HPLC PDF

LIQUID CHROMATOGRAPHY READING ASSIGNMENT: 60-100 (several articles), 115-178 (ion chromatography) Several Techniques o 1) Classical vs Modern (HPLC) o 2) Ion Exchange (Ion Chromatography) o 3) Gel Permeation Chromatography (GPC) (Size Exclusion Chromatography...

LIQUID CHROMATOGRAPHY READING ASSIGNMENT: 60-100 (several articles), 115-178 (ion chromatography) Several Techniques o 1) Classical vs Modern (HPLC) o 2) Ion Exchange (Ion Chromatography) o 3) Gel Permeation Chromatography (GPC) (Size Exclusion Chromatography (SEC)) o 4) Chiral Separations (Enantiomeric separations) o 5) Planar Techniques (TLC & Planar) Comparison of Techniques Parameter Classical HPLC UHPLC Partial Size of 75-600 Roughly 300 mesh 1.7 μ packing (diameter: μ, mesh) 30-200 50 μ to 10 μ to 5 μ (standard today) Packing Material Standard Special Special Particle Size Spread 20-30% Less than 10% Less than 10% (average of all particle diameters) (sigma, as % of Median) Column Length (cm) 10-200 cm 5-25 cm 5-25 cm Column Diameter 1 – 5 cm 0.1 – 1.0 cm 0.1 – 1.0 cm (cm) 0.1 & 0.2 (microbore) 0.1 & 0.2 (microbore) average (0.46 cm) average (0.46 cm) with MS (0.21 cm) with MS (0.21 cm) compromised with compromised with 0.3 cm 0.3 cm Column Inlet Pressure (1.0-0.1) atm 20 – 400 atm 20 – 800 atm (atm) 90% done at 1.0 atm Usually 80 – 200 atm Theoretical Plates (n) 2 – 50 1000 – 100000 1000 – 100000 Sample Size (g) 0.1 – 10 g 0.01 – 0.000001 g 0.01 – 0.000001 g  Separation Speed Less than 0.05 5 – 100 plates/sec More than HPLC (plates per sec) (n/s) Analysis Time (hrs) 1 – 20 hrs 0.05 – 1 hr 0.05 – 1 hr Cost CHEAP EXPENSIVE EXPENSIVE $20,000 $1,000,000 (2/3 is MS) High Pressure Liquid Chromatography (HPLC) o Diagram (Schematic in Phone) 11 1 9 6 5 7 10 2 12 3 8 4 Components o 1) Solvents ▪ A) One solvent If you pump one solvent, it is called Isocratic ▪ B) Gradient If you pump a gradient, it is still a gradient, but it is a solvent gradient Make a Gradient  AIR TIGHT HOSE 1 L 𝑀𝑒𝑂𝐻 𝐻2 𝑂 AIR TIGHT HOSE HOT PLATE TO COLUMN Graph Composition with Time 2L mL OF SOLVENT 1L TO COLUMN 100 𝐻2 𝑂 0 0 100 𝑀𝑒𝑂𝐻 % OF SOLVENT Two types of HPLC o FIGURE 55 (READING #1) ▪ Costs more ▪ For every solvent, you have an individual pump Programmer makes the mixture of solvents (50-50, 100-0, etc) Not as accurate as making by hand, but it is faster o NO MIXING CHAMBER ▪ Instead there is a mixing T ▪ Ten microliter is typical ▪ Have to withstand really high pressure ▪ Solvents go in, terbently mixed, mixture comes out bottom ▪ Low dead volume SOLVENT 1 SOLVENT 2 ▪ Give good mixing MIXTURE o FIGURE 56 (READING #1) ▪ Single Pump Pumps both solvent 1 and solvent 2 o Proportioning values (solenoid values) from both solvents make this possible o Valuing system that control the proportion of each solvent o Can have 3-4 solvents, 1 pump, and many proportioning values o Less expensive o One pump o Solenoid values are cheap o Work ok, most of the time o Less accurate Need mixing chamber o Between 1 mL to 2 mL of volume ▪ No dead volume o Ex. Pump 1 mL of solvent by One minute  ▪ One minute delay o By 2 minutes ▪ Half a minute delay o Purity of Solvents ▪ No such thing as a pure solvent Maybe 99.5%, 99.75% ▪ What does purity mean? 100%, impossible could be spectroscopically pure o do not absorb by UV free of water o buy dry ether (purified to remove water) free of peroxides o create instability (explosive) free of conducting solutes (ions) o electrochemical reaction o Ion Chromatography ▪ Do not want things that conduct free of electroactive solutes o do not want any reaction to occur free from other solvents or additives o Purity for HPLC ▪ Depends on the solvent used Organic o Remove water and other solvents/additives  ▪ Ex. Pentane, hexane (normal phase) How do they get pure solvents? o Frontal Analysis (Frontal Chromatography) ▪ The analyte and solvent are continuously pumped into column Breakthroughs at different parts Step wise chromatogram ▪ Done with huge amounts of solvents and big columns COLLECT FIGURE 3 (READING #1) PURIFIED SOLVENT o Plot concentration of contaminant (y) by milliters of eluate (x) (IN [𝐶𝑂𝑁𝑇𝐴𝑀𝐼𝑁𝐴𝑁𝑇] PHONE) BREAKTHROUGH mL ELUATE o 2 different phases ▪ Normal Phase Traditionally, normal way to do Chromatography Polar SP o Ex. Silica Gel (most common) ▪ Bare silica gel o Ex. Aluminum Oxide Nonpolar MP o Ex. Hexane o Ex. Heptane o Ex. Isopropyl Alcohol (IPA) (modifier) o Ex. 100% Ethanol (modifier)  Chromatogram POLAR NONPOLAR SOLUTES SOLUTES o Polar elute last o Nonpolar elute first ▪ Reverse Phase Most popular mode of HPLC Nonpolar SP o Ex. C18 bonded to silica gel o Ex. C8 o Ex. C4 (reduce hydrophobicity) Polar MP o Ex. Water o Ex. Methanol o Ex. Acetonitrile Chromatogram NONPOLAR POLAR SOLUTES SOLUTES o Polar elute first o Nonpolar elute last FIGURE 5 (READING #1)  o UV spectrum of solvent after 3 purifications o Curve 1: absorbing at 270, 290, 310 o Curve 2: absorbing at lower wavelengths o Curve 3: other impurities (discard) o Effect of Modifier vs Retention ▪ Normal Phase 𝑘 Highest retention: 100% heptane 100 𝐻𝐸𝑃𝑇𝐴𝑁𝐸 0 0 Lowest 100 IPA 𝐼𝑃𝐴retention: 100% ▪ Reverse Phase 100 𝐻2 𝑂 0 T 0 𝑀𝑒𝑂𝐻 100 I Highest retention: 100% water Lowest retention: 100% MeOH/can ▪ Exception Amino acids/Peptides/Sugars (same solvents as RP) 𝑘  HILIC 100 𝐻2 𝑂 0 0 𝑀𝑒𝑂𝐻 100 𝐴𝑐𝑁 Do not come out as they are insoluble Using AcN (concentration (40%-100%) HILIC mode (hydrophilic interaction liquid chromatography) o Reverse Phase Mobile Phases ▪ Problem Water is major components Not pure water (usually buffer inside) o 1) can cause corrosion (phosphate, etc) o 2) can cause precipitation (rinse it out and let it sit in pure organic solvent ALWAYS) o 3) can cause microbial growth (time to grow) Do not make mobile phase 100% aqueous, put 5-10% organic ▪ Air bubbles AIR BUBBLE Chromatogram PEAK Problem in single pump HPLC o Low pressure mixing chamber ▪ Fix: pump Helium (helium degasser might be malfunctioning) o Characteristics of Solvent ▪ Viscosity Higher the viscosity, the higher the back pressure Narrower columns, smaller packing sizes o Increases back-pressure Change solvent mixture o Changes viscosity o Reverse phase is dominant o Mixtures of water with immiscible organic solvents tend to increase pressure and viscosity (pressure and viscosity of mixture is higher than both compounds in mixture) o Acetonitrile does not increase as higher as other alcohols, but does have a higher viscosity than water o Mix alcohol with water, viscosity is higher (maxes at 50-50) (stays maxed out until 80%) ▪ Boiling Point Not usually need to worry about unless at high temperature Low BP are less viscous than high BP More volatile solvent evaporates o May think you have a 50-50, after 1 day, could be 60-40, 45- 55 ▪ FIX: seal it (do not let things evaporate) ▪ Miscibility Problem when HPLC is switched between phases (NP to RP and vice versa) Always tell if two immiscible solvents hit pump when back-pressure skyrockets (could be clog or immiscible solvents)  o FIX: shut it down and figure out the cause RP to NP o 1) get all water and solvent out o 2) Pump with water (no phosphate buffer) o 3) Pump water out with miscible solvent (isopropanol, etc) o 4) Pump in new mixture o 5) Pump in buffer salts NP to RP o 1) get all water out and solvent out o 2) Pump with hexane (no buffer) o 3) Pump in propanol o 4) Pump in new mixture o 5) Pump in buffer salts ▪ Polarity Eluotropic Strength o Refers to solvents that elute faster to solvent that elute more slower o Depends on stationary phase o Strong RP is weaker in NP ▪ Ex. Methanol < AcN < IPA (RP) IPA is more polar than Methanol ▪ Ex. IPA < AcN < Alcohol (NP) Alcohol is less polar than IPA *BE FAMILIAR WITH GENERAL SOLVENTS FOR NP AND RP AND THEIR PROPERTIES* ▪ Dielectric Constant Listed as one of the parameters, but not important for chromatography o Does not describe organic solvent reactions Definition o Ability of solvent to separate 2 oppositely charged ions ▪ Ex. Sodium Chloride (NaCl) NaCl ⟶ Na+ + Cl− Water o Has a higher dielectric constant as it can dissociate NaCl Hexane  o Has a lower dielectric constant as it cannot dissociate NaCl o Classification of Solvents by Empirical Polarity Scale ▪ Organic Chemists Used simple reaction (Sn1 ) to measure in different solvents and rank them o Ex. Alkane and Halide ▪ First step: dissociation of ions ▪ Used Solvatochromic Dye Reichard’s Dye Produced ET scale o Did not use a reaction (dissolve solvent in dye and measure with UV spectrum and determine where the maximum lies) ▪ Went from 810 nm to 453 nm (shift of 357 nm (Hicksochromic shift)) o 2) Pump Programmer o 3) High Pressure Pump ▪ 3 Types 1) Piston o Constant volume pump o Has pulsation o High pressure when pushed in, low pressure when pushed out o Reduces background noise o Smooth baseline ▪ Pulse Dampening Mechanism A) Use restrictors B) Use flexible tubing  Baseline now looks like o 95% of HPLC o Typical Schematic (Reciprocating Pump) FLOW OUTLET CHECK VALVE PUMP DRIVE (PISTON) INLET CHECK VALUE FLOW FILTER o Sucking solvent in through bottom ▪ Filter to filter solvent When selecting o If plastic: consider solvent compatibility ▪ Do not want to dissolve filter o What causes flow is the outlet and inlet check valve ▪ Cannot put an outlet in an inlet slot and vice versa Threaded in opposite direction from each other ▪ Each has a ruby ball No corrosion, hard to scratch Can’t seal properly (leak occurs) o See by Pressure varies o Piston  ▪ Made of sapphire No corrosion, hard to scratch Seal: scratch it (leak occurs) Change every few wks o Fix ▪Shut down machine ▪Sonicate the piston or check value in acid ▪If not, solicate it in 10−2 M HNO3 (always works) 2) Syringe o Constant volume pump o Holds a lot of solvent o Drive shaft that pushes piston o Can get to high pressure ▪ Smooth baseline o Difficult to do gradient ▪ Unless you have more than one syringe o HPLC vs GC ▪ GC Smooth tip ▪ HPLC Blunt tip Stop on injector to stop the nose of the syringe from touching the channel of the injector ▪ If use GC in HPLC Sharp tip hits channel, channel is scratched, leak in injector 3) Gas/Pneumatic o Constant pressure pump o 4) Presaturator ▪ Put before the injector Schematic GUARD COLUMN PRESATURATOR DETECTOR  ▪ 95% of particles usually used are silica gel Like glass, dissolved in base o High surface are, pH around 7, slowly begins to dissolve ▪ Solid form: silicitic acid (SiO(OH)3) o 5) Pressure o 6) Injector ▪ Manual or Automated ▪ 6 port Injector valve Schematic (in phone) o Position #1 SAMPLE INLET SAMPLE LOOP SAMPLE OUTLET MOBILE PHASE IN COLUMN o 6 holes drilled through ▪ Screw in 6 different things o Inside stainless steel shell ▪ Granitized Teflon cord that can be rotated 1/3 of a turn Can only turn it one way then back the way it was ▪ 1) Load sample into sample loop Volume injected is dependent of solvent volume o 5,10,20,100,200 um Analytical: 5-20 um injection loop o Want to fill entire volume of sample loop ▪ 2) Mobile phase is pumped in and goes through column Smooth baseline with minimal noise and pressure ▪ 3) When sample goes in, rotate 1/3 of a turn Channels turn and now connect  o Sample can now go through the loop and into the column ▪ 4) Turn it back while column is running ▪ 5) Repeat for each sample o 7) Column ▪ Straight precision or stainless steel tube No irregularities on tube o Very smooth, mirror-like surface ▪ Length: 25 cm, 5 cm, 10 cm, 5 cm ▪ Diameter: 0.46 cm, 0.3 cm, 0.21 cm, [0.1-0.01] ▪ Lose 40% of theoretical plates from 0.46 to 0.21 Wall effects o Where the particles line up at the capillary, disrupts packing on the walls for a few layers o Lose efficiency ▪ Maintain most of the efficiency from 0.46 to 0.3 Low enough flow that mass spec will have no trouble ▪ [0.1-0.01] True capillary columns o Efficiency is really low What if you want a longer column? o Do not coil LC columns ▪ Lose efficiency ▪ Due to different flow rates of liquids (one liquid is flowing faster than the other) o Put two columns together with zero dead volume fitting ▪ Packed with silica gel How to make silica gel? o Comes in irregular & spherical formats ▪ Today’s HPLC use spherical silica gel o Two starting materials ▪ 1) Salicic Acid Si(OH)4 (pka = 8.94, pka2 = 13.2) One of three prime nutrients in ocean (others are phosphate & nitrate) Dirt cheap o Tends to have other metals in it (Fe,Al)  ▪ Used to Wash it to rid of other metals ▪ Now, they start with organic derivative ▪ 2) Si(OEt)4 Put in water (H2 O: nucleophile) o OEt groups come off, back to Si(OH)4 ▪ Ionized o A) Put in a beaker o B) Stir it o C) Reacts with itself ▪ Si − O − Si bond ▪ Repeats till you form a polymer ▪ Forms Colloidal Silica ▪ Colloidal: 10−4 cm size So small it will stay in suspension regardless of gravity Can be in any state of matter in liquid (as long LOWER pH as it is of colloidal size) o D) Change pH EVAPORATE ▪ Causes particle to flocculate SPRAY Neutralizes charge of charged particles Creates larger particles that sediment out o Can be of any size depending on stirring rate and etc. ▪ Silica Gel Pore size is very important FLOCCULATE o Depends on how long the gel is allowed to sit ▪ Long time (ripening): pores begin to “build bridges” and the more its ripened, the smaller the pore size is Want to control diameter and pore size of particles Strengths and Weaknesses of Silica Gel o Strengths ▪ Good mechanical strength But brittle  ▪ Easily made in variety of diameters and pore sizes Depending on how long the gel is allowed to ripen ▪ Good binding chemistry Lots of good bonding o Organosilanes ▪ Known relationship between pore size and surface area High pore size: small surface area Low pore size: large surface area ▪ pkas of silanol groups not all Si-OH groups o ex. Silica surface with 3 bonds and single silanol ▪ pka: 3.5 o ex. Silica surface with 2 bonds and a single silanol ▪ pka: between 3.5 and 7.5 o ex. Silica surface with 1 bond and a single silanol ▪ pka: 7.5 pkas range from 3.5 to 7.5 o multiple ionizable groups: higher pka o Weaknesses ▪ Silica gel dissolves in alkaline pHs In base, dissolve to give free silicic acid  Greater surface area: greater ability to dissolve ▪ Resistant to strong acid But when reacted to form a siloxane bond, the Si-C bond will cleave o < pH of 2 o Use pH between 2 and 7/8 Si-C o Hydrolyzes in strong acid Si-N-C and Si-O-C o Hydrolyzes with water o No cleaving except in rare circumstances Odds and Ends o Other Supports for LC ▪ Alumina Stable in basic pHs No good bonding chemistry (strictly NP) Polymerized polymer if RP, reduces theoretical plates Dissolves in acid, stable in base Can control diameter and pore size ▪ Zirconia Stable in basic pHs No good bonding chemistry (strictly NP) Polymerized polymer if RP, reduce theoretical plates Stable in acid and base Can control diameter and pore size ▪ Graphitized Carbon Graphite o Benzene rings fused together How to make it o 1) Load silica gel with organic molecules ▪ Use low weight polymers o 2) Filter off solvent and stick gel in oven (under nitrogen/argon) up to 600-800 C (no oxygen)  ▪ Carbon cooks and forms graphite o 3) Put in strong base (sodium fluoride) ▪ Silica gel is dissolved away ▪ Mirror image of silica gel now is graphite Can control diameter and pore size ▪ Inner Surface RP ▪ Monolithic Stationary Phases Made the same way as silica gel o Makes porous monolithic column ▪ As long as 10 cm Two types of columns o Silica Gel ▪ Narrower the diameter, the better they are ▪ Less backpressure o Polymeric Particle ▪ Fused together ▪ Done with very narrow capillary columns ▪ Less backpressure Can control diameter and pore size ▪ Pore Size vs Surface Area vs Strength A° M2 Strength of Particle Surface Area ( ) g 60 Roughly 350 120 Roughly 200 300 Roughly 60 1000 Roughly 10 ▪ Diameter of Particles 20 u, 10 u, 5 u, 3 u, 1.7 u (Fully porous) o Pores go through the entire particle ▪ Smaller the diameter, more theoretical plates, but more back pressure o 20 u or : Analytical ▪ Cannot dry-pack MUST use slurry pack o Use Slurry solvent (close in density to particles), pack in column till full 1.7 u o Usually can only use a 10 cm long column ▪ Great efficiency, but lose plates due to smaller size of column Coreshell or Superficially porous particles o Used today o Solid core o Colloidal silica layer is just on the outside o 2.7 u diameter ▪ Give same efficiency as 1.7 u fully porous, but less back pressure ▪ Bonding Chemistries RP o C18, C8 , C4, Diphenyl, etc o Use organosilanes ▪ Direct C − Si bond Fairly stable ▪ Cl, OEt Leaving groups for synthesis ▪ Cl vs OEt Cl o HCl is a biproduct ▪ Reactive with C, breaks C − Si bond ▪ Do not want this o Add dry pyridine to reaction solvent to mop up acid OEt o Do not need pyridine o Bond to silica gel ▪ Outside of silica particle lies Si − OH groups Nucleophilic  o When reacted, attacks silicon on organosilane and leaving group “pops” off o Covalent bonding occurs Reacts commonly in dry toluene (BP = 110.6 K) o Dean Stark Trap (MBA) ▪ Toluene + H2 O ⟶ 84.1 C More acidic than alcohols (Alcohol pka = 13- 15) A) Monomeric o Ex. Organosilane with one OEt ▪ One reacting group One and only C18 produced o Carbon-loading ▪ A) Create particle and send off for analysis Gives 8 – 12 % C o Different pore sizes B) Polymeric o Ex. Organosilane with three OEt ▪ Multiple reacting groups One C18 is produced o Add a little water and an OH replaces the OEt ▪ Now the organosilane can react with it to create a longer chain ▪ Repeats o Carbon-Loading ▪ 20 – 80% C Thicker stationary phase Lower efficiency C) End capping reagent o Not all C18 will react due to being “sandwiched” in ▪ Deep in pores o In typical reaction: 80-90% Si − OH react ▪ Beneficial or not depending on circumstances o Smaller organosilane ▪ Ex. Cl or OEt leaving group  Can sneak into restrictive spacing and “cap it” o Carbon-loading ▪ 9 – 13% C D) Diol o Normal Phase ▪ Has epoxide group on organosiloxane ▪ Wash through dilute acid Hydrolyzed epoxide (creates OH groups) ▪ Polar instead of hydrophobic E) Chiral o Covered later F) Internal Surface RP ENZYME o Si − O − C bond ▪ Easier to cleave ▪ Outside and inside the particle o Took enzyme ▪ Hydrolyzes outside bond (too big to get inside) Silanol groups o Inside ▪ Polar C18 phase o Outside ▪ Polar silanol phase o Why? ▪ Analyze proteins and peptides ▪ Reverse phase is hydrophobic Protein denatures Irreversibly coats particles ▪ Proteins come out in dead volume o 8) Oven o 9) Temperature o 10) Detector ▪ Optical Detector  Ex. UV-VIS (Absorbance) o PDA ▪ Expensive More PDAs: more expensive ▪ Ex. Diode Array (DAD) Must have a good computer o Has to handle a lot of data o Can measure all wavelengths of light in 10 ms o Can do entire spectrum instantly ▪ Chromatogram (all wavelengths) ▪ Measured at 254 nm ▪ Click on one peak ▪ Gives data for UV spectrum ▪ Helps to identify compounds o 3D Plot ▪ Chromatogram and UV spectrum simultaneously ▪ Helps identify peaks  ▪ Measure tail end of both peaks ▪ Identical: same compound ▪ Different: different compounds Limitations o Not as sensitive as PMT ▪ PMT: better signal-to-noise ratio o Need computer ▪ Majority of problems are with software ▪ Light Source UV/Vis ▪ Various Lens ▪ Shutter between Light source and lens Shutter closed: no light Shutter open: light comes through (signal) ▪ Flow cell All wavelengths go through at same time o Measured in mL: want as small as possible ▪ limits broadening ▪ Slit Eliminates stray light ▪ Monochromator After flow cell o First done with no sample o Go through slit, hit grating o Goes through PDA ▪ Narrower the photodiode: the smaller the wavelength resolution ▪ Records data hv o Variable WavePHOTODIODE (VWD) ARRAY (PDA) Ex. Refractive Index (RI) o No absorbance/chromophore o Ex. Differential Refractive Detector ▪ Schematic SAMPLE OUT IN hv DETECTOR CONCAVE MIRROR OUT IN REFERENCE ▪ Universal detector Does not consume/destroy sample ▪ Used to be 2nd most popular with HPLC ▪ Limitations Less sensitivity with compounds with UV chromophore o Better with no UV chromophore ▪ Measures difference of light Passes through two wedge shaped cells Sample is run in one side Reference is run in another side Peak: sample is dissolved in mobile phase o Refracts light differently ▪ Angle (bending of light) will be different Detector detects position of light on the device as the light beam comes through the cell ▪ Must be well insulated  Temperatures cause refraction of light ▪ Tough to run gradient Run is isocratic o If sample is changed, reference has to be changed EXACTLY THE SAME Ex. Circular Dichroism (CD) Ex. Fluorescence (emission in UV-VIS) o Molecule must have a fluorophore (must fluoresce) o Schematic LIGHT SOURCE MONOCHROMATOR FLOW CELL (XENON ARC LAMP) DETECTOR SLIT MONOCHROMATOR READOUT o 10^2 – 10^3 more sensitive than UV ▪ Equal or better than MS o Compound must fluoresce ▪ Must have fluorophore o Signal-to-Noise ratio is better than UV o No analyte: no signal ▪ Background is as close to 0 as possible Ex. Evaporative Light Scattering o Schematic COLUMN DETECTOR  AIR OR 𝑁2 o Used for nonvolatile compound with little to no UV absorbance o Molecule must have no chromophore ▪ No absorbance o Limitations ▪ Buffer Creates huge background FIX: volatile buffers ▪ Destructive Others o Charged Aerosol (CAD) o Schematic CORONA DISCHARGE COLUMN ELECTROMETER AIR OR 𝑁2 ▪ Expensive ▪ No absorbance/chromophore o Limitations ▪ Buffer Creates huge background FIX: volatile buffers ▪ Destructive o Electrical Chemical Detector ▪ Pete Kissinger (Purdue) ▪ Dennis Johnson (Iowa State) ▪ Operate at constant potential Constant oxidizing or reducing power ▪ Electrodes exposed to solution coming out of column  Has current o Total current = background current + any current caused by the presence of an electroactive substance Sometimes called Amperometric Detectors o Measuring current ▪ Factors in Equations that cause signal Relating current to signal o Fluid flow rate (U) ▪ How fast fluid is pumped past electrodes o Kinematic viscosity of solvent (v) ▪ Ratio of fluid’s viscosity to its density o Solute’s diffusion coefficient (D) o Flow cell’s dimension (I) ▪ Two kind of flow cells Low efficiency flow cell o Only oxidizes or reduces about 1% of sample as it flows by electrodes High efficiency flow cell o Oxidizes or reduces about 100% of sample as it flows by electrodes ▪ Stronger signal o Do not need to stop as much as Low Limitations o Sample can “gum up” electrodes ▪ FIX: reverse potential, let stuff run out, and reverse it back o After several hours, stop experiment, and clean electrodes Compounds o Anything that can be oxidized or reduced ▪ Amines (R − NH) ▪ Thiols (R − SH) ▪ Alcohols (R − OH) ▪ Carboxylic Acids (R − COOH)  ▪ Aldehydes (R − C(= O) − H) o Mass Spectrometer (MS) ▪ Need volatile buffers as well o Electrical Conductivity Detector ▪ Ion Chromatography o Chiroptical ▪ Chiral Separations PDA, VWD, CD o Molecule must absorb light ▪ Absorbance Detection Wavelength (λ) o Visible ▪ 400 nm – 800 nm (typically 750 nm) o UV ▪ 200 nm – 400 nm o Vacuum UV ▪ < 200 nm – 10 nm Can only be achieved with a vacuum ▪ Absorption Intensities Beer’s Law o A= ε∙b∙c o A: absorbance o ε: molar extinction coefficient ▪ Function of λ ▪ Proportional to absorption cross section (electron diffraction) and probability of that transition ▪ 105: large ε (high absorbance) ▪ 101: small ε (low absorbance) o b: pathlength o c: concentration ▪ Instrumentation for UV-VIS (VWD) What do we need? o Light source ▪ Deuterium lamp (UV) 3-5 times higher intensity o Higher absorbance than H2 lamps 190 – 600 nm ▪ Tungsten Lamp (VIS) Incandescent o Slit  ▪ Picks one beam of light Depends on narrowness of slit ▪ Do not want stray light! o Monochromator ▪ Isolates single λ ▪ Prism ROY G BIV emitted Not used o Expensive ▪ Grating Measuring UV light: groves on grating are shorter Measuring IR light: groves on grating are longer Groves made of plastic coated with metal o Sample (Flow Cell) ▪ Capillary tubing (1 cm diameter) Measure absorbance as sample is continuously pumped through column o Reference Beam o Beam Splitter o Reference Detector o Sample Detector o Mirrors o Cuvette o PMT ▪ Measures electronic output Schematic (Simple Classic) PRISM COLUMALING SLIT LENS GRATING LIGHT SOURCE CUVETTE  SLIT PMT ▪ Nomenclature of Electronic Transitions Molecule (Formaldehyde) (CH2 = O) o Found in cars o Lowest energy transition: ▪ If bathochromic: n ⟶ π∗ ▪ If hypsochromic: n ⟶ σ∗ o Next energy transition: π ⟶ π∗ or σ ⟶ σ∗ o Highest energy transition: σ ⟶ σ∗ Aromatic compounds o π ⟶ π∗ *KNOW HOW TO PREDICT ACID/BASE, HYDROPHOBIC/HYDROPHILIC, AND TRANSITIONS OF A COMPOUND FROM STRUCTURE* 254 nm o Historical: Mercury light sources Compound λ (nm) Type of electronic transition CH4 122 σ ⟶ σ∗ CH3 CH2 135 σ ⟶ σ∗ CH3 Cl 173 n ⟶ σ∗ CH3 Br 204 n ⟶ σ∗ CH3 I 258 n ⟶ σ∗ CH2 = CH2 162 QUANTUM −(CH2 = CH2 ) 217 QUANTUM −(CH2 = CH2 )2 300 QUANTUM −(CH2 = CH2 )5 330 QUANTUM CH2 = O (Formaldehyde) 280 n ⟶ π∗  CS2 (Disulfide) 500 n ⟶ π∗ NH2 = O 650 n ⟶ π∗ C ≡ C (Acetylene) 178 π ⟶ π∗ Aromatics C6 H6 (Benzene) 256 π ⟶ π∗ C6 H5 NH2 (Aniline) 280 π ⟶ π∗ C6 H5 OH (Phenol) 270 π ⟶ π∗ Solvent + Analyte o Solvents can shift wavelengths of absorption ▪ Solvatochromism Polarity affects λMAX HYPERCHROMISM HYPOCHROMISM Abs BLUE SHIFT RED SHIFT (HYPSOCHROMIC) (BATHOCHROMIC) λ Systematically vary the solvent polarity o Monitor the λMAX shift ▪ Red: Bathochromic π ⟶ π∗ transition ▪ Blue: Hypsochromic n ⟶ π∗ transition n ⟶ σ∗ transition Why do we need to know this? o Pharma Abs ▪ Peak Purity  ▪ Charge-Transfer Bands t If you have an electron donor molecule (D) If you have an electron acceptor molecule (A) If in solution together o They can form a weak complex ▪ D + A ⇌ D+ A− ▪ Absorbance Spectrum CHARGE D A TRANSFER BAND ▪ Electrons from D goes to orbitals of A Band of lower energy forms ▪ Ex. Benzene + I2 ⇌ Benzene+ + I − ▪ Ex. Fe3+ + SCN − ▪ Transition Metals Colors Ex. Copper Sulfate (green) ALONG AXES Ex. Iron Oxide (orange) 5 d orbitals d-d TRANSITONS o Orbital splitting Ex. Nickel BETWEEN AXES Ex. Chromium E is smaller (large λ) o Metals cannot be used directly o 11) Recorder/Computer o 12) Waste