Chem. 230 – 11/4 Lecture

1. Chem. 230 – 11/4 Lecture

2. Announcements I • Exam 1 today • No Class Next Tuesday • 11/18 and 11/25 on remaining topics • Special Topics Presentations • Sign up for Presentation • Dates: • 11/25 (one group – if desired, not required) • 12/2 (4 or 5 groups – also have exam 4) • 12/9 (5 or 6 groups) • Need to prepare reading material (link to journal or photocopies in folder) one week before presentations

3. Announcements II • Today’s Lecture • HPLC • Aerosol-Based Detectors (in more detail) • Quantification • Performance Measures • Sensitivity Considerations • LOD and LOQ Calculations • Data Smoothing and Integration • Methods of Calibration • Mass Spectrometry (Introduction and instruments)

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

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

6. 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

7. 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

8. 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

9. 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

10. 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) 

11. 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

12. 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

13. 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).

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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.

22. Aerosol-Based Detectors for HPLCReferences For Applications: (See my faculty web page for CAD references) Foods: Asa, D., Carbohydrate and oligosaccharide analysis with a universal HPLC detector, Am. Laboratory, 38, 16, 18, 2006. Moreau, R. A..  The analysis of lipids via HPLC with a charged aerosol detector, Lipids, 41, 727-734, 2006. Lísa, M., F. Lynen, M. Holčapek, and P. Sandra, Quantitation of triacylglycerols from plant oils using charged aerosol detection with gradient compensation Pharmaceuticals: Loughlin, J., H. Phan, M. Wan, S. Guo, K. May and B. Lin, Evaluation of charged aerosol detection (CAD) as a complementary technique for high-throughput LC-MS-UV-ELSD analysis of drug discovery screening libraries, Am. Laboratory, 39, 24-27, 2007. Sun, P., X. Wang, L. Alquier, C. A. Maryanoff, Determination of relative response factors of impurities in paclitaxel with high performance liquid chromatography equipped with ultraviolet and charged aerosol detectors, J. Chromatogr., A, 1177, 87-91, 2008. Biotechnology: Inagaki, S., J.Z. Min, and T. Toyo’oka, Direct detection method of oligosaccharides by high-performance liquid chromatography with charged aerosol detection, Biomed. Chromatgr., 21, 338-342, 2007. Atmospheric Aerosols: Dixon, R. W. and G. Baltzell, Determination of levoglucosan in atmospheric aerosols using high performance liquid chromatography with aerosol charge detection, J. Chromatogr.A, 1109, 214-221, 2006.

23. Aerosol-Based Detectors for HPLCQuestions For a complicated sample with several analytes present at moderate concentrations (around 50 μg mL-1), is it advantageous to use an ELSD (vs. a UV Detector) 1) if the compounds are weak absorbers, 2) if the compounds are strong absorbers? What instrument components will ELSD and CNLSD have in common that are not present in CAD? ABDs can not detect volatile analytes. How should weakly absorbing volatile compounds be determined? With a single calibration standard (over different concentrations), is it possible to estimate concentrations of unknown compounds (e.g. for compounds without any standards)? and under what conditions? Protein concentration can be estimated by looking at absorption from aromatic amino acids? Why might using an ABD be a better way of quantifying unknown proteins?

24. Quantitation in ChromatographyOverview • Performance Measures • Detector Response • Levels of Detection and Quantification • Data Smoothing • Integration • Calibration Methods

25. Quantitation in ChromatographyPerformance Measures • Precision • How reproducible a measurement is • Accuracy • How close measured concentration is to true value • Sensitivity • The ability to measure small concentrations or amounts of analyte • Selectivity • Can be an issue in quantification when overlapping/interfering peaks occur • % Recovery • % of analyte added to sample that is measured in sample

26. Quantitation in ChromatographyDetector Response • Concentration Type vs. Mass Flow Type • In concentration type, signal depends on analyte in sample cell; so generally flow independent • In mass flow type, signal depends on mass transport to detector (e.g. in FID without compounds entering flame, no signal will result) • Note: for some mass flow (HPLC-ABDs and HPLC-MS) transport efficiency depends on liquid flow so signal is not directly proportional to flow rate Mass Flow Detector Concentration Detector flow on flow off flow off flow on Time Time

27. Quantitation in ChromatographyDetector Response • Concentration Type - examples: • PID (GC) • UV-Vis (HPLC) • Fluorescence (HPLC) • Mass Flow Type - examples: • FID (GC) • NPD (GC)

28. Quantitation in ChromatographyDetector Response • Detector Signal • Depends on concentration of analyte or mass of analyte reaching detector • Most (but not all) detectors give linear response over portion of detectable range • Detector Noise • Present in all detectors • High and low frequency types • Ability to Detect Small Quantities Depends on Signal (Peak Height) to Noise Ratios

29. Quantitation in ChromatographyLevels of Detection and Quantification • Noise can have high and low frequency parts • Ways of defining noise • peak to peak (roughly 5σ) • standard deviation (more accurate way) • Signal = peak height high frequency component peak to peak noise low frequency component

30. Quantitation in ChromatographyLevels of Detection and Quantification • Limit of Detection (LOD): • minimum detectable signal can be defined as S/Npeak-to-peak = 2 or S/σ = 3.3 • minimum detectable concentration = concentration needed to get S/Npeak-to-peak = 2 or S/σ = 3.3 • Calculate as 2N/m where m = slope in peak height vs. conc. calibration plot • Minimum detectable quantity = (minimum detectable conc.)(injection volume) • Limit of Quantification (LOQ): • Calculated in similar fashion as LOD • Lowest concentration to give an “reasonable” conc. (e.g. can be “auto-integrated” using software) • Typically 5∙LOD

31. Quantitation in ChromatographyData Smoothing • Data should be digitized with a frequency ~20/peak width • High frequency noise (where fnoise >> fsignal) can be removed by filtering • see example below • note: overfiltering results in reduction of signal and loss of resolution • overfiltering result also can occur if detector response is too slow (or cell volume is too large • Difficult to remove noise with frequency similar to or lower than peaks

32. Quantitation in ChromatographyIntegration • Integration of peak should give: • peak height • peak area • peak width (often just peak area/peak height) • Difficulty comes from determining if a peak is a peak (or just noise), and when to “start” the peak and “end” the peak. • Can use “auto integration” or “manual integration” but not these noise spikes we want to pick up this peak

33. Quantitation in ChromatographyIntegration • Other issues in integration (besides noise peaks) • start and ends to peaks • how to split overlapping peaks

34. Quantitation in ChromatographyIntegration • Peak Height vs. Peak Area • Reasons for using peak area • peak area is independent of retention time (assuming linear response), while the peak height will decrease with an increase in retention time • peak area is independent of peak width, while the peak height will decrease if the column is overloaded (non-linear response) • Reasons for using peak height • Integration errors tend to be smaller if samples are close to the detection limits

35. Quantitation in ChromatographyLOD/LOQ example • Determine the LODs and LOQ for the following example. Determine it for the 4.6 min peak if the concentration is 0.4 ng μL-1. Use the 3.3 and 2N LOD defintions.

36. Quantitation in ChromatographyCalibration Methods • External Standard • most common method • standards run separately and calibration curve prepared • samples run, from peak areas, concentrations are determined • best results if unknown concentration comes out in calibration standard range • Internal Standard • Common for GC with manual injection (imprecisely known sample volume) • Useful if slow drift in detector response • Standard added to sample; calibration and sample determination based on peak area ratio • F = constant where A = area and C = conc. (X = analyte, S = internal standard) Area Concentration AX/AS Conc. X (constant conc. S)

37. Quantitation in ChromatographyCalibration Methods Standard Addition Used when sample matrix affects response to analytes Commonly needed for LC-MS with complicated samples Standard is added to sample (usually in multiple increments) Needed if slope is affected by matrix Concentration is determined by extrapolation (= |X-intercept|) Surrogate Standards Used when actual standard is not available Should use structurally similar compounds as standards Will work with some detector types (FID, RI, ABDs) standards in water Area Analyte Concentration Concentration Added

38. QuantitationAdditional (Recovery Standards + Questions) Recovery Standards Principle of use is similar to standard addition Standard (same as analyte or related compound) added to sample, then measured (in addition to direct measurement of sample) Useful for determining losses during extractions, derivatization, and with matrix effects

39. QuantitationSome Questions/Problems Does increasing the flow rate improve the sensitivity of a method? Does the use of standard addition make more sense when using a selective detector or a universal detector? Is a matrix effect more likely with a simple sample or a complex sample? Why is the internal standard calibration more common when using manual injection than injection with an autosampler?

40. QuantitationSome Questions/Problems 5. A scientist is using GC-FID to quantitate hydrocarbons. The FID is expected to generate equal peak areas for equal numbers of carbons (if substances are similar). Determine the concentrations of compounds X and Y based on the calibration standard (1-octanol). X = hydroxycyclohexane and Y = hydroxypentane.

41. QuantitationSome More Questions/Problems 6. A chemist is using HPLC with fluorescence detection. He wants to see if a compound co-eluting with a peak is quenching (decreasing) the fluorescence signal. A set of calibration standards gives a slope of 79 mL μg-1 and an intercept of 3. The unknown gives a signal of 193 when diluted 4 mL to 5 mL (using 1 mL of water). When 1.0 mL of a 5.0 μg mL-1 standard is added to 4.0 mL of the unknown, it gives a signal of 265. What is the concentration of the unknown compound and is a significant quenching (more than 10% drop in signal) occurring?

42. QuantitationSome More Questions/Problems 7. A chemist is testing an extraction process for removing DDT from fish fat. 8.0 g of fat is first dissolved in 50 mL of 25% methylene chloride in hexane. The 50 mL is divided into two 25 mL portions, one of which is spiked by adding 2.0 mL of 25.0 ng mL-1 DDT. Each portion is run through a phenyl type SPE cartridge and the trapped DDT is eluted with 5.0 mL 100% methylene chloride. The methylene chloride is evaporated off, and the sample is redissolved in 0.5 mL of hexane and injected onto a GC. The un-spiked sample gives a DDT conc. (in 0.5 mL of hexane) of 63 ng mL-1, while the spiked sample gives a DDT conc. of 148 ng mL-1. What is the % recovery? What was the original conc. of DDT in the fat in ppb?

43. Mass SpectrometeryOverview Applications of Mass Spectrometry Mass Spectrometer Components GC-MS LC-MS Other Applications

44. Mass SpectrometeryApplications Direct Analysis of Samples Most common with liquid or solid samples Reduces sample preparation Main problem: interfering analytes Off-line Analysis of Samples Samples can be separated through low or high efficiency separations More laborious Chromatographic Detectors generally most desired type since this allows resolution of overlapping peaks

45. Mass SpectrometeryApplications Purposes of Mass Spectrometry Quantitative Analysis (essentially used as any other chromatographic detector) Advantages: selective detector (only compounds giving same ion fragments will overlap) overlapping peaks with same ion fragment can be resolved (through deconvolution methods) semi-universal detector (almost all gases and many solutes in liquid will ionize) very good sensitivity Disadvantages cost requires standards for quantification

46. Mass SpectrometeryApplications Purposes of Mass Spectrometry - continued Qualitative Analysis/Confirmation of Identity With ionization method giving fragmentation, few compounds will produce the same fragmentation pattern Even for ionization methods that don’t cause fragmentation, the parent ion mass to charge data gives information about the compound identity. Some degree of elemental determination can be made based on isotopic abundances (e.g. determination of # of Cl atoms in small molecules). Additional information can be obtained from MS-MS (further fragmentation of ions) and from high resolution mass spectrometry (molecular formula) if those options are available. Isotopic Analysis Mass spectrometry allows analysis of the % of specific isotopes present in compounds (although this is normally done by dedicated instruments) An example of this use is in drug testing to determine if testosterone is naturally produced or synthetic

47. Mass SpectrometeryInstrumentation Main Components: Ion source (more details on subsequent slides) Analyzer (more details on subsequent slides) Detector: most common is electron multiplier Detection Process: Ion strikes anode Electrons are ejected Ejected electrons hit dynodes causing a cascade of electron releases Current of electrons hitting cathode is measured Anode Dynodes Cathode M+ e- e- I