Chem. 133 – 5/3 Lecture

Announcements Lab – Term Project Progress Report Due Today – Last Assignments: Term Project Poster and Peer Review Grading (Friday, May 20 th at 8:30 am in Alumni Center) Quiz and Homework (Thursday) – Can skip Additional Problem 2c – part on resolution (will not get to that today) Exam 3 – May 10 th – Covering: Atomic Spectroscopy (atomization and spectrometers), NMR, Mass Spectrometers, Chromatography (at least through band broadening) Today’s Lecture – Chromatography Partitioning (partitioning in chromatography) Differential partitioning and separation Band Broadening

Chromatography Separation Theory: The good, the bad and the ugly The Good: Separation based on differential partitioning (differences in K) The Bad: Band Broadening (limits separation efficiency and dilutes analytes) The Ugly: Non-ideal behavior (causes non- Gaussian peak shapes)

Chromatography Partition Theory Partitioning in Chromatographic Columns – K = [X] s /[X] m where s is for stationary phase and m is for mobile phase – Above equation is designed where mobile and stationary phases are liquids, but a related equation can be used with other phases – K value affects how long it takes a solute to go through column because the solute is only moving when it is in the mobile phase – Solutes with larger K values (e.g. Y below) move through columns more slowly X Y

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 (every compound spends this time in the mobile phase)

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

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) = ( min.)/2.37 min. = 1.09

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

Chromatography Retention Factor Values Practical k values – ~0.5 to ~10 – Small k values → interference more likely – 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

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 – Increasing “strong” solvent decreases k – Strong solvent is one more like stationary phase – for reversed-phase HPLC (non-polar stationary phase, polar mobile phase), stronger solvent is less polar (methanol in example) – Increasing % methanol decreases k – opposite change needed in normal phase HPLC (polar stationary phase) Polarity Index non-polar polar C18 water methanol Analyte X SiOH hexane 2-propanol

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? What change could be made to improve the analysis?

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

Chromatography Reading Chromatograms Determination of parameters from reading chromatogram (HPLC example)  (for 1 st 2 peaks) = k B / k A = t RB ’ / t RA ’ = (5.757 – 2.374)/(4.958 – 2.374) = 1.31

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 – e.g. have different heats of vaporization) – 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.

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)

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

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 most common

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

Chromatography Measurement of Efficiency Measuring N and H is valid under isocratic/isothermal conditions Later eluting peaks normally used to avoid effects from extra-column broadening (from injector, detector, etc.) 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