1. Chemical Separation Techniques Robert E. Synovec, Professor Department of Chemistry University of Washington CHEM 429 / 529

2. Lecture 1 Course Introduction

3. Goal – Chromatography and Related Techniques “Obtain chemical information (our primary focus) and purification (eg. organic chemistry) by chemical separation in time and space (distance), optimized to minimize required time and instrumental requirements.”

4. Analytical Chemistry and Separation Science CHEM 429/529 Start Here Data Information

5. CHEM 429/529: Integration of ideas and technologies aimed to address challenging chemical analysis problems Chemical Separation Techniques Separation Mechanisms: Chemical & Physical Instrument Design Material Science, Nanotechnology, Microfabrication Fluid & Flow Dynamics Detection & Reagent Chemistry Data Analysis Methodologies

6. Capillary Chromatography Example “There are significant demands placed upon sample injection, separation conditions, and detection in order to provide the impressive resolution with excellent detection sensitivity and selectivity.” Why is the separation of such similar compounds possible? How is the separation optimized? How are injection, separation and detection integrated together to facilitate the goal? How has the instrumentation been miniaturized to benefit the analysis?

7. Lecture 2 Chromatography – Concept and fundamental equations, illustration of separation mechanism, instrumentation, data and band broadening (BB), with thermodynamics and mass transfer kinetics.

8. CHROMATOGRAPHY DEFINITIONS AND PRINCIPLES Chromatography is a chemical analysis method based upon the physical separation in time/space of the chemical species in a mixture (i.e., analytes and interferences). Separation occurs in a column filled or coated with a stationary phase in conjunction with a mobile phase. Differential migration, and thus separation, occurs due to the relative time a given species spends in the stationary phase relative to the mobile phase, governed by a distribution constant, K D. Thus, separated species have different K D values. The retention time, t R, is the total time a given species spends in the mobile phase and the stationary phase. The separated species are detected by a suitable device and the resulting data is called a chromatogram. Information is obtained from chromatograms.

9. Waste Pump Sample Injection Valve Computer Chromatographic Column (Stationary Phase) Detector Waste Mobile Phase Reservoir Rotates Instrumentation Applied (shown for Liquid Chromatography) Time, min 0510152025303540 Signal Chromatogram

10. Chromatography Method Summary HPLC – High performance liquid chromatography. Utilizes a liquid mobile phase and various stationary phases depending upon the separation mechanism: Partitioning, Adsorption/Desorption, Chiral, Hydrophobic Interaction, Size Exclusion, Ion Exchange, etc. GC – Gas Chromatography. A carrier gas is the mobile phase. The stationary phase is generally a thin polymer. Separation is based upon analyte volatility (boiling point) and analyte interaction with the stationary phase. SFC – Supercritical Fluid Chromatography. The mobile phase is generally supercritical carbon dioxide possibly with a polar additive such as methanol. The stationary phase is similar to those used in GC. CE – Capillary Electrophoresis. The mobile phase must contain ions, and there is either no stationary phase or the capillary is filled with a gel.

11. Chromatography Detectors HPLC, SFC and CE (liquid phase) Absorbance (single  to full spectrum) Fluorescence Electrochemical and Conductivity Mass Spectrometry (MS) …. Most definitive identification Refractive Index (RI) Polarimetry (optical activity) Light Scattering GC (gas phase) Flame Ionization (FID) Electron Capture (ECD) Thermal Conductivity (TCD) Mass Spectrometry (MS) ….. Most definitive identification

12. Chromatography Equations - - - - - Relating Thermodynamics to Separation (1)Distribution Constant: [analyte] Stationary Phase K D = --------------------------------- [analyte] Mobile Phase where [species] = molar concentration (2) Retention Time: t R = t Stationary Phase + t Mobile Phase t R = t StationaryPhase + t 0 ……..where t 0 = dead time, time in mobile phase only

13. More Chromatography Equations - - - - - Relating Thermodynamics to Separation (3)Retention Factor (aka, capacity factor) : moles analyte in SP k’ = ------------------------------ ……at any instant in time moles analyte in MP Summing all time analyte spends either in SP or MP……. k’ = t SP / t MP k’ = (t R - t 0 ) / t 0 = K D (Volume SP / Volume MP ) …recall t R = t SP + t 0 and t MP = t 0 Unique K D results in a unique t R, and resolved analyte peaks !

14. (4) Chemical Selectivity,  Therefore, t R is often unique for a given analyte and volumetric flow rate F, and is used for identification. While t R changes with F, the retention volume V R is constant: V R = t R F (thermodynamic quantity).  is a measure of selectivity, for analytes 1 and 2:  is independent of Volume SP / Volume MP (called the phase volume ratio), and independent of F, so allows simple comparison of different chromatographic systems.

15. Separation Illustration Injection Separation Detection (Chromatogram) Partition Interaction (like solvent extraction) Sample mixture with analytes: A, B, and C Polarity scale, ,  MP =  A >  B >  C >  SP H 2 O/acetonitrile “alkane” “likes are dissolved by (attracted to) likes”

16. Hildebrand Solubility Parameter,  Solvent Name  Perfluoroalkanes~6.0 n-Alkanes~8.0 Cyclohexane 8.2 Ethyl acetate 8.9 Toluene 8.9 Tetrahydrofuran 9.1 Benzene 9.2 Chloroform 9.3 Acetone 9.6 Anisole 9.7 Chlorobenzene 9.7 Propanol12.0 Acetonitrile12.1 Phenol12.1 Ethanol12.7 Methanol14.5 Ethylene glycol17.0 Water23.4 More polar Less polar

17. Miscibility of Different Solvent Pairs

18. Stationary Phase To Detector Direction of Mobile Phase Flow Step 1 – Injection of Sample Mixture: A, B, C Capillary Column Cross-Section Mobile Phase b b b b b c c c c c a a a a a Column Inlet Column Outlet

19. Step 2 – Separation of Sample Mixture: A, B, C some time later after injection…. To Detector Direction of Mobile Phase Flow b b b b b c c c c c a a a a a K D,C > K D,B > K D,A t R,C > t R,B > t R,A

20. Key Issues Achieving the desired separation is the GOAL. Positions in column and thus the t R ’s, are correlated to K D ’s, driven by thermodynamics (or other retention interactions). However, the peak band widths of the separated analytes are determined by diffusion-driven and adsorption/desorption driven kinetics of analyte mass transfer within the MP, within the SP, and between the MP and SP. Can’t get something for nothing! The separation comes at a price, but much research development has gone into obtaining desired separations with minimal peak broadening.

21. Waste Pump Sample Injection Valve Computer Chromatographic Column (Stationary Phase) Detector Waste Mobile Phase Reservoir Rotates Instrumentation Applied (shown for Liquid Chromatography) Time, min 0510152025303540 Signal Chromatogram

22. Step 3 – Detection of Sample Mixture: A, B, C “CHROMATOGRAM”

23. Separation leads to Band Broadening (BB) due to “slow” mass transfer between MP and SP. BB diminishes the ability to extract useful information. Measure of BB

24. Information in Chromatograms Analyte identification is provided by the retention time t R, i.e., the thermodynamic basis for selectivity in separation. A unique K D provides a unique t R. Match the t R of an“unknown” peak in a sample chromatogram with “known” peak in a standard chromatogram. Also, maybe couple with the standard addition method. Identification is strengthened by using a detector that provides additional analyte selectivity (eg., unique absorbance spectrum, mass spectrum, or optical activity). Analyte quantification is provided by the peak height or area, which are proportional to the injected analyte concentration (the quantity of interest in many applications). Alternatively, complex chromatograms may provide information using pattern recognition or other multivariate chemometric approaches.

25. Detector Selectivity LC of urine, with two detectors in series, but each detector ”sees” the same sample differently. Upper chromatogram is absorbance detection. Lower chromatogram is optical activity detection (polarimetry) More selective!! Abs OA, 

26. Lecture 3 Motivations to study band broadening (BB): C(t),  S(t), LOD, R s and N.

27. Detection of Sample Mixture: A, B, C “CHROMATOGRAM”

28. The GOAL of analyte separation is accompanied by peak band broadening (BB), due to on-column mass transfer effects and off-column effects. Issues to Study: (1)Analyte Concentration Dilution, which impacts Detectability (2) Analyte and Interferent Resolving Capability, which impacts Chemical Selectivity (3) Time of Analysis, optimization of (1) and (2) while ensuring confident analyte identification and quantification

29. Separation Leads to Dilution C(t R ) < C inj We want to determine C inj, but C(t R ) is measured. How are they related?

30. Detected Peak Model: Gaussian-Like Peaks C(t) = analyte concentration as function of time…….  t = standard deviation of peak (time units). Much work has gone into minimizing this quantity: BAND BROADENING. Peak Width (BB): 95% peak area = +/- 2  t = w b When = (Dilution Factor) C inj Peak Model:

31. Gaussian Peak Model When does the model hold and when does it fail? Finding the “sweet spot” of V inj to optimize detection sensitivity (maximize peak concentration), while keeping the analyte peak width at a minimum.

32. Limit-of-detection (LOD) An analytical figure-of-merit, which occurs when the signal  S(t R ) disappears into the detected noise, and is numerically defined as follows:  S(t R ) = 3  N where  N is the standard deviation of the baseline noise

33. Importance of Dilution: Effect on LOD  N = standard deviation of baseline noise

34. Lecture 4 Motivations to study band broadening (BB): C(t),  S(t), LOD, R s and N.

35. Resolution in Chromatographic Systems Further impetus to minimize band broadening Resolution = R S R S = time difference two adjacent peaks average base width of the two peaks Relative Signal Time, t RSRS

36. SEPARATION EFFICIENCY, N Quantities of Interest t R = retention time W b, peak width at base W b = 4  t = 1.7 W 1/2 Three equivalent ways to calculate N: N = ( t R /  t ) 2 N = 16 ( t R / W b ) 2 N = 5.54 ( t R / W 1/2 ) 2 b N is a quantitative metric for how well a chromatographic system is performing, and relates to BB and resolution R S

37. 0510152025303540 5 6 7 8 9 10 11 12 13 Time, seconds Signal Gas Chromatography Data, with t 0 = 7 seconds Chlorobenzene Anisole Benzene

38. Plate Theory, a means to study BB contributions Conceptually divide column of length L into N “extraction segments” of shorter length H ……. H = L / N = plate height L H N = 10 extraction units ….. Higher N implies faster mass transfer, thus lower BB, and smaller H Time domain (t), to/from distance domain (x): N = ( t R /  t ) 2 = ( L /  (x) ) 2 = L 2 /  (x) 2  (x) 2 = length variance of analyte peak band at the end of the column Plate Height, H = L / N =  (x) 2 / L  to RATE THEORY

39. x-axis along column length 0L Band Broadening and Column Efficiency, N Improving N requires examination of band broadening contributions.  (x) 2 = length variance  variances are additive Flow direction, u 4  (x)  Van Deemter and other variations u = MP linear flow velocity  F H ext  injection, detection, tubing, electronics…all off-column BB Minimize by good instrument design and good plumbing

40. Lecture 5 BB Theory – molecular basis of on-column BB terms, define and illustrate the contributions to H, With fluid dynamics: convection and diffusion.

41. Hydrodynamic Principles and Slow Mass Transfer in the Mobile Phase Observed: Stationary Phase +R -R 0 - t = 0  t later r x, Flow Analyte Concentration Profiles: Band broadening due to flow ! C(x) u x (r) = linear flow velocity along x-axis, at radius r. u x (r) = u max (1-(r/R) 2 ) u ave = 1/2(u max ) u x (r) r 0 -R+R Laminar Flow: Parabolic Flow Profile u max

42. Molecular Diffusion allows analyte molecules to be influenced by a broader range of u x (r), diminishing band broadening by this mechanism Low D m, so C m u dominates, since u > u opt, analyte position “fixed” in flow: InitiallyWith flow, little diffusion “Ideal” D m, with each molecule influenced by ~ u ave, and if u ave = u opt (at H min ): InitiallyWith flow and ideal diffusion radial longitudinal C(x) narrower InitiallyWith flow and excessive diffusion High D m, so B/u dominates, since u < u opt, analyte diffuses “too much”:

43. Lecture 6 Materials for capillary and packed columns, comprehensive BB equation (Giddings), fluid mechanics for separations, separation performance: Gas vs. Liquid mobile phase behavior. Trade-offs in chromatography…. looking toward optimization issues.

47. Reynold’s Number, Re Re = inertial forces / viscous forces Turbulent Flow: Inertial forces dominate, and convection assists diffusion in radial mass transfer of analyte in mobile phase to/from stationary phase Laminar Flow: Viscous forces dominate, with smooth, constant flow lines, and diffusion controls radial mass transfer of analyte in mobile phase to/from stationary phase Transition Fluid Mechanics in Chromatography

48. Lecture 7 Separation performance: Gas vs. Liquid mobile phase behavior. Optimization issues in chromatography: chemical selectivity, resolution, instrumental performance and analysis time.

50. 0510152025303540 5 6 7 8 9 10 11 12 13 Time, seconds Signal Gas Chromatography Data, with t 0 = 7 seconds Chlorobenzene Anisole Benzene

51. Lecture 8 Optimization issues in chromatography: chemical selectivity, resolution, instrumental performance and analysis time.

53. Abstract

58. H vs. u plots for LC at various temperatures for polystyrene 9,000 g/mol (PS). Same k’ at each temperature since size-exclusion separation.

59. High temperature, high speed LC (B = 9000PS) Analysis Times 30 seconds at 150 o C 120 seconds at 25 o C Key Point Resolution and pressure kept constant with ¼ th the analysis time at 150 o C !

60. Expanded version of 150 o C separation