High-Performance Liquid Chromatography HPLC, when GC won’t cut it!!!
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Transcript of High-Performance Liquid Chromatography HPLC, when GC won’t cut it!!!
Components
• Mobile phase reservoirs• HPLC Pump(s)• Mixing valves• Sample injector (manual or auto)• Column• Detector• Plumming• Mobile phase waste container
HPLC-UV
Mobile Phases
A and B
HPLC Pump
syringe
6-port valveSample
loopHPLC column
Detector
MP waste
Jacket for controlling column temperature
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
• Length (5-15 cm); much shorter than GC column• Diameter (4 mm down to 50m)• Particle size (3, 5, or 10 m)• 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 L/min
Reversed phase stationary phase
• Most common; n-octyldecyl, C18
Si-O-Si-(CH2)17-CH3
CH3
CH3
Si-O
-Si-(
CH
2)17
-CH
3
CH
3
CH
3
Si-O-Si-(CH2)17-CH3
CH3
CH3
Si-O
-Si-(C
H2 )
17 -CH
3
CH
3
CH
3
Reverse-phase mobile phases
• 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
0 5 10 15 20 25 30 35 40 45
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
11.36 17.23
12.57
12.74
17.68
36.21
1.21 15.13 24.95
24.53
22.462.54
3.01 21.735.43 6.14 25.20
20.41 48.5527.31 37.1829.53 32.43 40.11 45.43
RP-HPLC Separation of a Tryptic Digest of BSA
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
– 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?Ift/tG > 0.25, then a gradient is appropriate
t = time between first and last peak
tG = time of gradient
RT: 0.00 - 35.03
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
8.59
10.02
12.56
3.152.99 13.933.6014.612.38
5.53 16.89 17.787.53 20.031.70 22.71 32.8429.14 31.2525.24 27.28
NL:2.27E8
Base Peak F: + c ESI Full ms [ 300.00-1300.00] MS chem361gbsaf
t = 22-8 = 14 min
tG = 22-4 min = 18 min
t/tG = 14/18 = 0.63 > 0.25
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 chromatography
• Separation of small ionic species– PO4
3+, SO42-, BrO3-, NO2-, F-, Cl-, ect
– Mg2+, Na+, Ca2+, Li+, Ba2+, ect– -Detected by differences in conductivity
Size Exclusion Chromatography• 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
Wall of columnavidin
biotin
linker
iodoacetamide
CS A T WM
PA