Chem. 230

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Chem. 230 10/07 Lecture
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Announcements I

• Second Homework Set Due
• Exam 2
• Next Week
• You can bring a 3 x 5 note card with notes
  (front and back) to the exam
• I will provide constants but no equations
• Topics Covered
• Simple Separations vs. Chromatography
• Chromatographic Theory (Basic definitions of
  parameters, meaning of parameters, how to read
  chromatograms, rate theory)
• Intermolecular Forces Their Effects
• Optimization

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Announcements II

• Should Sign Up for Presentation Topic Today
• Todays Topics
• Optimization (last topic on Exam I)
• Gas Chromatography
• Comparison of methods
• Historical Development
• Column types
• Analytes and Samples
• Instrumentation (mobile and stationary phases,
  flow control, injection?)

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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/solutes will
  dictate many of the requirements (e.g. how many
  solutes are being separated and in what
  concentration? what is the purpose of the
  separation?, 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 greater likelihood of column
  overloading
• Trade-offs in lengthening column (N L/H)
• Longer times due to more column (can be
  considerably longer for HPLC due to pressure
  limits)

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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 d/ave(w) 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 (for problems in
  this class we will assume no change in a with
  change in T or solvent composition)

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Chromatographic TheoryGraphical Representation
Smaller H (narrower peaks)
Initial Separation
Larger k - separation increases more than width
Increased alpha (more retention of 2nd compound)
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Chromatographic TheoryOptimization Back to 1st
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, kY 4.5 and a 1.13.
• What change is needed in N, k, and a to get RS
  1.5?

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Chromatographic TheoryOptimization 2nd Example

• tM 1 min, tX 2 min, wX 0.1 min, tY 2.1
  min, wY 0.105 so RS 0.98, a 1.1, kY 1.1
• With small initial k values, increasing k helps
  more
• After k gt 5, only minor increases in resolution
  possible

Maximum RS
Baseline Resolved
Start Point
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Chromatographic TheoryOptimization Changes in
a - I

• In GC analysis on a DB-1 (non-polar) GC column,
  the compounds acetone (KOW 0.58, bp 56C)
  elutes at 7.82 min while diethyl ether (KOW
  7.76, bp 34.6C) elutes at 7.97 min. Peak
  widths are around 0.2 min. If the unretained
  time is 1.00 min., this is a difficult separation
  with this column.
• Occasionally, changing T to change k will also
  increase a (more on this on next slide)
• Suggest a column switch (aimed at increasing a to
  improve the separation).

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Chromatographic TheoryOptimization Changes in
a - II

• Changes in a with T
• Example alkanes and toluene
• In Plot, most alkanes show similar temp.
  retention behavior (similar slopes no overlap)
• If two alkanes overlap (e.g. two branched
  alkanes), there is not much chance in increasing
  a (since both have same Dret/DT)
• If a separation of octane and toluene had been
  performed at 150, coeluting peaks would be
  observed
• Decreasing T would lead to improvement because
  different slopes lead to a change in a

note if chromatogram started at 200C, one would
be disappointed by initial change
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Chromatographic TheoryOptimization Changes in
a - III

• In HPLC, it is possible to change the mobile
  phase to affect solute solvent interactions and
  retention.
• For example, if molecules A and B are separated
  by normal phase HPLC using 15 2-propanol/85
  hexane and are found to co-elute, solvent changes
  may resolve.
• One might expect that changing solvent to 25
  toluene 75 hexane will increase affinity of
  compound B for mobile phase relative to compound
  A (due to compound B being aromatic) leading to
  increase retention of B

compound A
compound B
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Chromatographic TheoryOptimization Changes in
a - IV

• The two compounds below are found to give
  retention times of 8.91 and 9.02 min. (aniline
  and benzaldehyde, respectively) when separated
  using HPLC on a C18 column using 60 methanol/40
  water vs. an unretained time of 1.62 min.
• There is an easy way to increase a for this
  separation. How can the mobile phase be changed
  to increase a?

NH2
O
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Chromatographic TheoryOptimization Some
Questions

• Indicate how the chromatograms could be improved?

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Chromatographic TheoryReview Questions

• What is the most common way to increase retention
  of analytes in gas chromatography?
• a) decrease flow rate
• b) decrease temperature
• c) increase flow rate
• d) use carrier gas with larger molecular weight
• Increasing the flow rate in chromatography will
  increase which term in the van Deemter equation.
  (Give name or term).
• What type of intermolecular force is typically
  the most important for analyte stationary phase
  in reversed phase HPLC?
• An obviously tailing peak is observed in a
  chromatogram. The concentration of the standard
  is decreased by a factor of 10 and the sample is
  re-injected. The tailing looks about the same.
  What can be concluded about the source of
  tailing? List one other possible source of the
  tailing. (added later)

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Chromatographic TheoryOptimization Some
Questions

1. Why is it usually more difficult to improve the
   separation factor (a) when there are a larger
   number of analytes/contaminants?
2. Both using a longer column or using a column of
   smaller H will improve resolutions. Which method
   will lead to a better chromatogram? Why?
3. RS 0.93 and kB 2.7. What is the maximum RS
   value just by changing kB?
4. An initial run of two standards at moderate
   concentrations results in RS 1.9, kA 3.3 and
   kB 4.0. Why might an analytical chemist and a
   prep chemist change k in opposite directions?

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Gas ChromatographyOverview of Topics

• Comparison of mobile phases (Chapter 6)
• History, analyte stationary phase interaction
  (Section 7.1)
• Instrumentation (Section 7.2, 7.3)
• Stationary phase (Section 7.4)
• Temperature issues (Section 7.6)

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Gas ChromatographyComparison of Mobile Phases

• Two key differences between GC and LC
• No analyte mobile phase interaction in GC
• Temperature is routinely changed (and always
  controlled) in GC
• Effects of gases (vs. liquids)
• Much higher diffusivity (larger B term of van
  Deemter equation but very small CM term)
• Lower viscosity of gases (backpressure is not as
  big an issue)
• Much lower density (capacity of column is a big
  issue with liquid samples)
• Gases are compressible

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Gas ChromatographyCompressibility of Gases

• The volume flow rate will not be a constant along
  a column because as the pressure drops, the
  volume increases
• There are various ways to calculate average flow
  rates which we will not go into

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Gas ChromatographyAdvantages vs. HPLC

• Main practical advantage comes from high N values
  (although H is usually larger) achieved with open
  tubular columns.
• Another advantage comes from being able to use
  quite long columns (60 m vs. 250 mm for HPLC)
  because backpressure is not a major issue
• Other advantages have to do with instrument cost
  and better detectors
• Main disadvantage is for analysis of non-volatile
  compounds

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Gas ChromatographyDevelopment and Theory

• Initially, GC was developed to improve upon
  fractional distillations
• In fractional distillations, the liquid at each
  plate is a mixture of analytes
• In gas chromatography analytes are present, but
  stationary phase interactions are dominant and
  analyte X and Y generally dont interact

X
Y
Liquid (or solid) stationary phase interacts with
x and y
Y
X
Liquid at each plate is mixture of distillates
(only X and Y)
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Gas ChromatographyDevelopment and Theory

• Types of Columns
• Packed Columns
• Older type of column
• Both solid and liquid stationary phase
• Best column for preparatory GC and for use with
  thermal conductivity detectors
• Sometimes used for very specific applications
  (low production volume less of an issue)
• Open Tubular Columns
• More modern columns
• Much better analytical performance (large N
  values)
• Most common in wall coated format (WCOT)
• Variety of diameters (0.25 to 0.53 mm most
  common) allow high resolution vs. easier
  injection
• Stationary phases are mainly bonded of varying
  amounts of polarity
• Good reliability
• Disadvantages harder to make and less capacity

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Gas ChromatographyDevelopment and Theory

• Retention of Compounds
• KC value depends on
• Volatility
• Polarity of analyte vs. polarity of stationary
  phase
• Measure of volatility
• Best measure is vapor pressure at temperature
• Boiling point temperature is used more frequently
• Depends on molecules size and polarity
• Polarity in separations
• Compounds of similar polarity as stationary phase
  will be more retained than similar compounds of
  different polarity if their boiling points are
  the same (ether vs. acetone example)

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Gas ChromatographyDevelopment and Theory

• Application of GC
• Gas samples
• Somewhat different equipment (injector and oven
  range) is needed vs. liquid samples
• Liquid samples
• Compounds must be volatile (plus small amounts of
  non-volatile interferences)
• Compounds must be stable at GC temperatures
• Separations are better for less polar compounds
• Issues occur for very volatile and low volatility
  samples (due to min and max temperatures)

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Gas ChromatographyDevelopment and Theory

• Application of GC
• Extension to non-volatile, thermally labile
  compounds
• Derivatization example fatty acids are highly
  polar and do not produce narrow peak with
  non-polar columns, but they can be reacted to
  produce fatty acid methyl ester (same reaction
  used to produce biodiesel) that are volatile and
  stable
• Pyrolysis GC non-volatile samples are heated
  and breakdown products are measured by GC. This
  give information about compounds building
  blocks

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Gas ChromatographyStationary Phase

• Selection of stationary phase affects k and a
  values
• Main concerns of stationary phase are polarity,
  functional groups, maximum operating temperature,
  and column bleed (loss of stationary phase due to
  decomposition)
• More polar columns suffer from lower maximum
  temperatures and greater column bleed

Type Functional Groups Polarity
OV-1 methyl Non-polar
OV-17 50 methyl/50 phenyl Somewhat polar
OV-225 Cyanopropyl, methyl, and phenyl More polar
carbowax Ether groups polar
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GC InstrumentationMobile Phase

• Since there is no mobile phase analyte
  interaction in GC, why does the mobile phase
  matter?
• Affects diffusion
• Smallest MW gases diffuse faster
• van Deemter B term at low flow rates (fast is
  worse) and C term at higher flow rates (fast is
  better)
• Hmin not affected much, but umin affected by gas
  chosen
• Smallest MW allows fastest runs at min. H
• Detector requirements
• He is most common (inert, safe gas with high
  diffusivity for better efficiency at high flow
  rate)
• H2 also can be used with even better efficiency,
  but is less safe

CO2 min
H2 min
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GC InstrumentationSome Questions

• If a set of compounds in a sample could be
  analyzed by GC or HPLC what would be two reasons
  for picking GC?
• What is a concern in analyzing a liquid sample
  that has numerous highly volatile compounds?
• In the case of the situation in question 2, would
  you want a column with the stationary similar to
  or different from the polarity of the analytes?
• What is one way in which low volatility samples
  can be analyzed by GC?
• In response to high He prices, a lab director
  says that no more He can be purchased. Would you
  want to use Ne or N2? (assuming reasonable prices
  for both of those gases)? What other change
  would be needed to get reasonable separations
  with Ne or N2 carrier gases?
• How is the retention of polar compounds affected
  by switching from He to H2 as a carrier gas?

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GC InstrumentationFlow Control

• Flow can be controlled by regulating inlet
  pressure (either constant pressure or
  compensation for constant linear velocity).
• Equipment consists of valves for regulating
  pressure (constant pressure) in older instruments
  or electronic pressure control (solenoid valve
  opens or closes in response to pressure).
• Flow rate is typically checked at detector using
  bubble meter.

Pressure Transducer
Solenoid valve
Soap film
soap
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GC InstrumentationSample Injection

• Several types of injectors are available and
  choice of injector depends on sample phase,
  analyte concentration, and other sample
  properties
• The most common injectors are designed for
  liquids (but can be used for gases)
• Injectors for gases only can be used for gases
• Liquids require much smaller volumes (1 µL, a
  typical liquid injection volume, is equivalent to
  1 mL after evaporation) and column overloading
  is common
• Column overloading is most common with narrow
  diameter OT columns and least common with packed
  columns
• Most injectors are heated (except on-column)

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GC InstrumentationSample Injection Gas Samples
6 port valve

• Fixed Loop Injectors
• A loop of fixed volume is filled with a gas
• The injection valve is twisted so that the mobile
  phase pushes the gases in the loop into the
  column
• Very similar to most common injections in HPLC
  (Covered later)
• Very reproducible injection

He in
To GC column
Waste
Gas sample in
INJECT POSITION
LOAD POSITION
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GC InstrumentationSample Injection Gas Samples

• Specialized Injectors (Fixed loop injectors with
  trapping capability)
• Best for trace analysis
• In place of loop is a trap (adsorbant or cold
  trap) so that all gas sent into loop gets
  trapped, then injected
• These allow injection of greater volumes but may
  require removal of interferents (oxygen, water)
  and require better quantitative control of gases
  (careful volume or pressure monitoring)
• Thermal trapping (cool to trap, then hot to
  desorb) can increase efficiency
• Other ways to inject gas samples (using injectors
  designed for liquids)
• Direct syringe injection (samples at higher
  concentrations)
• Solid phase microextraction (SPME with fibers
  exposed to gas samples)