Transcript HPLC - University of Massachusetts Boston

High-Performance Liquid
Chromatography
HPLC, when GC won’t cut it!!!
Components
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Mobile phase reservoirs
HPLC Pump(s)
Mixing valves
Sample injector (manual or auto)
Column
Detector
Plumming
Mobile phase waste container
HPLC-UV
HPLC
Pump
Jacket for
controlling column
temperature
Mobile
Phases
Sample
loop
6-port
valve
HPLC
column
A and B
syringe
MP waste
Detector
HPLC Separations
• Different analytes have different equilibria
between the mobile phase and stationary phase
• Equilibrium is dynamic; thus we can view it as a
given analyte molecule spending a fraction of
time dissolved in the mobile phase
• Since different solutes gave different fractions, a
separation of the analytes occur as they are
pushed through the column by the mobile phase
Types of HPLC
• Reverse-phase (polar mobile phase/non-polar
stationary phase/somewhat polar analytes)
• Normal Phase (non-polar mobile phase/polar
stationary phase/non-polar analytes)
• Adsorption (non-polar mobile phase/polar
stationary phase/non-polar analytes); isomer
separation
• Ion-Exchange (salts/ionic stationary phase)
• Size-exclusion (aqueous/gel for large MW
solutes, >104)
Columns
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Length (5-15 cm); much shorter than GC column
Diameter (4 mm down to 50mm)
Particle size (3, 5, or 10 mm)
Different phases bonded to silica
Typically detection limit is decreased by
decreasing the column diameter
• Optimal linear flow rate conserved; so optimal
volumetric flow rate decreases with the square
of the radius
• 4 mm/ 1.0 mL/min; 1 mm/60 mL/min
Reversed phase stationary phase
CH3
Si-O-Si-(CH2)17-CH3
CH3
• Most common; n-octyldecyl, C18
CH3
CH3
Si-O-Si-(CH2)17-CH3
Si-O-Si-(CH2)17-CH3
CH3
CH3
CH3
Si-O-Si-(CH2)17-CH3
CH3
P
P
C18 Phase designed to retain very
polar compounds
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 or acetonitrile
RP-HPLC Separation of a Tryptic Digest of BSA
11.36
100
17.23
95
90
Relative Abundance
85
12.57
80
12.74
75
70
65
60
55
50
45
17.68
40
36.21
35
1.21
30
24.95
15.13
25
20
24.53
22.46
2.54
15
3.01 5.43
6.14
10
21.73
25.20
20.41
5
27.31 29.53 32.43
37.18 40.11
45.43
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10
15
20
25
Time (min)
30
35
40
45
48.55
HPLC Method Development
• Isocratic, Fig 25-25 Harris
• Find the best methanol separation
• Use Table 25-25 to guide you in finding the best
acetonitrile and THF separations
• Based on separations try binary mixtures
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Methanol, 38 %
Acetonitrile, 30 %
THF, 22 %
19 % MeOH/15% acetonitrile, 15 % acetonitrile/11%
THF, 19 % MeOH/11% THF
– Trinary mixture, 13:10:7
• Temperature/computer simulations
Gradients
• First step
– long, simple gradient
– Adjust accordingly
– Can become complex
• Do you need a gradient?
If Dt/tG > 0.25, then a gradient is appropriate
Dt = time between first and last peak
tG = time of gradient
RT: 0.00 - 35.03
NL:
2.27E8
Bas e Peak F: +
c ESI Full m s [
300.001300.00] MS
chem 361gbs af
8.59
100
95
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85
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10.02
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35
30
25
20
15
12.56
10
2.99 3.15 3.60
2.38
5.53
1.70
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0
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13.93
14.61
16.89
7.53
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10
12
14
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17.78 20.03
18
Tim e (m in)
20
22.71
22
Dt = 22-8 = 14 min
tG = 22-4 min = 18 min
Dt/tG = 14/18 = 0.63 > 0.25
25.24
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27.28
28
29.14
30
31.25
32.84
32
34
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
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