Designer

B. Basic Instrumental Methods

Our practical work in this unit will mainly cover the most widely used methods of analysis such as spectrometry , potentiometry and separation analysis ; we will introduce the basic concepts of these topics in a concise and clear form , we will also present 2-3 experiments for each topic .

1. Spectrometry :

Spectroscopic analytical methods involve absorption or emission of electromagnetic radiation by an analyte . The amount of energy absorbed or emitted from the electromagnetic radiation is related to the concentration of the analyte .

There are various spectroscopic methods of analysis depending on the region of the electromagnetic spectrum being used ; these include gama-rays , ultraviolet (UV) , visible , infrared (IR) , microwave and radio frequency (RF) .

Electromagnetic radiation is considered to be form of energy that travels through the space at tremendous velocity , associated with wavelength , frequency , velocity and amplitude . But this model of description fails to account for its interaction with matter in terms of absorption and emission behavior . Better explanation can be obtained by considering the electromagnetic radiation as discrete packets called photons or quanta in which the energy of the photon is directly proportion to its frequency . This dual nature of explanation is applicable to all elementary particles such as electrons , proton and associated particles. We mentioned that electromagnetic radiation is designated on the base its wavelength (λ) which the distance between two consecutive crests or troughs (one complete cycle) .

The frequency (f) is defined as the number of complete cycles passing a fixed place per unit of time . Another term to describe the wave is the wave number () which is the number of waves in a given distance or the reciprocal of the wavelength. The relationship between the wavelength (λ) and frequency (f) is ,

λ = (1)

where C is the speed of light ( 3x108 m/sec) .

λ = wavelength in cm.

f = frequency per second (sec-1) .

the wave number , (2)

different units are used to express the above terms :

Interconversion can be easily made between these units .

2. Nature of the light

We described briefly the dual nature of the electromagnetic radiation and it is more helpful to consider light as consisting of photons with specific energy that can be related to its frequency , wavelength and wavenumber.

(3) substituting the frequency in equation (1) ,

(4)

E = energy of the photon in ergs. h = Planck's constant = 6.62x10-27 erg-sec.

3. Interaction of matter and radiant energy

In this wide electromagnetic spectrum , we will be mainly concerned with UV-Visible region which extends from 200-750 nm . Visible region is the only region of the spectrum sensitive to the human eyes and can be seen as colors . White light consists of combination of all wavelengths in the visible region so when the white light passes through an object , certain wavelength will be absorbed leaving the other wavelengths transmitted which can be seen as colors .

Absorbed wavelength(nm)	Color absorbed	Transmitted color (complementary color)
380 - 450	Violet	yellow-green
450 - 495	Blue	Yellow
495 - 570	Green	Violet
570 - 590	Yellow	Blue
590 - 620	Orange	Green-blue
620 - 750	Red	blue-green

By using visible light , we can perform qualitative and quantitative analytical methods based on the absorption and emission of the visible light by the analyte . This analytical application can be extended to other regions of the electromagnetic spectrum such as x-ray , IR , microwave and the radio frequency ; but our analytical scope will be will be limited to UV-Visible region .

Application of electromagnetic waves in chemical analysis – as we already mentioned – is based on absorption of the radiant energy by the molecule raising its internal energy to higher level . This absorbed energy may affect the molecule in different ways such as rotational , vibrational , and electronic transitions . In order these processes to take place , specific wavelength with discrete energy is needed .

At room temperature , the molecule is in its lowest energy state called ground state with energy state E0 . If the molecule is exposed to a form of radiant energy , it will be affected according to the wavelength of that radiant energy and become in excited state . The amount of energy absorbed can be measured and related to the nature and the amount of the substance present in the sample ; this is the essence of the spectroscopic analysis .

4. Uv – visible spectrometry

In the following discussion , we will concentrate on colorimetric and spectrophotometric analysis and selected experiments based on these topics will be presented . In colorimetry , the concentration of the analyte is determined by utilizing the relative absorption of the white light by the analyte and result is compared with a sample of known concentration . Photoelectric colorimeters generally employ visible light of narrow range of wavelengths produced by passing white light through filters to transmit the specific wavelength in which the analyte absorbs most .

Spectrophotometric analysis use more extended range of the electromagnetic radiation that covers the whole of the ultraviolet region and near – infrared . In this region , specific wavelength of very small bandwidth is selected . There are two main designs of spectrophotometers ; double beam that contain two light paths , one for the sample and the other for the reference .

Components of the spectrophotometr.

Double been spectrophotometer

Single beam spectrophotometer

Single – beam spectrophotometer contains one light path in which the intensity of the light path is measured before and after the sample is introduced . Nearly all the spectroscopic instruments that employ UV/visible and IR regions are of a same design and consist of the following parts :

A. Source of radiant energy, the most common light source in spectrophotometric measurements is the Tungsten Lamp which is tungsten filament in a glass envelope with a wavelength range of 330 – 900 nm . Deuterium lamps are regularly used in the ultraviolet region with a range 200 – 450 nm ; in any case the source must provide sufficient light intensity to give measurable output .

B. Monochromator or filter, which is an optical device to select the required wavelengths and isolate the region of the spectrum for measurement . Monochromators use optical dispersion units like prism or gratings to separate the colors of the light and guide the selected wavelength to an exit slit .

Monochromator parts

C. Cuvettes, are containers of the sample , they are small tubes rectangular or square in shape made up of transparent material such as glass , plastic or special quartz . They are designed to hold samples for spectroscopic determinations . Quartz cuvettes convenient to be used in all uv/visible region while glass cuvettes are suitable for the visible region .

Plastic cuvettes glass cuvette quartz cuvette

D. Detector, is a device which transforms the light absorbed or emitted into proportional electric signal and finally into the read out unit which displays the result .

Quantitative measurement of the analyte concentration

We already described the effect of radiant energy on a matter in which fraction of the energy can be absorbed by the matter . The amount of the energy absorbed by an analyte can be quantitatively related to its concentration. There is mathematical relationship between the energy absorbed and the concentration of a single analyte or mixtures of two analytes . This relationship is known is known as Beer – Lambert's law and states that " the absorbance (A) of a light absorbed by a sample is directly proportional to the concentration(c) of the solution sample in use and to the length (b) of the path .

I0 = incident light I = transmitted light

A by introducing a constant, A = (5)

= molar extinction coefficient, or molar absorptivity.

In terms of intensity of light, A =

This is known as Beer – Lambert law

The length of the path ,b , is expressed in centimeters and the concentration, c , of the sample solution is expressed in moles per liter .The absorptivity depends on the nature of the absorbing material .

Equation (5) can be written as, A = KC , which explains direct proportionality between the absorbance and the concentration of the analyte .

The concentration of unknown analyte can be calculated using this above relationship. In almost all analytical instrumentations , series of known concentrations are prepared and their instrumental response is recorded and a curve of concentrations against the instrument response is constructed , see the fig. below.

This curve is called calibration or standard curve . The concentration of the analyte is directly calculated by extrapolating the obtained signal of the unknown .

Atomic Spectrometry

Atomic spectrometry is analytical method based on absorption or emission of the electromagnetic radiation by the atoms . when molecules absorb electromagnetic radiation , they exhibit variety of microscopic motions such as rotation , vibration or electronic transition depending on the wavelength of radiant energy .

In the case of atoms , only electronic transitions take place when radiant energy is absorbed . Electrons exist in different energy levels in an atom called orbitals ; they can move between orbitals by absorbing or emitting amount of energy equal to the difference between the orbitals . This quantized energy is in the form of photons .

In this section, we shall describe an analytical approach in which atoms are excited in different techniques and the resulting emission is measured (flame emissin) , or the atoms are exposed to particular radiant energy and the amount of absorbed energy is measured (absorption photometry) .

Emission spectroscopy

Emission spectroscopy is a method of great importance in analytical chemistry ; in this method the intensity of a light emitted from different sources like flame , plasma , arc of a particular wavelength is used to determine the amount of an element in a sample .

One of the important instruments used in this analysis is the spectrograph. In this instrument , high voltage is applied to vaporize the solid sample (soil, rock etc .) . The atomic vapor of each element become excited and the electrons will be raised to higher energy level ; but these excitations have short lifetime and / back to ground state emitting photons .

Each element may have several electronic transitions displaying different light emissions each representing discrete wavelength . The light emitted will be dispersed by a grating and since there may be several wavelengths, the suitable detector is a photographic film which can easily be developed.

The intensity of these emitted lights depends on time of exposure , the concentration of the element besides other factors .

Prism spectrograph parts

Spectrograph differs from the spectrometer in the sense that spectrograph separates light on the base of its frequency spectrum and record the signal on camera film ; while spectrometer displays the intensity of light based on its wavelength and display the reading as an electronic signal .

Flame photometry ,

Flame photometry is analytical system used to determine certain metals like sodium , potassium , lithium and calcium . It is kind of emission spectroscopy in which the source of excitation is a flame . In this analytical technique , the solution sample is introduced into the flame at constant rate ; the procedure is simple and cheap in comparison to other flame emission techniques like ICP (inductive coupled plasma) , which employ monochromators for analyzing the emitted light .

In flame photometry , the sample is introduced into the flame as a fine spray . After the evaporation of the solvent , the element disintegrates into free gaseous atoms in which some of them utilize the energy of the flame and get excited into higher energy state .These excited atoms return back to ground state by emitting photons of distinctive wavelength , guided by the lenses through the slit to a filter and then to the detector .

The main internal components are:

1. Atomizer: Is the part of the flame photometer that supplies homogenous solution into the flame at balanced rate .

2. Optical system: Consist of lens that focus and transmit light to the slit that passes the light to the filter .

3. Color filters: These filters isolate the wavelength to be measured from unwanted emissions .

4. Photo detector: It changes the intensity of the emitted radiation into proportional electric signal .

Atomic Absorption spectrometry

Atomic absorption spectrometry is analytical method for quantitative determination of elements in a solution using the absorption of optical radiation by free atoms in gaseous state , the method is closely related to the flame photometry .

The method employs the theoretical aspect of absorption spectroscopy to analyze sample solutions . In this procedure , analyte of known concentration is prepared to set up relationship between the observed absorbance and the concentration of the analyte on the base of Beer-Lambert law .

· Principles

In this method the sample solution is aspirated into a flame ; the atoms of element in the sample is converted to atomic vapor . These vaporized atoms are mostly in ground state so radiant energy is applied to excite these atoms to promote the electrons to higher energy orbitals . The source of the radiant energy is from an element of specific wavelength .

· Instrumentation

The main parts of ordinary atomic absorption spectrophotometry:

A. Source of radiant energy: Which is a hollow cathode lamp that emits sharp line of specific wavelength for each element . These sharp lines are passed through the flame to be absorbed by the atoms of the element and become excited into higher energy state .

Hollow cathode lamp

B. Atomizer The most common atomizer used nowadays in atomic absorption spectrophotometry are flame or electrothermal atomizers . Flame sources usually come from air acetylene and nitrous oxide – acetylene . Electrothermal atomization comes from graphite furnace which consists of graphite tube open at both ends and small hole at the center for holding the sample .

Fuel	Oxidant	Temperature (oc)
Natural gas	Air	1700 - 1900
H2	Air	200 – 2100
Acetylene (C2H2)	Air	2100 – 2300
Acetylene (C2H2)	Nitrous oxide (N2O)	2600 - 2900
Acetylene (C2H2)	Oxygen (O2)	3060 – 3150

C. Burners head

Burner heads are usually made of a metal that can resist any corrosion that take place due to strong acids and different gases being used in the analysis . There are diverse shapes and sizes employed in this analytical technique depending on the sensitivity and the nature of the fuel at use ; the head burner can be moved up and down and sideways to obtain the best sensitivity for a given set of conditions .

different types of burners

D. Importance of atomic absorption

This instrument plays very important role in analytical chemistry . It is used for determining the concentration of a particular element in a sample . It can be used for determination of over 70 elements in solution form or in solid form through electrothermal vaporization process.

It has wide application in the field of clinical chemistry determining metals in biological fluids and tissues . It also has significant application in pharmacology , pharmaceutics and toxicology .

Potentiometric titrations

We discussed in chapter (6) the formation of potential when two electrodes are immersed in a solution containing ions and how this potential is related to the concentration of these ionic species . In this section , we shall briefly explain how the electrode is applied to determine the concentration of an analyte . Any analytical procedure based on electrode potential measurements is known as potentiometry

Potentiometric titration is the titration in which the end point is detected by the change in electrode potential . In classical solution analysis , the end point of the titration is indicated by specific indicator usually characterized by color change .

In these methods there must be two electrodes - separated or combined together - they are referred as indicating and reference electrodes . The potential of the indicating electrode depends on the concentration of the ion to be measured while the reference electrode has known and stable potential . Various types of electrodes are used for different types of titrations , such as acid-base titration , redox titration , precipitation and complexometric titrations . The procedures of these practical topics has been discussed in wet chemistry section.

In acid-base titrations , the indicator electrode is the glass electrode which is ion-selective electrode specific for hydrogen ions. The glass electrode consists of silver - silver chloride electrode in a solution of 0.1M HCl contained in a special glass membrane . The glass membrane is selectively permeable to hydrogen ions , the potential developed in this process depends on the concentration of the hydrogen ions in the solution to be tested. The potential of the glass electrode is measured against reference electrode .

Glass electrode (indicated) calomel electrode (reference)

Reference electrode in pH titration is saturated calomel electrode (SCE) which is based on the reaction between elemental mercury and Hg(I)Cl.

The most important information in potentiometric titration is the location of the end point after the result data is obtained . In this process , no ordinary chemical indicator is needed . There are several methods applied to determine the end point of the potentiometric titration . The simplest method is the one formulated by plotting the potential (pH) as a function of the reagent volume . The inflection point is denoted by steep rising of the curve from which virtual estimation of the end point can be made.

different end point detections .

The second method to detect the end point is to plot the first derivative of the curve , against volume (v) of the titrant . The maximum of this curve corresponds to the inflection point .

· Selected experiments (N.B: In performing these experiments, consult with your instructor).

1. Spectrophotometry.

Experiment 1-1 , spectrophotometric determination of Iron(II)

We have seen in chapter 9 that one of the properties of the transition metals is the formation of co-ordination complex . Fe2+ complexes with 1,10 – phenanthroline forming deep red color . To reduce any traces of Fe3+ to F2+ and prevent further oxidation , hydroxylammonium chloride is added .

Colorless Red

Requirements:

A. 10 ppm Fe (dissolve 0.0702 g of ferrous ammonium sulfate into 1-liter volumetric flask , dissolve in sufficient water , add 2.5 ml conc. H2SO4 dilute to the mark with distilled water and mix thoroughly .

B. 1,10 – phenanthroline (100 mg/100 ml water .

C. Hydroxylammonium chloride solution (10 g/100 ml water .

D. Sodium acetate solution ( 10 g /100 ml water .

E. Spectrophotometer

Procedure

Prepare a calibration solution by pipetting 1 ml , 2 ml , 4 ml , 8 ml , 20 ml of the standard solution into five 100 ml -volumetric flasks . Take another two 100 ml-volumetric flasks , one for the blank containing 50 ml distilled water and other for the unknown sample . To each of these flasks add 1 ml of hydroxylammonium chloride , 5 ml of 1,10-phenanthroline and 8.0 ml of sodium acetate solution ( to adjust the pH) . Wait for few minutes to fully develop the color . Prepare the calibration curve and determine the unknown at 510 nm .

NB: consult your instructor for the operation of the spectrophotometer.

Experiment 1- 2 spectrophotometric determination of available phosphorus in soil

Requirements:

A. Ammonium paramolybdate [(NH4)6Mo7O24.4H2O] . Dissolve 12.0 g in 250 ml distilled water .

B. Potassium antimony tartrate ( KSbO.C4H4O6) . Dissolve 0.2908 g in 100 ml distilled water.

C. 5 N H2SO4 .

D. Reagent A : [ add reagent (a) and (b) to 1liter of 5N H2SO4 in 2-liter volumetric flask , mix well and dilute to the mark with distilled water .

E. Reagent B : [ dissolve 1.06 g of ascorbic acid in 200 ml of reagent A .

N.B: reagent B should be prepared as required since it does not keep more than 24 hrs.

F. Standard solution : Weigh accurately 0.4393 g of KH2PO4 , dissolve in 500 ml H2O and dilute to 1liter . take 20 ml of that solution and dilute to 1 liter , the concentration becomes 2 ppm .

Procedure

Weigh 5 g air – dried soil into conical flasks , add 100 ml distilled water . Shake for 15 minutes in a horizontal shaker . Filter in Whatman filter paper 42 . Pipette 10 ml of the filterate into 50 ml volumetric flask . Add 4 ml of reagent B and dilute to the mark . Prepare the calibration solution by pipetting 0 ml , 2ml , 4 ml , 6 ml , 8 ml into five 50 ml – volumetric flasks . Add 4 ml to each flask , leave for 15 minutes for the color to develop . determine the unknown at 750 or 882 nm . Calculate the amount of P in ppm .

Experiment 1 –3 Determination of total carbohydrate in a sample .

Requirements :

a) Phenol solution (5%) .

b) Conc. Sulfuric acid

c) Standard solution (glucose , 200 ppm) .

d) Sample solution (consult the instructor for preparation) .

Prepare six 100 ml- volumetric flasks , one for the blank , four for the calibration solution and one for the unknown sample . Transfer 0 ml , 5 ml , 10 ml , 20 ml and 40 ml into five 100-volumetric flasks , corresponding to 0 ppm , 10 ppm , 20 ppm , 40 ppm , 80 ppm . Add 2ml of the phenol solution to each flask followed by carefully adding 5 ml of the conc. Sulfuric acid . Allow the flasks to stand 10 minutes ; then mix well and let it stand for another 10 minutes . Take the absorbance at 600 nm . Calculate the concentration of the unknown from the calibration curve and determine the amount of carbohydrate in the sample .

2. Atomic spectrometry

Experiment 2-1:

determination of Na and K in certain fruits by flame photometer .

Requirements :

a) Flame photometer

b) Dried Fruits : Dates , orange and pepper.

c) Standard solution of Na and K ( 0ppm , 5ppm , 10ppm , 15ppm , 20ppm) .

d) Deionized water .

e) Conc. HCl & Conc. HNO3.

Procedure:

Collect a representative sample of each dry fruit separately , wash with deionized water and dry in an oven at 50oC .Powder finely each fruit sample with a blander ; take 2g accurately from each powdered sample into separate 100ml volumetric flask (well cleaned and dried) . Add 10ml of HCl/HNO3 solution (2:1) and few boiling chips . Digest the samples on a hotplate until you get clear solution in a fume chamber . Cool the samples and dilute with deionized water to the mark , leave them overnight to get clear supernatant solution . Take 10ml of the clear solution and dilute to 100ml . Make a blank solution by digesting a solution of HCl/HNO3/H2O (2:1:4) . Setup the flame photometer and create a calibration curve using the standard solutions .

Determine the Na and K content of each fruit from the calibration curve . Clean the sprayer with deionized water after each determination .

Experiment 2-2:

Determination of trace elements ( Fe , Cu , Mn , Zn) in a plant sample by atomic absorption spectrophotometer .

Requirements:

a) Grinded dry plant sample .

b) Atomic absorption spectrophotometer .

c) Perchloric acid (70%) .

d) Nitric acid conc.

e) Standard solution of the above metals (1000ppm) as follows :

Cu = 1.0 g of copper metal dissolved in 50 ml 5N HNO3 dilute to liter or 3.8 g of Cu(NO3)2.3H2O in 200 ml deionized water , dilute to 1liter .

Fe = 1.0 g of the metal dissolved in 20 ml of 5N HCl dilute to liter or 4.84 g FeCl3 . 6H2O , dilute to 1liter .

Mn = 1.0 g of the metal dissolved in 50 ml conc. HCl dilute to 1liter or 3.6 g MnCl2.4H2O in conc. HCl , dilute to 1liter .

Zn = 1.0 g of the metal dissolved in 30 ml of 5N HCl dilute to 1liter or 10 . 245 g of zinc oxide (ZnO) in 5 ml deionized water followed by 25 ml of 5N HCl dilute to 1liter .

You can prepare one standard solution that contains all the four metals . Take 20 ml of each standard solution into 1liter volumetric flask and dilute to the mark , this solution contains 20 ppm of each element . Prepare a calibration solution by taking 5 ml , 10 ml , 15ml , 20 , 25 ml into five 50-ml volumetric flasks and dilute to the mark with deionized water ; this dilution corresponds to 2ppm 4ppm , 6ppm , 8ppm and 10ppm respectively .

Procedure ,

Weigh out accurately 0.5 g of the prepared plant sample and transfer it to dry clean 100 ml – volumetric flask add 10 ml mixture of conc. Nitric acid / perchloric acid (2:1 respectively) add several boiling chips . Heat the mixture gently for the first 3minutes on hotplate in the fume chamber , then increase the heating power until clear solution is formed and dense fumes appear . Take the sample and let it cool for 15 minutes .

Add 40 ml deionized water shake well and dilute to the mark . Leave it stand still until all the particles settle and clear supernatant is formed (it may take more than five hours ) . Use this supernatant solution to determine the trace metals . Draw the standard curve from the solutions already prepared and find the amount of each element in the plant sample from the obtained standard curve .

3 – Potentiometry

Experiment 3 – 1 .

Acid – base titration

Requirements:

1. 0.1N HCl (8.4 ml of 37% HCl dilute to 1liter).

2. 0.1N NaOH ( dissolve 4.0 g in distilled water and dilute to 1liter) .

3. Buffer solution pH 7.0 and pH 4.0

4. 50 ml burette .

5. pH meter , with glass and camel electrodes .

6. magnetic stirrer

Procedure:

a) Transfer 25 ml of the HCl into 400-ml beaker , add enough distilled water to avoid touching of electrode tips on the bed of the beaker ; put in the magnetic stirrer in the solution and place the beaker on a hotplate with magnetic stirring system .

b) Fill the burette with the sodium hydroxide solution up to top of the mark . Open the burette to remove the air bubbles , clamp the burette properly and adjust the volume .

c) Set up the pH meter and calibrate with buffers 7.0 & 4.0 (consult your instructor to help you ) , electrodes must be conditioned to be ready for the measurement .

d) Clean the tips of the electrodes with distilled water and immerse them in solution , stir the solution gently , stop the stirrer for few seconds and take the reading in pH or millivolt scale .

e) Add 2 ml of the NaOH solution from the burette , stir for 30 sec. stop , wait for another few seconds and take reading . Continue adding the NaOH solution in 1 ml portion and record the readings .

f) Plot a graph of pH values as co-ordinates and the volume added as the abscissa . The equivalence point corresponds to the steepest point of the curve .

There is another way of locating the end point more precisely by the first derivative (/V of the data .

Experiment 3 – 2:

Determination of iron (II) by potentiometric titration .

Requirements:

a) Platinum electrode (indicator electrode ) .

b) Calomel electrode ( reference electrode ) .

c) 0.1N potassium dichromate solution (dissolve 4.9 g of dried K2Cr2O7 in 200 ml distilled water and dilute to 1liter ) .

d) 0.1 N ammonium iron (II) sulphate solution (dissolve 39.2 g in 250ml distilled water , add 3.5 ml conc. H2SO4 shake well and dilute to 1liter).

e) Prepared pH-meter .

Place 25 ml of the ammonium iron (II) sulfate solution into 400 – ml beaker add enough water to raise the level of the solution so that electrode tips will not be damaged . Fill the burette with the dichromate solution to highest level possible , open the burette to remove the bubbles if any and fix the volume to specific number . Immerse the two electrodes into the solution and set the pH-meter in mV measurement . Proceed the titration as in the case of experiment 3 – 1. Construct potential – volume curve and calculate the concentration of the ammonium iron(II) sulphate and compare the result with the actual weight employed .