Ist Part for Record

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Expt.No:

Date:

pH METER

Aim: - To calibrate the given pH meter and measure pH of the given solution.

Theory: - pH is defined as the negative logarithm of hydrogen ion concentration in a

solution.

pH = -log [H+]

A pH meter is the instrument used to measure and adjust the pH of the given solution

to the desired level. The instrument works like a potentiometer which measures the voltage

difference between two electrodes placed in a solution. The two electrodes used are calomel

electrode and a glass electrode. The calomel electrode is the external reference electrode,

whose electrical potential is always constant, where as the glass electrode is the standard test

electrode whose electrical potential depends on the pH of the test solution. The electromotive

force (emf) of the complete cell (E) is given by the expression.

E=Eref - Eglass

Where

Eref = Potential of reference (Calomel) electrode which at normal temperature is +

0.250V.

Eglass = Potential of the test electrode which depends on the pH of the test

solution.

The pH of the test solution can be calculated from the equation.

pH = Eglass - Eref at 25o C

0.0591

pH = Eglass - 0.250v at 25o C

0.0591

The Calomel electrode: It contains mercury, mercuric chloride and saturated solution of

potassium chloride. Each of these compounds exists in ionized state although the extent of

ionization may vary by temperature. Their dissociation constants are:

a) Hg Hg+ + e-



[Hg+] [e-]

K a =

[Hg]

b) Hg2Cl2 2Hg+ + 2Cl-

[2Hg+] [2Cl-]

K b =

[Hg2Cl2]

The calomel electrode is dipped in saturated solution of potassium chloride (its ionization is

negligible). The electrical constant between the calomel electrode and the test solution is

achieved by the potassium chloride salt bridge through a fine capillary in the glass casing

known as porous plug.

The Glass electrode: The glass electrode contains silver, silver chloride and 0.1M HCl

solutions. Their dissociation constants are as follows:

c) Ag Ag+ + e-

[Ag+][e-]

K c =

[Ag]

d) AgCl Ag+ + Cl-

[Ag+][Cl-]

K d =

[AgCl]

The electrode is dipped in 0.1M HCl solution.



FIG. 1 CALOMEL ELECTRODE



1. Because the concentration HCl in the glass electrodes changes by repeated use, the pH

meter should be calibrated against the standard solution of known pH every time.

2. The bulb containing HCl is very sensitive & hence should be handled very carefully.

3. After each time the electrode is taken out from solution, it should be rinsed with distilled

water and wiped gently with tissue paper.

Expt .No:

Date:

TITRATION OF STRONG ACID WITH STRONG BASE

Aim: To titrate the strong acid with strong base and to determine the pK a value of the acid by

using the pH meter.

Requirements: pH meter with combined glass electrode, beakers, glass rods, standard buffer

solutions, tissue paper etc.

Reagents required: 0.1M HCl, 0.1M NaOH, standard buffer solutions of pH 4, pH 9.2 and

distilled water

Procedure:

1. 50 ml of 0.1M Hcl was prepared and taken in a 250 ml beaker.

2. 100 ml of 0.1M NaoH was prepared.

3. pH electrode was calibrated to the pH of the two standard buffer solutions viz., buffer with

pH 4 and buffer with pH 9.2.

4. The electrode was then washed with distilled water and cleaned gently with tissue paper.

5. Now the pH probe was dipped in the beaker containing 50 ml of 0.1M HCl and pH is noted.

6. 2 ml of 0.1M NaOH was added, mixed thoroughly with a glass rod and the pH was noted.

7. The above steps were repeated until the pH rises approximately to 10.

8. A graph was plotted with volume of NaOH on X - axis and pH on Y - axis. All the points

were connected by a smooth line and this gives the titration curve for the acid.

8. The mid point of the inflection gives the pK a of the acid.

Observations:

Stock solution concentration =

S.NO. Volume of acid

taken

Volume of base

to be added (ml)

Total volume of

base added (ml)

Change in the

pH



(ml)

1 50 0 0

2 50 2 2

3 50 2 4

4 50 2 6

5 50 2 86 50 2 10

7 50 2 12

8 50 2 14

9 50 2 16

n 50 2 18

Result:

The given strong acid was titrated with strong base and the pK a value of the given acid viz.,

HCl from graph is found to be ………….

Precautions:

1. The acid and the base solutions are prepared carefully considering the molarity of the

commercially supplied reagent.

2. The titration is done carefully to ensure thorough mixing of the base with the acid.

3. The pH value is read accurately.

4. The PH electrode is cleaned every time with distilled water and wiped with tissue

paper before taking next reading.

Expt.No:

Date:

COLORIMETER – BEER LAMBERT’S LAW

Introduction: Colorimeter is one of the useful and non-destructive instruments for measuring

the amount of biochemical solute in solution. Biological chemicals tend to absorb light in the

visible range, which causes transitions in the energy state of valency electrons that are lastingfor 10 - 100 ns, especially for the electrons that are involved in conjugated orbitals. It measures

the intensities of the light before and after it has passed through a coloured solution and hence

used to determine the concentration of known (standard) as well as unknown samples.

It is based on two laws of absorption: 1. Beer’s Law

2. Lambert’s Law

Lambert’s Law: It states that the amount of light absorbed is proportional to the thickness of

the absorbing material and is independent of the incident light. To understand this statement, let

us assume that thickness ‘b’ has the ability to absorb 50% incident intensity of light passing



through it. If the intensity of the radiation incident upon such a thickness is assigned a value of

‘1’, the outcome i.e. the transmitted beam will have a value of 0.5. If we now place a second

equal thickness b, it will absorb 50% of the transmitted beam i.e. 50% of 0.5. Thus the second

transmitted beam will have a value of 0.25.

This is clearly an exponential function and may be expressed as

I = Io e-kb

where I = intensity of transmitted light.

Io = Intensity of incident light.

b = the absorbing thickness better known as path length.

k = the linear absorption coefficient of absorbing material.

Beer’s Law: This law states that the amount of absorbed light by a material is proportional to

the number of absorbing molecules. The relationship between absorbance of the light incident

is directly proportional or dependent on concentration.

K c = 2.303 log10 Io/I

Where c = the concentration of absorbing molecules.

Io = Intensity of incident light

I = Intensity of transmitted light

Beer – Lambert’s Law: This combined law states that the amount of light absorbed is

proportional to the concentration of absorbing substance and to the thickness of the absorbing

material (Path length). The quantity (Io/I) is known as absorbance or optical density while the

reverse I / Io is known as transmittance T.

Absorbance A = Log10 I0/I = ε bc

Where

ε = molar absorptivity coefficient (SI unit: m2 mol-1 );

b = path length;

c = concentration of absorbing molecules



Deviations From Beer – Lambert’s Law:

The following factors may cause the violation of Beer- Lambert’s law.

1. Deviation from Beer’s law occurs when high sample concentrations are being measured.

One effect of high concentration is that the molecules may dimerize. It’s not necessary that the

absorption spectra of dimmers are the same as that of monomers. If the spectra differ, the

absorption coefficient will also undergo a change leading to a positive or a negative deviation.

High concentrations may lead to aggregation of molecules and these large aggregates may

scatter light. With the result, the light reaching the detector is lesser & thus two important

phenomena are contributing to reduction in the transmitted intensity. Absorption & Scattering

lead to positive deviation. Low concentrations may also lead to deviation. Most significant

point to note here is about the proteins. When proteins are denatured at their low

concentrations, their absorption spectrum may be deviated.

2. Instrumentation limitation may also result in deviations. Lambert’s law is obeyed only for

monochromatic light, which is not realized in practice. The filters used in colorimeters pass a

broad band of light.

Ex: 640 nm red filter passes light which ranges from 625nm to 650nm.

3. Temperature effects, the degree of solubility, dissociation and association properties of the

solute, hydration and several such other factors may be responsible for deviation. Therefore

absorbance must be measured at constant temperatures.

4. Sample instability may also cause deviation; this is seen in some coloured samples which

are unstable. As the color changes, the absorption also will change.

5. Fluorescence: Some substances fluoresce and as a result along with transmitted intensity of

light, the fluorescent intensity also reaches the detector.

6. Turbidity: Turbid solution always ends up giving higher absorbance than what is determined

by the color.

Expt. No:

Date:

VERIFICATION OF BEER LAMBERT’S LAW

Aim: To verify Beer-Lambert’s law for the given concentration of the solution.

Apparatus required: Colorimeter, cuvettes, power supply unit, test tubes, test tube stand,

pipette, tissue paper etc.



Chemicals required: 5% CuSO4 solution, distilled water.

Procedure:

1. 5% CuSo4 solution was prepared using distilled water. This serves as the stock solution.

2. Clean dry test tubes were taken and labeled as T1,T2,……… T10. Another clean dry test

tube labeled as control was taken.

3. The colorimeter was switched on just before the preparation of the test solutions of known

concentration for warming up the instrument.

4. In the control test tube, no CuSO4 solution was taken and in the experimental tubes

labeled T1,T2,……… T10 , 0.5ml of CuSo4 to 5ml of CuSo4 solution were taken

respectively with the gradual increment of 0.5ml.

5. Add distilled water to each of the test tubes so as to make the final volume to 5ml and mix

thoroughly.

6. The control with 5ml of distilled water and with no CuSo4 solution serves as a blank.

7. The colorimeter is calibrated to zero with the blank after setting the wave length to 640nm.

8. Absorbance of the solutions in experimental tubes is noted down at 640nm.

9. A standard graph was plotted with absorbance on y-axis and concentration of CuSO4

solution on x-axis.

Observations: Stock solution concentration = 5%

S.NOVolume of

CuSO4 (ml)

Distilled water

added (ml)

Concentration of

CuSO4

(mg/ml)

Absorbance at

640nm

Blank 0.0 5.0 - 0.0

1 0.5 4.5

2 1.0 4.0

3 1.5 3.54 2.0 3.0

5 2.5 2.5

6 3.0 2.0

7 3.5 1.5

8 4.0 1.0

9 4.5 0.5

10 5.0 0.0

Formula & Calculations:

Test OD



Concentration Of CuSO4 in test solution = X Std. Concentration

Standard OD

Result:

1. The concentration of CuSO4 in the given test solution from calculations is

…………. mg/ml.

2. The concentration of CuSO4 in the given test solution from graph is

………….mg/ml.

Precautions:

1. Colorimeter should be calibrated and kept for 15 to 20 min. on for warming up.

2. Every time the cuvettes should be cleaned with tissue paper so as to remove the

water on outer walls before placing it in colorimeter.

3. All the weighing must be done accurately.

4. Standard graph should be prepared carefully to interpret concentration of

unknown solution accurately.

Expt.No:

Date:

VISIBLE SPECTROPHOTOMETRY

Theory: In order to obtain an absorption spectrum, it is necessary to measure the absorbance

of a substance at a known series of wave lengths. The instruments that are used to study the

absorption or emission of electromagnetic radiation as a function of wave lengths are called

Spectrophotometers. Visible spectrophotometry is mainly used for quantitative analysis and

serves as an auxiliary tool for structural elucidation. The visible light has wave length ranging

b/w 400nm and 800nm.

Instrumentation: A spectrophotometer essentially consists of the following parts:

1. Source of light



2. A monochromator

3. Sample holder

4. Detector

1. Source of Light:

The visible spectrum ranges from 400nm to 800nm. Hence any lamp source

which gives adequate intensity of radiation over the entire wave length region can be used.

Such a kind of light is called polychromatic or heterochromatic light. The polychromatic light

reflected back using a plane mirror passes through an entrance slit, a condensing lens and

then falls on to a monochromator. This disperses the light and the desired wave length is

focused on the exit slit using the wavelength selector.

The requirements for the source of light are:

1. It should provide continuous radiation from 400nm-800nm.

2. It should provide adequate intensity.

3. It should be stable.

4. It should also be free from fluctuation.

The commonly used sources of light are-

(a) Tungsten lamp: As it satisfies the above criteria, this lamp finds place in

spectrophotometer. The lamp consists of a tungsten filament in a vacuum bulb similar to the

used domestically but offers sufficient intensity.

(b) Carbon arc lamp: This is used for a source of very high intensity. It also provides

an entire range of visible spectrum.

2. Filters and Monochromators:

As the natural source of light obtained is polychromatic in nature and as the

requirement for a spectrophotometer is monochromatic light, a filter or a monochromator

should be used. It converts polychromatic light into monochromatic light with varying

efficiency.

3. Sample Holder/Sample Cells:

These are commonly called cuvettes and are used to hold a sample solution.

Their geometry as well as material varies with the instrument and the nature of the sample.

The material of the sample cell should not absorb the wave length that’s being observed.

4. Detectors:

The detectors used in uv-visible spectrophotometers can be called as

photometric detectors. When a radiation is passed through a sample cell, a part of it will be



absorbed by the sample solution & the rest will be transmitted. This transmitted radiation

falls on the detector & the intensity of absorbed radiation can be determined and displayed. In

these instruments the light energy is converted to electrical signal which can be read or

recorded.

In a spectrophotometer the light source emits white light which then is allowed to fall

on a silver mirror with the help of a shutter. The reflected light from the mirror passes

through an entrance slit and a condensing lens. This lens renders the light into parallel beams

and these beams fall on a monochromator. The monochromator disperses the light into its

component wavelengths and the desired wavelength is selected using the wavelength

selector. Now the selected beam of monochromatic light passes again through a lens to a light

free compartment where the sample is kept in a cuvette.

Figure depicting the path of light in a Spectrophotometer



Detailed path of light in a Spectrophotometer

After passing through the sample, the transmitted light falls on a photomultiplier (PMT).

The PMT converts the light energy into electrical energy, which is then amplified, measured

and recorded on the analog/digital read out.

Applications: Spectrophotometers are used in

1. Quality control for testing the purity of the substance.

2. Quantitative analysis.

3. Determination of Ligand / metal ratio in metallic complexes.

4. Structure elucidation of organic compounds.

5. Determination of pK a of indicators etc.

Spectrophotometric Titrations:

It is the titration in which the absorbance of a reactant or a product or both are followed as

function of added titrant. The advantages are:

(a) The end – point is sharp.(b) No interference from other absorbing spieces etc.



Conditions to be observed:

1. The titrant or the reactant or the product should be able to absorb light.

2. Beer’s law must be obeyed under experimental conditions.

3. Titrant must be strong, as the error due to volume change must be minimized.

4. Absorbance is monitored at λ max of titrant, reactant or product.

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