Chem 230 Lecture Announcements and Topics

Chem.
230 – 10/21 Lecture

Announcements I
• Exam 2 Results
– Ave = 46.5 SD = 8
– similar average, but less
variability
– distribution
• Chem 253 –
Environmental Chem
offered next semester
Announcements II
• Posted HW Set #3 (original posting had
incorrect due date)
• Application Abstract due Today – I should be
able to check your and give you an o.k. by next
week
• 2D GC references posted on website
• Today’s Lecture
– GCXGC Questions
– SFC
– HPLC (start)
GC
GC x GC Questions
1. Why is the second dimension in GC x GC limited in resolution?
2. For which column in GC x GC is it important to use columns of
small diameter and thin films?
3. Why is overloading on narrow diameter 2nd dimension columns
less important than when using the same column with 1D GC?
4. How many dimensions does a GC x GC x MS chromatogram have?
5. Why does the peak capacity of a two dimensional technique
somewhat over express how many compounds can be resolved?
6. Why are two hot/cold jets needed in the modulator?
7. For what type of samples is GC x GC particularly useful?
a) Complex samples with compounds of similar polarity
b) Complex samples with a variety of compound polarity
c) Complex samples with a small number of analytes (e.g. pesticides in
food)
d) Related compounds with different sized alkyl groups R(CH2)nCH3
Supercritical Fluid Chromatography
= SFC liquid + gas supercritical fluid
• What is a supercritical
fluid? heat
– has properties intermediate
between a liquid and a gas
supercritical
– Defined by region P – T solid fluid
phase diagram critical P
– Phase boundary between liquid
liquid and gas disappears
Pressure
critical point
at critical point
– Demonstrated by heating gas
two phase system
– Fluid must operate above initial state
critical T and critical P
Temperature critical T
• Mobile Phase
– Supercritical fluid
– Most common fluids are:
• 100% CO2 (31.3°C critical temp. + 71 bar critical temperature)
• Mixture of CO2 and polar modifier (e.g. methanol), where modifier
added to adjust retention
– Properties vs. gases and liquids
• densities a little below liquids (solute – solvent interactions matter,
unlike ideal gases)
• viscosities a little greater than gases – allowing minimal pressure
drop vs. liquids
• diffusion coefficients closer to but greater than liquids, minimizing
C-term broadening vs. liquids
– Properties generally result in efficient separations that can be
applied to a greater class of compounds than by GC
• Stationary Phase
– Since carbon dioxide is non-polar, polar stationary
phase is most common
– Both OT columns (smaller diameters since diffusion
is slower than in GC) and packed columns can be
used
– Columns can be set up to take advantage of smaller
pressure drops of supercritical fluids vs. liquids of
using smaller particle sizes (or OT columns) as well
as longer column
• Instrumentation
– Equipment is somewhat specialized since it requires:
• pumps to reach supercritical pressures
• a way to add polar modifiers
• heaters to keep temperatures supercritical
• a restrictor after the column or detector to ensure pressure
remains high
• all fittings must be able to handle higher pressures
– Gradients can be set up by density programming
– Both GC type (e.g. FID) and many HPLC detectors (e.g UV
detection) can be used with some modifications). However,
FID is limited to 100% CO2 mobile phase
– Fraction collection (which is popular) uses cyclones to trap
solids released upon degassing
– The combination of specific equipment requirements and
limited market has made instrumentation expensive
• Applications
– Analyses where neither GC nor HPLC functions well (e.g.
polymers without chromophores – polyethylene glycol)
– Recent interest has been in high value preparative separations:
• SFC uses expensive packing material (e.g. chiral stationary
phases) more efficiently than HPLC
• SFC solvent is cheaper than HPLC
• Post column solvent removal occurs with chromatography (as
opposed to in additional step)
• Limitations
– Expensive equipment (partly a result of limited market)
– Limited mobile phases
Questions
1. How is the lower viscosity of SFs an advantage over
liquids in terms of chromatography?
2. Why are smaller diameter OT columns used in SFC
than in GC?
3. A drug manufacturer currently is using preparative
HPLC to isolate a single enantiomer of a chiral
mixture. List two advantages and one disadvantage
in going to SFC.
4. SFC is being used with 98% CO2/2% methanol to
separate polyethylene glycols with a polar stationary
phase. The separation of different sized polymers is
good but the retention factors are somewhat high
causing the separation to take too long. What can be
changed to decrease the separation time?
Liquid Chromatography
Classification of Types
• Classification based on pressure:
– Low pressure (gravity based, preparative)
– Moderate pressure (pressure from compressed gases in “flash
chromatography” or from low pressure pumps, also mainly
preparative)
– High pressure (high performance or HPLC)
– Ultra-high pressure (UPLC)
• Classification based on separation mechanism:
– Normal-phase (polar stationary phase, less polar mobile phase)
– Reversed-phase (non-polar stationary phase, more polar mobile
phase)
– Size exclusion chromatography (separation of molecules based
on size)
– Ion exchange chromatography (exchange of ions)
– Other types (ligand exchange and affinity)
Stationary Phases – Packing Material Geometry
• No geometry comparable to
OT GC
• Packed Columns
– Irregularly shaped particles
(older technology)
• Larger A term Advantage: H ~ kdp
• Less robust due to particles breaking
– Spherically shaped particles Disadvantage: P = k/d2p
• Smaller particles: decrease H (constant column dimensions)
(smaller A and C terms), but
increase back pressure dp (μm) tR (min) N P (bar)
5 30 25,000 19
3 18 42,000 87
Ultra performance LC 1.5 9 83,000 700
(UPLC) for needed P
Stationary Phases – Packing Material Geometry
• Packed Columns (cont.)
Dense core
– Superficially porous spheres
• Reduces C term without
Porous outer shell
increasing P (much)
• Less sample capacity
• Monolithic Columns
– Single “web” of polymer
(usually silica based) rather
than separate particles
– Advantageous because of
high efficiency with relatively
low back pressure
Stationary Phases – Packing Material Composition
• Packing Material Composition
– Silica based packing material
– Most commonly used material (many advances tried first with
silica)
– Available unbonded (normal phase), a wide variety of bonded
phases and silica hydride (relatively new)
– Main disadvantages are stability of silica (in normal phase) and
of Si-OR bonds (limits pH to 2 to 8)
• Polymeric (all hydrocarbon) packing material
– Usually styrene-divinylbenzene
– Poorer performance (efficiency, maximum pressure)
– Common with ion-exchange and size exclusion
– “Bonded Phase” contains ion exchange sites
Stationary Phases – Packing Material Composition
• Other packing material
– Zirconia
• More temperature/solvent stable than silica
• Both with and without bonded phases
– Porous graphitic carbon
• non-bonded reversed phase (similar to phenyl groups)
• highly stable
– Silica – polymeric hybrid particles
• Silica core with polymer between silica and bonded phase
• Improves stability of silica (possible to run from pH 0 to 14)
Silica-Based Normal Phase
• Historically, normal phase HPLC was the first type
regularly used and the stationary phase was (uncoated,
non-bonded) silica
• Stationary phase = silica surface
• Silica surface is somewhat heterogeneous (can have Si-
OH groups, Si-O- groups, Si-O-Si groups, and adsorbed
water)
• Use has decreased due to replacement by bonded phase
• Main disadvantage is slow equilibration (especially with
regards to water) and requirement of consistent water%
in mobile phase
• Mobile phases
– Normal phase requires mobile phases of low polarity
(to be opposite to stationary phase)
– Most common with hexane with polar additive
although wide variety of solvents possible (but only
low % water)
– Weak solvent = hexane (or other less polar solvent)
– Strong solvent = polar additive (e.g. 2-propanol)
– Increasing strong solvent decreases retention
• Mobile Phase
– Other Factors in solvent selection:
• Selectivity (different solvents will have different
solvent – analyte interactions; best to choose
solvent that emphasizes analyte differences)
• Solvent viscosity (low viscosity means smaller back
pressure for given flow rate)
• Solvent miscibility
• Detector limitations (e.g. wavelength cut-offs for
UV detection)
• Compatibility with column packing and tubing
• Advantages:
– Simpler stationary phase
– Useful for wide range of analyte polarities
• Disadvantages:
– Long stabilization times
– Retention time drifts
– Must control % water in solvents
Bonded Phases
• Most common on silica substrate but also
common with other substrates
• Bonded layer is stationary phase
• Common bonded phases
– Reversed phase:
• C18 (most common by far of all bonded phases)
• C8 (shorter alkyl chain)
• Phenyl (better retention of aromatic groups)
• Other groups (e.g. cholesterol)
– Normal phase or hydrophilic interaction (HILIC):
• Cyano (most stable)
• Diol (glycophase in text)
• Amino (least stable)
Bonded Phases
• Quality of column (particularly
(CH2)17CH3
for C18) depends on: Side group
– % of SiOH bonded to C18 group

– % of remaining SiOH left (many
groups “end capped”) R Si R
– Bulk of side groups CH3
O O
• Normal phase stationary phase
has a greater tendency to Si Si
decompose
Bulk SiO2
End capped Si
C18 Phase
• Stationary Phase
– Very non-polar group; but base of C18 is polar (similar
in polarity to octanol)
– Some columns use imbedded polar groups
• Mobile Phase
– Polar mobile phase needed
– Most often water with polar additive (acetonitrile,
methanol, and tetrahydrofuran (THF) most common)
– Water is weaker solvent, organic modifier is stronger
solvent
– Except for columns with imbedded polar groups,
minimum % organic is 10% (needed to “wet” C18
phase)
Polar Bonded Phase
• Groups like –CN (cyano) provide separations similar to
unbonded silica but reach equilibrium faster and have
more consistent chromatograms
• Hydrophillic interaction chromatography (HILIC) refers to
polar stationary phase with water present in mobile
phase (usually in small amounts)
– The mechanism in HILIC is somewhat different because the
stationary phase has a higher % water (due to strong attraction
to polar stationary phase) than the mobile phase.
– Analyte interaction can occur with stationary phase or with
associated water
Note: Penetration of mobile phase into stationary phase can also
occur to some extent with other bonded phases (even with C18),
especially with stronger solvents
Bonded Phases
• Advantages:
– Faster equilibration (vs. silica)
– Absorption vs. adsorption (less tailing)
– Greater stationary phase volume
• Disadvantages:
– Often have reduced range of analyte polarities (very
polar analytes are very weakly retained by C18)
– Column bleed (if bonded layer starts degrading)
Gradient Elutions
• Similar to temperature
programming in GC Elution of glucose oligosaccharides: glucose
• Program increases % strong (monomer) to maltoheptaose (7 units)
solvent during run
• Example for HILIC (Normal Phase)
HPLC to right
• Advantageous when wide variety
of analyte polarities (plus peak
width and S/N advantages)
• Disadvantages: Isocratic run (~62% ACN/38% water)
– see those mentioned for GC plus
– Additionally column equilibration
takes time (gradient run took
longer when including
equilibration time)
– Requires more equipment
Gradient run (65% ACN to 50% ACN)

Size Exclusion Chromatography
• Basis for separation: large medium
– Analyte inclusion in pores
– Smaller analytes fit in pores and small
are retained more
– Larger analytes pass around
packing material
• Used for separation of polymers
based on size
• Variety of packing geometries
can be used (best if weaker
analyte – stationary phase
interactions)
• Pore size is critical and affects
retention
Size Exclusion Chromatography
• Applications:
– Coarse separations
(monomers from oligomers
or oligomers from polymers)
– Determination of
polymer/oligomer molecular
weight
• When used for
determination of molecular
weight, standards must
also be run
• Standards should be
similar and have similar
retention
http://www.phenomenex.com/cms400min/litlib/brands/asahipakgfcolumns.pdf
Ion Exchange Chromatography (IC)
• Stationary phase is made of ionic groups attached to polymeric
support
• Ionic groups have opposite charge of solute ions being separated
(anion exchange requires cationic groups and cations require
anionic groups)
• Anion exchange can be “strong” –NR3+ (permanent charge) or
“weak”, -NH3+ (charged at low pH)
• Cation exchange also uses permanently charged (e.g. –SO3-) or
groups charged at higher pH ( –CO2-)
• Intermolecular forces are very strong (so strong that without an
exchanging ion, K is impractically large)
• Mobile phase is aqueous buffer (cations or anions needed to
exchange with those on column)
• Strong eluents are higher concentrations and/or more strongly
bound ions
IC
Separation of Ions
• Strength of ion-ion interaction Cl- SO4=
depends on: A -
A-
– Charge (stronger for more highly
charged) A-
– Ion size (smaller hydrated size A-
interacts more strongly)
• Cation ranking: A-
Li+ (weak) < H+ < NH4+ < Mg2+ < Ca2+
• Anion ranking: A-
F- (weak) < OH- < Cl- < NO3- < SO42-
A-
• pH also affects ion forms and
elution
Note: cartoon ignores effect of mobile phase
counter ions
Other Stationary Phases
• Ligand Exchange (e.g. sugar
separation) HO-R
SO3-
– retention based on ligand sticking
to metal (or visa-versa) Ca2+
• Affinity Chromatography/ SO3-

Molecular Imprinted Stationary
Phases
– Stationary phase designed toward
retention of specific groups or
compounds stationary
phase
• Zwitterionic Phases
– contains both + and - groups analyte
1. Why is bonded silica generally the first type of stationary phase
considered?
2. What pH range could be used if separating an organic weak acid
(RCO2H) with a pKa of 5.2 using a silica based C18 column?
3. What type of reversed-phase stationary phase could be used to separate
alkyl amines (bases with conjugate acids of pKa of about 9)?
4. Is it possible to run some silica-based columns at extreme pH values?
5. What type of packing material should be chosen for high temperature
separations?
6. List two examples of stationary phases that could be used with normal
phase HPLC.
7. Which of the following additives could be used to retain ClO4- on a C18
column?
a) CH3NH2 b) (CH3CH2CH2CH2)4N+
c) CH3SO3- d) CH3(CH2)9SO3-
Some More Questions
8. In a silica based NP-HPLC separation with hexane and ethyl
acetate, the % of which solvent should be increased to decrease
retention?
9. What can cause tailing in poor quality C18 columns?
10. A silica based NP-HPLC separation is using ether/2-propanol to
elute steroids. The retention factors are too small leading to
resolution issues even when using 100% ether. Suggest an
eluent that would lead to better retention.
11. What are the advantages and disadvantages of using bonded
polar phases vs. silica?
12. What types of compounds are least retained in size-exclusion
chromatography?
13. What groups are present on the surface of packing material in a
cation exchange column?
Instrumentation – Mobile Phase Delivery
• Mobile Phase Selection
– See slide 18 of lecture for factors influencing selection
of mobile phase
– Solvents must meet purity requirements (for column
and detector functions)
– Solvent selectivity issue is important because:
• Changing solvent affects retention for different analytes
differently
• HPLC is less efficient than GC so often more likely to have
overlapping peaks
• Changes in pH also are important for acidic/basic compounds
Less retention
• Example of solvent changes OH
CH3
OH
H3C
to affect selectivity: More
O
O O
CH3
– RP-HPLC Separation of retention

syringols from guaiacols R R
– Difference is in 2nd MeOH Syringols Guaiacols

group
– Water/Acetonitrile eluents
produce poor HPLC-UV
HPLC Sample
Sample11(MeOH/0.1%TFA)
(ACN/0.1%TFA) acetovanillone
acetosyringone
syringol/guaiacol separation 350
350
acetosyringone
acetovanillone
cinnamic
cinnamicacid
acid
300
300 isoeugenol
isoeugenol
factors 250
250
Absorbance
Absorbance syringic
syringicacid
acid
200
200
– Water/Methanol works better 150
150
100
100
(although greater retention 5050

00
-50
-50
with MeOH of syringol is 00 55 1010 1515 20 20
counter intuitive) Time

Time (minutes)
(minutes)
• Optimization of Mobile
Phase Composition Acetonitrile
– Separation should be (40% in water)
perfomed on three different
water/organic systems 20% ACN, 25%
– Then additional MeOH, water
separations can be carried
out using 3 component
THF (30% in
mobile phases Methanol (50% water)
in water)
– Patterns in retention can be
used to optimize mobile
phase composition
• Mobile Phase Selection – pH
Buffering O O
– In reversed-phase HPLC,
solute generally must be non- OH O
-
ionized to be retained
– pH is adjusted by adding
buffer in water/organic retained unretained
modifier
– pH at pKa means retention
factor about half of non-
+
NHNH
2 3 -
ionized acid retention time O
O
– In ion-exchange S
chromatography, pH should O CH3
be in range needed to
produce ions
– In ion-pairing RP-HPLC, an Benzyl amine Ion pair reagent = pentane
(conj.
ion-pairing reagent is added sulfonic acid (sodium salt)
acid pKa = 9.35)
Non-ionized only at
high pH
• Solvent Flow
– HPLC requires high pressures and thus
specific pumps
– The solvent also needs low levels of dissolved
gases for pumps to function
– For the simplest “dedicated” HPLC, a single
solvent reservoir and pump is needed
– For gradients and/or more method
development work, switching between
different solvents is needed
• Pumps Check valves InOut
Stroke
Stroke
– Most pumps use two piston closed
open
heads 180º out of phase to
reduce pressure open
closed
fluctuations
– Solvents go into and out of
piston heads through one-
way “check valves”
– Exit check valve closes on
“in” stroke and entrance
check valve closes on “out”
stroke
pistons
• Example of pump with 16000
14000
12000
non-functioning check 10000
Signal (uV)
8000
valves
6000
4000
2000
0
• Fluctuation in -2000
7 7.5 8 8.5 9 9.5 10
pressure and signal

Time (min)
can occur 200
100
• Changes to retention
Signal (uV)
0
time also will occur -100
-200
-300
8 8.2 8.4 8.6 8.8 9 9.2
Time (min)
Bad check valve leaking

• Solvent Flow (for gradient/greater flexibility
operations)
– Dual Pumps (high pressure mixing)
– Low Pressure Mixing (stream “open” in proportion to
fraction)
To column
To column
pump
pumps Mixing chamber

Chem 230 Lecture Announcements and Topics

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Chem.

230 – 10/21 Lecture

– % of SiOH bonded to C18 group

Gradient run (65% ACN to 50% ACN)

• Affinity Chromatography/ SO3-

– RP-HPLC Separation of retention

– Difference is in 2nd MeOH Syringols Guaiacols

(although greater retention 5050

counter intuitive) Time

chromatography, pH should O CH3

non-functioning check 10000

pressure and signal

can occur 200

time also will occur -100

Bad check valve leaking

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