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Chem. 31 – 4/3 Lecture Announcements I • Lab Resubmissions – Cl lab – due today – Water Hardness – due Wednesday • Quiz 4 this Wednesday – On shape of precipitation curve – Spectroscopy – Chromatography: Extractions plus sections covered today Announcements II • Upcoming Assignments – Atomic Absorption Lab Report (4/12) – Additional Problem 3 (4/17) – Formal Lab Report (Part A – will post materials soon – due 4/17) – Exam 2 (4/19; about 1 lecture behind expectations) Announcements III • Today’s Lecture – Chapter 23 – Chromatography • Partitioning – liquid – liquid example – in chromatography • Chromatography Equipment • Transport and Retention in Chromatography 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 O H3C H3C O Compound A K = 7.59 Not very acidic OH Compound B K = 6.17 pKa = 4.62 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 1. 2. 3. 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? 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 Stationary phase (wall coating) Expanded View Stationary Phase Chemically bonded to packing material Packing Material Packing Material (solid) Stationary phase is surface (larger area than shown because its porous) Bonded phase (liquid-like) 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 Flow/Pressure Control Mobile Phase Reservoir Injector Signal to data recorder Detector Waste or fraction collection Chromatogram 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 Chromatography Reading Chromatograms • Determination of parameters from reading chromatogram (HPLC example) • tM = 2.374 min. (normally determined by finding 1st peak for unretained compounds – contaminant below) • VM = uV·tM = (1.0 mL/min)(2.37 min) = 2.37 mL Chromatography Some Questions 1. What are the required two phases in chromatography called? 2. List two ways in which a stationary phase is “attached” to a column? 3. List 3 main components of chromatographs. 4. A GC has the flow rate set to 2.0 mL/min. When methane is injected (it is not retained at all on the stationary phase), it takes 2.85 min to appear. What is the mobile phase volume? 5. What phase is the mobile phase in HPLC? Chromatography Partition and Retention • • • • • Partition Coefficient = K = [X]S/[X]M Higher K means slower elution K is constant if T and/or solvent remain constant K is not used that frequently in chromatography Retention Factor = k = main measure of partioning/retention in column • k = (moles X)S/(moles X)M = K(VS/VM) • Retention Factor is more commonly used because of ease in measuring, and since VM/VS = constant, k = constant·K (for a given column) • Note: kColumn1 ≠ k Column2 (if VM/VS changes) Chromatography Definition Section – Partition and Retention • Since the fraction of time a solute molecule spends in a given phase is proportional to the fraction of moles in that phase, k = (time in stationary phase)/(time in mobile phase) • Experimentally, k = (tR – tM)/tM • Note: t’R = tR – tM = adjusted retention time Chromatography Reading Chromatograms • 1st peak, tR = 4.958 min.; k = (4.958 - 2.374)/2.374 = 1.088 Chromatography What do all these Parameters Mean? • Large k value (or K value) means analyte prefers stationary phase • “Adjusting k (or K)” - in GC: – k value will depend on volatility and polarity (analyte vs stationary phase) – k value adjusted by changing T (most common) – higher T means less retention (lower k and K) Chromatography What do all these Parameters Mean? II • Adjusting k – in HPLC – k value will depend on analyte vs. mobile phase and stationary phase polarity – Oldest type (= Normal phase) stationary phase is polar and mobile phase is non-polar – Most common type (= Reversed phase) has nonpolar stationary phase and polar mobile phase – k value adjusted by changing mobile phase polarity – k value is decreased by making mobile phase more like stationary phase (= using “stronger” eluent) – In reversed phase HPLC, less polar mobile phase means less retention analyte more polar water methanol stationary phase less polar Chromatography What do all these Parameters Mean? III • Retention Factor is a more useful measure of partitioning because value is related to elution time • Compounds with larger K, will have larger k, and will elute later • Practical k values – ~0.5 to ~10 – Small k values → overlapping peaks likely – Large k values → must wait long time Chromatography More Questions 2. A chemist is analyzing samples by normal phase HPLC using a mobile phase containing 90% hexane and 10% 2propanol (2-propanol is the more polar solvent). The analysis is taking too long. How can she decrease k values? From the chromatogram to the right, calculate kX. Chromatogram 1 3.1 2.9 2.7 response 1. 2.5 2.3 2.1 1.9 1.7 1.5 0.0 0.5 1.0 1.5 2.0 2.5 time (min.) unretained peak X Y