Analytical Techniques

Spectroscopy
Dr. Muhamma Mansha

Spectroscopy is the use of the absorption, emission, or scattering of electromagnetic

radiation by matter to qualitatively or quantitatively analyze or study the matter or to study physical processes. The matter can be atoms, molecules, atomic or molecular ions, or solids. The interaction of radiation with matter can cause redirection of the radiation and/or transitions between the energy levels of the atoms or molecules.

Beer-Lambert Law: Beer-Lambert law or simply Beers law is the heart

of spectrometry. Consider the absorption of monochromatic radiation as in Figure. Incident radiation of radiant power P0 passes through a solution of an absorbing species a concentration c and path length b, and the transmitted radiation has radiant power P.

Transmittance (T), is defined as:

A new term absorbance is defined as :

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The figure shows the case of absorption of light through a sample cell and includes other processes that decreases the transmittance such as surface reflectance and scattering. For a monochromatic light absorbance is proportional to path length b, and concentration of absorbing species c.

Instrumentation
General aspects of UV-Vis spectrophotometers are given in the introductory document on UV-Vis spectroscopy. Single-beam spectrophotometers can utilize a fixed wavelength light source or a continuous source. The simplest instruments use a single-wavelength light source, such as a lightemitting diode (LED), a sample container, and a photodiode detector. Instruments with a continuous source have a dispersing element and aperture or slit to select a single wavelength before the light passes through the sample cell. (see schematic below).

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In either type of single-beam instrument, the instrument is calibrated with a reference cell containing only solvent to determine the Po value necessary for an absorbance measurement. Schematic of a wavelength-selectable, single-beam UV-Vis spectrophotometer

Ultraviolet/Visible (UV-Vis) Spectroscopy of Potassium Permanganate Potassium permanganate is used to kill bacteria in reclaimed water Use UV-Vis to ensure that the concentration of Potassium Permanganate is at acceptable limit Potassium permanganate (KMnO4) in solution is purple / violet color meaning maximum absorption should be at 500 550 nm, which is confirmed from the absorption spectrum of potassium permanganate at two different concentrations shown in the figure.

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Absorption Spectrum of Light4

Wavelength of maximum absorption (nm)
380 420 420 - 440 440 470 470 500 500 520 520 550 550 580 580 620 620 680 680 - 780

Color Absorbed

Color Observed (complementry)

Green-Yellow Yellow Orange Red Purple Violet Violet-Blue Blue Blue-Green Green

Violet Violet-Blue Blue Blue-Green Green Yellow-Green Yellow Orange Red Purple

Five known concentrations of KMnO4: 1ppm, 20ppm, 40ppm, 60ppm, 80ppm were prepared and absorbance readings were taken at 520 nm.
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The absorbance readings are shown in the table.

UV-Vis Absorbance Readings for Potassium Permanganate at 520 nm

Average A (after Standard 3 runs) Deviation (A) 0.015 0.004 0.256 0.520 0.753 1.046 0.462 0.001 0.004 0.002 0.001 0.001

1 ppm 20 ppm 40 ppm 60 ppm 80 ppm Unknown #4

The calibration curve is in accordance with Beers law from which the concentration of the unknown solution is determined.

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Atomic Absorption Spectrometry (AAS)

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Potentiometry

Introduction
Potentiometry is the field of electroanalytical chemistry in which potential is measured under the conditions of no current flow. The measured potential may then be used to determine the analytical quantity of interest, generally the concentration of some component of the analyte solution. The potential that develops in the electrochemical cell is the result of the free energy change that would occur if the chemical phenomena were to proceed until the equilibrium condition has been satisfied. This concept is typically introduced in quantitative analysis courses in relation to electrochemical cells that contain an anode and a cathode. For these electrochemical cells, the potential difference between the cathode electrode potential and the anode electrode potential is the potential of the electrochemical cell.

If the reaction is conducted under standard state conditions, this equation allows the calculation of the standard cell potential. When the reaction conditions are not standard state, however, one must utilize the Nernst equation to determine the cell potential. In the following equations Ox stands for oxidized species and Red for its reduced form. For example Cu2+ is oxidized form and Cu is reduced form.

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pH Meters
pH meter measures the pH of a solution using an ion-selective electrode (ISE) that responds to the H+ concentration of the solution. The pH electrode produces a voltage that is a function of the concentration of the H+ concentration, and making measurements with a pH meter is therefore a form of potentiometry.

The pH electrode is attached to electronics which convert the voltage to a pH reading and displays it on a meter. The difference in concentration of hydrogen ions on both sides of membrane causes the potential to develop. There are no half reactions and the Nernst equation can be written as: E = Eo 0.0592 log {[H+]internal / [H+]external } Since the internal [H+] is constant, it can be lumped into Eo E = E* + 0.0592 log [H+]external E = E* 0.0592 p[H+]

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Instrumentation
A pH meter consists of a H+-selective membrane, an internal reference electrode, an external reference electrode, and a meter with control electronics and display. Commercial pH electrodes usually combine both electrodes into one unit that is then attached to the pH meter. The potential scale is calibrated in pH units with each pH unit equal to 59.19 mV at 25 oC.The pH meter is adjusted with the calibration knob with the help of a standard buffer.

Picture of a pH meter

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Separation Techniques:
Chromatography is a separations method that relies on differences in partitioning behavior
between a flowing mobile phase and a stationary phase to separate the the components in a mixture. A column (or other support like paper) holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster.

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As the components elute from the column they can be quantified by a detector and/or collected for further analysis. An analytical instrument can be combined with a separation method for on-line analysis. Examples of such "hyphenated techniques" include gas and liquid chromatography with mass spectrometry (GC-MS and LC-MS). GC: It is applied to volatile organic compounds. The mobile phase is a gas and the stationary phase is usually a liquid on a solid support or sometimes a solid adsorbent.

Distribution of analytes between phases http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm The distribution of analytes between phases can often be described quite simply. An analyte is in equilibrium between the two phases; Amobile Astationary The equilibrium constant, K, is termed the partition coefficient; defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase. Page 11 of 15

K = [A]s/[A]M = cS /cM Where the bracketed terms are activities of solute A in the two phases. We shall substitute c to represent molar analytical concentration. Ideally the cs is directly proportional to cM over a wide concentration range of solute.

The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time (tR ). Each analyte in a sample will have a different retention time. The time taken for the mobile phase to pass through the column is called tM (In the diagram 19.3 it is shown as unretained peak.

A term called the retention factor, k', (also known as capacity factor in old literature) is often used to describe the migration rate of an analyte through a column. The retention factor for analyte A is defined as;

k'A = (t R - tM )/ tM
t R and tM are easily obtained from a chromatogram. When an analytes retention factor is less than one (note that it is never negative) elution is so fast that accurate determination of the retention time is very difficult. High retention factors (greater than 20) mean that elution takes a very long time. Ideally, the retention factor for an analyte is between one and five. We define a quantity called the selectivity factor, , which describes the separation of two species (A and B) on the column;

= k 'B / k 'A
When calculating the selectivity factor, species A elutes faster than species B. The selectivity factor is always greater than unity. The selectivity factor for two analytes in a column provides a measure of how well the column will separate the two.
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`The Theoretical Plate Model of Chromatography The plate model supposes that the chromatographic column is contains a large number of separate layers, called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next.

It is important to remember that the plates do not really exist; they are imaginary. The concept helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better). If the length of the column is L, then the HETP is HETP = L / N The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution;

where w1/2 is the peak width at half-height. However when peak width is measured at base line, the formula becomes:

As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture.

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Liquid Chromatography (LC): It is used to separate analytes in solution including metal ions. The mobile phase is a solvent and the stationary phase is a liquid on a solid support, a solid, or an ion-exchange resin.

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