Chem. 230 – 10/28 Lecture

1. Chem. 230 – 10/28 Lecture

2. Announcements I • HW Set #3 – due today (short answer problems have been posted) • Next Exam: Topics + format (still can bring 3” x 5” notecard) • Gas Chromatography • Supercritical Fluid Chromatography • HPLC (everything except detection) • Return Application Abstracts • missing a few • most looked good; a few seemed to be focused on technology rather than application to a particular problem (check to see if you need to improve on your abstract)

3. Announcements II • Today’s Lecture • HPLC • covered so far: classification, packing material geometry and composition, gradient elution, size exclusion chromatography, ion exchange chromatography • Instrumentation (mobile phase selection and delivery, injection, column dimensions, detection) • Aerosol-Based Detectors (in more detail)

4. 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

5. 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

6. 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)

7. 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 factor 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

8. 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 (through solvent degassing) 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

9. 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

10. 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

11. 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

12. Liquid ChromatographyInstrumentation – Injection Fixed Loop Injectors (see GC slides for diagram) Used in almost all cases For some injectors, partial filling of loop is possible (Vinj < Vloop), but then filling precision must be good Special injection valves needed for small injections (< 1 to 5 μL) Small injections often needed for microbore columns Other Injectors Traps replace loops (can be used if sample is in weak solvent) SPME (not as common as for GC but with solvent removing trapped compounds) SPME requires special injector

13. Liquid ChromatographyInstrumentation – Injection • Sample Matrix • Best chromatography solvent – should be weaker than mobile phase, particularly for larger volume injections • Remember, weaker solvent allows on-column concentrating • With traps, sample must have weaker solvent, but must be pulled off with significantly stronger solvent so pulled off in narrow injection plug • Other concern can be solvent miscibility and solute solubility (example: in reversed phase HPLC, water is a good solvent, but many compounds such as aromatic compounds have limited solubility in weakest solvents)

14. Liquid ChromatographyInstrumentation – Columns Column dimensions Length: balance between flow, pressure and efficiency Diameter: Choice depends on separation purpose Preparative for isolation of larger quantities Microbore usually results in smaller mass detection limits but greater concentration detection limits (good when limited sample) Special care is needed using microbore with sample injection, pump stability, and extra-column broadening (tubing diameter and fitting connections)

15. Liquid ChromatographyInstrumentation – Columns Column dimensions Equation for extra-column broadening: Extra-Column broadening is more of a problem when using 1) low H columns, 2) early eluting peaks (where Wcol is small) Demonstration of Extra-Column Broadening on Narrowest Bore Columns note: W can have time or volume dimensions, but hard to get very small volume W for tubing, and some detection

16. Liquid ChromatographySome Questions A student is running a RP-HPLC separation using methanol and water. The selectivity (a value) is not good. He decides to switch to ethanol in water. Is this a good decision? A chemist is planning on purchasing an HPLC instrument for developing isocratic analysis methods. Is there an advantage to being able to select multiple solvents? In order to decrease H in a column, which column or packing material dimension should be changed? and in which direction? Why would one want to go to a microbore HPLC system? Why is the decrease in H observed often less than predicted when using smaller diameter packing material or small diameter columns? If injecting large volumes of a sample containing trace levels of benzoic acid in water for a reversed phase separation, will it make any difference what the pH of the sample is? In anion exchange chromatography, what type of sample would allow on-column trapping? What type of samples would give broad peaks if using large injection volumes?

17. Liquid ChromatographyInstrumentation – Detectors Some Generalizations Relative to GC, HPLC detectors perform poorly and cost more Universal Type UV absorption (also considered selective) Refractive Index Aerosol-based detectors (will cover later) Conductivity (for ion chromatography) Selective Type Fluorescence Electrochemical Hyphenated Detectors Photodiode Array Detector (type of UV detector) Mass Spectrometer

18. Liquid ChromatographyInstrumentation – Detectors UV Absorption Detectors The most common type of detector Principle: absorption of ultraviolet (or visible) light Follows Beer’s Law: A = -log(I/Io) = εbC I = intensity of light (Io for blank) ε = molar absorptivity (constant) b = path length C = concentration Best results for 0.001 < A < 1 Fast response – sensitivity trade off in path length (can select cell volumes) Light beam b Cell

19. Liquid ChromatographyInstrumentation – Detectors UV Absorption Detectors Sensitivity to Compounds (ε values) Best for compounds with conjugated double bonds, aromatic groups or strongly absorbing functional groups (e.g. R-NO2, R-I, R-Br) Poor response for compounds with few or weakly absorbing functional groups (worst for R-CN, R-NH2, R-F; poor for R-OR’, R-OH, R-COOH, R-COOR’) Solvents: Requires use of solvents that absorb poorly in UV

20. Liquid ChromatographyInstrumentation – Detectors UV Absorption Detectors Wavelength Selection: Must choose λ > solvent cut-offs Most compounds absorb strongly at short wavelengths but many also absorb less moderately at longer wavelengths More sensitivity at shorter wavelengths (provided little mobile phase absorption) More selectivity at longer wavelengths solvent analyte 180 220 260 Wavelength (nm) Sensitive λ More selective λ

21. Liquid ChromatographyInstrumentation – Detectors UV Absorption Detectors General Properties Reasonably good (but variable) sensitivity Good linearity, reproducibility Good stability (but baseline drift and warm up time) Poor as a universal detector Types: Fixed wavelength (absorption at single wavelength) Variable wavelength (can select one wavelength using monochromator) Photodiode array (can measure at multiple wavelengths simultaneously) – these give some qualitative information and allow more peak overlap

22. Liquid ChromatographyInstrumentation – Detectors Application of UV Detection to Weak Absorbers Use short wavelengths (method must be selective; not always effective) Derivatize compounds to add strong absorber (common for amino acids, carbohydrates) Use indirect UV absorption (absorber added to eluent, analytes displace eluent and give negative peak)

23. Liquid ChromatographyInstrumentation – Detectors Refractive Index Detectors Principle: liquids with different refractive index will diffract light differently Composition will determine refractive index Any compound with a refractive index different than the solvent’s is detectable Advantage: Most universal detector (can detect weakly absorbing compounds) Disadvantages: Gradients are not possible Requires thermal stability Generally not very sensitive

24. Liquid ChromatographyInstrumentation – Ion Exchange Chromatography • Types of Instruments: • Single column • With analytical plus suppressor columns • Detection in Single Column Instruments • Other detection methods (fairly common) • Conductivity detection • Conductivity Detector • Resistance measured (AC circuit) • Conductivity = 1/(resistance) • Ions in solution create conductance • Conductivity depends on ion concentration and size From HPLC column Electronics Conductivity cell

25. Liquid ChromatographyInstrumentation – IC • Difficulties with single column instruments: • Both analytes and ion exchanger conduct electricity • High concentration of ion exchanger means high background conductance and difficulties in detecting small concentrations of analytes • Often large ions used as exchanger (such as potassium hydrogen phthalate for anion exchange)

26. Liquid ChromatographyInstrumentation – IC • Suppressor Columns • The purpose of the suppressor is to convert the ion exchanger to mostly non-ionic compounds • Example below with sodium bicarbonate eluent (Na+HCO3- is the ion exchanger) in anion exchange • In the suppressor column, Na+ is replaced with H+ • This converts conductive Na+HCO3- to non-conductive H2CO3 • NaCl is converted to HCl (still conductive) • Na2HPO4 is converted to H+H2PO4- (less conductive) • This reduces the baseline and increases sensitivity Na+HCO3- Suppressor Column Separation column To Conductivity detector H+Cl- Na+2HPO42- Na+Cl- H+ In; Na+ out

27. Liquid ChromatographyInstrumentation – Detectors Electrochemical Detectors Principle: Redox reactions occur at electrodes following column Potential cycle used to periodically oxidize/reduce analytes at electrode Current depends on concentration of analyte being reduced or oxidized (similar to A in UV detector) Electrode potential determines classes of compounds that are detectable (similar to λ in UV detector) From column Analyte electrode Referenceelectrode Voltage supply/ electrometer

28. Liquid ChromatographyInstrumentation – Detectors Electrochemical Detector Advantages: Very sensitive (limits of detection under 1 pg possible) Adjustable selectivity Wide range of compounds can be detected (including UV inactive compounds) Advantageous for microbore Disadvantages Electrode fouling Variable analyte response Requires ions to “complete circuit” Array Detectors: Can have multiple electrodes in detector (set to different potentials)

29. Liquid ChromatographyInstrumentation – Detectors Fluorescence Detectors Detection Principle: Light promotes molecules to excited electronic state Excited molecules transition from lowest excited state back to the ground state and emit light in the process Equipment: High intensity light source Filters or monochromators to select wavelengths (before and after cell) Sensitive light detector M + hν→ M* M*→ M*’ (lower vibrational level) M*’ → M + hν’ Light Source Filter or monochromator Light detector

30. Liquid ChromatographyInstrumentation – Detectors • Fluorescence Detectors • Advantages: • Greater sensitivity possible (for molecules with high fluorescence efficiencies) because easy to detect small signal against zero background (see below) • Much greater selectivity because few molecules fluoresce, particularly at selected wavelengths • Disadvantages: • Limited to relatively few molecules (although derivatization is also possible) Absorption of light Emission of light 95% transparent (equiv. to A = 0.022) Weak light in black background

31. Liquid ChromatographyDetector Questions A compound has an absorptivity of 493 M-1 cm-1 at 210 nm and 32 M-1 cm-1 at 280 nm. Why would one even consider setting the wavelength to 280 nm? Describe one way to use a UV detector for detecting weakly absorbing organic compounds. Describe how you could use a photodiode array detector to determine if the odd shaped peak below is from one or multiple compounds. A (254 nm) Time

32. Liquid ChromatographyMore Questions • Why is electrochemical detection difficult to use with non-bonded silica HPLC? • When weakly absorbing compounds are derivatized, it is more common to use fluorescent derivatizing agents. Why is this? • What is the advantage of using suppression in ion chromatography? • Why is suppressed ion chromatography not so useful for weak acid anions vs. strong acid anions?

33. Aerosol-Based Detectors for HPLC Example Advanced Method Presentation

34. Aerosol-Based Detectors for HPLC Outline Introduction to Technology Theory Including Three Types of Detectors Advantages and Disadvantages of ABDs Some Applications Conclusions References

35. Aerosol-Based Detectors for HPLCIntroduction Limitations of Conventional Detectors UV Absorption Detectors: Not very universal Poor sensitivity for many classes of compounds (carbohydrates, fats, amino acids, dicarboxylic acids, etc.) Refractive Index Detectors: Low and somewhat variable sensitivity Not gradient compatible Mass Spectrometer Detectors: Not all compounds ionize readily Expensive, large, expensive to operate

36. Aerosol-Based Detectors for HPLCIntroduction Processes in Aerosol-Based Detectors: Effluent from column is nebulized producing spray of solvent and solute Spray droplets are heated in an oven, evaporating solvent gas and producing aerosol particles from solute Aerosol passes to an aerosol detector to produce a signal Nebulizer Spray Chamber N2(g) HPLCColumn Oven Aerosol Detector particle droplet

37. Aerosol-Based Detectors for HPLCIntroduction Mobile Phase Requirements Solvent must be volatile (and cause little column bleed) Analyte Requirements Works best if analyte is non-volatile Semi-volatile compounds give reduced response

38. Aerosol-Based Detectors for HPLCTheory Nebulization produces a distribution of drop sizes Solvent viscosity and surface tension can affect distribution of droplet sizes Evaporation shifts this to distribution of particle sizes based on: where: dd, dp are drop and particle diameters, C is mass concentration, and ρp is particle density Size Distributions

39. Aerosol-Based Detectors for HPLCTheory Types of Aerosol-Based Detectors Depends on method of detecting aerosol particles Evaporative Light Scattering Detection (ELSD) (Charlesworth, J. M. Anal. Chem.1978, 50, 1414) Condensation Nucleation Light Scattering Detection (CNLSD) (Allen, L. B.; Koropchak, J. A. Anal. Chem.1993, 65, 841) Charged Aerosol Detector (CAD)/Aerosol Charge Detector (Dixon, R. W.; Peterson, D. S. Anal. Chem., 2002, 74, 2930) 

40. Aerosol-Based Detectors for HPLCTheory ELSD principles Detection by light-scattering by particles Efficient detection when dp ~ λ; less efficient at other sizes Non-linear response results At low concentrations, dp < λ so sensitivity is poor (detection limits of around 0.1 to 1 μg mL-1) Detector concentration Expanded Region

41. Aerosol-Based Detectors for HPLCTheory Condesation Nucleation Light Scattering Detection Detection principle also uses particle light-scattering but overcomes poor detection of small particles by growing small particles to bigger particles by condensation of vapor on to particles This technology is very sensitive (a single 3 nm particle can be detected) This can translate to very low detection limits (~10 ppb or ~50 pg) under optimal conditions Commercialized recently Particles In Butanol condensor To light-scattering detector

42. Aerosol-Based Detectors for HPLCTheory Charged Aerosol Detection Particles charged as aerosol jet collides with ion-rich jet from corona discharge (commercial version) Charged particles are collected on a filter with charge passed to electrometer (current measured) In another version, particles are charged as they pass near a corona discharge region Sensitivity has equalled CNLSD (at least at standard HPLC flows) Large response range and linearity at lower concentrations Aerosol In Corona Discharge Wire Ion Filter (negatively charged rod) To Electrometer Aerosol Filter Gamache et al., LCGC North America (2005).

43. Aerosol-Based Detectors for HPLCAdvantages and Disadvantages Advantages: Better performing universal detectors than refractive index detectors Universal response for non-volatile analytes CNLSD and CAD sensitivity is similar to typical UV sensitivity Disadvantages: Requires analytes of low-volatility, volatile mobile phases CNLSD and CAD are often limited by solvent purity and column bleed Non-linear calibration often is needed Cost is higher than UV Detectors

44. Aerosol-Based Detectors for HPLCSome Applications Food ELSD has been used extensively to characterize carbohydrates and lipids. Methodology requires no derivatizations and allows analysis of whole lipids (as opposed to just fatty acids) Polymers (with SEC) Useful for polymers without chromophores Pharmaceutical Industry ABDs are useful for assessing contaminants in pharmaceutical products Biotechnology and Environmental Samples Greater potential with CNLSD and CAD for analyzing low concentration samples (some carbohydrate examples) Analysis of Cations, Anions and Neutrals Use in combination with zwitterionic stationary phase allows simultaneous detection of three categories in single run

45. Aerosol-Based Detectors for HPLCTriglyceride Example • By Lísa et al (J. Chromatogr. A, 1176 (2007) 135-142). • Homogenous trigylcerides shown above without (left) and with “gradient compensation” (right) • Gradient compensation allows response to remain proportional to area with a gradient • An alternative is to use a 2 dimensional calibration (Hutchinson et al., J. Chromatogr. A, 1217 (2010) 7418-7427) • Gradient compensation uses 2 additional pumps pumping eluent after the column to produce a constant eluent composition • Plant oil samples shown below

46. Aerosol-Based Detectors for HPLCPaclitaxel Example • By Sun et al. (J. Chromatogr. A, 1177 (2008) 87-91). • Looked at impurities in paclitaxel (a anti-cancer natural product from Pacific yew tree) using UV and CAD • Shown in upper figure (standards – highest and stressed paclitaxel – lower) • Paclitaxel impurity response shown to be uniform by CAD but not by UV detection • Pharmaceutical impurity analysis used for determining acceptable pharmaceuticals • If no standards available, CAD provides better estimation of impurity levels

47. Aerosol-Based Detectors for HPLCSmoke Tracer Example • My work (published in Dixon and Baltzell and Ward et al. – see my research webpage) • Detected levoglucosan and related monosaccharide anhydrides • These are thermal breakdown products from cellulose and hemicellulose • It was possible to use the levoglucosan concentrations to estimate the total particulate matter (2.5) derived from woodsmoke cellulose levoglucosan Chico Winter Air Sample mannosan levoglucosan

48. Aerosol-Based Detectors for HPLCGlycan Profiling oligosaccharides • My more recent work (with Thomas Peavy, Biological Sciences) also preliminary work done by Ignaki et al. • Glycans (glycoprotein oligosaccharides) are difficult to quantify • Glycans are post-translational modifications and composition can depend on host organism/cells • Profiles change in cancer cells • Standards are unavailable or expensive • Currently running surrogate standards to prepare multi-dimensional calibration (depending on mass concentration and retention time) • Test standards show errors of ~0 to 25% Peptide backbone Frog Egg example

49. Aerosol-Based Detectors for HPLCConclusions ABDs have been replacing RID as a universal detector (at least for non-volatile compounds) ABDs can be used without exact standards for quantification (much as an FID is used in GC) Biggest limitations are volatility/non-volatility requirements, cost, and linearity

50. Aerosol-Based Detectors for HPLCReferences ELSD Text (p. 247-248) Charlesworth, J. M., Evaporative analyzer as a mass detector for liquid chromatography, Anal. Chem., 50, 1978, 1414-1420. Review: Koropchak et al., Fundamental Aspects of Aerosol-Based Light-Scattering Detectors for Separations, Adv. Chromatogr. 40, 2000, 275. CNLSD Allen, L. B. and J. A. Koropchak, Condensation nucleation light scattering: A new approach to development of high-sensitivity, universal detectors for separations, Anal. Chem., 65, 1993, 841-844. Same review listed for ELSD CAD Dixon, R. W. and D. S. Peterson, Development and testing of a detection method for liquid chromatography based on aerosol charging, Anal. Chem., 74, 2002, 2930-2937. Gamache, P.H., R.S. McCarthy, S.M. Freeto, D.J. Asa, M.J. Woodcock, K. Laws, and R.O. Cole, HPLC analysis of nonvolatile analytes using charged aerosol detection, LCGC North America, 23, 150, 152, 154, 156, 158, 160-161, 2005.