INSTRUMENTAL ANALYSIS (I) INTRODUCTION Classification of Analytical Methods Qualitative instrumental analysis is that measured property indicates presence of analyte in matrix Quantitative instrumental analysis is that magnitude of measured property is proportional to concentration of analyte in matrix Species of interest: All constituents including analyte and Matrix-analyte (concomitants) Often need pretreatment - chemical extraction, distillation, separation, precipitation (A) Classical: Qualitative - identification by color, indicators, boiling points, odors Quantitative - mass or volume (e.g. gravimetric, volumetric) (B) Instrumental: Qualitative - chromatography, electrophoresis and identification by measuring physical property (e.g. spectroscopy, electrode potential) Quantitative - measuring property and determining relationship to concentration (e.g. spectrophotometry, mass spectrometry) Often, same instrumental method used for qualitative and quantitative analysis Types of Instrumental Methods Property Example MethodRadiation emission Emission spectroscopy - fluorescence,

phosphorescence, luminescence Radiation absorption Absorption spectroscopy - spectrophotometry, photometry,

nuclear magnetic resonance, electron spin resonance

Radiation scattering Turbidity, Raman Radiation refraction Refractometry, interferometry Radiation diffraction X-ray, electron Radiation rotation Polarimetry, circular dichroism Electrical potential Potentiometry Electrical charge Coulometry Electrical current Voltammetry - amperometry, polarography Electrical resistance Conductometry Mass Gravimetry Mass-to-charge ratio Mass spectrometry Thermal Thermal gravimetry, calorimetry Rate of reaction Stopped flow, flow injection analysis Radioactivity Activation, isotope dilution (Often combined with chromatographic or electrophoretic methods)

Example: Spectrophotometry Instrument: spectrophotometer Stimulus: monochromatic light energy Analytical response: light absorption Transducer: photocell Data: electrical current Data processor: current meter Readout: meter scale Data Domains: way of encoding analytical response in electrical or

non-electrical signals. Interdomain conversions transform information from one domain to another.

Detector (general): device that indicates change in environment Transducer (specific): device that converts non-electrical to electrical data Sensor (specific): device that converts chemical to electrical data Non-Electrical Domains Electrical Domains

Physical (light intensity, color) Chemical (pH) Scale Position (length) Number (objects)

Current Voltage Charge Frequency Pulse width Phase Count Serial Parallel

Time - vary with time (frequency, phase, pulse width) Analog - continuously variable magnitude (current, voltage, charge) Digital - discrete values (count, serial, parallel, number*)

Digital Binary Data Advantages (1) easy to store (2) not susceptible to noise How to choose an analytical method? How good is measurement? How reproducible? - Precision How close to true value? - Accuracy/Bias How small a difference can be measured? - Sensitivity What range of amounts? - Dynamic Range How much interference? - Selectivity Precision - Indeterminate or random errors

Accuracy - Determinate errors (operator, method, instrumental)

Sensitivity

(larger slope of calibration curve m, more sensitive measurement) Detection Limit Signal must be bigger than random noise of blank

From statistics k=3 or more (at 95% confidence level)

Dynamic Range At detection limit we can say confidently analyte is present but cannot perform reliable quantitation Level of quantitation (LOQ): k=10 Limit of linearity (LOL): when signal is no longer proportional to concentration

Selectivity: No analytical method is completely free from interference by concomitants. Best method is more sensitive to analyte than interfering species (interferent).

k's vary between 0 (no selectivity) and large number (very selective). Calibration methods Basis of quantitative analysis is magnitude of measured property is proportional to concentration of analyte

Calibration curves (working or analytical curves)

Calibration expression is Absorbance = slope [Analyte (ppm)] + intercept

SPECTROCHEMICAL TECHNIQUES INTRODUCTION Methods of analysis based on the interaction of LIGHT with matter. LIGHT is an Electromagnetic (EM) radiation:

Speed of light, c = 2.99782 x 108 m / s .Frequency is always fixed but velocity can vary!

H = Planck's Constant = 6.626x10-34 J·s

3. Atoms, ions and molecules exist in certain energy states only

E0 = ground state E1, E2 , E3 … = excited states

Excitation can be electronic, vibrational or rotational Energy levels for atoms, ions or molecules different.

2. When an atom, ion or molecule changes energy state, it absorbs or emits energy equal to the energy difference

ΔE = E1 – E0 3. The wavelength or frequency of radiation absorbed or emitted during a transition is proportional to ΔE

EMISSION SPECTRA Have you noticed the bright yellow light emitted when crystals of NaCl fall in the flame in your cooker at home? This is emission

Plot of emission intensity vs. ν or λ called emission spectrum Atom: line emission spectra Inner shell (core) electrons (1s¬2p) – x-rays photons Outer shell (valence) electrons (3d¬4p) – UV/vis photons Molecule: vibrational and rotational transitions - band emission spectra

Continuum Spectra: A piece of iron in the fire: first become red then white, Why? Very broad band spectra in Emission from solids produced by blackbody radiation – thermal excitation and relaxation of many vibrational (and rotational) levels. Blackbody Spectrum Absorption Spectra Plot of Absorbance vs. ν or λ called absorption spectrum just as in emission spectra an atom, ion or molecule can only absorb radiation if energy matches separation between two energy states

Atoms: No vibrational or rotational energy levels - sharp line spectra with few features

For example: Na 3s → 3p 589.0, 589.6 nm (yellow) Na 3s → 5p 285.0, 285.1 nm (UV)

Visible enough energy for valence (bonding) excitations UV and x-ray enough energy for core (inner) excitations Molecules: Electronic, vibrational and rotational energy levels - broad band spectra with many features ΔE = ΔEelectronic + ΔEvibrational + ΔErotational

For each electronic state - many vibrational states For each vibrational state - many rotational states

→ many features Absorption spectra affected by

(1) number of atoms in molecule more features

(2) solvent molecules blurred features

Effect of Chemical State Emission

produces emission at same wavelength as absorption (common for atoms)

Excitation methods:

EM radiation

SPECTROSCOPY AND INSTRUMENTATION (IR, visible and UV)

Examples:

T=1.00 (100 %T), A=0.00 T=0.10 (10 %T), A=1.00 T=0.001 (0.1 %T), A=3.00

Determination of concentration from Absorbance measurements; Absorbance is directly proportional to the concentration, c, and the path length, b, of the absorbing species; BEER-LAMBERT'S LAW:

A ∝c and A ∝b so A ∝b ⋅c A = a ⋅ b⋅ c

proportionality constant, a = absorptivity - units (L/g·cm) If units of concentration are M (mol/L) then use molar absorptivity ε

Absorbance is additive

in a mixture of 2 components

So what is the Beer–Lambert law used to calculate? The molar absorption coefficient, ε and λ max which is the wavelength at which maximum absorption occurs. PHOTOMETRIC ERROR At low absorbance values, I (incident) ≈ I (transmitted) Thus, error is relatively large. At high absorbance values,

I (transmitted) too small not measured accurately.

Relative error in A vs %T of KMnO4 solutions at 523 nm where ε = 2400. Lowest error needs %T = 38,6% = A value ≈ 0.43 A. Working is possible within -/+ 15% which is absorbance 0.2 – 0.8 as best measurement range. INSTRUMENTS:

(1) Radiation Sources: Tungsten Lamp == Visible; Deuterium lamp == UV; Globar Silicon Carbide == IR.

(2) Sample Containers and Optics:

• Cuvettes Made of Glass 400-3000 nm (vis-near IR)

Silica/quartz 200-3000 nm (UV-near IR) NaCl 200-15,000 nm (UV-far IR)

(3) wavelength selector Filters: Absorption filter - colored glass or dye between two glass plates; Interference filter – two thin sheets of metal sandwiched between glass plates, separated by transparent material. Interference for transmitted wave through 1st layer and reflected from 2nd layer Monochromators: consists of Entrance slit, Collimating lens or mirror; Dispersion element (prism or grating), Focusing lens or mirror and Exit slit Typical Prism Monochromator

(4) radiation detector (A) Photovoltaic cells (B) Phototube: radiation falls on a sensitive Plate which produces electron.

(C) Photomultiplier tube (PMT) (D) Thermal detectors – sensitive to IR (λ>750 nm)

(5) Signal processor and readout Double Beam systems:

a. Light from the source is segmented by a rotating segmented mirror which allows radiation through to the sample and reflects it through the reference cell alternately.

b. The two beams are recombined by a rotating mirror in phase with the first rotating mirror.

c. The combined beam is allowed to fall on the detector in which the reference signal is electronically subtracted from the sample signal before it is sent to the recording system.

Wavelengths of colors of visible light

Applications of UV-Vis Spectrometry: UV/VIS IS USED FOR QUALITATIVE AND QUANTITATIVE DETERMINATIONS Organic molecules π (bonding) molecular orbital as in

n (non-bonding) atomic orbital as in

Absorption of UV light energy cause transition of π (bonding electrons) and n (non-bonding) to π* (Antibonding) molecular orbital

Inorganic Ions Most transition metal ions are colored (absorb in UV-vis) due to d→ d electronic transitions. Inorganic ions can be quantitatively determined by spectrophotometry by reaction with some ligands to produce colored complexes which have clear absorption in the visible region.

Remember: • Solution absorbs red appears blue-green • Solution absorbs blue-green appears red Ligand Field Strengths: Chromium can give different colors and hence absorb at different wavelengths when reacts with different ligands.

Vis UV I-<Br-<Cl-<F-<OH-<C2O4

2-~H2O<SCN-<NH3<en<NO2-<CN "Spectrochemical Series" Chemical kinetics of a chemical reaction, Reactant or product absorbs radiation at different wavelength. Measuring A with time. Follow the formation of the product or the removal of the reactant from solution.

Absorption of light by biological molecules: Chlorophyll a solutions absorb blue and red light and are green in color. DNA solutions absorb light in the ultraviolet and are colorless. Oxyhemoglobin solutions absorb blue light and are red in color. Solvent Effects: Solvent effects mean UV-Vis not reliable for qualitative but excellent for quantitative analysis. For good analysis the sample must have these properties: 1. Stability in solution; 2. Adherence to Beer's law, 3. Large molar absorptivities, 4. Sufficient separation of the desired analyte absorbance wavelength from interfering substances. If Not, the substance is usually converted into a new species suitable for quantitative spectrophotometry. SAMPLE + CHROMOGENIC REAGENT → UV-VIS ABSORBING PRODUCT

Example: In the determination of iron we convert it into iron(III)-thiocyanate complex, distinguished red color. We follow the procedure:

1. Pipet 5 mL of the stock Fe+3 solution (~0.001 M) and 3 mL of saturated NH4SCN into a clean 100 mL volumetric flask and dilute to volume.

2. Place some of the solution in the cuvett of the UV/Vis; 3. Scan the solution with the UV-VIS (Change the wavelength at certain

speed). 4. Record the wavelength of maximum absorbance. 5. Measure the absorbance, 6. Make 4 - 5 standard concentration solutions of Fe(III) (1.0 X 10-3 M to

1.0 X 10-5 M and measure absorbance at the wavelength of maximum absorbance.

7. Plot absorbance versus concentration.

8. Treat unknown sample solutions in the same way (Thiocyanate); 9. Measure the absorbance of unknown sample solutions. 10. Refer these absorbance values to their related concentrations in the

calibration graph. ATOMIC ABSORPTION SPECTROMETRY • AAS Atomic absorption spectrometry (AAS) is an analytical technique that measures the concentrations of metals. It is so sensitive that it can measure down to parts per billion (μg/L) in a sample. The technique makes use of the wavelengths of light specifically absorbed by an element. The method is based on the absorption of radiation by free atoms. Atomic absorption spectrometry has many uses: Clinical analysis. Analysing metals in biological fluids such as blood and urine. Environmental analysis. Monitoring our environment – eg finding out the levels of various elements in rivers, seawater, drinking water, air, petrol and drinks such as wine, beer and fruit drinks.

Pharmaceuticals. In some pharmaceutical manufacturing processes, minute quantities of a catalyst used in the process (usually a metal) are sometimes present in the final product. By using AAS the amount of catalyst present can be determined. Industry. Many raw materials are examined and AAS is widely used to check that the major elements are present and that toxic impurities are lower than specified – eg in concrete, where calcium is a major constituent, the lead level should be low because it is toxic. Thus, the most important step is to produce free atoms of metals (M) from sample solution (or Solid) ATOMIZATION: Flames premix burner; sample (1–2 mL) + oxidant + fuel :

air + acetylene; 2400–2700 K acetylene + nitrous oxide; 2900–3100 K, refractory elements

Nebulization; aerosol (narrow size distribution and smallest sizes are desirable) rich flame; excess C tends to reduce MO and MOH into M lean flame; excess oxidant, hotter need careful optimization Flame aspiration Typical burner and spray chamber. Aflame of Acetylene/air (giving a temperature of 2200 –2400 °C) or Acetylene /dinitrogen oxide (2600 – 2800 °C) are often used. A flexible capillary tube connects the solution to the nebuliser. At the tip of the capillary, the solution is ‘nebulised’ – ie broken into small drops. The larger drops fall out and drain off while smaller ones vaporise in the flame. Only ca 1% of the sample is nebulised.

• simplest atomization • laminar flow burner – • best for reproducibility (precision) (<1%) • low sensitivity, short time in beam

Electrothermal Atomizers: For improved sensitivity atomization is performed by a graphite furnace in which a drop (1 – 100 μL) of the sample solution is placed. The tube is heated electrically by passing a current through it in a pre-programmed series of steps: 30 – 40 sec at 150 °C (Drying) 30 sec at 600 °C (Ashing and charring of organic material and convert sample into oxides at fast heating rate 5–10 sec at 2000 – 2500 °C (atomization) To produce free atoms Finally heating the tube to a still higher temperature – ca 2700 °C – cleans it ready for the next sample. During this heating cycle the graphite tube is flushed with argon gas to prevent the tube burning away. Inelectrothermal atomisation almost 100% of the sample is atomised. This makes the technique much more sensitive than flame AAS. • Light source: Hollow Cathode Lamp, Each Element has specific Lamp.

• 300 V applied between anode (+) and metal (Na, Fe, Mn, ) cathode (-) • Ar ions bombard cathode and sputter cathode atoms • Fraction of sputtered atoms excited, then fluoresce

INSTRUMENTAL SET UP

Hydride generator

Some elements form volatile hydrides LIKE As, Se, Te, and Sn. They are useful to improve sensitivity of AAS determination of these elements. The sample solution reacted with NaBH4 in acid medium. As3+ + NaBH4 → AsH3 (Arsenic trihydride, Arsine) The liberated hydride is transported to an atomizer by an inert carrier gas or by other means. The advantage of this technique over flame AAS is the separation and enrichment of the element to be determined and the significantly lower detection limit resulting from greater efficiency of sample introduction. This method may have more interference effects than the flame AAS system.

Mercury is converted first into Hg (II) and then allowed to react with a reducing agent such as SnCl2 to produce elemental Hg. Air or nitrogen is allowed to pass through the solution to carry the hg produced into the absorption cell (glass tube with quartz windows placed in the radiation path and AA is measured. TYPES OF INTERFERENCE Spectral interference; overlapping signal from absorption or emission of unwanted species (molecules or radicals) Correction: choose another wavelength or use D2 lamp for flame signals Ionization: Higher atomization temperature cause ionization of analytes like Na or K; Na → Na+ + e Correction: add ionization suppresser, easily ionizable atoms like CsCl (to increase electrons in the flame to reverse the direction of ionization reaction of the analyte ionization, Le Châtelier’s principle. Atomic emission spectroscopy is very sensitive to temperature fluctuations. Atomic absorption spectroscopy is less sensitive to temperature fluctuations. Chemical interference; Some metals cannot be determined in the presence of other species due to the formation of thermally stable compounds and can not be atomized. Example: Ca can not be determined in the presence of phosphate, because of the formation of refractory calcium phosphate and decreases the atomization of Ca and the amount of atoms produced are very low. Correction: A releasing agents is added like La3+, salt solution LaCl3 to allow chemical exchange Ca3(PO4)2 + 2LaCl3 → 3CaCl2 + 2LaPO4 CaCl2 is easily atomized

EDTA may also be used for other metals. Rich flame may also be used to give high temperature able to atomize refractory materials. Matrix modification With electrothermal atomization, chemical modifiers can be added which react with an interfering substance in the sample to make it more volatile than the analyte compound. This volatile component vaporizes at a relatively low temperature and is removed during the low and medium temperature stages of electrothermal atomization. Example: For the determination of heavy metals in tobacco leaves, 0.5 – 1.0 g of the tobacco leaves are digested with a mixture of 1 : 10 HClO4 : HNO3 until complete dissolution. The resulted solutions are diluted into 100 mL with distilled water, in volumetric flasks. The instrument is calibrated by analyzing standard solutions of the metals: Na, K, Ca, Mg, Cu, Mn, Fe, Ni, Co and Zn. The standard solutions are prepared by serial dilution from a 1000 ppm (mg/mL). A calibration graph is prepared by aspiration of the standard solutions (1, 2, 4, 10 and 20 ppm) and plotting the absorbance versus concentration. The sample solutions are thensamples When the concentration is very low, preconcentration procedures are applied like solvent extraction, evaporation. When the matrix is complex the technique of standard addition is used. Double beam spectrometers are successfully used in AAS. SAMPLE PREPARATION Sample preparation is often simple, and the chemical form of the element is usually unimportant. This is because atomisation converts the sample into free atoms irrespective of its initial state. The sample is weighed and made into a solution by suitable dilution. Elements in biological fluids such as urine and blood are often measured simply after a dilution of the original sample CALIBRATION Standard solutions are measured first Aspirated and their AA signals are measured The unknown sample solution is then Analysed. The value of the unknown is used To determine the concentration of the metal in the unknown solution.

STANDARD ADDITION In some cases, the composition of the sample solution is very complicated and the expected interferences are high. Here a known amounts of the standard solution are added to the sample solution. The AA is measured for the untreated sample solution and those treated with the standard added to them. A calibration graph of the type shown in the figure. The concentration of the sample is represented by the negative intercept on the concentration axis.

INFRARED SPECTROMETRY ~750-3000 nm A spectrophotometric method for the determination of composition and structure of compounds depending on the absorption of radiation in the INFRARED region of wavelengths, 2 – 50 μm (μm = 10-6 m). It is preferable to express the region by frequency units: Wave number = Number of waves in one cm; 1 cm-1 = 10000 / wavelength.

Energy of IR cause vibrational or rotational excitation. Molecule must have change in dipole moment due to vibration or rotation to absorb IR radiation. Molecules with permanent dipole moments (μ) are IR active

HCl, H2O, NO Atoms, O2, H2, Cl2

IR active IR inactive

Types of Molecular Vibrations:

Stretch: change in bond length symmetric asymmetric Bend: change in bond angle: scissoring, wagging; rocking, twisting/torsion

Example in ν(C-O) methanol 1034 cm-1

ethanol 1053 cm-1 butanol 1105 cm-1

INSTRUMENTATION: Dispersive Grating IR Instruments: Similar to UV-Vis spectrophotometer, BUT sample after source and before monochromator in IR (sample after monochromator in UV-Vis - less incident light) Double beam is used and useful to eliminate atmospheric gas interference. RADIATION SOURCES

Nernst Glower heated rare earth oxide rod (~1500 K) 1-10 μm

DETECTORS Photoconducting PbS, HgCdTe light sensitive, resistance thermometer at 77 K fast and sensitive

Now Fourier Transform (FTIR) Instruments The main component in the FTIR spectrometer is an interferometer. This device splits and recombines a beam of light such that the recombined beam produces a wavelength-dependent interference pattern or an interferogram. The Michelson interferometer is most commonly used. Computer needed to turn complex signal into spectrum. They have two advantages:

(1) Few optics, no slits == high intensity (2) Simultaneously measure all spectrum at once saves time

APPLICATIONS IR (especially FTIR) very widely used for qualitative analysis of gases, liquids and solids IR mostly used for rapid qualitative but not quantitative analysis Sample preparation Liquids are placed in liquid cell of short pathlength (0.015-1 mm). Solids are dissolved in a solvent. Solids (2 - 4 mg) is grinded and mixed with KBr to make semi-transparent pellet; or: grind and mix with Nujol (hydrocarbon oil) to form mull. One drop is placed between NaCl plates. water (attacks windows) Qualitative Analysis: Step One Identify functional groups (group frequency region) Step Two Compare with standard spectra with these groups. (fingerprint region)

Group Frequencies: • Approximately calculated from masses and spring constants • Variations due to coupling • Compared to correlation charts/databases

CHROMATOGRAPHIC SEPARATION Introduction: 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 for TLC, see below) 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. The sample is separated into zones or bands

Elution Chromatography: flushing of sample through column by continual mobile phase (eluent) addition migration rate µ fraction time spent in mobile phase Planar chromatography - flat stationary phase, mobile phase moves through capillary action or gravity Column chromatography - tube of stationary phase, mobile phase moves by pressure or gravity

Gas Chromatography A method for the separation of components (mainly organic compounds) of a homogeneous mixture and to determine the composition of the mixture. It is based on: - Injection of sample mixture into a column packed with certain materials

(stationary phase); - Passing a fluid/gas (the mobile phase) through the column. - Components of the mixture held strongly or weakly to the stationary phase. - Mobile phase pass to other end of the column taking away the weakly bound

component first to the outlet of the column. Thus, the components will be separated according to the boiling point, molecular weight, polarity, ability to for hydrogen bonding with the stationary phase. The components will appear et the end of the column at different times, tR , the retention time.

Two major types • Gas-solid chromatography -- (stationary phase: solid) • Gas-liquid chromatography -- (stationary phase liquid that is fixed or immobilized on a certain support material) Retention Volume, VR: The volume of the carrier gas which succeed to separate the component.

F = average volumetric flow rate (mL/min) F can be estimated by measuring flow rate exiting the column using soap bubble meter (some gases dissolving in soap solution) - often scales with vapor pressure (constant polarity analytes) GC Instrumentation Carrier gas: He (common), N2, H2 F=25-150 mL/min packed column F=1-25 mL/min open tubular column Column: 2-50 m coiled stainless steel/glass/Teflon Oven: 0-400 °C ~ average boiling point of sample accurate to <1 °C Detectors: FID, TCD, ECD, (MS)

Sample injection: Sample in liquid form is injected using microsyringe. The injector must be kept at some high temperature to change into vapour phase for easy analysis. - direct injection into heated port (>Toven) (i) 1-20 μL packed column (ii) 10-3 μL capillary column

Carrier gas is allowed to enter the system after the injection port. This will lead the vapours of the sample to move through the column. To make sure that the components reached the end of the column (separated), a detector system must be attached to the end of the column. GC Detectors: for reasonable GC analysis the detector need to be:

• Sensitive (10-8-10-15 g solute/s) • Operate at high T (0-400 °C) • Stable and reproducible • Linear response

Desire • Wide dynamic range • Fast response • Simple (reliable) • Nondestructive • Uniform response to all analytes

Flame Ionization Detector (FID): The separated components are allowed to enter a flame (Hydrogen/air) to be burned and changed into radicals which will be ionized by the flame. The ions will be detected by electrical conduction to give signal that a component is separated. The FID is: • Sensitive (10-13 g/s) • Wide dynamic range (107) • Weakly sensitive to carbonyl, amine, alcohol, amine groups • Not sensitive to non-combustibles - H2O, CO2, SO2, NOx • Destructive Thermal Conductivity Detector (TCD) The conduction of heat from certain filament by carrier gas (mobile phase) He N2 or H2, will be lowered when the organics reached the detector because the thermal conductivity of them is lower than that of the carrier gas. Thus, the organics will cause Temperature rise in filament. The TCD is characterized by:

• Wide dynamic range (105) • Nondestructive • Insensitive (10-8 g/s) - non-uniform

Column Stationary Phases: Packed

• liquid coated silica particles (<100-300 μm diameter) in glass tube • best for large scale but slow and inefficient.

Immobilized Liquid Stationary Phases: • low volatility and thermally stable • chemically inert (reversible interactions with solvent) • chemically attached to support (prevent "bleeding")

Many based on polysiloxanes or polyethylene glycol (PEG):

Some common Stationary phases for gas liquid chromatography

Stationary phases usually bonded by covalent linking to support and/or cross-linked by polymerization reactions after bonding to join individual stationary phase molecules non-polar analytes retained preferentially on Non-polar stationary phases polar analytes retained preferentially on Polar stationary phases Capillary/Open Tubular • wall-coated (WCOT) <1 μm thick liquid coating on inside of silica tube • support-coated (SCOT) 30 μm thick coating of liquid coated support on inside of silica tube • best for speed and efficiency but only small samples • Film thickness (0.1-5 μm) affects retention and resolution - thicker films for volatile analytes, poorer resolutions • Chiral phases being developed for enantiomer separation (pharmaceuticals) Temperature Programming: When a mixture of various boiling points (BP) components is to be analysed, at low column temperature the High BP will elute after very long time because the vapor pressure analyte increases as column temperature is raised. Elution will be faster. Meanwhile, low BP components will elute rapidly at high column temperature without good resolution (Separation is bad).

To solve this problem we must raise column temperature during separation – temperature programming - separates species with wide range of polarities or vapor pressures

Liquid Chromatography A method for the separation of components (solids or high BP, high molecular weight liquid organic compounds or other compounds) of a homogeneous mixture and to determine the composition of the mixture. It is based on the injection of the sample solution (i.e. solids must be dissolved in certain solvent) mixture into a column packed with certain materials (stationary phase) and passing a liquid (the mobile phase) through the column. The components of the mixture will be held strongly or weakly to the stationary phase. The liquid mobile phase will pass at high pressure through the column. It will take away the weakly bound component first to the outlet of the column. Thus, the components will be separated according to the boiling point, molecular weight, polarity, ability to form hydrogen bonding with the stationary phase. The components will appear et the end of the column at different times, tR , the retention time.

Instrumentation for HPLC:

• For reasonable analysis times, moderate flow rate required but small particles of the stationary phase (1-10 �m) • Solvent forced through column 1000-5000 psi – (i.e. instrument is more complicated than in GC.) • Solvents must be degasses to remove air bubbles "sparging" • High purity solvents Single mobile phase composition - isocratic elution Programmed mobile phase composition - gradient elution: Solvent polarity (composition) continuously varied or stepped (See page 8a) • Up to 10,000 psi, small internal volumes Sample injection

• Similar to FIA, GC • Introduce small sample (0.1-100 �L) without depressurization • Microsyringe/septum system (only <1500 psi)

HPLC COLUMNS: Heavy-wall glass, stainless steel and plastic are among materials that can:

a. withstand high pressures. b. resist the chemical action of the mobile phase. c. 10-30 cm long; d. 4-10 mm internal diameter; e. 1-10 μm particle size - 40,000-60,000 plates/m f. Well-packed because channels would cause peak broadening and a

decrease in efficiency. Usually a short guard column is placed in front of the analytical column. This serves to increase the life of the analytical column by removing particulate matter and contaminants from the solvents.

High Speed Isocratic Separation

Detectors: All properties previously discussed and Bulk property detectors - measure property of mobile phase

(refractive index, dielectric constant, density) Solute property detectors - measure property of solute not present in mobile phase

(UV absorbance, fluorescence, IR absorbance) Normal phase HPLC nonpolar solvent/polar column Interaction : Adsorption Packing materials : Polar ex. Silica gel

Silica-NH2 Silica-CN Silica-OH

Mobile phase : Non-polar ex.n-Hex/CH2CL2 iso-Oct/IPA iso-Oct/AcOEt

Sample : Fat-soluble Different polarity

Reversed phase HPLC Interaction : Hydrophobic Packing materials : Non-polar ex. Silica-C18

Silica-C8 Polymer

Mobile phase : Polar ex.MeOH/H2O CH3CN/H2O MeOH/Buffer sol.

Sample : Having different length of carbon chain Comparison of Reversed Phase and Normal Phase

Normal phase Reversed phase Stationary phase High polarity Low polarity Mobile phase Low polarity High polarity Interaction Adsorption Hydrophobic Elution order Low to High

(Polarity) Short to Long (Length of Carbon chain)

Reversed-phase HPLC most common (high polarity solvent, high polarity solutes elute first) R is C8 or C18 hydrocarbon faster elution higher resolution Column Optimization in HPLC: More difficult than GC - in GC mobile phase just transported solute - in HPLC mobile phase interacts with solute Analyte Polarity: hydrocarbons< ethers< esters< ketones< aldehydes <amines<alcohols Stationary Phase Choice: Choose column with similar polarity to analyte for maximum interaction Mobile Phase Choice: Polar ("strong") solvent interacts most with polar analyte (solute) - elutes faster but less resolution Size Exclusion Chromatography (SEC)

GPC and GFC Non-aqueous SEC : GPC (Gel Permeation Chromatography) Interaction : Gel permeation Packing : Cross-Linked porous Polystyrene

Mobile phase: Organic solvent (THF, CHCl3, DMF) Sample: Molecular weight distribution of polymer

SyntheticOligomerseparation Aqueous SEC : GFC (Gel Filtration Chromatography) Interaction : Gel permeation Packing : Hydrophilic silica gel / Hydrophilic porous polymer Mobile phase: Buffer solution Sample: Separation of Water-soluble polymers (proteins, nucleic acid, sugar)

oligomers

SEC Separation mechanism

Column: AApakNaII-H Mobile phase: Sodium citrate buffer

Stepwise gradient Detection: OPA post label

Ex 345nm Em 445nm Sample: Sake

Amino Acid Analysis

ELECTROCHEMICAL METHODS Many electroanalytical methods are available. They are fast and inexpensive. They are based knowledge about oxidation states, stoichiometry, rates, charge transfer and equilibrium constants. Electrochemical Cells: The composition of electrochemical Cells is based on Oxidation and reduction (redox) reactions. We need to separate species to prevent direct reaction (Fig 1). Most electrochemical Cells contain external wires (electrons carry current), ion solutions (ions carry current) and interfaces or junctions. All contain complete electrical circuit and conducting electrodes (metal, carbon)

Fig. 1: Electrochemical Cells: Electrons are transferred at electrode surface at liquid/solid interface. The potential difference (voltage) is measure of tendency to move to Equilibrium. Galvanic cell - cell develops spontaneous potential difference

Convention: Reduction at Cathode Oxidation at Anode

Galvanic cell - Zn anode (negative), Cu cathode (positive) Electrolytic cells - require potential difference greater than galvanic potential difference (to drive away from equilibrium)

Electrolytic cell Electrolytic cell - Zn cathode (positive), Cu anode (negative) Many chemically reversible cells are available. The short-Hand Cell notation places the Anode on Left with liquid-liquid interface

Galvanic cell as written Electrolytic cell if reversed Not all cells have liquid-liquid junctions

Electrode Potentials: • Cell potential is difference between anode and cathode potential Ecell = �Ecathode - ��Eanode when half-reactions written as reductions with electrons on left Example:

Galvanic cell Ecell = �Ecathode - ��Eanode potential on each electrode Can't be measured independently - only differences. The standard reference electrode is usually standard hydrogen electrode (SHE), Fig 3. The SHE is assigned to 0.000 V.

It can be anode or cathode. Pt does not take part in reaction. The Pt electrode is coated with fine particles (Pt black) to provide large surface area. However, the SHE is cumbersome to operate. Alternative reference electrodes are: • Ag/AgCl electrode

• Calomel electrode

Electrode and Standard Electrode Potentials (E and E0): How do we know which way reaction will go spontaneously? Use electrode potentials, E (potential of electrode versus SHE) to find Eanode and Ecathode. Then find Ecell. But electrode potential varies with activity of ion aX = γX . [X]

aX = activity γX =activity coefficient and [X] = concentration γX varies with presence of other ions (ionic strength)

Note: activity of pure liquid or solid in excess=1.00 Note: use pressure (atm) for gases If a=1.00 M, the electrode potential, E, becomes standard electrode potential, E0

Cell containing Cu/Cu2+ and Cd/Cd2+ called couple

(1) Cu2+ + 2e- → Cu spontaneously forward Cd2+ + 2e- → Cd spontaneously backward (Cd → Cd2++2e-) (2) e- flow towards Cu electrode (cathode/positive electrode) e- flow away from Cd electrode (anode/negative electrode) (3) Cu2+ good electron acceptor (oxidizing agent) Cd good electron donor (reducing agent)

The most positive E or E0 spontaneously forward forming cathode Calculation of Cell Potentials, Ecell: Ecell = �Ecathode - ��Eanode when written as reductions Example:

Zn reaction spontaneously backward - forms negative electrode - place of oxidation – anode

If a = 1.00 M, E = E0:

Ecell = �Ecathode - ��Eanode = + ��0.337 - ��(�0.763) = + ��1.100 V

Spontaneous reaction is galvanic

Ecell indicates if reaction is spontaneous as written Ecell positive - reaction forward Ecell negative - reaction backwards Electrode potential is related to position of equilibrium

If reaction is long way from thermodynamic equilibrium, K will change with time Eventually, concentrations reach equilibrium values and K stops changing (true equilibrium constant Keq) In general:

In principle, can calculate E and Ecell from E0 for any activity from Nernst equation:

• E= E0 when log quotient in Nernst equation is unity • E0 is relative to SHE • E0 is measure of driving force for half-cell reduction

Limitations of Standard Electrode Potentials:

(1) E0 is temperature dependent (2) Substitution of concentration for activity always introduces error. Error is worse at high ionic strength (3) Formation of complexes, association, dissociation alter E0

Formal potentials (E0') apply for specific reactions when specifying ALL concentrations

POTENTIOMETRY REFERENCE ELECTRODES:

• reversible • little hysteresis • follows Nernst equation • stable potential with time

Saturated Calomel Electrode (SCE):

Half-cell for Calomel Electrode:

Position of equilibrium affected by aCl- from KCl so E0 depends on aCl- Most common saturated calomel electrode SCE ([Cl-]~4.5 M) Silver/Silver Chloride Electrode: Similar construction to calomel

• Ag wire coated with AgCl • solution of KCl sat'd with AgCl

Again depends on aCl-, but commonly sat'd (~3.5 M)

Potential vs. SHE ¯

Which one?

• Ag/AgCl better for uncontrolled temperature (lower T coefficient) • Ag reacts with more ions

Precautions in Use:

• Level of liquid inside reference electrode above analyte level to minimize contamination • Plugging problematic if ion reacts with solution to make solid (e.g. AgCl in Cl- determination)

Measuring Concentration using Electrodes: Indicator Electrodes for Ions: Electrode used with reference electrode to measure potential of unknown solution

• potential proportional to ion activity • specific (one ion) or selective (several ions)

Ecell = Eindicator - Ereference Two general types - metallic and membrane electrodes Metallic Indicator Electrodes: I. Electrodes of the first kind

- respond directly to changing activity of electrode ion Example: Copper indicator electrode

BUT other ions can be reduced at Cu surface - those with higher +ve E0 (better oxidizing agents than Cu) Ag, Hg, Pd...

In general, electrodes of first kind:

• simple • not very selective • some metals easily oxidized (deaerated solutions) • some metals (Zn, Cd) dissolve in acidic solutions

Electrodes of the second kind - respond to changes in ion activity through formation of complex Example: Silver works as halide indicator electrode if coated with silver halide Silver wire in KCl (sat'd) forms AgCl layer on surface

• Electrodes of the third kind - respond to changes of different ion than metal electrode Membrane (or Ion Selective) Electrodes: Membrane:

• Low solubility - solids, semi-solids and polymers • Some electrical conductivity - often by doping • Selectivity - part of membrane binds/reacts with analyte

Two general types - crystalline and non-crystalline membranes

• Non-crystalline membranes: Glass - silicate glasses for H+, Na+ Liquid - liquid ion exchanger for Ca2+ Immobilized liquid - liquid/PVC matrix for Ca2+ and NO3-

• Crystalline membranes: Single crystal - LaF3 for F- Polycrystalline or mixed crystal - AgS for S2- and Ag+

GLASS MEMBRANE ELECTRODES:

Combination pIon electrode (ref + ind) Contains two (reference) electrodes - glass membrane is pH sensitive Glass Membrane Structure: SiO4

4- framework with charge balancing cations - SiO2 72 %, Na2O 22 %, CaO 6 % In aqueous solution, ion exchange reaction at surface

• H+ carries current near surface • Na+ carries current in interior • Ca2+ carries no current (immobile)

Surface where more dissociation occurs becomes negatively charge with respect to other surface Alkaline Error: At high pH, glass electrode indicates pH less than true value Low [H+] means membrane exchanges with alkali metal ions in solution too

Most accurate 0-10 (0.01-0.03 pH units) Interference in Glass Membrane Electrodes: Sensitive to

• H+ • alkali metal ions

Selectivity coefficients (kX/Y) measure sensitivity to other ions Range between 0 (no interference) to 1 (as sensitive to alkali and hydrogen ions) to >1 (large interference)

Glass Electrodes for Other Ions: Maximize kH/Na for other ions by modifying glass surface (usually adding Al2O3 or B2O3) Possible to make glass membrane electrodes for Na+, K+, NH4+, Cs+, Rb+, Li+, Ag+ ...

Crystalline Membrane Electrodes: • Usually ionic compound • Single crystal • Crushed powder, melted and formed • Sometimes doped (Li+) to increase conductivity • Operation similar to glass membrane

Presence of F- analyte pushes equilibrium right, reduces +ve charge on electrode surface

Liquid Membrane Electrodes:

• Based on potential that develops across two immiscible liquids with different affinities for analyte • Porous membrane used to separate liquids

Example: Calcium dialkyl phosphate insoluble in water, but binds Ca2+ strongly Molecule Selective Electrodes:

• Gas Sensing Probes • Biocatalytic Membranes

Gas Sensing Probes:

Simple electrochemical cell with two reference electrodes and gas permeable PTFE membrane

allows small gas molecules to pass and dissolve into internal solution Analyte not in direct contact with either electrode - dissolved

POTENTIOMETRIC TITRATIONS Titrations can be followed potentiometrically; Reaction involves removal or addition of some ion for which an electrode is available.

Changing Potential of the indicating electrode during titration (Nernst Eq.). Titration curve: Potential vs. mL titrant. At Eq. point, slope is steepest. Concentration cell is used: Two sides, identical electrodes, in the reference side a solution similar to that expected at the Eq. point; with addition of titrant the potential difference decreases until reach zero.

INSTRUMENTATION Nernst eq. is valid when no current passes through the cell. Precision potentiometer Now the PH meters or p-ion meters are used for this purpose. VOLTAMMETRY

Electrochemistry techniques based on current (i) measurement as function of voltage (Eappl)

THREE ELECTRODES Working electrode (microelectrode) place where redox occurs

surface area few mm2 to limit current flow Reference electrode constant potential reference (SCE) Counter electrode inert material (Hg, Pt) plays no part in

redox but completes circuit Supporting electrolyte alkali metal salt (e.g. KNO3) does not react with

electrodes but has conductivity Why not use 2 electrodes? OK in potentiometry - very small currents. Now, want to measure current (larger=better) but

• potential drops when current is taken from electrode (IR drop) • must minimize current withdrawn from reference electrode surface

Potentiostat (voltage source) drives cell

• supplies whatever voltage needed between working and counter electrodes to maintain specific voltage between working and reference electrode

NOTE:

• Almost all current carried between working and counter electrodes • Voltage measured between working and reference electrodes • Analyte dissolved in cell not at electrode surface!

Microelectrodes C, Au, Pt, Hg each useful in certain solutions/voltage ranges

At -ve limit, oxidation of water

At +ve limit, reduction of water

Varies with material/solution due to different overpotentials Overpotential ŋ always reduces theoretical cell potential when current is flowing ŋ = Ecurrent - Eequilibrium Overpotential (overvoltage) develops as a result of electrode polarization:

• concentration polarization - mass transport limited • adsorption/desorption polarization - rate of surface attach/detachment • charge-transfer polarization - rate of redox reaction • reaction polarization - rate of redox reaction of intermediate in redox reaction

Overpotential means must apply greater potential before redox chemistry occurs

Hg particularly useful

high overpotential at -ve limit easy to prepare clean surface

Hg Microelectrodes: Dropping Mercury Electrode

(DME)

Voltammograms The potential applied to the cell in continuously increased, usually in the negative direction (DME becoming more negative with respect to SCE) and the current is recorded. The voltammetric waves are graphs of current (i) vs. applied voltage (Eappl). The position of the wave (half wave potential, E1/2) is characteristic of the analyte (reduced) and the height (Diffusion Current) is directly proportional to Concentration (See the Fig.).

Hg microelectrode is cathode -ve terminal in above

Increase in current at potential at which A can be reduced (reaction demands electrons, supplied by potentiostat) Two important points • Half wave potential (E1/2) is close to E0 for reduction reaction

• Limiting current (il) proportional to analyte concentration (really, activity) il = k× cA Current is just measure of rate at which species can be brought to electrode surface Two methods:

Stirred - hydrodynamic voltammetry Unstirred - polarography (dropping Hg electrode)

Single voltammogram can quantitatively record many species provided enough separation between waves

Problems with dissolved O2 - must purge (sparge) solutions

POLAROGRAPHY First voltammetric technique Differs from hydrodynamic

• unstirred (diffusion dominates) • DME is used as working electrode current varies as drop grows then falls

off

Linear Scan Polarography:

Unstirred - only diffusion - currents smaller Diffusion Current (Ilkovic Equation)

n = number of electrons; cA = analyte conc (mM); t = drop time (s) D = diffusion coefficient (cm2·s-1); m = flow rate of Hg (mg/s) Residual Current

• redox reactions of impurities in solution • charging of Hg drop

(non-faradaic current/non-redox current)

Advantages of DME (compared to planar electrodes):

• clean surface generated • clean surface generated • rapid achievement of constant current during drop growth • remixing of solution when drop falls • high Hg overvoltage means even metals with high -ve E0 can be measured without H2 formation

Disadvantages of DME:

• Hg easily oxidized, limited use as anode (E< +0.4 V)

• nonfaradaic residual currents limit detection to >10-5 M • cumbersome to use (toxic mercury) • sometimes produce current maxima for unclear reasons (use maxima suppressor)

Pulse Polarography: Differential Pulse Polarography (DPP): Conventional polarography somewhat limited by nonfaradaic currents DPP relies on two measurements when difference between faradaic and nonfaradaic currents are largest. Detection limits 10-7-10-8 M.

POLAROGRAPHY Qualitative Analysis: Since the half wave potential is characteristic of the species undergoing reduction or oxidation at the DME, it can be used for its identification (fig). Portion AB and DF are extended, a tangent is drawn to the curve at point C. Line GH is bisected, and a line JK is drawn parallel to AB and DF. The abscissa of the point of intersection of JK with the curve gives the value E1/2. The selection of the supporting electrolyte is very important because of the different tendency of ions to complex formation: Quantitative Analysis The magnitude of diffusion current is related to the concentration of the reducible species through the Elkovic Equation. It is impractical to measure the factors in the Equation and a comparison method is preferable. The equation may be id = K C K is evaluated for a standard solution, and then the value of id for the unknown solution is measured to evaluate the concentration.

For example, Suppose Cd is to be determined in a sample of Zn. A 0.1 g sample is dissolved in HCl, a few drops of Triton X-100 added, and the solution was diluted to a known volume with 1.0 M KCl. A portion is placed in the polarographic cell and sparged with N2 to remove dissolved oxygen. Polarogram is recorded. The potential range from about -0.4 to 0.8 V versus SCE is suitableA wave at about -0.64 V in this experiment (Qualitative: Cd is present or not). The reduction potential of Zn is far negative (No inter- ferences). Another polarogram is recorded for a standard Cd solution. The values of id for both standard and unknown solutions from the graph. The conc. Of the unknown can be calculated.

TYPES OF ELECTRODES

1. Metal in equilibrium with its ions: The oxidized form Aox is the cation, and the reduced form Ared is the free metal. The E0 values are given relative to saturated Calomel electrode, SCE, at 25oC. The electrolyte is a solution of a salt of the metal with anion of a strong mineral acid, SO4, NO3, and ClO4 are suitable. Ea = Ea0 + RT/nF Ln(Aox) Because the activity of a pure solid element is taken = 1.0. The more active metals (Na, K, ….) cannot be used because they are not stable in water. Less active metals, (Ag…) may not give good response in dil. Solutions. 2. A metal in eq with a saturated solution of a slightly soluble salt

Ag/ AgCl (s) Mostly as reference electrodes

3. Ametal in Eq. with two slightly soluble salts with a common anion

Ag2S → 2 Ag + S2- CdS → Cd + S2- This half cell can be used to measure the activity of Cd ions. The second salt must be slightly more soluble than the first. On e widely used electrode has eq. between EDTA, Hg2+ ions and the ions of a di, tri, or tetravalent metal, the slightly dissociated complexes play the same roles as the slightly soluble sulfide salts in the above example.

CONCENTRATION CELL: Cell with two identical electrodes dipping into solutions similar in every thing except the concentration of ion to which the electrodes aare sensitive. Ag/AgCl electrode in an unknown solution of chloride Other Ag/AgCl electrode in standard soln of chloride, = 0.1 M Es = + 0.222 – 0.0591 Log 0.1 = + 0.281 V For the Unknown if the conc. 0.15 M Ex = 0.222 – 0.0591 Log 0.15 = +0.271 V Ecell = Es – Ex = 0.01 V by subtracting the more negative from the more positive potential.

POTENTIOMETRY

Suppose the meter indicates a 0.450 V difference in potential between the electrode: ESCE = + 0.246 V By subtraction Log [Ag] = (Ecell –E0

Ag + 0.246)/2,303 (RT/nF) = (0.450 – 0.799 + 0.246)/0.059 = - 1.74 Thus, [Ag] = 0.018 M Consider a cell made of Pt electrode dipping into a solution 0.1 M in Fe(II)SO4. The KCl salt bridge from an SCE is inserted directly into the same solution, since no harmful interaction can occur. A potential difference of 0.395 V is observed ar 25oC. The goal is to fine the percentage of Fe(II) that has been converted by air oxidation to Fe(III). Starting with the two half cell potentials, and for n = 1: Ept = E0

Fe3+/Fe2+ + RT/nF Ln[Fe3+]/[Fe2+] ESCE = + 0.246 V Ecell = Ept - ESCE = E0

Fe3+/Fe2+ - ESCE + 0,0591 Log [Fe3+]/[Fe2+] From which Log [Fe3+]/[Fe2+] was calculated = 0.0063 = 0.63%. Therefore, 0.63 percent of Fe(II) has been oxidized by air. Potentiometric Titration: Titration of Ce(IV) with Fe(II): Fe3+ + e Fe2+

Ce4+ + e Ce3+

Reference half cell is SCE Therefore 2 Fe3+ + 2Hg + 2Cl- → 2 Fe2+ + Hg2Cl2 And 2 Ce4+ + 2Hg + 2Cl- → 2 Ce3+ + Hg2Cl2 Fe2+ in solution and Ce4+ is the titrant

CONDUCTOMETRY A non-Faradaic quantity that can be used successfully for analysis, the Electric Conductance. A cell consisting of two Pt electrodes in an ionic solution. If a constant DC potential is applied on this network, nothing will happen if the voltage is small enough that no electrochemical process can occur. If the voltage is higher then current will flow through the resistance of the solution. The conductance of a solution in Siemense is the reciprocal of the resistance. It is related to the ratio of the area (a) of the electrodes and to the distance between them, (d), and to the total ionic concentration: L = 10-3 (a/d) ziCi λi a/d is a geometric property of the cell, and its reciprocal is cell constant, θ, with units of cm-1. Ci is the molar conc. Of ion i with charge zi and λi I is the equivalent ionic conductivity. The summation covers all ions of both signs. λ is a property of ions that gives quantitatively information about their relative contribution to the conductance of a solution. λ0 is the limiting value of λ at zero concentration, infinite dilution. It differs from ionic mobility by the factor F (Faraday constant). For some applications, it is necessary to know the cell constant θ . It is generally determined from measurements on solutions of known specific conductance L θ = K. For calibration purposes: KCl Conc., g KCl/Kg of solution K, S.cm 71.1352 0.11134 7.41913 0.01285 0.74526 0.0014088 Instrumentation: To measure electrolytic conductance, we use the wheatstone bridge modified for ac operation. The circuit is supplied by different resistances to be selected by a switch to give precise ratios of 0.1, 1 or 10. The unknown resistance is that of the conductance cell. Applications: Most applications are concerned with aqueous solutions. Water is very poor conductor. Water stills and deionizers are usually provided with conductance monitors. Solutions of strong electrolytes show a nearly linear increase of conductance with concentration up to about 10 to 20% by weight. At higher concentrations, the

conductance decrease again, as interionic attraction hinders free movement of ions through the solution. Conductometric Titrations

1. Significant difference in conductance between original solution and the reagent;

2. The distance between electrodes does not change during titration; 3. Necessary to correct for the dilution due to addition of reagents besides

the consumption of the reactants. 4. Hydrolysis or partial solubility of precipitates may cause departure from

linearity. 5. No need to determine the cell constant, since relative values are enough

to determine end point.

Example: Titration of HCl by NaOH

mL NaOH mL NaOH

L

H+ OH- L

Na+

Cl-

Overall shape Details of the conductance