Transcript HPLC

HPLC
when GC won’t cut it!!!
Types of HPLC
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Reverse-phase (water/MeOH-soluble)
Normal Phase (very polar)
Adsorption (very non-polar)
Ion-Exchange (ionic)
Size-exclusion (large MW, >104)
C18 Phase designed to retain very
polar compounds
CH3
Si-O-Si-(CH2)17-CH3
CH3
P
P
Reverse-phase mobile phases
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Water
Methanol
Acetonitrile
THF
Additives, salts, acids, bases
Ion pairing
Gradients in reverse-phase
• For complex mixtures
• Polar
non-polar
– Buffer A 100 % H2O
– Buffer B 100 % MeOH
Separation Efficiency
• Van Deempter H = a + b/v + cv
– H – plate height
– A – multiple paths
– B – longitudinal diffusion
– C – equilibrium
• Ideal v
Key Variables effecting HPLC
separation and sensitivity
• Minimizing H
– Smaller ID of packing beads (3-10 mm)
• Maximizing N - number of extraction
events
– Longer columns N = L/H
• Maximizing sensitivity
– Smaller ID columns
– Band concentration vs. sample capacity
Parameters used to describe
retention of analyte
• Capacity factor - k`
– (tr-tm)/tm, where tr is the retention time of the
solute and tm is the void retention –
independent of column length
• Selectivity factor – a = k`a/ k`b = tr`a/tr`b
– Selectivity of a column for the separation of
component A and B
• Resolution = (tra – trb)/W =
N1/2/4(a/(a-1))2 ((1+k`b)/k`b)2
Normal Phase
• Bare silica
– Mobile phases, (ethyl acetate/ hexane)
• HILIC columns
– Attach polar groups to silica
– Methanol to water
Ion Exchange
• Ion exchange resins
– Strong cation, -SO3-H+
– Weak cation, - COO-H+
– Strong anion, - N(CH3)3+OH– Weak anion, - NH3+OH-
• Bound to polystyrene support
• Mechanism
– RSO3-H+ + P
RSO3-P+ + H+
Ion Exchange Gradients
• Mobile Phase A – H2O
• Mobile Phase B – 500 mM NaAc
Single ion chromatography
• Separation of small ionic species
– PO43+, SO42-, BrO3-, NO2-, F-, Cl-, ect
– Mg2+, Na+, Ca2+, Li+, Ba2+, ect
– -Detected by differences in conductivity
Size Exclusion Chromatography
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Stationary phase is a gel
Fractionates sample on the basis of size
Elution volume vs. molecular weight
Pore size of the gel defines the MW range
Exclusion limit – (10 6), permeation limit
(103)
• Ve = V0 + KVi
• Large molecules can not diffuse into the
pore, Ve = V0
Stationary and Mobile phases
• Gel filtration – hydrophilic packing (styrene
and divinylbenzene) and aqueous mobile
phase
• Gel permeation –hydrophobic packing
(sulfanated divinylbenzenes and
polyacrylamides) and non-polar organic
mobile phases
Affinity Chromatography
• A “handle” is attached to a solid support,
which is packed into a column
• This handle selectively binds to a certain
analyte or group of analytes
• Examples
– Antibodies to capture specific proteins
– avidin binds to biotin
ICAT reagent
• Selectively capture cysteine-containing peptides
A
M
S
C
A
T
W
P
iodoacetamide
linker
biotin
avidin
Wall of column
TLC
• Glass plates coated with thin layer of
coated particles
• Apply sample with capillary tube or
syringe or fancy applicators
• Develop plate
• Rf = dr /dm, retardation factor