1. Chem. 133 – 5/5 Lecture

2. Announcements Lab Report 2.4 due Thursday – can turn in today for reduction of late penalties Term Project Progress Report – late (no penalty) if turned in today Thursday – last quiz (basically bonus) and homework (one regular + two bonus) due Thursday Today’s Lecture Chromatography Partitioning/Retention Band Broadening (last topic on Exam 3)

3. Chromatography More on Stationary Phases Open Tubular (end on, cross section view) Column Wall Mobile phase Stationary phase (wall coating) Packed column (side view) (e.g. Silica in normal phase HPLC) Packing Material Stationary phase is outer surface Bonded phase (liquid-like)Expanded View Stationary Phase Chemically bonded to packing material Packing Material View showing pores

4. Chromatography Basis for Separation The partition coefficient (K) is not used that much in chromatography In its place is k, the retention factor k = n s /n m where n = moles of analyte (in stationary and mobile phases) k is used because it is easily measured t r = retention time = total time spent on column t m = time required for mobile phase to flow through column

5. Chromatography Parameters from Chromatograms Determination of parameters from reading chromatogram (HPLC example) t M = 2.37 min. (normally determined by finding 1 st peak for unretained compounds – contaminant below) 1 st peak, t R = 4.96 min. k (1 st peak) = (4.96 -2.37 min.)/2.37 min. = 1.09

6. Chromatography Flow – Volume – Time Relationship Chromatographic parameters can be expressed in terms of volume or time V = F · t where F = volume flow rate t m also can be determined as V m /F k can be related to K through volumes: note: V s is often hard to measure k can be increased by increasing K or V s /V m

7. Chromatography Capacity Factor Values Practical k values –~0.5 to ~10 –Small k values → usually poor selectivity –Large k values → must wait long time Changing k values –Can change: V m /V s – requires column change so less desired K – this can be an “ adjustment ” without needing a column change

8. Chromatography Changing k k adjustment in GC –changes to oven temperature (T) –at low T, compounds are less volatile and spend more time in stationary phase, so k is larger at low T k adjustment in HPLC –for reversed-phase HPLC (non-polar stationary phase, polar mobile phase), increasing mobile phase polarity (greater % water or decreased % methanol/acetonitrile) causes analytes of intermediate polarity to spend more time in stationary phase –opposite change needed in normal phase HPLC (polar stationary phase) Polarity Index non-polar polar C18 water methanol Analyte X SiOH hexane 2-propanol

9. Chromatography Some Questions 1.List 3 main components of chromatographs. 2.A chemist purchases a new open tubular GC column that is identical to the old GC column except for having a greater film thickness of stationary phase. How will the following parameters will be affected (assuming column run as before): K, k, t M, t R (component X)? 3.What “easy” change can be made to increase k in GC? In normal phase HPLC using a hexane/ethylacetate mobile phase? 4.A GC is operated close to the maximum column temperature and for a desired analyte, k = 10. Is this good?

10. Chromatography Selectivity Selectivity refers to the ability to separate one component from another component Separation, and selectivity, arise from components with different K values Example: separation of phenol (K ow = 32) from 1- butanol (K ow = 5.7) using reversed-phase HPLC Because of lower K value, 1-butanol would expect to be eluted first OH CH 3 CH 2 CH 2 CH 2 OH butanolphenol

11. Chromatography Selectivity - Continued Selectivity is given by  = relative retention (also called selectivity coefficient)  = k y /k x (where t r (y) > t r (x)) A larger  value means a better separation. An  value close to 1 means a difficult separation. Note that  = K y /K x also applies

12. Chromatography Selectivity - Continued How can  be increased? –Not always easy to increase –In GC, a new column often is needed (  only changes if K x and K y change with T differently) –Example: Separation of hexane from acetone Both have similar boiling points With a weakly polar column  is near 1, but going to a polar column will cause greater retention of acetone.

13. Chromatography Selectivity – in HPLC How can  be increased? –Mobile phase changes often can be used in HPLC (no need for column change) –Possible changes: change in pH (e.g. adjust retention of weak acids by changing % in ion form) different analyte – solvent interactions for reversed phase, 3 common organic solvents are acetonitrile, methanol, and tetrahydrofuran (THF)

14. Chromatography Column Efficiency – The Bad Original theory developed from number of simple separation steps (e.g. from fractional distillation columns) N = number of theoretical plates (or now plate number) = best absolute measure N = 16(t r /w) 2 or = 5.55(t r /w 1/2 ) 2 –w = peak width at baseline –w 1/2 = peak width at half height

15. Chromatography Shape of Chromatographic Peak Gaussian Distribution Normal Distribution Area = 1 Widths –σ (std deviation) –w = 4σ –w 1/2 = 2.35σ –w’ = Area/y max = 2.51σ (commonly given by integrators) Gaussian Shape (Supposedly) 2σ2σ Inflection lines w Height Half Height w 1/2

16. Chromatography Column Efficiency Good efficiency means: –Large N value –Late eluting peaks still have narrow peak widths Relative measure of efficiency = H = Plate height = L/N where L = column length H = length of column needed to get a plate number of 1 Smaller H means greater efficiency Note: H is independent of L, N depends on L large N Value low N value

17. Chromatography Measurement of Efficiency Measuring N and H is valid under isocratic conditions Later eluting peaks normally used to avoid effects from extra-column broadening Example: N = 16(14.6/0.9) 2 = 4200 (vs. ~3000 for pk 3) H = L/N = 250 mm/4200 = 0.06 mm W ~ 0.9 min

18. Chromatography Causes of Band Broadening There are three major causes of band broadening (according to theory) These depend on the linear velocity (u = L/t m ) Given by van Deemter Equation: –where H = Plate Height, and A, B, and C are “ constants ”

19. Chromatography Band Broadening u H Most efficient velocity A term B term C term

20. Chromatography Band Broadening “ Constant ” Terms –A term: This is due to “ eddy diffusion ” or multiple paths –Independent of u –Smaller A term for: a) small particles, or b) no particles (best) X X X dispersion