1. ANALYTICAL CHEMISTRY CHEM 3811 CHAPTER 21 DR. AUGUSTINE OFORI AGYEMAN Assistant professor of chemistry Department of natural sciences Clayton state university

2. CHAPTER 21 CHROMATOGRAPHY AND MASS SPECTROMETRY

3. CHROMATOGRAPHY - The most powerful tool for separating mixtures - Used for both qualitative and quantitative analysis

4. CHROMATOGRAPHY Comprises of Two Phases Stationary Phase - A solid or liquid packed in a column (does not move) Mobile Phase - A gas or liquid that passes through the column

5. CHROMATOGRAPHY - A column is packed with the stationary phase - Mobile phase passes through the stationary phase - Separation process involves the interaction of the mobile phase (a mixture) with the stationary phase

6. CHROMATOGRAPHY A B eluent eluate

7. CHROMATOGRAPHY Adsorption - Occurs when a solute sticks to the surface of another species - Consider a mixture containing solutes A and B - A is more strongly adsorbed to the stationary phase than B - A moves down the column more slowly than B - B comes out of column before A

8. CHROMATOGRAPHY Elution - The process of passing a liquid or gas through a column Eluent - Fluid entering the column Eluate - Fluid exiting the column

9. CHROMATOGRAPHY Gas Chromatography (GC) - Mobile phase is a gas Liquid Chromatograpgy - Mobile phase is a liquid

10. CHROMATOGRAPHY Solutes may be retarded by the stationary phase based on various interactions - Surface adsorption - Relative solubility - Charge Chromatography is classified based on the type of interactions

11. CHROMATOGRAPHY Adsorption Chromatography - Stationary phase is a solid - Mobile phase is a liquid or a gas - Solute adsorbs to the surface of the solid particles

12. CHROMATOGRAPHY Partition Chromatography - Stationary phase is a thin liquid coated on the surface of a solid support - Mobile phase is a liquid or a gas - Solute equilibrates between the stationary and mobile phases

13. CHROMATOGRAPHY Ion-exchange Chromatography - Allows separation of ions and polar molecules - Ionic groups are covalently attached to a stationary solid phase - Mobile phase is a liquid - Ionic solutes are electrostatically attracted to the stationary phase

14. CHROMATOGRAPHY Size Exclusion Chromatography (Gel Filtration, Gel Permeation) - Solutes are separated based on size - Stationary phase has small pores that exclude large molecules - Small molecules enter the pores so spend more time in column - Large molecules come out of column before small molecules

15. CHROMATOGRAPHY Affinity Chromatography - Very selective - Based on specific interactions between a type of solute molecule and another molecule covalently attached to the stationary phase

16. THE CHROMATOGRAM - Detector response as a function of time or elution volume - Different peaks correspond to different eluates Retention Time (t r ) - Time taken by a solute to reach detector after injection

17. THE CHROMATOGRAM trtr trtr h 1/2h w 1/2 = 2.35σ w = 4σ Detector response Time

18. THE CHROMATOGRAM - An ideal chromatogram has a Gaussian shape - h = height of peak - σ = the standard deviation of the peak - w = base width = 4σ - w 1/2 = width at half height (w at 1/2h) = 2.35σ - t r and w can be measured in time or volume units

19. THEORETICAL PLATES - Imaginary way to picture the separation process - Imaginary discrete sections of the chromatography column - Though the process is continuous - Retention of solutes can be described by the number of equilibrium steps (theoretical plates)

20. THEORETICAL PLATES The number of theoretical plates on a column (N) The Plate Height (H) - The length of one plate H = L/N L = the length of column

21. THEORETICAL PLATES - The higher the N the narrower the bandwidth - The higher the N better the separation - The smaller the H the narrower the peaks - The smaller the H the better the separation

22. To Test a Column for Degradation - Inject standards periodically - Look for Peak asymmetry Change in number of plates THEORETICAL PLATES

23. - Peak separation (Δt r ) divided by the average peak width (w av ) - Better resolution implies more complete separation between neighboring peaks RESOLUTION - Doubling the length of a column (2L) increases resolution by √2

24. QUALITATIVE ANALYSIS - Identify peaks by comparing retention times to those of authentic samples - Unknown sample is “spiked” (authentic sample is added) - The relative size of a peak will increase if the authentic sample is identical to one of the components - Different compounds may have the same retention time - It is more likely for different compounds to have different retention times on different stationary phases

25. QUANTATIVE ANALYSIS - Chromatographic peak area is proportional to quantity of solute - A good measure of solute concentration is obtained by using internal standards - Internal standards eliminate the effect of variable conditions - Conditions mostly vary from run to run

26. QUANTATIVE ANALYSIS Conditions Include - Sample injection errors or changes - Column changes - Detector variations

27. QUANTATIVE ANALYSIS Internal Standard Method - Concentration of analyte (c analyte ) can be determined using the concentration of internal standard (c IS ) and both peak areas

28. SCALING UP Analytical Chromatography - For small-scale analysis Preparative Chromatography - For large-scale analysis

29. SCALING UP - A developed procedure for analytical chromatography can be scaled up and used for preparative chromatography - Maintain column length and increase cross-sectional area - Volume flow rate should also be increased by the same factor

30. BAND BROADENING

31. May be due to Diffusion - Diffusion of solute molecules away from the center of the band in both directions - Longitudinal diffusion - The faster the flow rate the sharper the peaks - Broadening is inversely proportion to flow rate

32. BAND BROADENING May be due to Solute Equilibration - If solute equilibrates slowly between mobile and stationary phases - Solute in stationary phase tends to lag behind solute in mobile phase - Broadening is directly proportional to flow rate

33. BAND BROADENING May be due to Irregular Flow Paths - Occurs since column is packed with solid particles - There are random multiple paths for solute particles - These multiple paths are unequal - Independent of flow rate

34. BAND BROADENING van Deemter Equation - The plate height equation as a result of the three band broadening mechanisms Multiple paths Longitudinal diffusion Equilibration time

35. BAND BROADENING van Deemter Equation u = flow rate A, B and C are constants dependent on - Column - Stationary phase - Mobile phase - Temperature

36. OPEN TUBULAR COLUMN - Hollow capillary column - Inner wall is coated with thin layer of stationary phase - Gives better separation than packed column No multiple paths (A = 0) Can be much longer (gives less resistance to gas flow) Smaller plate height - Only useful for analytical chromatography (can only handle small samples due to less stationary phase)

37. ASSYMETRIC PEAKS - When a band is overloaded by too much solute - Band emerges gradually in front - An abrupt cut off is observed behind the concentration region - Overloading leaves very little trails of solute behind the concentrated region

38. ASSYMETRIC PEAKS - Tailing is when the trailing part is elongated - Occurs when the stationary phase is strongly polar has highly adsorptive sites (-OH groups) Salinization - Chemical treatment to reduce tailing - Converts -OH groups to nonpolar -OSi(CH 3 ) 3 groups - Column should be replaced when tailing increases

39. MASS SPECTROMETRY - Measures the masses and abundances of ions in the gas phase - Detector is sensitive to low analyte concentrations - Distinguishes different substances with the same retention time - Used for both qualitative and quantitative analysis

40. MASS SPECTROMETRY - Molecules are converted to ions prior to separation - Molecules entering the ionization chamber of a mass spectrometer are converted into ions - Ions are separated based on mass-to-charge ratio (m/z)

41. MASS SPECTROMETRY Two Common Methods of Ionization Electron Ionization (EI) - Electrons emitted from a hot filament are accelerated by 70 V - Molecules are ionized by striking electrons as they absorb energy M + e - → M + + e - + e - M + is called the molecular ion M + breaks into fragments after ionization

42. MASS SPECTROMETRY Two Common Methods of Ionization Electron Ionization (EI) - The most intense peak from fragments is called the base peak - Other peaks are expressed as percentages of the base peak intensity

43. MASS SPECTROMETRY Two Common Methods of Ionization Chemical Ionization (CI) - Ionization chamber contains a reagent gas (CH 4 ) - Pressure is maintained at about 1 mbar - Energetic electrons convert gas into a variety of products

44. MASS SPECTROMETRY Two Common Methods of Ionization Chemical Ionization (CI) CH 4 + e - → CH 4 + + 2e - CH 4 + + CH 4 → CH 5 + + CH 3 CH 5 + then protonates the analyte CH 5 + + M → CH 4 + MH + - Fragmentation is less than EI

45. MASS SPECTROMETRY Types of Mass Spectrometers (Analyzers) Electrostatic Magnetic Time of flight Ion trap (quadrupole ion storage) Quadrupole mass spectrometer

46. THE MASS SPECTRUM - Fragmentation patterns from the mass spectrum provide information about the structure of analyte molecule Nominal Mass - Integer mass of the species with the most abundant isotope of each element For benzene (C 6 H 6 ) - The most abundant isotopes are 12 C and 1 H Norminal mass = (6 x 12) + ( 6 x 1) = 78

47. THE MASS SPECTRUM Isotope Pattern - Information is obtained from relative intensities at M+1 and M + - M+1 is one mass unit above the molecular ion Nitrogen Rule - Used to propose composition of molecular ions - Odd nominal mass implies compound has odd number of N atoms - Even nominal mass implies compound has even number of N atoms