Chem. 230

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Chem. 230 9/30 Lecture
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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 2
lt36 3
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Announcements II

• Second Homework Set Are Online (due 10/7)
• Todays 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 TheoryQuestions on Definitions

1. List 3 main components of chromatographs.
2. 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?
3. 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), ß, a.

5
Chromatographic TheoryQuestions on Definitions

1. What easy change can be made to increase KC in
   GC? In HPLC?
2. A GC is operated close to the maximum column
   temperature and for a desired analyte, k 10.
   Is this good?
3. If a new column for problem 8 could be purchased,
   what would be changed?
4. In reversed-phase HPLC, the mobile phase is 90
   H2O, 10 ACN and k 10, is this good?
5. Column A is 100 mm long with H 0.024 mm.
   Column B is 250 mm long with H 0.090 mm. Which
   column will give more efficient separations
   (under conditions for determining H)?

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Chromatographic TheoryQuestions 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 a

Unretained pk
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Chromatographic TheoryRate 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 TheoryRate Theory
Most efficient velocity
H
C term
B term
A term
U
9
Chromatographic TheoryRate 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 areaporosity
X
X
X
dispersion
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Chromatographic TheoryRate 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

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Chromatographic TheoryRate 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
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Chromatographic TheoryRate 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
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Chromatographic TheoryRate Theory

• More generalities
• Often run at u values greater than minimum H
  (saves on time reduces time based s 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
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Chromatographic TheoryRate 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?

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Chromatographic TheoryEffects 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

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Chromatographic TheoryIntermolecular 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
NC-CH3
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Chromatographic TheoryIntermolecular 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

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Chromatographic TheoryIntermolecular 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

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Chromatographic TheoryIntermolecular 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)

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Chromatographic TheoryIntermolecular 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.
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Chromatographic TheoryIntermolecular 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
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Chromatographic TheoryIntermolecular 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)
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Chromatographic TheoryIntermolecular 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

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Chromatographic TheoryIntermolecular Forces
Odd Peak Shapes

• Other Reasons for Odd Peak Shapes
• Large volume injections
• Example 1.0 mL/min. 0.1 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
X
X
In strong solvent
X
Analytes stick on column until stronger mobile
phase arives
X
X
X
In weak solvent
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Chromatographic TheoryIntermolecular Forces
Odd Peak Shapes

• Analyte exists in multiple forms
• Example maltotetraose (glu1?4glu1?4glu)
• Has 3 forms (a, ß, or aldehyde on right glu)
• a and ß 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
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Chromatographic TheoryIntermolecular Forces
Some Questions

1. Describe the dominant forces involving the
   molecules to the right in interacting with
   non-polar molecules? in interacting with polar
   molecules
2. 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. C181)

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Chromatographic TheoryIntermolecular Forces
Some Questions

1. Describe the dominant forces involving the
   molecules to the right in interacting with
   non-polar molecules? in interacting with polar
   molecules
2. 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. C181)

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Chromatographic TheoryIntermolecular Forces
Some Questions

1. 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?
2. 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?

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Chromatographic TheoryIntermolecular Forces
Some Questions

1. 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?
2. 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.

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Chromatographic TheoryIntermolecular Forces
Some Questions

1. 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?
2. Normal phase HPLC is used to separate esters. Is
   better peak shape expected if hexane or methanol
   is the solvent? Why?

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Chromatographic TheoryOptimization - 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)

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Chromatographic TheoryOptimization 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)

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Chromatographic TheoryOptimization 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)

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Chromatographic TheoryOptimization 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 0.94. Also, N
  1024 and H 100 mm/1024 0.097 mm
• Lets 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 2N100 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 21-0.5 (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

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Chromatographic TheoryOptimization 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 a
  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
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Chromatographic TheoryOptimization Resolution
Equation

• Dont 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 a also