Chem. 230 – 10/21 Lecture

1. Chem. 230 – 10/21 Lecture

2. Announcements I • Exam 2 Results • Ave = 46.5 SD = 8 • similar average, but less variability • distribution • Chem 253 – Environmental Chem offered next semester

3. Announcements II • Posted HW Set #3 (original posting had incorrect due date) • Application Abstract due Today – I should be able to check your and give you an o.k. by next week • 2D GC references posted on website • Today’s Lecture • GCXGC Questions • SFC • HPLC (start)

4. GCGC x GC Questions Why is the second dimension in GC x GC limited in resolution? For which column in GC x GC is it important to use columns of small diameter and thin films? Why is overloading on narrow diameter 2nd dimension columns less important than when using the same column with 1D GC? How many dimensions does a GC x GC x MS chromatogram have? Why does the peak capacity of a two dimensional technique somewhat over express how many compounds can be resolved? Why are two hot/cold jets needed in the modulator? For what type of samples is GC x GC particularly useful? Complex samples with compounds of similar polarity Complex samples with a variety of compound polarity Complex samples with a small number of analytes (e.g. pesticides in food) Related compounds with different sized alkyl groups R(CH2)nCH3

5. Supercritical Fluid Chromatography= SFC supercritical fluid liquid + gas • What is a supercritical fluid? • has properties intermediate between a liquid and a gas • Defined by region P – T phase diagram • Phase boundary between liquid and gas disappears at critical point • Demonstrated by heating two phase system • Fluid must operate above critical T and critical P heat supercritical fluid solid critical P liquid Pressure critical point gas initial state Temperature critical T

6. Supercritical Fluid Chromatography • Mobile Phase • Supercritical fluid • Most common fluids are: • 100% CO2 (31.3°C critical temp. + 71 bar critical temperature) • Mixture of CO2 and polar modifier (e.g. methanol), where modifier added to adjust retention • Properties vs. gases and liquids • densities a little below liquids (solute – solvent interactions matter, unlike ideal gases) • viscosities a little greater than gases – allowing minimal pressure drop vs. liquids • diffusion coefficients closer to but greater than liquids, minimizing C-term broadening vs. liquids • Properties generally result in efficient separations that can be applied to a greater class of compounds than by GC

7. Supercritical Fluid Chromatography • Stationary Phase • Since carbon dioxide is non-polar, polar stationary phase is most common • Both OT columns (smaller diameters since diffusion is slower than in GC) and packed columns can be used • Columns can be set up to take advantage of smaller pressure drops of supercritical fluids vs. liquids of using smaller particle sizes (or OT columns) as well as longer column

8. Supercritical Fluid Chromatography • Instrumentation • Equipment is somewhat specialized since it requires: • pumps to reach supercritical pressures • a way to add polar modifiers • heaters to keep temperatures supercritical • a restrictor after the column or detector to ensure pressure remains high • all fittings must be able to handle higher pressures • Gradients can be set up by density programming • Both GC type (e.g. FID) and many HPLC detectors (e.g UV detection) can be used with some modifications). However, FID is limited to 100% CO2 mobile phase • Fraction collection (which is popular) uses cyclones to trap solids released upon degassing • The combination of specific equipment requirements and limited market has made instrumentation expensive

9. Supercritical Fluid Chromatography • Applications • Analyses where neither GC nor HPLC functions well (e.g. polymers without chromophores – polyethylene glycol) • Recent interest has been in high value preparative separations: • SFC uses expensive packing material (e.g. chiral stationary phases) more efficiently than HPLC • SFC solvent is cheaper than HPLC • Post column solvent removal occurs with chromatography (as opposed to in additional step) • Limitations • Expensive equipment (partly a result of limited market) • Limited mobile phases

10. Supercritical Fluid ChromatographyQuestions • How is the lower viscosity of SFs an advantage over liquids in terms of chromatography? • Why are smaller diameter OT columns used in SFC than in GC? • A drug manufacturer currently is using preparative HPLC to isolate a single enantiomer of a chiral mixture. List two advantages and one disadvantage in going to SFC. • SFC is being used with 98% CO2/2% methanol to separate polyethylene glycols with a polar stationary phase. The separation of different sized polymers is good but the retention factors are somewhat high causing the separation to take too long. What can be changed to decrease the separation time?

11. Liquid ChromatographyClassification of Types • Classification based on pressure: • Low pressure (gravity based, preparative) • Moderate pressure (pressure from compressed gases in “flash chromatography” or from low pressure pumps, also mainly preparative) • High pressure (high performance or HPLC) • Ultra-high pressure (UPLC) • Classification based on separation mechanism: • Normal-phase (polar stationary phase, less polar mobile phase) • Reversed-phase (non-polar stationary phase, more polar mobile phase) • Size exclusion chromatography (separation of molecules based on size) • Ion exchange chromatography (exchange of ions) • Other types (ligand exchange and affinity)

12. Liquid ChromatographyStationary Phases – Packing Material Geometry • No geometry comparable to OT GC • Packed Columns • Irregularly shaped particles (older technology) • Larger A term • Less robust due to particles breaking • Spherically shaped particles • Smaller particles: decrease H (smaller A and C terms), but increase back pressure Advantage: H ~ kdp Disadvantage: P = k/d2p (constant column dimensions) Ultra performance LC (UPLC) for needed P

13. Liquid ChromatographyStationary Phases – Packing Material Geometry • Packed Columns (cont.) • Superficially porous spheres • Reduces C term without increasing P (much) • Less sample capacity • Monolithic Columns • Single “web” of polymer (usually silica based) rather than separate particles • Advantageous because of high efficiency with relatively low back pressure Dense core Porous outer shell

14. Liquid ChromatographyStationary Phases – Packing Material Composition • Packing Material Composition • Silica based packing material • Most commonly used material (many advances tried first with silica) • Available unbonded (normal phase), a wide variety of bonded phases and silica hydride (relatively new) • Main disadvantages are stability of silica (in normal phase) and of Si-OR bonds (limits pH to 2 to 8) • Polymeric (all hydrocarbon) packing material • Usually styrene-divinylbenzene • Poorer performance (efficiency, maximum pressure) • Common with ion-exchange and size exclusion • “Bonded Phase” contains ion exchange sites

15. Liquid ChromatographyStationary Phases – Packing Material Composition • Other packing material • Zirconia • More temperature/solvent stable than silica • Both with and without bonded phases • Porous graphitic carbon • non-bonded reversed phase (similar to phenyl groups) • highly stable • Silica – polymeric hybrid particles • Silica core with polymer between silica and bonded phase • Improves stability of silica (possible to run from pH 0 to 14)

16. Liquid ChromatographySilica-Based Normal Phase • Historically, normal phase HPLC was the first type regularly used and the stationary phase was (uncoated, non-bonded) silica • Stationary phase = silica surface • Silica surface is somewhat heterogeneous (can have Si-OH groups, Si-O- groups, Si-O-Si groups, and adsorbed water) • Use has decreased due to replacement by bonded phase • Main disadvantage is slow equilibration (especially with regards to water) and requirement of consistent water% in mobile phase

17. Liquid ChromatographySilica-Based Normal Phase • Mobile phases • Normal phase requires mobile phases of low polarity (to be opposite to stationary phase) • Most common with hexane with polar additive although wide variety of solvents possible (but only low % water) • Weak solvent = hexane (or other less polar solvent) • Strong solvent = polar additive (e.g. 2-propanol) • Increasing strong solvent decreases retention

18. Liquid ChromatographySilica-Based Normal Phase • Mobile Phase • Other Factors in solvent selection: • Selectivity (different solvents will have different solvent – analyte interactions; best to choose solvent that emphasizes analyte differences) • Solvent viscosity (low viscosity means smaller back pressure for given flow rate) • Solvent miscibility • Detector limitations (e.g. wavelength cut-offs for UV detection) • Compatibility with column packing and tubing

19. Liquid ChromatographySilica-Based Normal Phase • Advantages: • Simpler stationary phase • Useful for wide range of analyte polarities • Disadvantages: • Long stabilization times • Retention time drifts • Must control % water in solvents

20. Liquid ChromatographyBonded Phases • Most common on silica substrate but also common with other substrates • Bonded layer is stationary phase • Common bonded phases • Reversed phase: • C18 (most common by far of all bonded phases) • C8 (shorter alkyl chain) • Phenyl (better retention of aromatic groups) • Other groups (e.g. cholesterol) • Normal phase or hydrophilic interaction (HILIC): • Cyano (most stable) • Diol (glycophase in text) • Amino (least stable)

21. Liquid ChromatographyBonded Phases • Quality of column (particularly for C18) depends on: • % of SiOH bonded to C18 group • % of remaining SiOH left (many groups “end capped”) • Bulk of side groups • Normal phase stationary phase has a greater tendency to decompose Side group (CH2)17CH3 R Si R CH3 O O Si Si Bulk SiO2 End capped Si

22. Liquid ChromatographyC18 Phase • Stationary Phase • Very non-polar group; but base of C18 is polar (similar in polarity to octanol) • Some columns use imbedded polar groups • Mobile Phase • Polar mobile phase needed • Most often water with polar additive (acetonitrile, methanol, and tetrahydrofuran (THF) most common) • Water is weaker solvent, organic modifier is stronger solvent • Except for columns with imbedded polar groups, minimum % organic is 10% (needed to “wet” C18 phase)

23. Liquid ChromatographyPolar Bonded Phase • Groups like –CN (cyano) provide separations similar to unbonded silica but reach equilibrium faster and have more consistent chromatograms • Hydrophillic interaction chromatography (HILIC) refers to polar stationary phase with water present in mobile phase (usually in small amounts) • The mechanism in HILIC is somewhat different because the stationary phase has a higher % water (due to strong attraction to polar stationary phase) than the mobile phase. • Analyte interaction can occur with stationary phase or with associated water Note: Penetration of mobile phase into stationary phase can also occur to some extent with other bonded phases (even with C18), especially with stronger solvents

24. Liquid ChromatographyBonded Phases • Advantages: • Faster equilibration (vs. silica) • Absorption vs. adsorption (less tailing) • Greater stationary phase volume • Disadvantages: • Often have reduced range of analyte polarities (very polar analytes are very weakly retained by C18) • Column bleed (if bonded layer starts degrading)

25. Liquid ChromatographyGradient Elutions • Similar to temperature programming in GC • Program increases % strong solvent during run • Example for HILIC (Normal Phase) HPLC to right • Advantageous when wide variety of analyte polarities (plus peak width and S/N advantages) • Disadvantages: • see those mentioned for GC plus • Additionally column equilibration takes time (gradient run took longer when including equilibration time) • Requires more equipment Elution of glucose oligosaccharides: glucose (monomer) to maltoheptaose (7 units) Isocratic run (~62% ACN/38% water) Gradient run (65% ACN to 50% ACN)

26. Liquid ChromatographySize Exclusion Chromatography large • Basis for separation: • Analyte inclusion in pores • Smaller analytes fit in pores and are retained more • Larger analytes pass around packing material • Used for separation of polymers based on size • Variety of packing geometries can be used (best if weaker analyte – stationary phase interactions) • Pore size is critical and affects retention medium small

27. Liquid ChromatographySize Exclusion Chromatography • Applications: • Coarse separations (monomers from oligomers or oligomers from polymers) • Determination of polymer/oligomer molecular weight • When used for determination of molecular weight, standards must also be run • Standards should be similar and have similar retention http://www.phenomenex.com/cms400min/litlib/brands/asahipakgfcolumns.pdf

28. Liquid ChromatographyIon Exchange Chromatography (IC) • Stationary phase is made of ionic groups attached to polymeric support • Ionic groups have opposite charge of solute ions being separated (anion exchange requires cationic groups and cations require anionic groups) • Anion exchange can be “strong” –NR3+ (permanent charge) or “weak”, -NH3+ (charged at low pH) • Cation exchange also uses permanently charged (e.g. –SO3-) or groups charged at higher pH ( –CO2-) • Intermolecular forces are very strong (so strong that without an exchanging ion, K is impractically large) • Mobile phase is aqueous buffer (cations or anions needed to exchange with those on column) • Strong eluents are higher concentrations and/or more strongly bound ions

29. Liquid ChromatographyIC Separation of Ions • Strength of ion-ion interaction depends on: • Charge (stronger for more highly charged) • Ion size (smaller hydrated size interacts more strongly) • Cation ranking: Li+ (weak) < H+ < NH4+ < Mg2+ < Ca2+ • Anion ranking: F- (weak) < OH- < Cl- < NO3- < SO42- • pH also affects ion forms and elution Cl- SO4= A- A- A- A- A- A- A- Note: cartoon ignores effect of mobile phase counter ions

30. Liquid ChromatographyOther Stationary Phases • Ligand Exchange (e.g. sugar separation) • retention based on ligand sticking to metal (or visa-versa) • Affinity Chromatography/ Molecular Imprinted Stationary Phases • Stationary phase designed toward retention of specific groups or compounds • Zwitterionic Phases • contains both + and - groups HO-R SO3- Ca2+ SO3- stationary phase analyte

31. Liquid Chromatography • Why is bonded silica generally the first type of stationary phase considered? • What pH range could be used if separating an organic weak acid (RCO2H) with a pKa of 5.2 using a silica based C18 column? • What type of reversed-phase stationary phase could be used to separate alkyl amines (bases with conjugate acids of pKa of about 9)? • Is it possible to run some silica-based columns at extreme pH values? • What type of packing material should be chosen for high temperature separations? • List two examples of stationary phases that could be used with normal phase HPLC. • Which of the following additives could be used to retain ClO4- on a C18 column? a) CH3NH2 b) (CH3CH2CH2CH2)4N+ c) CH3SO3- d) CH3(CH2)9SO3-

32. Liquid ChromatographySome More Questions • In a silica based NP-HPLC separation with hexane and ethyl acetate, the % of which solvent should be increased to decrease retention? • What can cause tailing in poor quality C18 columns? • A silica based NP-HPLC separation is using ether/2-propanol to elute steroids. The retention factors are too small leading to resolution issues even when using 100% ether. Suggest an eluent that would lead to better retention. • What are the advantages and disadvantages of using bonded polar phases vs. silica? • What types of compounds are least retained in size-exclusion chromatography? • What groups are present on the surface of packing material in a cation exchange column?

33. Liquid ChromatographyInstrumentation – Mobile Phase Delivery Mobile Phase Selection See slide 18 of lecture for factors influencing selection of mobile phase Solvents must meet purity requirements (for column and detector functions) Solvent selectivity issue is important because: Changing solvent affects retention for different analytes differently HPLC is less efficient than GC so often more likely to have overlapping peaks Changes in pH also are important for acidic/basic compounds

34. Liquid ChromatographyInstrumentation – Mobile Phase Delivery Example of solvent changes to affect selectivity: RP-HPLC Separation of syringols from guaiacols Difference is in 2nd MeOH group Water/Acetonitrile eluents produce poor syringol/guaiacol separation factors Water/Methanol works better (although greater retention with MeOH of syringol is counter intuitive) Less retention More retention Syringols Guaiacols

35. Liquid ChromatographyInstrumentation – Mobile Phase Delivery Optimization of Mobile Phase Composition Separation should be perfomed on three different water/organic systems Then additional separations can be carried out using 3 component mobile phases Patterns in retention can be used to optimize mobile phase composition Acetonitrile (40% in water) 20% ACN, 25% MeOH, water THF (30% in water) Methanol (50% in water)

36. Liquid ChromatographyInstrumentation – Mobile Phase Delivery Mobile Phase Selection – pH Buffering In reversed-phase HPLC, solute generally must be non-ionized to be retained pH is adjusted by adding buffer in water/organic modifier pH at pKa means retention factor about half of non-ionized acid retention time In ion-exchange chromatography, pH should be in range needed to produce ions In ion-pairing RP-HPLC, an ion-pairing reagent is added retained unretained Ion pair reagent = pentane sulfonic acid (sodium salt) Benzyl amine (conj. acid pKa = 9.35) Non-ionized only at high pH

37. Liquid ChromatographyInstrumentation – Mobile Phase Delivery Solvent Flow HPLC requires high pressures and thus specific pumps The solvent also needs low levels of dissolved gases for pumps to function For the simplest “dedicated” HPLC, a single solvent reservoir and pump is needed For gradients and/or more method development work, switching between different solvents is needed

38. Liquid ChromatographyInstrumentation – Mobile Phase Delivery Pumps Most pumps use two piston heads 180º out of phase to reduce pressure fluctuations Solvents go into and out of piston heads through one-way “check valves” Exit check valve closes on “in” stroke and entrance check valve closes on “out” stroke In Stroke Check valves Out Stroke closed open closed open pistons

39. Liquid ChromatographyInstrumentation – Mobile Phase Delivery Example of pump with non-functioning check valves Fluctuation in pressure and signal can occur Changes to retention time also will occur Bad check valve leaking

40. Liquid ChromatographyInstrumentation – Mobile Phase Delivery Solvent Flow (for gradient/greater flexibility operations) Dual Pumps (high pressure mixing) Low Pressure Mixing (stream “open” in proportion to fraction) To column To column pump Mixing chamber pumps