CHEM 429 / 529 Chemical Separation Techniques

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

Lecture 1 Course Introduction

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.

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

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

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?

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

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.

Signal Instrumentation Applied (shown for Liquid Chromatography) Sample Injection Rotates Injection Valve Computer Waste Waste Chromatographic Column (Stationary Phase) Detector Chromatogram Mobile Pump Phase Reservoir 0 5 10 15 20 25 30 35 40 Time, min

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.

Chromatography Detectors HPLC, SFC and CE (liquid phase) Absorbance (single l 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

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

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 recall t R = t SP + t 0 and t MP = t 0 k = (t R - t 0 ) / t 0 = K D (Volume SP / Volume MP ) Unique K D results in a unique t R, and resolved analyte peaks!

(4) Chemical Selectivity, a 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). a is a measure of selectivity, for analytes 1 and 2: a is independent of Volume SP / Volume MP (called the phase volume ratio), and independent of F, so allows simple comparison of different chromatographic systems.

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

Hildebrand Solubility Parameter, d Less polar More polar 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 Propanol 12.0 Acetonitrile 12.1 Phenol 12.1 Ethanol 12.7 Methanol 14.5 Ethylene glycol 17.0 Water 23.4 d

Miscibility of Different Solvent Pairs

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

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

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.

Step 3 Detection of Sample Mixture: A, B, C CHROMATOGRAM

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

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.

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

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

Detection of Sample Mixture: A, B, C CHROMATOGRAM

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

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?

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

Gaussian Peak Model When does the model hold and when does it fail? 6 5 4 3 2 Peak no. 1 1 2 2 3 3 4 4 5 6 6 8 Relative V inj 1 Finding the sweet spot of V inj to optimize detection sensitivity (maximize peak concentration), while keeping the analyte peak width at a minimum.

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

Importance of Dilution: Effect on LOD s N = standard deviation of baseline noise

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

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

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

Gas Chromatography Data, with t 0 = 7 seconds Signal 13 12 11 10 9 8 7 6 Benzene Chlorobenzene Anisole 5 0 5 10 15 20 25 30 35 40 Time, seconds

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 N = 10 extraction units.. L Higher N implies faster mass transfer, thus lower BB, and smaller H Time domain (t), to/from distance domain (x): N = ( t R / s t ) 2 = ( L / s(x) ) 2 = L 2 / s(x) 2 s(x) 2 = length variance of analyte peak band at the end of the column Plate Height, H = L / N = s(x) 2 / L to RATE THEORY H

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

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

Hydrodynamic Principles and Slow Mass Transfer in the Mobile Phase Observed: +R 0 - -R t = 0 Analyte Concentration Profiles: C(x) Stationary Phase Dt later r x, Flow Stationary Phase Band broadening due to flow! Laminar Flow: Parabolic Flow Profile 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 u max

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: Initially With flow, little diffusion Ideal D m, with each molecule influenced by ~ u ave, and if u ave = u opt (at H min ): longitudinal radial Initially With flow and ideal diffusion C(x) narrower High D m, so B/u dominates, since u < u opt, analyte diffuses too much : Initially With flow and excessive diffusion

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.

Fluid Mechanics in Chromatography 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: Transition Viscous forces dominate, with smooth, constant flow lines, and diffusion controls radial mass transfer of analyte in mobile phase to/from stationary phase

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

Gas Chromatography Data, with t 0 = 7 seconds Signal 13 12 11 10 9 8 7 6 Benzene Chlorobenzene Anisole 5 0 5 10 15 20 25 30 35 40 Time, seconds

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

Abstract

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.

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!

Expanded version of 150 o C separation