instrumental analysisscbaghdad.edu.iq/sc/files/lectures/chem/ana4o.pdfapplication and verification...

Instrumental Analysis Lab (1)4 th stage

Prof.Dr. Nagam S. Turky

Ass. Lecturer Huda M.NafeaLecturer Ghada F. Hussin

Spectrophotometric determination of Manganese (Mn7+) and Chromium (Cr6+)

in admixture of potassium permanganate and potassium dichromate

Experiment No.1

human eye can see only these colors

Instrumentation

• Visible Region

٦

Instrumentation (Spectrophotometers)

A single beam spectrophotometer

Wavelength selector

The above essential features of a spectrophotometer shows that polychromatic light from a source separated into narrow band of wavelength (nearly monochromatic light) by a wavelength selector, passed through the sample compartment and the transmitted intensity, P, after the sample is measured by a detector

In a single beam instrument, the light beam follows a single path from the source, to the monochromator, to the sample cell and finally to the detector

Beer-Lambert’s law

A abc=

a – Molar absorptivityUnits of molar absorptivity:

cA∝ bA∝bcA∝

))()(( cofunitbofunitaofunitAofunit =

))(( cofunitbofunitAofunitaofunit =

۸

Selection of wavelengthAbsorbance measurements are always carried out at fixed wavelength (using monochromatic light). When a wavelength is chosen for quantitative analysis, three factors should be considered

1. Wavelength should be chosen to give the highest possible sensitivity. This can be achieved by selecting λmax or in general the wavelengths at which the absorptivity is relatively high.

λmax

λmax - wavelength where maximum absorbance occurs

• total absorbance of a solution is equal to the sum of absorbances of the individual component presentAtotal = AM + AN…

= εMcMl + εNcNl …

Analysis of Mixtures of absorbing substances:

•By measuring the absorbance at 2 wavelength (λ1

and λ2) A1 = ε1M c1

M l + ε1N c1

N l (at λ1)

A2 = ε2M c2

M l + ε2N c2

N l (at λ2)

Spectrophotometric determination of Manganese (Mn7+) and Chromium (Cr6+) in admixture of potassium permanganate and potassium dichromate

Experiment No.1

Procedure:

1- Two solutions already prepared; KMnO4 and K2Cr2O7 .2- Transfer with the aid of graduated pipette 4ml of KMnO4 solution to a

volumetric flask (50ml) dilute to the mark with distilled water (representstandard solution A).

3- Transfer with the aid of graduated pipette 4ml of K2Cr2O7 solution to avolumetric flask (50ml) dilute to the mark with distilled water (representstandard solution B).

4- Dilute the unknown given solution (available in the (50ml) volumetric flask)dilute to the mark.

5- Fill the first glass cell with reference solution (i.e. distilled water), this solutionused for Zero adjustment the spectrophotometer. The second glass cell is usedfor standard solution A. The third glass cell with standard solution B; while thefourth cell is used for the unknown solution.

6- Record and tabulate the absorbance reading each 10 nm increments within therange 400-570 nm.

Results and Calculations:

1- With the aid of the absorbance table of wavelength in nanometers, plotabsorbance curve versus wavelength in nanometers for solutions A&B&

unknown.Find the wavelength of maximum absorbance (λ max).

2- Find absorbance for the unknown solution at wavelength of maximum absorbance (λ max).

3- Calculate concentration of MnO4- & Cr2O7

-2 in admixture using Beers Low for mixture.

A total at λ max.1 = AMn + ACr (1) at λmax.1A total at λ max.2 = AMn + ACr (1) at λmax.2

Application and Verification of Beer's Law using

Spectrophotometer

Experiment No.2

• Spectrophotometero Components of optical instruments

1. Sources2. Wavelength selectors (filters, monochromators)3. Sample containers4. Detectors5. Readout devices

• Single and double beam instruments

• Applications of Spectrophotometry

o Spectrophotometry is more suited for quantitative analysis rather than qualitative one

٤

Instrumentation

• Visible Region

Fundamentals of Spectrophotometry

Absorption of Light

3.) Beer’s Law The relative amount of light making it through the sample (P/Po) is known as the

transmittance (T)

oPPT =

×=

oPPT% 100Percent transmittance

T has a range of 0 to 1, %T has a range of 0 to 100%

Fundamentals of Spectrophotometry

Absorption of Light

3.) Beer’s Law Absorbance (A) is the relative amount of light absorbed by the sample and is related

to transmittance (T)- Absorbance is sometimes called optical density (OD)

)()( 100/T%logTlogPP

logAo

−=−=

−=

A has a range of 0 to infinity

Interaction of light with matter

Iin Iout

Definition of absorbance & transmittance

Absorbance Transmittance

Absorbance (A) and transmittance (T) are unitless

s

%100% ×=out

in

IIT

in

out

IIA log=

Beer-Lambert’s law

Iin

Iin

Iout [2]

Iout [1]

Absorbance Vs. Concentration (c )

Iin

Iin

Iout [2]

Iout [1]

Absorbance Vs. Path length (b)

Path length- the distance that light travels in the sample

Unit of path length = cmUnit of concentration = M

s

s

s

s

This is not true at high concentrations

cA∝ bA∝

Application and Verification of Beer's Law using Spectrophotometer

Experiment No.2

ProcedureThe intensity of incident radiation Po for each light emitting diode (LED) (Red, Orange, Greenish yellow, Green, White and Blue).

1- Set the instrument on Red (or R) the red light absorb at 650-700 nm, then the intensity is measured (Po) for an (Nothing is placed in the path of light).

2- Place a plastic slice at the absorption position and the transmitted light is measured (represented by P) (remaining of the incident light), followed by the addition of another slice and P is measured again; repeat the experiment for 33 slices.

3- Arrange table for calculating transparency, % transparency %T. it is known that the absorbance is:

%Tlog2A or

TlogA of 100PPT% ,

PPT

oo

−=

−=×==

For the purpose of determining the λmax for blue slice thereby using layer scanning (using different λ for different sourced which shows the possibility of exposing and λ for any transparat solid subject using this spectrophotometer).

a = Absortivity.ε = molar absortivity.A = Absorbance.

Slice no.

Slice thickness (b) mm

Transmitted light (P)

1

2

-

oPPT = 100

PPT%

o

×=TA log−= %Tlog2A −=

Instrumental Analysis Lab (1) 4 th stage



Determination of phosphoric acid and hydrochloric acid via pH

measurements

Experiment No.3

pH - Meter

Determination of phosphoric acid and hydrochloric acid via pH measurements

Experiment No.3

Procedure:1- Glass electrode (single or combined) should be combined using two buffer

solutions; mainly solution having pH=4.01 and a solution having pH=9.18

2- The unknown solution which is a mixture of H3PO4 and HCl. Small amount of distilled water should be added whenever it is necessary so that the electrodeshould be immersed inside the solution. The solution is stirred with the aid ofa magnetic stirrer.The initial reading should be taken using the pH- meter.

3- Sodium hydroxide (NaOH) should be added while constant stirring isachieved the pH-reading should be read. (Addition should be inverselyproportioned with the range pH changes).

4- Base addition should be follows:

NaOH pH1ml, 1ml, …….. 3.50.2ml, 0.2ml, …. 6.51ml, 1ml, .…….. 8.50.2ml, 0.2ml, …. 10.21ml, 1ml, ……... 11.3

5- pH values should be drawn versus added base volume; then calculate from equivalence point the concentration HCl and H3PO4 in mM.

Conductometric titration for a strong acid (HCl)

Experiment No.4

4

Conductivity measurements

1.ElectrodesTwo parallel platinized Pt. foil electrodes or Pt. black with

electrodeposited a porous Pt. film which increases the surface area of the electrodes and further reduces faradaic polarization.

2.Primary standard solutionsPrimary standard KCl solution ,at 25℃, 7.419g of KCl in 1000g of solution has a specific conductivity of 0.01286Ω-1/cm.

CONDUCTOMETRIC TITRATIONS:

The determination of end point of a titration bymeans of conductivity measurements are knownas conductometric titrations.

5

Conductometric titration for a strong acid (HCl)

Experiment No.4

Procedure:1- The unknown acid solution (HCl) to which 50ml of distilled watered added.

2- Immerse the clean conductivity cell in the unknown solution ensuring that the solution covers the platinum plates of the conductivity cell, then mix thesolution well and record the conductivity value.

3- Titrate the unknown solution by adding a successive amount (0.5ml each time) of NaOH solution to the unknown solution then mix the solution welland record the conductivity value after each addition.

Results & Calculations:1- Tabulate your results volume of sodium hydroxide added versus conductivity

value. 2- Draw the curve of volume of NaOH added solution versus conductivity.

3- Calculate the concentration of unknown acid.

Colorimetric determination of Iron (Fe3+) using ammonium

thiocyanate

Experiment No.5

When a sample only absorbs light of a single wavelength the eye seesCOMPLEMENTARY colours.

VISIBLE SPECTROSCOPY

LOW HIGH

Wavelength Range AbsorbedColour Absorbed Colour Seen By Eye

380 - 430 Violet Yellow - Green

430 - 480 Blue Yellow

480 - 490 Green - Blue Orange

490 - 500 Blue - Green Red

500 - 560 Green Purple

560 - 580 Yellow - Green Violet

580 - 590 Yellow Blue

590 - 610 Orange Green - Blue

610 - 750 Red Blue - Green

IMPORTANCE OF THE BEER LAMBERT LAW

A = Ecl but if E and l are constant

ABSORBANCE ∝ CONCENTRATION and should be linear relationship

Prepare standards of the analyte to be quantified at known concentrations and measure absorbance at a specified wavelength.

Prepare calibration curve.

From measuring absorbance of sample

Concentration of analyte in samplecan be obtained from the calibration curve

E can be obtained from the slope of the calibration curve for a given wavelength (λ)

x

x

x

x

x

AB

SOR

BA

NC

E A

T 30

0n

m

CONCENTRATION (moles litre-1 )

Increasing [Fe2+]

Absorbance is directly proportional to concentration of Fe+2

Colorimetric determination of Iron (Fe3+) using ammonium thiocyanate

Experiment No.5

Procedure:1- Prepare (100ml) of iron (III) stock solution having the concentration of

starting with ammonium ferrous sulphate following the steps:a) Add approximately 10ml of distilled water then with using graduated cylinder 5ml of

nitric acid to the calculated weighted ammonium ferrous sulphate.b) Heat the solution above (in step a) cautiously and gently for three minute.c) Cool the solution then transfer it quantitatively with precision to a volumetric

flask of 100ml, then continue to the mark, close and mix well.2- Transfer with the aid of graduated pipette (5ml) the following volumes (1, 2, 3,

4, 5ml) successively to five different volumetric flasks (size 50ml).3- To the five volumetric flasks and the unknown solution add (5ml) with the aid of graduated pipette (size 5ml) thiocyanate solution then continue to the mark with distilled water. Close and mix well.4- Record the absorbance for the solution using 400nm (blue filter), 500nm

(green filter), 600nm (red filter).5- Find the unknown concentration for Fe+3 from the drawn calibration curve.

Determination of Chloride using chloride selective electrode

Experiment No.6

Ion Selective Electrode (I.S.E.)

• I.S.E. consists of a thin membrane across which only the intended ion can be transported.

• The transport of ions from a high conc. to a low one through a selective binding with some sites within the membrane creates a potential difference.

Ion Selective Electrode

ElectrodeBody

IonSensitive

Area

ElectricalConnection

SensingElectrode

ReferenceElectrode

Current Flow

Meter

ElectrochemicalMeasuring System

Determination of Chloride using chloride selective electrode

Experiment No.6

Procedure:

1- 10ml of the chloride unknown concentration in a 250ml – beaker. Enoughdistilled water is added so that the electrode is immersed in a proper way.

2- Chloride selective electrode where it is immersed in the unknown solution. Itis attached with the instrument at position cave G.

3- In another beaker, a solution of NaNO3. The saturated calomelelectrode is attached with the instrument at position cave R.

4- The two beakers placed adjacent to each other. A salt bridge is used to connect

the two solutions (NaNO3 is used as salt bridge).

5- The unknown solution is titrated against AgNO3 solution as follows:

AgNO3 V1ml, 1ml, …….. 6ml0.5ml, 0.5ml, …. 9ml0.2ml, 0.2ml, …. 10ml0.1ml, 0.1ml, …. 11ml0.2ml, 0.2ml, …. 12ml

6- The potential is recorded for each addition, a table is arranging for these values.

7- A graph is drowning for the potential in mV and the volume of AgNO3. Find the concentration of the unknown solution by two curves.

8- Tabulate your data according to the following table:

E(mv) V(ml) AgNO3 ΔE (mv)E2-E1

ΔV (ml)V2-V1

ΔE / ΔV V avgV1+V2 / 2

Estimation of the complex structure by mole ratio and

the continuous variation method

Experiment No.7

4

Absorption of Light

Beer-Lambert Law: A = εbc

5

(1,10-phenantholine)iron(II)

N N

1,10-Phenanthroline

Fe2+ + 3phen orange- red complex

Visible spectrum of Fe complex

λmax

Estimation of the complex structure by mole ratio and the continuous variation method

Experiment No.7

Procedure:1- Prepare 100ml of ammonium ferrous sulphate.2- Add to series of solutions (25ml capacity) a constant volume of 2ml of

prepared iron sulphate solution. 3- Add to each volumetric flask 2ml of the buffer solution (pH=5) and 1ml of hydroxylamine

hydrochloride . 4- Add variable volumes (1, 2, 3, 4, 6, 8, 10ml) of 1,10 phenanathroline5- Dilute all solutions to the mark with distilled water then mix well and leave a

side for 10 minute for complete the reaction. 6- Read the Absorbance (A) at 510 nm 7- Repeat steps 2-6 using variable volumes of each iron solution (1, 1.5, 2, 2.5, 3,

5, 7ml) and 1,10 phenanathroline (9, 8.5, 8, 7.5, 7, 5, 3ml) with 5x10-4 molaron the find volume to be constant volume of 10ml.

Results & Calculations:

1- Tabulate your results in a table include volume of each ferrous solution (Vm) and the volume of 1,10 phenanathroline (VL) against absorbance for eachmethod.

2- Draw the value of Vm/VL versus absorbance for the first method.3- Draw the value of Vm / (Vm+VL) against the value of absorbance for the second

Photometric titration of permanganate with oxalate

Experiment No.8

Photometric titration• Photometric titration is titration method in

which end point of titration (e.p.t) isdetermined by photometry orspectrophotometry method.

• Method is based on determination e.p.t onjump of solution absorbance in equivalencepoint.

Some of the typical titration curves are:

a

b

c

d

a)Where titrant alone absorbs ex: titration of arsenic(III) with bromate-bromide, where absorbance is measured at the wavelength where the bromine absorbs

b)Where the product of the reaction absorbs ex: titration of Copper(II) with EDTA

c) Where species is converted into non-absorbing product ex: titration of p-toluidine in butanol with perchloric acid

d) Where a colored species is converted to a colorless product by a colored titrant ex: bromination of a red dyestuff

Photometric titration of permanganate with oxalate

Experiment No.8

Procedure:1- Prepare a standard solution of sodium oxalate Na2C2O4 from a 0.1M

standard solution in a volumetric flask in 100ml.2- Prepare 7 volumetric flasks of 25ml capacity. The first one is the reference

one.3- To each volumetric flask add of unknown concentration of KMnO4 solution then of – H2SO4 is added (the catalyst). The first volumetric flask is continued to the mark with distilled water, while the rest (six volumetric flasks), 0.5, 1, 1.5, 2, 2.5, 3 ml of 0.01M oxalate solution is added to volumetric flask no. 2,3,4,5,6,and 7 successively, followed by the continuation of the volume to the mark with distilled water.4- The solution transferred to large supplied tube for the purpose of heating for a

while until the disappearance of the color of the one of the tube (pinkcoloration of KMnO4). Then all tubes are removed from heating, and cooled.

5- Absorbance is measured at 525nm, starting from tube no. 1 (reference tube).6- A relation of absorbance and the volume of Na2C2O4 for finding the unknown

concentration of KMnO4.

Colorimetric determination of aspirin in pharmaceutical

preparation

Experiment No.9

Visual Observations – Because colorimetry is based on inspection of materials with the human eye, it is necessary to review aspects of visible light.

Visible light is the narrow range of electromagnetic waves with the wavelength of 400-700 nm.

Colorimetry

Simple Spectrophotometer Schematic

The lamp emits all colors of light (i.e., white light). The monochromator selects one wavelength and that

wavelength is sent through the sample. The detector detects the wavelength of light that has

passed through the sample.The amplifier increases the signal so that it is easier to

read against the background noise.

Colorimetric determination of aspirin in pharmaceation preparation

Experiment No.9

Procedure: 1- Prepare of aspirin solution in a final volume of 100ml as follows:•Add sodium hydroxide solution to the weighted aspirin material.•Heat the mixture until boiling and leave it to boil for 2 minutes.•Leave the solution to cool then transfer to a volumetric flask capacity 100ml and complete the volume to the mark by distilled water.2- Add to a series of volumetric flasks (50 ml capacity) the various volumes of the prepared solution in the previous step.3- Add to flask including the unknown flask of ferric chloride solution .

4- Dilute the solution to the mark by distilled water, mix well then record theabsorbance value of the solution at 525nm.

5- Find the concentration of the unknown and the prepared pharmaceutical drug from the graph and from linear equation.

Sulphate ion determination using Turbidimetric method

Experiment No.10

Turbidimetry deals with measurement ofIntensity of transmitted light . Nephelometry deals with measurement of

Intensity of scattered light. Turbidometric measurements are made at

180o from the incident light beam. In Nephelometry, the intensity of the

scattered light is measured, usually at r ightangles to the incident light beam.

TURBIDITY MEASUREMENT

• Turbidity does not mean suspended solidsmeasurement, but a effect determination of lightrefracted through solids.

• As light of a known intensity is passed through asample some of the light will be scattered byparticles in the sample. A detector is placed at 90° tothe sample to collect the scattered light. Theintensity of the scattered light is compared to that ofthe source. This value is reported as a NephelometricTurbidity Unit, or NTU.

• LED light source emits at a range close to infrared. In this range absorption is minimized, and turbidity measurements are more accurate.

Sulphate ion determination using Turbidimetric method

Experiment No.10

Procedure:1- Preparation of series of solutions in a gradual increase in volume from sodium sulphate in 25 ml volumetric flask.2- To each flask and unknown solution add of constant volume of BaCl2 and 5 ml of the glycerol-alcohol solution and dilute to 25 ml with distilled water and shaken gently in order to obtain a uniform particle size .Note : - A white precipitate will appear SO4

2- + Ba 2+ ↔ BaSO4- Each flask should be shaken at the same rate and the same number of

times to reproduce the measurements .3- Plot the calibration graph NTU versus sulphate concentration .4- Find the concentration of the unknown NTU from the calibration graph .

instrumental analysisscbaghdad.edu.iq/sc/files/lectures/chem/ana4o.pdfapplication and verification...

Documents