Exploring Chemical Analysis 3e

1
Intro to HPLC

2
Modes of HPLC

• Reversed Phase
• Normal Phase
• Ion Pair
• Ion Exchange
• Size Exclusion

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Modes of HPLC
Compounds Separated Mode Stationary Phase Mobile Phase
Neutral, Weak acids bases Reverse phase C-18, C-8, C-4, phenyl, amino, cyano Water/Organic/Modifiers
Water insoluble cmpds Normal phase Silica, Cyano, amino, propyl, diol Organics
Ionics, acids, bases Ion Pair (a type of reverse) C-18, C-8 Water/Organic Ion/pair reagent
Ionics, inorganic ions Ion exchange Anion or cation exchange resin Aqueous/ buffer/ counter ion
High MW cmpds Size exclusion Polystyrene silica gel filtration aqueous gel permeation organic
4
Reversed Phase

• Most commonly used HPLC mode.
• Excellent for water soluble compounds.
• Can analyze neutral cmpds, weak acids and bases.
• Good for MW less than 2000 amu
• Proteins, peptides

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Reversed Phase

• Typical runs are isocratic, same mobile phase
  composition throughout the run.
• Gradient runs are also used, where the mobile
  phase composition varies (becomes stronger)
  through the run.

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Reversed Phase

• Stationary phase is less polar than the mobile
  phase.
• Polar solutes are eluted earlier than nonpolar
  solutes.
• Typical columns have a silica support to which
  the stationary phase is covalently attached.
• Polymer stationary phases may be used for
  extremely high or low pH values.

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Reversed Phase Columns

• C-18 (octadecylsilyl)
• C-8 (octasilyl)
• C-4 (butyl)
• Phenyl
• Cyano
• Amino

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Reversed Phase Columns

• For neutral compounds, water and MeOH or
  acetonitrile are the most common mobile phases.
• The exact -composition would of course be
  optimized to get the best resolution in the
  shortest time.
• MeOH and acetonitrile will not necessarily give
  the same elution order for solutes or of course
  the same retention times.

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Reversed Phase Columns

• For weak acids, if just water and MeOH or
  acetonitrile are used, the peak shape will be
  extremely ugly, with a double peak.
• The retention time may also be too short.
• Therefore, a buffer to modify the pH is used
  glacial acetic acid at a low pH (at least 2 pH
  units below the pKa) is commonly used.
• At a low pH, the acid is in the free acid form
  (protonated form) instead of the deprotonated
  form.

10
Reversed Phase Columns

• For weak bases, a similar problem occurs.
• The base will be attracted to the silanol groups
  on the stationary phase and thus give weird
  retention times and ugly peaks.
• So a base modifier like TEA (triethylamine) is
  added.
• TEA acts as a competitor for the silanol groups,
  lowering the probability that your base is
  attracted.
• So better peaks.

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Reversed Phase Columns

• For a mixture of solutes, can we predict the
  elution order? (at least roughly?)
• Predict based on
• polarity or water solubility
• degrees of unsaturation
• branching
• numbers of carbon atoms

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Elution Order Polarity

• Clearly the more polar the cmpd, the earlier it
  elutes.
• General order from most polar to least
• carboxy acids
• alcohols and phenols
• amines, aldehydes, and ketones
• hydrocarbons

13
Elution Order D. of Unsat.

• Unsaturated cmpds containing pi bonds are more
  acidic than saturated cmpds.
• So the more unsaturated the cmpd, the earlier it
  will elute.
• It would still typically elute after amines.

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Elution Order Branching

• Hydrocarbon branching decreases retention time.
• Due to steric hindrance, they would have less
  interactions with the stationary phase.

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Elution Order Number of Carbons

• The more carbon atoms, the more nonpolar a
  molecule becomes in general.
• Thus the retention time increases for increasing
  C atoms.

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Elution Order for Reversed Phase

• Here is the general guidelines for predicting
  elution order
• ions come out with void volume
• strong or moderately strong acids
• weak acids
• weak bases
• weakly polar (ketones, aldehydes, then
  unsaturated)
• nonpolar

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Reversed Phase Mobile Phase

• Although MeOH and acetonitrile are the most
  common, THF and IPA are also used.
• What do you want in the organic portion of the
  mobile phase?
• water soluble
• low viscosity which means low pressure
• low UV detection or interference
• solutes are soluble in it
• unreactive

18
Reversed Phase Mobile Phase

• THF is unstable with time
• IPA is very viscous
• MeOH is more viscous and has a higher UV-cutoff
  than Acetonitrile
• MeOH cutoff for UV interference is 210 nm
• Acetonitrile is 190 nm
• Acetonitrile is preferred choice (except right
  now due to global shortage)

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Reversed Phase Mobile Phase

• What is the order of the strength of the
  typical solvents for the mobile phase?
• Stronger solvents will give shorter retention
  times.
• Strength from weakest
• water/buffers
• water
• MeOH
• acetonitrile
• IPA
• THF

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Reversed Phase Column

• You know that there are different stationary
  phases available for columns.
• Columns also have different lengths, particle
  sizes of the stationary phase, pore sizes, and
  column diameter (and other differences such as
  endcapping to deactivate the silanol groups).
• These differences lead to different retention
  times of solutes.

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Reversed Phase Column

• The longer the column, the longer the retention
  time. But it can be good for method development
  to use a longer column until you know what is in
  your mixture.
• Smaller diameter columns mean shorter retention
  times.
• Smaller particle sizes mean shorter retention
  times.
• But pressure can be an issue with the shorter and
  smaller columns use lower flow rates.

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Ion Pair HPLC Reversed Phase

• Using Reverse phase HPLC, strong acids and bases
  can be separated.
• This subset of reverse phase is called ion pair
  chromatography.
• An ion-pair reagent is added to the mobile phase.
• This reagent has a hydrophobic tail and a cation
  or anion group elsewhere.

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Ion Pair HPLC Reversed Phase

• To separate bases, an ion-pair reagent that has
  an anion group is used.
• Common
• alkyl sulfonates (-SO3- ending)
• TFA (trifluoroacetic acid)
• other moderately strong or strong alkyl acids)

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Ion Pair HPLC Reversed Phase

• To separate acids, an ion-pair reagent that has
  an cation group is used.
• Common
• tetramethylammonium phosphate (so quarternary
  amine)
• quarternary alkyl amines (phosphate or acetate)
• triethylammonium acetate
• TEA

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Ion Pair HPLC Reversed Phase

• By adding an ion-pair reagent, the hydrophobic
  tail interacts with the stationary phase, leaving
  the ionic ending pointing out into the mobile
  phase.
• Thus, very polar solutes can interact with the
  charged ion-pair and the retention time is
  increased.

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Ion Exchange HPLC and IC

• Ion exchange is used to separate out organic and
  inorganic ions.
• The stationary phase is a polymer or silica
  surface with charged groups bonded to the
  surface.
• The charged groups may be cations or anions.
• Typical charged groups include
• sulfonates (-)
• carboxymethyl (-)
• quarternary amines ()
• diethylaminoethyl ()

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Ion Exchange HPLC and IC

• The mobile phase contains counter ions and
  buffers and organic modifiers.
• The counter ions in the mobile phase compete with
  the solute ions in the sample for interactions
  with the ionic endings on the stationary phase.
• The more counter ions present, the less able the
  solute ions are able to interact with the
  stationary phase ions.
• So retention time depends on the concentration of
  the counter ions.

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Ion Exchange HPLC and IC

• Ion exchange is useful for the analysis of wines,
  fruit drinks, other foods, etc.
• IC is used to analyze trace ions like halides,
  nitrates, phosphates, sulfates, and metal ions.

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Normal Phase HPLC

• Normal phase HPLC was the first mode of HPLC
  developed.
• It is adsorption chromatography.
• Now, the mobile phase is organic.
• The stationary phase is more polar than the
  mobile phase.
• The more nonpolar the cmpd, the shorter the
  retention time.

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Normal Phase HPLC

• It is useful to separate
• water insoluble cmpds
• structural isomers
• moderately polar to nonpolar cmpds

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Normal Phase HPLC

• In the column, the polar end groups on the
  stationary phase interact with the solute
  molecules.
• Again, the solvent can compete with solute
  molecules.
• The solvent or solute particles are adsorbed on
  the surface via H-bonding, dipole-dipole forces,
  etc.
• The stronger the solute interacts, the longer the
  retention time.

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Normal Phase HPLC Elution Order

• It is basically backwards of reverse phase.
• The more polar functional groups on the solute,
  the longer the retention time.

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Normal Phase HPLC Mobile Phase

• Fluoroalkanes are weakest solvent (long RT)
• n-pentane (common)
• n-hexane (common)
• chlorobutane
• benzene
• xylene
• chloroform
• toluene (common)
• methylene chloride
• ethyl acetate
• THF
• Acetonitrile
• MeOH

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Size Exclusion HPLC

• Size exclusion is used for high MW cmpds
• gt 10000 amu
• It is also called gel filtration (GFC) or gel
  permeation chromatography (GPC) depending on the
  mobile phase and stationary phase.
• GFC uses silica columns and aqueous buffer mobile
  phases. It is used for biochemical applications.
• GPC uses polymer stationary phases and organic
  mobile phases (like THF) to separate large MW
  organic cmpds, like polymers.
• The separation is based on size of the solute.

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Size Exclusion HPLC

• Whatever, the stationary phase, it contains pores
  of a very defined size.
• Solutes which are too big to enter the pores are
  eluted first.
• Solutes that kind of fit but get kind of stuck,
  elute next.
• Solutes that are small enough to enter the pores
  and take a picnic break or take the scenic route
  down the column, elute last!
• Different pore size columns are available for
  different MW separations.

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Back to Reverse Phase HPLC!

• Lets now go back and discuss the resolution
  between peaks.
• Of course, this applies also to the other HPLC
  modes, with some exceptions and modifications.
• Remember that we want a R of at least 1.5 to
  separate peaks sufficiently with no overlap, but
  we also want reasonable overall analysis times.
• You learned how to calculate R between two peaks
  on a chromatogram.
• But there is another equation.

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Resolution of Solutes in Reverse Phase

• There is a master equation for resolution

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Resolution of Solutes

• What parameters are important in the resolution
  between peaks?
• Based on the previous equation, there are 3 major
  parameters
• Efficiency (related to the theoretical plates)
• Capacity (retention factor)
• Selectivity

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Capacity, k

• This is also called the retention factor.
• It is both the interaction of the solute with the
  mobile and the stationary phases.
• So it is about the equilibration of a solute
  particle partitioning between the two phases.

41
Capacity, k

• It is related to the retention time.
• Mathematically
• The longer the RT, the higher the capacity
  factor.
• You want it to between 1 to 5 (best for overall
  resolution) or between 2 and 10 for complicated
  mixtures.

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Capacity, k

• The best way to change the capacity is to change
  the mobile phase and temperature.
• The higher the -water in the mobile phase, the
  weaker it is, and the longer the retention
  times.
• Increasing the organic content of the mobile
  phase weakens the interactions of the solute with
  the stationary phase (nonpolar) and lowers the RT
  and k.
• Rule of thumb a 10 increase in O-content
  decreases k by a factor of 2 to 3.
• As increasing T tends to decrease RT, k would
  also decrease.

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Efficiency

• Efficiency is related to N, the theoretical
  number of plates in the column.
• It is related to the width of a peak.
• Narrower peaks have a higher efficiency.
• So for a higher efficiency, we need to lower the
  tendency of peaks to spread in a column.
• We want to increase N, the number of plates.

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Efficiency

• What is N mathematically?
• N is ideally 5000 to 25000.

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Efficiency

• Longer columns have more stationary phase and so
  have more theoretical plates, N.
• They would give longer RTs so would raise N in
  the equation.
• Smaller particle sizes (3-5 microns) lower
  diffusion effects and so would increase N.

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Selectivity, ?

• It is how a stationary phase differentiates
  between two different solute particles and
  separates them.
• So a higher selectivity means that the stationary
  phase can separate two solutes.
• A selectivity of 1 is no separation.
• It should be at least 1.2

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Selectivity, ?

• What is it mathematically?

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Selectivity, ?

• So what affects the selectivity?
• Clearly the type of stationary phase is
  important.
• So changing the type of column changes the
  selectivity.

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Selectivity, ?

• However, you can also change the selectivity by
  changes in the mobile phase and temperature.
• A different mobile phase can change the strength
  of the solutes interactions with the stationary
  phase.

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Selectivity, ?

• You can change
• the -composition of the mobile phase
• organic solvent in mobile phase
• buffer pH
• additives like TEA

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Selectivity, ?

• Temperature in the column can also affect
  selectivity
• Increased temp generally shortens retention times
• However, it can have a different affect on
  different solutes
• Some solutes will get closer at higher T, others
  will get further apart with higher T

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Overall Resolution

• The overall optimal resolution is a balancing act
  between the selectivity, capacity factor, and the
  efficiency.
• Once you have the right type of stationary phase
  and the right mobile composition you can play
  with the flow rate, column length, and column
  temperature to optimize the resolution.
• Gradient runs may also be employed to optimize
  overall resolution.