CHMB16 NOTES: Lectures 8-9

Lecture 8 Part 1: Chromatography

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Chromatography is the separation of analytes based on their migration patterns in 2 phases: mobile phase and
stationary phase
o Mobile
Gas, liquid, or supercritical fluid
Liquid has many subcategories
Carries the components of the mixture through the medium being used
o Stationary
Remains fixed in either a column or on a solid surface
Acts as a constraint on many components in a mixture by slowing them down
Immiscible to mobile phase
Column chromatography: stationary phase is held in a narrow tube; mobile phase is forced under pressure or
by gravity
o Used as a basis for modern instrumentation
o Good at separating complex mixtures

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Individual chemical compounds are purified from mixtures

Can be used for larger scale applications
Main advantage: low cost, can dispose the stationary phase
Fresh solvent (eluent) is added to the top of the column
All the materials are packed in vertical glass column
Column is packed with silica gel or alumina and saturated with solvent
Wool/cotton or a porous disk packed at bottom to prevent stationary phase from being washed out (think
of the spinach pigment lab)
Keep adding solvent until compound of interest
reached bottom of column
Drained in separate flask
Process is called elution
Produces chromatogram
Injected at t0
Peak height and peak areas are
proportional to concentration
Partition Coefficient: K
K = CS/CM
[Stationary]/[mobile]
Larger K = the more a molecule interacts
with the column, longer retention time
Example: Sample A and Sample B

A and B are retained differently in the column

B has a larger K value
o Takes longer to elute from column
o Longer retention time
o Retention time: since K cant be measured
directly, use retention time (t R) instead
The time it takes for retained species to
reach detector
Detector will see A first then B
tM = time for unretained molecule to reach detector, aka
dead time

Plate Theory
- the theory states that a chromatographic column contains a
large number of separate layers (theoretical plates) and
that both mobile and stationary phases are at equilibrium at
these platesdo not actually exist
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Liquid-liquid Extractions
K = Corg/Cwater
o Aqueous and organic layers
The column uses this same principle, only its a continuous
process
Divide column into segments (plates, use equilibrium units)
o More plates = greater efficiency, better at
separating

Column length, L, is equal to the number of theoretical plates times the height equivalent of the theoretical plate
o L = NH, N= L/H
o N = 5.54 (tR / W1/2)2
W1/2 is the width at half height of the peak
o N = 16(tR /W)2
W= width of base of peak
o Plate number is a measure of column efficiency, can help gauge how good a column is before you buy it
Look for sharper peaks, higher resolution

Sample Calculation

Calculate the number of theoretical plates, N and the plate height H, when the retention time is
20.40 minutes, peak width at half height (given in minutes) is 0.65 minutes.
N = 5.54 (tR / W1/2)2 5.54 (20.4/0.65)2 = 5457 plates
Problems with Plate Theory
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Assumes K is independent of concentration

Assumes rapid equilibrium relative to velocity of mobile phase (equilibrium between M and S phase is infinitely
fastnot true, causes band broadening)
o Solute can pass a plate without entering
Doesnt consider longitudinal diffusion (causes band broadening)
o Band broadening: reduces the efficiency of the separation, another way to say loss of efficiency
Pertains to both quality and accuracy of separation
Increases with increasing age of column
Doesnt take into account that equilibrium is a semi-continuous process throughout the column

RATE Theory
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Looks at the time taken for the solute to equilibrate between M and S phases of the column
Accounts for band broadening
Van Deemter Equation: H= A + B/ +C
o A, B and C are coefficients and is the velocity of mobile phase
o A: multiple flow paths through a column
Consider how the column in packed
There can be an infinite number of paths in which the solvent or liquids can travel
Shortest path = fastest elution
Look at diagram: Path 1 is shorter than Path 2
More paths = peak broadening in final chromatogram
If no packing in column, A =0
o B: longitudinal diffusion
Caused by the components natural migration from a place of high concentration to low
B=0 when t=0 (start)
During the separation, 0 < t < tR
Peak at detector: tR
B increases with time and temperature
Becomes significant at very low flow rates of mobile phase
o C: mass transfer in and out the stationary phase
Broadening of peaks is a function of mobile phase velocity because mobile phase molecules
move faster than those in the stationary phase
In plate theory the slow condition is assumed to hold throughout

Van Deemter equation helps explain the

optimum flow rate for separation on a column
Resolution
o The resolution of an elution in the measure of
how well 2 elution peaks can be differentiated in
chromatographic separation
Quantitative measure
Defined as the difference in retention
times between the 2 peaks divided by
the combined widths of the elution
peaks

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A = component A, elutes first

B = component B, elutes second
W = width of peaks
If RS = 1.0, then peaks touch with 4% overlap
Too big of an error to tolerate
If RS = 1.5, 0.3% overlap, acceptable

Resolution can be improved by increasing the number of plates, lengthening column

Increases time for analysis though

Planar chromatography: stationary phase is supported

on a flat plate; mobile phases moves via capillary action
o Good for organic synthesis (TLC plates)
o Quick, cheap
o Not universal/versatile
Thin Layer Chromatography (TLC)
o Important for identification and separation of
mixtures of organic compounds
o Also has roles in:
Following course of reaction
Analyzing fraction collected during
purification
Analyzing purity of a compound

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Components of a mixture are partitioned between adsorbant (stationary phase, usually silica gel) and
the solvent (mobile phase, flows through adsorbent)
Uses plates: plastic, glass, or aluminium sheet is coated with a thin layer of silica gel
Solution of analyte applied on plate with a capillary tube (small spot) 1 cm from bottom of plate
Developed in chamber containing mobile phase (developing solvent)
How it works:
Mobile phase moves up TLC plate via capillary action
Components dissolve in solvetn and move up the TLC plate
Different components move up at different rates, depends on intermolecular forces between the
component and the silica gel (stationary phase) and the components and the movile phase
Silica (stationary phase) is very polar
o Polar analytes will interact more strongly with the stationary phase and will move
up very slowly on the TLC plate
o Non-polar analytes wont interact as much with silica and will move up higher on
the plate
Visualization
Not all compounds being separated are colourful, so have to be visualized
UV (254) blacklight
o Adsorbent layer fluoresces green
o Analyte spots quench fluorescence, appears as dark spot
Iodine vapours are unspecific colours
Some reagents have specific colours
Once you're able to visualize spots, can determine Rf values
Short-wave UV
Good for some organic compounds, they illuminate or fluoresce
Iodine Vapour
Forms brown/yellow complexes with organic compounds
Fluorescent Indicators
Fluoresce when places under UV light
Silver Nitrate Spray
Good for alkyl halides, dark spots form upon exposure to light
Sulfuric Acid Spray + heat
Permanent charred spots are produced
Rf Values
The distance the center of the spot moved/ distance the solvent from moved
Both measured from the origin
Can be used to compared compounds to standards
Not a physical constant should only be used to compare spots on same sheet, run at the same
time
Same Rf value = may be identicalif different, not identical
Resolution
The separation between 2 analytes can be expressed as R S
If Rs is > 1, analytes are resolved
To improve. (for 2 closely migrating components)
Change elution strength of solvent
o If compounds are interacting with the stationary phase too much, and therefore,
too slow, add non-polar solvent to reduce interaction
Decrease diameter of analyte spots
o Smaller and less concentrated spots
Change stationary phase
o Find a stationary phase that interacts more selectively with one component than
the other

Lecture 8 Part 2: Gas Chromatography (GC)

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Sample is vaporized by injection into a heated system

Eluted through a column by inter gaseous mobile phase, then detected
2 Types

Gas-Solid (GSC)
Not used that much because theres semi-permanent retention of polar molecules (gases)
Called tailing of elution peaks
Gas-Liquid (GLC)
Most common
Martin and Synge (1941)
Analyte is put between gaseous mobile phase and a liquid phase on the walls of capillary tubing
How it works
GAS is the mobile phase (carrier gas)
o Must be chemically inert (noble gases are good for this; He, Ar, N 2, etc.)
o Depends on detector
o Sample (analyte) doesnt interact with carrier gas
Partitioning controlled by temperature

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Today: flow rates controlled by GC computer instead of bubble meter

Sample Injection
Inject with micro syringe, quantity is in L
Injector is usually 50C hotter than boiling point of sample, should be
hotter than column as well
Spitless mode: all the sample goes into column, split vent is closed
Split Mode: only a fraction of the sample reaches the column,
dependant on split ratio, split vent is open
Done to prevent detector/column overload

If split ratio is 100:1, only 1% of sample is injected and 99% goes to waste/vent
Columns
o Temperature needs precise regulation
o Housed in GC oven
o 2 Types
Packed
Older, poor resolution
Stationary phase was thin liquid film on fine solid support
Some used non-coated solid stationary phases (GSC) rare though
Open Tubular/Capillary
Modern
Stationary phase is a liquid film that coats inside of very long capillary tube (100 m)
Better resolution, 300,000 plates

WCOT: primary type of GC column used

High resolution, especially compared to packed
Smaller capacity, but not an issue if sufficient
amount of analyte is available
Can analyze ppt to ppb concentration for volatile
liquids
Immobilized Liquid Stationary Phase
When picking what column coating to use,
consider:
Low volatility
Thermal stability (100C greater than operating
temperature)
Chemically inert
Polar analytes need polar stationary phases
(-CN, -CO, -OH)
Same with non-polar (hydrocarbons, dialkyl
siloxanes)

Common Capillary
Column Stationary
Phases
(Diphenyl)x (dimethyl)x-1
polysiloxane
o Generally, non polar
Cyanopropylphenyl dimethyl
polysiloxane
o Intermediate polarity
Biscyanopropyl
cyanopropylphenyl polysiloxane,
carbowax (polyethylene glycol)
o Very polar

Other Considerations
- Longer column length = greater number of plates
o But brand broadening observed if analytes remain on column for too long
o Increased length requires more time and money
- Increased stationary phase thickness + diameter = increased sample capacity
o Drawback: longer analysis time and more column bleed (signal in background)
Temperature Programming
- Easiest way to alter GC separation is to alter the temperature of the oven
o Can alter pressure of carrier gas as well, but not commonly done
- Isothermal: constant temperature,
- Gradient: varied temperature
- Changing temperatures causes change in equilibrium (rate) of the analyte (stationary mobile phase)
o More or less time is spent in the stationary phase
o Greater difference in times between analytes = better separation
- If temperature is too high, peaks can co-elute
o Poor resolution
- If temperature is too low, can still get a good elution
o Decent resolution, long separation time
DETECTORS
- Lots of variation
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Ideal detectors are/ have:

Sensitive
Stable
Good precision
Linear response over
several orders of magnitude
of conc
Temp range: room-400C
Rapid response time
Note: no one detector has
all these qualities

Types of Detectors
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Flame Ionization Detector (FID)

o Most commonly used
o When analyte exits column, directed to air-hydrogen flame
o Most organic compounds produce ions and electrons when pyrolyzed (flamed)
o Electrical current applied between burner tip and collector causes ions and electrons to move towards
collector, producing weak current

 Weak signal is amplified and measured

Advantages
Signal is proportional to # reduced carbon atoms in flame
Not affected by flow rate
High sensitivity, low noise
Large linear response range
Easy to use
Rugged
Cheap (is)
o Disadvantages
Destructive
Not good for inorganics (CO and CO2 hard to detect)
Carbonyl, alcohol functional groups give weaker signals
Thermal Conductivity Detector (TCD)
o Earliest types, not popular today
o Uses heated metal/ semi-conductor wire (gold, tungsten, platinum)
o Electrical resistance of wire depends on thermal conductivity of gas, and analyte present in gas
o Advantages
Simple, easy to use, reliable
Universal response (organic/inorganic)
Large linear range
Non-destructive
Older ones have built in TCD
o Disadvantages
Low sensitivity
Cant use with capillary columns because amount of analyte is small
Electron Capture Detector (ECD)
o Sample from column passes over radioactive emitter, absorbed on metal foils
o Electrons from emitter ionize carrier gas (N2) cause burst of electrons to be released and measured from
electrode
Burst produces current, gives signal signal
o Good for environmental samples with halogens
Advantages
o Good for molecules with electronegative atoms
o Used for chlorinated pesticides
o Insensitive to amines, alcohols, hydrocarbons
o Highly sensitive
o Doesnt alter sample significantly
Disadvantages
o Narrow linear range
o Radioactive
o Can be limiting, as it is highly selective
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Photoionization Detector (PID)

- Column effluent irradiated with intense UV light source
- Ionizes molecules
- Measures ions with electrodes in detector cell
- Selectivity based on how easily molecules are ionized
- Not universal, most sensitive for aromatic hydrocarbons and organosulfur compounds that photoionize easily
GC can be combined with mass spectrometry as well.
Lecture 9 Part 1: Modern Liquid Chromatography (LC)
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Old: glass columns, stationary phase was solid particles coated with adsorbed liquid, slow flow rates, long
separations
o Pressure was applied simply to increase flow rates (think of how we used the bulbs on the disposable
pipettes to make the spinach pigments go down the tube faster)
o Large plate height and poor separation

Smaller particle size of stationary phase = better and faster separations

High Performance Liquid Chromatography (HPLC)
o High pressure LC is another applicable name as well
o Most widely used of all analytical separation methods
o Uses high pressure to push the mobile phase through a column containing a stationary phase
o Separated complex mixtures with high resolution
o Basic components:
Reservoirs: solvents, with degasser, usually multiple exist to create gradient elution (changing
polarity of the mobile phase during a run; 2 or more solvents of different polarities varied
throughout the separation) have a degassing unit built to prevent air bubbles entering system
(called sparging)
Pumping system: pressure goes up to 6000 psi
Injection system: small sample volumes (0.1 - 500 L)
Sample injection can be limiting factor for precision of HPLC
Slow injection cause band broadening, sample volume must be small
Column can become saturated
Manual or (new) autosamplers exist
Sampling loops useful injection tool as well
Columns
Main (analytical column) where sample goes other columns exist as well
Guard Column: increase lifespan of analytical column by removing particles,
contaminants, and anything else that would irreversibly bind to analytical column
o Small, disposable
Columns are usually packed
HPLC categorized by types of stationary phases found in columns
o Adsorption: only silica/alumina stationary phase, no additional (silica gel
column used in orgo labs are an example)
Solute is adsorbed on surface of stationary phase
Oldest types
Mobile phase is liquid, stationary phase is solid
Column is washed with strong solvent weakly binding molecules are
washed off first, strongly bindings ones wash off last
DISADVANTAGE: some solutes permanently adsorb and cant be
washed off
o Partition: stationary phase coated onto a solid support (silica, alumina,
divinylbenzene resin)
Solute is dissolved in liquid phase coated on surface of solid support
Most widely used HPLC method
Both stationary and mobile phases are liquids
Immiscible with each other
Stationary attached to a solid packing support material via a
chemical bond
Tetramethylsilane (TMS): organosilicon used to make column surface
hydrophobic, C-18 hydrocarbon
The -Si-OH groups of this molecule act as attachment sites for
the bonded (stationary) phase
Endcapping: replacing accessible silanol (-Si-OH) groups in a bonded
stationary phase with trimethylsilyl groups
Prevents tailing of a polar compounds peak
Covers up unreacted OH on silica surface to prevent unwanted
interactions with polar molecules and ion exchange
2 Types
Normal Phase (NPLC)
o Binds polar R groups
Amino, cyano, and diol groups
o Polar stationary phase
o Non-polar mobile phase
Reversed Phase (RPLC)

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Cation Exchange: column surface is
negatively charged, cations are
retained.
Applications: separation of drugs,
metabolites, serums, food
preservatives,
vitamins, sugars, etc.

of all HPLC today is with reverse phase

Binds non-polar R groups
Octadecyl, octyl, phenyl
Non-polar stationary phase (C8, C18, hydrocarbons)
The column is non-polar
Hydrophobic analytes stick to it
Polar mobile phase
Solvents; H2O, methanol, acetonitrile
Polar solutes elute first
Increasing organic fraction (organic : water solvent)
causes more hydrophobic compounds to elute
ADVANTAGE:
Water can be used as mobile phase
Cheap, nontoxic, UV transparent, biologically
compatible

Ion Exchange: use charged resins (+/-)

Cations are covalently attached to stationary phase; mobile anions are
held near the cations
In anion exchange resin, only anions attracted to it
Separation is based on charge
Uses polymer beads containing cationic or anionic groups
Anionic molecules bind to cationic media, cationic molecules washed
through column
Elution via salt gradient or pH gradient
Example: anion exchange

Size Exclusion: polymer networks

Small molecules penetrate pore of particles (think of mesh strainers)
Large molecules are excluded
When molecules are in pores, they are trapped and removed from the
mobile phase
Molecules larger than the pore size are excluded and have no retention
Elute first
Smallest molecules trapped in pores for the longest time
Elute last
Packing materials: styrene-divinyl benzene copolymers
Pore size controlled by extent of cross-linking
ADVANTAGES:
Short, well-defined separation times
Narrow bands
No sample loss (theres no chemical interaction between packing
material and analyte)
DISADVANTAGES
Short separation time; limited number of bands
Not effective in separating molecules of identical size
o Need ~10% difference in molecular mass to get
separation

Detectors
Many types: UV, IR, refractive, fluorescence, mass spec, etc.
Universal or analyte-specific
Most are non-destructive, but mass spec ones are
Standard Absorbance Detector (SAD)
o Single beam UV-VIS instrument (245 nm typically)
o Non-destructive, but non-universal (not all compounds absorb light)
o Z-shape flow cell
Diode Array Detectors (DAD): used when full UV-VIS spectral info is desired
o More common, versatile
o Non-destructive, but non-universal (only works for adsorbing analytes)
o Scans range of wavelengths
o Helps confirm identity of molecule
o Can obtain entire chromatogram at one wavelength or different wavelengths
Fluorescence Detectors
o Very selective because only a few molecules naturally fluoresce
o Can label molecules with fluorescence, so only they can be detected
o POLYAROMATIC HYDROCARBONS******* (fluoresce =)
Affinity Chromatography: covalent bonding of reagent (affinity ligand) to solid packing material such
as agarose or porous glass
Affinity ligand examples: antibodies, enzyme inhibitors, etc.
When mixture passes through column, only analyte that selectively binds to affinity ligand is
retained, molecules that dont bind pass through
Analyte eluted by pH gradient or ionic strength
ADVANTAGE: extreme specificity
One kind of molecule in a complex mixture becomes attached to a molecule that is covalently
bound to stationary phase
Applications: protein purification, nucleic acid purification, etc.
ADVANTAGES (HPLC):
Sensitive
Quantitative
Can be automated
Works on non-volatile and thermally instable molecules
Applicable to a variety of compounds:
Amino acids, proteins, nucleic acids, drugs, organics/inorganics, etc.
Changing the polarity of the mobile phase alters the separation
Isocratic vs. Gradient Elution

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Gel Permeation
Gel beads have pores
Look at diagram
Can be used to separate amino acid/protein mixtures

Isocratic: constant mobile phase composition (100%, MeOH, 50% H2O, etc.)
Can usually use one pump
Simple (no mixing chamber required)
Limited flexibility, not used in research
o Used mostly in industrial chemistry or routine analysis
Have little control as to how peaks are eluted
o Depends on how column an analyte interact chemically
Gradient: varying mobile phase composition (20% MeOH, 80% H2O or 90:10, etc.)
Multiple pumps, output from each is mixed together
Changing mobile phase changes polarity
Can elute compounds that were stuck on column
Column has to re-equilibrate to original conditions after each run
o Takes time
Cant control how the peaks are eluted by varying composition of the mobile phase
Ultra Performance Liquid Chromatography (UPLC)
o HPLC resolution can be increased by decreasing particle size of stationary phase
HPLC particle size ~ 5 m, UPLC, ~1.7 m
o Smaller particle size = increase of optimum flow rate to reach maximum N
o Requires a lot more pressure (up to 15,000 psi vs max 6000 psi)

Lecture 9 Part 2: Electrophoretic Separations

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Separation method based on differential rates of migration of a charged species in an applied DC field
First used to study blood proteins, now used to separate enzymes, hormones, antibodies, DNA, and RNA
Used to sequence DNA, human genome project
Methods
o Differential movement of charged species by attraction or repulsion in an electric field
o Traditionally done on polyacrylamide or agarose gels (gel electrophoresis)
o New method: separation occurs in narrow tubes or capillaries (capillary electrophoresis)
Simple instrumentation
Can possibly replace HPLC in many areas of chemistry
Development of Electroosmotic Flow
o The capillary is composed of silica, has OH groups in the form O Negatively charged fused silica surface (Si-O-)
Positive ions from buffer are attracted to negatively charged capillary wall
Under electric current, positive charges in double layer (see diagram,) move towards the
cathode
The double layer is produced by the cations
Electroosmotic flow (EOF): when voltage is applied across capillary, positively charged ions in
the diffuse layer move towards cathode, dragging the bulk solvent with them
Electrophoresis vs. Chromatography: Solvent Fronts (EOF vs. Laminar Flow)
o EOF
Flat solvent front
Minimizes band broadening
Narrows detection zone
Needs to be controlled sometimes
If pH is too high (basic), EOF can be too large/rapid
Some separations require lower EOF
Ways to change EOF
Change electric field
Alter pH
Adjust concentration or ionic strength of buffer
Change buffer temperature
Modify capillary wall
o Laminar Flow
Parabolic

Resistance to flow at surface

More diffusion/broader detection zone
In electrophoresis, molecules move through the solvent on the basis of their charges, slowed down by frictional
movement through the solvent
o A large, positively charged molecule will elute SLOWER than a small counterpart because there are
greater frictional forced acting upon it
o NEGATIVELY charged molecules are moving against the EOF
o Migration rate:

E = field strength (V cm-1)

e = electrophoretic mobility (cm2 V-1s-1)

electrophoretic mobility is proportional to ionic charge and inversely proportional to frictional

factors
electric field only acts on ions (neutral species arent separated)
- Joule Heating: generation of heat by passing and electrical current through a metal
o Limits ability of electrophoresis to use high of an electric field as possible
o Causes non-uniform temperature gradients and changes in viscosity
Causes laminar flow lower efficiency and resolution
o Can be eliminated with liquid cooling of capillary (in modern instruments only)
- Detection
o UV: most common, somewhat universal, but doesnt work on carbs
o Laser-induced fluorescence: sensitive, but limited to fluorescent species
o MS: best universal detector, doesnt work with buffers
- Other Types of CE
o Capillary Zone Electrophoresis (CZE)
Sample injected into capillary containing buffer
Source and destination vials contain same buffer
High voltage applied to separate samples into zones
Positively charged, neg, and neutral
o Micellar Electrokinetic Chromatography (MEKC)
Separates hydrophobic/ionic interactions with micelles
Charged and uncharged species can be separated
o Capillary Isoelectric Focusing (CIEF)
Uses neutral capillary to get rid of electroosmotic flow
pH gradient created in capillary
Analyte will move while it has a charge
When it loses its charge (neutralizes) and no longer moves = isoelectric focusing
Separation: positive charged particles moves towards the area of high pH, negatively charged
move towards area with low pH
o Capillary Isotachophoresis (CITP)
Used for sample concentration prior to additional analysis
CEC vs. HPLC
- Less band broadening
- Efficienct
- Faster
- Compatible with MS