High-Performance Liquid Chromatography HPLC, when GC won

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High-Performance Liquid ChromatographyHPLC,
when GC wont cut it!!!
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Components

• Mobile phase reservoirs
• HPLC Pump(s)
• Mixing valves
• Sample injector (manual or auto)
• Column
• Detector
• Plumming
• Mobile phase waste container

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HPLC-UV
HPLC Pump
Jacket for controlling column temperature
6-port valve
Mobile Phases A and B
Sample loop
HPLC column
syringe
MP waste
Detector
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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

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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,
  gt104)

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Columns

• 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

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Reversed phase stationary phase

• Most common n-octyldecyl, C18

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C18 Phase designed to retain very polar compounds
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Reverse-phase mobile phases

• Water
• Methanol
• Acetonitrile
• THF
• Additives, salts, acids, bases
• Ion pairing

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Gradients in reverse-phase

• For complex mixtures
• Polar non-polar
• Buffer A 100 H2O
• Buffer B 100 MeOH or acetonitrile

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RP-HPLC Separation of a Tryptic Digest of BSA
11.36
17.23
100
95
90
85
12.57
80
12.74
75
70
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Relative Abundance
60
55
50
45
17.68
40
36.21
35
1.21
24.95
15.13
30
25
24.53
20
22.46
2.54
15
3.01
21.73
5.43
6.14
25.20
10
20.41
48.55
27.31
37.18
5
29.53
32.43
40.11
45.43
0
0
5
10
15
20
25
30
35
40
45
Time (min)
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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, 13107
• Temperature/computer simulations

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Gradients

• First step
• long, simple gradient
• Adjust accordingly
• Can become complex
• Do you need a gradient?
• If Dt/tG gt 0.25, then a gradient is appropriate
• Dt time between first and last peak
• tG time of gradient

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• Dt 22-8 14 min
• tG 22-4 min 18 min
• Dt/tG 14/18 0.63 gt 0.25

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Normal Phase

• Bare silica
• Mobile phases, (ethyl acetate/ hexane)
• HILIC columns
• Attach polar groups to silica
• Methanol to water

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Ion Exchange

• Ion exchange resins
• Strong cation, -SO3-H
• Weak cation, - COO-H
• Strong anion, - N(CH3)3OH-
• Weak anion, - NH3OH-
• Bound to polystyrene support
• Mechanism
• RSO3-H P RSO3-P H

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Ion Exchange Gradients

• Mobile Phase A H2O
• Mobile Phase B 500 mM NaAc

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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

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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

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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

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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

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ICAT reagent

• Selectively capture cysteine-containing peptides

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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