Chem. 31 – 10/30 Lecture

Announcements I AA Lab – Due today Quiz 4 Today’s Lecture On Wednesday On Acids part of Ch. 6/Chapter 7 materials/Chapter 18 materials (extractions) Today’s Lecture Chapter 18 – Spectroscopy Beer’s Law Spectrometers

Announcements II Today’s Lecture – cont. Chapter 23 – Chromatography Overview Partitioning (in extractions and chromatography) Chromatographs Transport (if time)

Spectroscopy Beer’s Law Transmittance = T = P/Po Absorbance = A = -logT sample in cuvette Light source Absorbance used because it is proportional to concentration A = εbC Where ε = molar absorptivity and b = path length (usually in cm) and C = concentration (M) Light intensity in = Po Light intensity out = P b ε = constant for given compound at specific λ value

Spectroscopy Beer’s Law Question Half of the 284 nm light is absorbed when benzoic acid at a concentration of 0.0080 M is in a cuvette with a path length of 0.50 cm. What is the molar absorptivity of benzoic acid at this wavelength?

Spectroscopy More on Beer’s Law Useful for determination of analyte concentrations Some limitations Law not valid for high concentrations Deviations to law appear to occur when multiple wavelengths of light used or when multiple species exist but absorb light differently Uncertainties are lowest when 0.1 < A < 1 Example of deviations to Beer’s Law: Unbuffered Indicator with ε(In-) = 300 M-1 cm-1, ε(HIn) = 20 M-1 cm-1; pKa = 4.0 HIn ↔ H+ + In-

Spectroscopy Spectrometers light detector – measures light intensity by converting it to an electrical signal sample in cuvette Data processor light source light discriminator: monochromator (passes only a small range of wavelengths) Components can look very different in different types of spectrometers, but spectrometers will have all of the major components (except other methods of wavelength discrimination may replace monochromators)

Spectroscopy Example Measurement: Ozone Ozone (O3) is a pollutant (lower atmosphere) and in stratosphere provides UV protection Instrument is used for measurement at station or in airplane compares absorbance through sample cell vs. absorbance through reference cell Can also make measurements remotely (e.g. absorbance between two skyscrapers) light source (l = 254 nm) chopper air in reference cell sample cell O3 scrubber light detector

Chromatography – Ch. 23 Introduction Purpose of Chromatography To separate and detect components of a mixture Analytical chemists are more interested in the detection part Advantages of Chromatography Can handle more complex samples than typical spectroscopic methods Also results in purification of mixtures (if desired) Disadvantage of Chromatography Separation takes time (so generally not as fast as pure spectroscopic methods) Basis for Separation: differential partitioning between a stationary and a mobile phase

Chromatography Partitioning – Ch. 23 Sect. 1 Covering to understand partitioning in chromatography Partitioning can occur between any two phases (as long as one phase is a fluid) Liquid-liquid is chosen as an example Partitioning governed by equilibrium equation X(org) X(aq) K = partition coefficient (a constant) note: technically, upper conc. is for “raffinate” phase while lower is for “extractant” phase

Chromatography Partitioning Partitioning coefficient depends on stability in solvents (related to solubility in solvents) Most common rule is likes dissolve likes Example of water – hexane partitioning Other Effects on Partitioning K gives distribution if compound does not react further in either phase However, compounds may react further (e.g. acid HA → H+ + A- in aqueous phase) Ions (e.g. A-) will be found almost exclusively in aqueous phase Distribution coefficient (D) gives ratio of total species concentration (only covering qualitatively) larger K Although both have K > 1, the OH group makes right molecule more polar (favors water more vs left)

Chromatography Partitioning Example of Effect of Aqueous Reactions on Compound Distribution Compound A is nearly as polar as B However, acidity affects distribution between water and organic layer Compound B will undergo dissociation in water: HA ↔ H+ + A- Distribution of B given by: D = [HA]org/{[HA] + [A-]}aq Compound A Compound B K = 7.59 K = 6.17 pKa = 4.62 Not very acidic If aqueous phase is buffered at pH > pKa (e.g. pH = 6), most of B will be in anion form and very little of B will be in organic phase With a low pH buffer, D ~ K

Chromatography Partitioning - Questions A compound with an octanol water partition coefficient of 52 is placed in a separatory funnel with water and octanol and shaken. The concentration of it in octanol is found to be 0.150 M. What is its concentration in water? It is desired to separate the following two compounds: CH3(CH2)3OH and CH3(CH2)3NH2. The two compounds have similar KOW values (around 11) but the second compound is basic. What can be done to separate the two? 3. It is desired to transfer butanol (left compound in #2) from water to an organic phase. Would it be transferred most efficiently using 1-octanol, a less polar solvent (e.g. octane), or a more polar solvent (e.g. 1-hexanol) as the organic solvent?

Chromatography Partitioning in Chromatography Separation Occurs in Column Partitioning Requires Two Phases: Mobile phase fluid flowing through the column type of fluid determines type of chromatography fluid = gas means gas chromatography (GC) liquid chromatography (high performance liquid chromatography or HPLC) supercritical fluid (SFC) [supercritical fluid = fluid at high temperature and pressure with properties intermediate between liquid and gas] Stationary phase (solid or liquid within column) most commonly liquid-like substance on solid support

Chromatography More on Stationary Phases Open Tubular – in GC (end on, cross section view) Packed column (side view) (e.g. Silica in normal phase HPLC) Column Wall Mobile phase Packing Material (solid) Stationary phase is surface (larger area than shown because its porous) Stationary phase (wall coating) Expanded View Bonded phase (liquid-like) Stationary Phase Chemically bonded to packing material Packing Material

Chromatography Overview – The Good, the Bad, and the Ugly The Good: Differential partitioning of solute between a mobile and a stationary phases The Bad: Band broadening The Ugly: Non-ideal peak shapes (we will see this in the GC lab) More realistic picture Concentration profile

Chromatography Equipment Chromatograph = instrument Chromatogram = detection vs. time (vol.) plot Chromatograph Components Sample In Chromatographic Column Detector Flow/Pressure Control Mobile Phase Reservoir Waste or fraction collection Injector Chromatogram Signal to data recorder

Chromatography Flow – Volume Relation Relationship between volume (used with gravity columns) and time (most common with more advanced instruments): V = t·uV V = volume passing through column part in time t at flow rate uV Also, VR = tR·uV where R refers to retention time/volume (time it takes component to go through column or volume of solvent needed to elute compound) Can also use linear velocity (ux) ux = L/tR where L = column length

Chromatography More on Volume Hold-up volume = VM = volume occupied by mobile phase in column Stationary phase volume = VS Calculation of VM: VM = tM·uV, where tM = time needed for unretained compounds to elute from column Unretained compound = compound 100% in mobile phase