1. Chem. 230 – 9/30 Lecture

2. Announcements I Quiz 1 Results Solutions have been posted
See class distribution
Large number of high scores (best ever # 90%+)
Also significant numbers of low scores
Score Range N
60-62 (100%+) 2
54-60 7
48-53 3
42-47 4
36-41
<36 2

3. Announcements II Second Homework Set Are Online (due 10/7)
Today’s Topics – Mainly Chromatographic Theory
Basic definitions (more questions)
Rate Theory (cause of band broadening – Sect. 3.2)
Intermolecular Forces and Their Effects on Chromatography (Sect. 4.1)
Optimization – if time

4. Chromatographic Theory Questions on Definitions
List 3 main components of chromatographs.
A chemist perform trial runs on a 4.6 mm diameter column with a flow rate of 1.4 mL/min. She then wants to scale up to a 15 mm diameter column (to isolate large quantities of compounds) of same length. What should be the flow rate to keep u (mobile phase velocity) constant?
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. Which parameters will be affected: KC, k, tM, tR(component X), β, a.

5. Chromatographic Theory Questions on Definitions
What “easy” change can be made to increase KC in GC? In HPLC?
A GC is operated close to the maximum column temperature and for a desired analyte, k = 10. Is this good?
If a new column for problem 8 could be purchased, what would be changed?
In reversed-phase HPLC, the mobile phase is 90% H2O, 10% ACN and k = 10, is this good?
Column A is 100 mm long with H = mm. Column B is 250 mm long with H = mm. Which column will give more efficient separations (under conditions for determining H)?

6. Chromatographic Theory Questions on Definitions
Given the two chromatograms to the right:
Which column shows a larger N value?
Which shows better resolution (1st 2 peaks top chromatogram)?
Which shows better selectivity (larger a; 1st 2 peaks on top)?
Should be able to calculate k, N, RS, and α
Unretained pk

7. Chromatographic Theory Rate Theory
We have covered parameters measuring column efficiency, but not covered yet what factors influence efficiency
In order to improve column efficiency, we must understand what causes band broadening (or dispersion)
van Deemter Equation (simpler form)
where H = Plate Height
u = linear velocity
and A, B, and C are “constants”

8. Chromatographic Theory Rate Theory
Most efficient velocity H
C term
B term
A term U

9. Chromatographic Theory Rate Theory
Inside of column
(one quarter shown)
How is u determined?
u = L/tM
u = F/A* (A* = effective cross-sectional area)
“Constant” Terms
A term: This is due to eddy diffusion
Eddy diffusion results from multiple paths
Shaded area = cross-sectional area = area*porosity X X X
dispersion

10. Chromatographic Theory Rate Theory
A Term
Independent of u
Smaller A term for: a) small particles, b) spherical particles, or c) no particles (near zero)
Small particles (trend in HPLC) results in greater pressure drop and lower flow rates

11. Chromatographic Theory Rate Theory
B Term – Molecular Diffusion
Molecular diffusion is caused by random motions of molecules
Larger for smaller molecules
Much larger for gases
Dispersion increases with time spent in mobile phase
Slower flow means more time in mobile phase
at start X X X
Band broadening

12. Chromatographic Theory Rate Theory
C term – Mass transfer to and within the stationary phase
Analyte molecules in stationary phase are not moving and get left behind
The greater u, the more dispersion occurs
Less dispersion for smaller particles and thinner films of stationary phase
Less dispersion for solute capable of faster diffusion (smaller molecules) X X
dispersion
Column particle

13. Chromatographic Theory Rate Theory
More generalities
Often run at u values greater than minimum H (saves on time; reduces time based σ which can increase sensitivity depending on detector)
For open tubular GC, A term is minimal, C term minimized by using smaller column diameters and stationary phase films
For packed columns, A and C terms are minimized by using small particle sizes
Low flow conditions
Higher flow conditions

14. Chromatographic Theory Rate Theory
Some Questions:
What are advantages and disadvantages of running chromatographs at high flow rates?
Why is GC usually operated closer to the minimum H value than HPLC?
Which term is nearly negligible in open tubular GC?
How can H be decreased in HPLC? In open tubular GC?

15. Chromatographic Theory Effects of Intermolecular Forces
Phases in which intermolecular forces are important: solid surfaces, liquids, liquid-like layers, supercritical fluids (weaker)
In ideal gases, there are no intermolecular forces (mostly valid in GC)
Intermolecular forces affect:
Adsorption (partitioning to surface)
Phase Partitioning
Non-Gausian Peak Shapes

16. Chromatographic Theory Intermolecular Forces – Types of Interactions
Interactions by decreasing strength
Ion – Ion Interactions
Strong attractive force between oppositely charged ions
Of importance for ion exchange chromatography (ionic solute and stationary phase)
Also important in ion-pairing used in reversed-phase HPLC
Very strong forces (cause extremely large K values in absence of competitors)
From a practical standpoint, can not remove solute ions from stationary phase except by ion replacement (ion-exchange)
Ion – Dipole Interactions
Attractive force between ion and partial charge of dipole
d d+ M+
:N=C-CH3

17. Chromatographic Theory Intermolecular Forces – Types of Interactions
Interactions by decreasing strength – cont.
Ion – Dipole Interactions – cont.
Determines strength of ionic solute – solvent interactions, ionic solute – polar stationary phase interactions, and polar solute – ionic stationary phase interactions
Important for some specific columns (e.g. ligand exchange for sugars or Ag+ for alkenes)
Metal – Ligand Interactions
ion – ion or ion – dipole interaction, but also involve d orbitals

18. Chromatographic Theory Intermolecular Forces – Types of Interactions
Interactions by decreasing strength – continued (non-ionic interactions = van der Waal interactions)
Van der Waals Forces
dipole – dipole interactions (requires two molecules with dipole moments)
important for solute – solvent (especially reversed phase HPLC) and solute – stationary phase (especially normal phase HPLC)
Hydrogen bonding is a particularly strong dipole-dipole type of bonding
dipole – induced dipole interactions
induced dipoles occur in molecules with no net dipole moment
larger, more electron rich molecules can get induced dipoles more readily
induced dipole – induced dipole interactions (London Forces)
occur in the complete absence of dipole moments
also occur in all molecules, but of less importance for polar molecules

19. Chromatographic Theory Intermolecular Forces – Types of Interactions
Modeling interactions
Somewhat of a one-dimensional model can be made by assigning a single value related to polarity for analytes, stationary phases, and mobile phases (See section 4.3)
These models neglect some interactions however (e.g. effects of whether an analyte can hydrogen bond with a solvent)

20. Chromatographic Theory Intermolecular Forces – Asymmetric Peaks
More than one possible cause (e.g. extra-column dispersion)
One common cause is sample or analyte overloading of column
Analyte loading shown →
More common with solid stationary phase
More common with open tubular GC; less common with HPLC
5% by mass ea.
20% by mass ea.

21. Chromatographic Theory Intermolecular Forces – Asymmetric Peaks
Low Concentrations
Most common for solid stationary phase and GC because
Less stationary phase (vs. liquid)
GC behavior somewhat like distillations
At low concentrations, column “sites” mostly not occupied by analyte
As conc. increase, % sites occupied by analyte increases, causing change in analyte – stationary phase interaction
Active sites
analyte X
High Concentrations
New analyte X X X X X

22. Chromatographic Theory Intermolecular Forces – Asymmetric Peaks
As concentration increase, interactions go from analyte – active site to analyte – analyte
If interaction is Langmuir type (weak analyte – analyte vs. strong analyte – active site), tailing occurs (blocking of active sites causes additional analyte to elute early)
If interaction is anti-Langmuir type (stronger analyte – analyte interactions), fronting occurs (additional analyte sticks longer)
Tailing peak (up fast, down slow)
Fronting peak (up slow, down fast)

23. Chromatographic Theory Intermolecular Forces – Asymmetric Peaks
If tailing is caused by saturation of stationary phase, changing amount of analyte injected will change amount of tailing and retention times

24. Chromatographic Theory Intermolecular Forces – Odd Peak Shapes
Other Reasons for Odd Peak Shapes
Large volume injections
Example: 1.0 mL/min mL injection
Injection plug time = 0.1 min = 6 s (so no peaks narrower than 6 s unless on-column trapping is used)
Injections at high temp./in strong solvents
Will not partition to stationary phase until mobile phase mixes in
In strong solvent X X X
Analytes stick on column until stronger mobile phase arives
In weak solvent X X X

25. Chromatographic Theory Intermolecular Forces – Odd Peak Shapes
Analyte exists in multiple forms
Example: maltotetraose (glu[1→4]glu[1→4]glu)
Has 3 forms (α, β, or aldehyde on right glu)
α and β forms migrate at different rates
At low T, interconversion is slow relative to tR. At high T, interconversion is faster
Extra-column broadening/turbulent flow
Multiple types of stationary phase
Low T
High T X X
Polar groups OH OH
Non-polar groups

26. Chromatographic Theory Intermolecular Forces – Some Questions
Describe the dominant forces involving the molecules to the right in interacting with non-polar molecules? in interacting with polar molecules
How does going from DB-1 (100% methyl stationary phase) to DB-17 (50% methyl – 50% phenyl) in GC affect elution of fatty acid methyl esters? (e.g. C16 vs. C18 vs. C18:1)

27. Chromatographic Theory Intermolecular Forces – Some Questions
Describe the dominant forces involving the molecules to the right in interacting with non-polar molecules? in interacting with polar molecules
How does going from DB-1 (100% methyl stationary phase) to DB-17 (50% methyl – 50% phenyl) in GC affect elution of fatty acid methyl esters? (e.g. C16 vs. C18 vs. C18:1)

28. Chromatographic Theory Intermolecular Forces – Some Questions
Silica has many SiOH groups on the surface (pKa ~2). What interactions will occur with the analyte phenol, C6H5OH, if the eluent is a mixture of hexane and 2-propanol?
Sugars are often separated on amino columns. A sugar that has a carboxylic acid group in place of an OH group will have extremely large retention times (at least at neutral pH values). What does this say about the state of the amino groups?

29. Chromatographic Theory Intermolecular Forces – Some Questions
In reversed phase HPLC with a C18 column, benzene and methoxybenzene (anisole) have very similar retention times. What are the differences in the interactions between the two solutes and mobile phases and stationary phases?
A heavily used non-polar GC column is used to separate non-polar to polar columns. Polar compounds are observed to tail. A new column replaces the old column, tailing stops, and the polar compounds elute sooner. Explain the observations.

30. Chromatographic Theory Intermolecular Forces – Some Questions
A megabore GC column (d = 0.53 mm) is replaced with an 0.25 mm diameter column in order to improve resolution of constituents from a sample. However, when the same sample is injected into the 0.25 mm diameter, little improvement in resolution and poor peak shape is seen. What is a possible reason? How can this be tested?
Normal phase HPLC is used to separate esters. Is better peak shape expected if hexane or methanol is the solvent? Why?

31. Chromatographic Theory Optimization - Overview
How does “method development” work?
Goal of method development is to select and improve a chromatographic method to meet the purposes of the application
Specific samples and analytes will dictate many of the requirements (e.g. how many analytes are being analyzed for and in what concentration?, what other compounds will be present?)
Coarse method selection (e.g. GC vs HPLC and selection of column type and detectors) is often based on past work or can be based on initial assessment showing problems (e.g. 20 compounds all with k between 0.2 and 2.0 with no easy way to increase k)
Optimization then involves making equipment work as well as possible (or limiting equipment changes)

32. Chromatographic Theory Optimization – What are we optimizing?
Ideally, we want sufficient resolution (Rs of 1.5 or greater for analyte/solute of interest peaks)
We also want the separation performed in a minimum amount of time
Other parameters may also be of importance:
sufficient quantity if performing “prep” scale separation
sufficient sensitivity for detection (covered more with instrumentation and quantitation)
ability to identify unknowns (e.g. with MS detection)

33. Chromatographic Theory Optimization – Some trade offs
Flow rate at minimum H vs. higher flow rates (covered with van Deemter Equation) – low flow rate not always desired because of time required and sometimes smaller S/N
Maximum flow rate often based on column/instrument damage – this can set flow rate
Trade-offs in reducing H
In packed columns, going to small particle sizes results in greater back-pressure (harder to keep high flow)
In GC, small column and film diameters means less capacity and can require longer analysis times
Trade-offs in lengthening column (N = L/H)
Longer times due to more column (often not proportional since backpressure at same flow rate will be higher)

34. Chromatographic Theory Optimization – Improved Resolution Through Increased Column Length
Example:
Compounds X and Y are separated on a 100 mm column. tM = 2 min, tX = 8 min, tY = 9 min, wX = 1 min, wY = 1.13 min, so RS = Also, N = 1024 and H = 100 mm/1024 = mm
Let’s increase L to 200 mm. Now, all times are doubled:
tM = 4 min, tX = 16 min, tY = 18 min. So DtR (or d) now = 2 min. Before considering widths, we must realize that N = L/H (where H is a constant for given packing material).
N200 mm = 2*N100 mm. Now, N = 16(tR/w)2 so w = (16tR2/N)0.5
w200 mm/w100 mm = (tR200 mm/tR100 mm)*(N100 mm/N200 mm)0.5
w200 mm/w100 mm = (2)*(0.5)0.5 = = (2)0.5
w200 mm = 1.41w100 mm
RS = 2/1.5 = 1.33
Or RS 200/RS 100 = d/wave = (d200/d100)*(w100/w200)= (L200/L100)*(L100/L200)0.5
So RS is proportional to (L)0.5

35. Chromatographic Theory Optimization – Resolution Equation
Increasing column length is not usually the most desired way to improve resolution (because required time increases and signal to noise ratio decreases)
Alternatively, k values can be increased (use lower T in GC or weaker solvents in HPLC); or α values can be increased (use different solvents in HPLC or column with better selectivity) but effect on RS is more complicated
Note: above equation is best used when deciding how to improve RS, not for calculating RS from chromatograms

36. Chromatographic Theory Optimization – Resolution Equation
Don’t use above equation for calculating Rs
How to improve resolution
Increase N (increase column length, use more efficient column)
Increase a (use more selective column or mobile phase)
Increase k values (increase retention)
Which way works best?
Increase in k is easiest (but best if k is initially small)
Increase in a is best, but often hardest
Often, changes in k lead to small, but unpredictable, changes in α also