the advantage of measuring Dehydrogenase activity.
20.7 ANSWERS
Self-Assessment Questions
1. a) The three categories of natural waters are ; atmospheric water, surface water and ground water. Of the three ground water is of good quality and with negligible microbial content. b) About 3/4 of the earths surface is occupied by water.
2. a) Aquifer is the pool of water saturating the pores in soil. Its an important source for surface water. b) Eutrophication is a term describing the addition of nutrients to waters resulting in massive microbial growth and depletion of oxygen.
3. a) Indicator organism is one whose presence in water proves that the water is polluted with fecal material and not fit to drink. b) Coliforms survive longer and are present in larger number than pathogens. Hence, coliforms are tested, for their detection is easy. c) Coliforms are Gram negative, non-sporulating, aerobic and facultatively anaerobic bacilli that produce acid and gas from the fermentation of lactose. d) Pathogens e) Coliforms and especially Escherichia coli are normal inhabitants of intestinal tracts of human and other warm blooded animals, whose body temperatures are around 37 0C.
4. a) coliforms. b) most probable number. c) E. coli forms metallic green colored colonies and Enterobacter red coloured colonies on EMB agar. d) BGLB, EMB e) 40 MPN per 100 cm3
5. a) Soils are characterized by horizons. b) Soil properties like organic and mineral content,colour, texture, structure, porosity and pH affect the numbers and kinds of microorganisms in soil. c) Bacteria are highest in number followed by fungi, algae,protozoa and viruses. d) N-cycle, C-cycle and S -cycle. e) Organic matter in soil governs the biological activity. High organic matter supports increased microflora and low organic matter show less microflora.
6. a) Microorganisms in soil are enumerated to know the kind and extent of microbial flora. b) When incubated at 300C thermophiles or psychrophiles would not be recovered. c) Enrichment culture technique. d) No.
89 7. a) CO2 b) Dehydrogenases Instrumental Methods c) Polymerase chain reaction of Analysis d) Stable
8. a) By dust particles b) By exposing a petridish with medium to air and incubating the plate for growth.
Terminal Questions
1. The earth’s supply of water undergoes continuous recycling, various stages constitute the hydrologic cycle. 2. Lactose 3. Chlorine 4. The MPN three step procedure includes presumptive, confirmed and completed tests which gives positive evidence of the presence or absence of coliforms and not the number of coliforms present. 5. The term 'presumptive' is used because gas formation in lactose broth at 37°C is a characteristic not only of coliforms but also of non-fecal coliform Enterobacter aerogenes and some Klebsiella species. 6. Klebsiella species 7. The advantages are: a) It is more direct, quicker (giving results in 18-24 hours) and able to cope with larger volumes of water sample. 8. Klebsiella, Escherichia, Enterobacter 9. Fermentation of lactose 10. The indicator organism should survive longer, be present in greater number and be harmless to humans. 11. Phosphorus, iron, potassium 12. a) F , b) T, c) T d) T 13. Direct microscopy, agar-plate count, & MPN 14. Identification of microorganisms without culturing them in a short time.
FURTHER READINGS
1. Ananthanarayan, R and C.K.Jayaram Paniker. 1978. Text Book of Microbiology. Orient Longman Ltd. Madras. 2. Harry W. Seeley Jr. and Paul. J. van Demark. 1975. Microbes in Action: A laboratory Manual of Microbiology. II Edition. W.H.Freeman and Company. 3. Ronald, M. Atlas. 1989. Microbiology: Fundamentals and Applications. II Edition. Maxwell Macmillan Intern. 4. Pelczar, M.J. Jr., Chan E.C.S and Kreig N.R. 1993. Microbiology- Concepts and Applications. McGraw Hill Inc. 5. Kathleen Talaro and Arthur Talaro. 1993. Foundations in Microbiology. W C B Pub. 6. SubbaRao N.S. 1977. Soil Microorganisms and Plant Growth. Oxford and IBH Pub. Co. 7. Alexander. M. 1985. Introduction to Soil Microbiology. II Edition Wiley Eastern Ltd. 8. Paul, E.A. and Clark, F.E. 1989. Soil Microbiology and Biochemistry.
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