HPLC BASICS

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HPLC BASICS
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Chromatography? ? ??? ????? ??? ? ??? ??? ????
??, ??, ???? ?? ???? ???? ?? ?? ??? ?? ????
???? ?? ???. ??, ??, ?? ?? ????? ??,??,?? ???
????. Injection Interaction
Elution abacbc
aabcbc a
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HPLC System? ??
Mobile Phase Reservoir
Pressure Gauge
Data System
Pump
Pulse Damper
Injector
Column
Detector
Fraction Collector
Gradient Device
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Solvent Delivery System (Pump) ??? ?????? ???
??? ?????? ???? ??
1. Pump? ?? a. ??? ??? ?? ??. b. ??? ?? ???
??? ??? ? ??? ?. 2. Waters Pump? ?? a. ???
??? ?? ?? b. Pulse ??? ?? system (Gear ????
Cam ???? Pulse? ??.) c. ??? ?? ?? ?? ?? ??. d.
Isocratic?? Gradient?? ??. e. ???? ??.
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FLOW PATH DIAGRAM
High sensitivity accessory
Transducer
Pump outlet
Solvent revervior filter
High pressure filter
Tee
Outlet check valve
Outlet check valve
Right pump head
Left pump head
Mechanical drive
Inlet check valve
Inlet check valve
Inlet manifold assembly
Solvent drawoff assembly
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??? ?????? ?? ??

• ??????? ( Isocratic Mode)
• ?????? ??? ??? ??? ???.
• ??????? ( Gradient Mode)
• ?????? ??? ??? ???? ??? ??? ? ?
• ??.
• - Single pump Low pressure
• - Multiple pump High pressure

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???? ???
pump
detector
----
column
injector
solvent resorvoir
- ?? ??? ??? ???? ? pump? ????? ??? ?? ???
??? ??? ??? ??? ???.
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???? ??? (Gradient Mode) Gradient Mode?
???? ??? ??? ??? ? ? ?? ?? ???, ??? ??
?? ? ? ?? ??? ???? ? ??. 1) Single pump.
Low pressure
pump
solvent A
mixer
solvent B
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2) Multiple pump
High pressure
pumpA
mixer
solvent A
pumpB
solvent B
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Injector
???? ??? loading?? Column?? ??? ??
Injector? ?? 1. Rheodyne injector (Valve
injector) 2. U6K injector (Syringe Valve
Mixed injector) 3. Automatic injector
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Rheodyne injector
1. ???? loop? ?? ???, ??? ??? loop ?? 2. Sample
loop ??? ??
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Auto Injector
1. Free injection ?? ??? ??? ?? 2. 96 Step? ????
?? 3. 4 mL vial 48?, 1 mL vial 96? ????
?? ?? ???? ????
?? 4. ???? ?? ??? ?? ?? ?? ? ?? 5. ???? ??
??? ?? ?? ??? ?? 6. Auto priming ? Auto
needle washing ? ???
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??

• ?? ??
• 3.5um, 4um, 5um, 6um, 7um, 10um, 37um, 55um, 75um
• ??
• Silica, Alumina, Silica-Bonded,
  Polymer(resin)-Bonded
• ?? Spherical, irregular
• Pore ?? 50 A 300 A

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???? ??

• Pore size Distribution
• Small Pore higher efficiency less band
  broadening
• Spherical or Irregular
• Irregular higher Surface area higher carbon
  load
• Spherical lower pressures longer life
• Spherical better structural stability longer
  life
• Particle size
• Smaller particle higher operating pressure
  higher efficiency

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??? ??

• ??? ?? ??
• Steel ??
• PEEK ??
• Radial-pak ??
• ????? ?? ??
• ????
• ????
• ????
• ??????

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??? ?? ??

• Steel ??
• Steel ???? ?? - End fitting ??
• PEEK (polyetheretherketone) ??
• ???? Radial-Pak ??????
• ??????

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1. Normal Phase(????)

• Functional group? polarity? ?? ????.
• ????? non-aqueous, non-polar solvent? ???? ???
  ? ??? ?? ??? ????.

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??????? ??

• Silica Phase
• Resolve Silica, uPorasil, Nova-pak Silica
• Bonded Phase
• Nova-pak CN, NH2
• u-Bondapak CN, NH2
• Resolve CN, NH2

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2. Reverse Phase(????)

• Bonded compound(C18, C8, CN, ph)? ?????? ????? ??
  ??
• ???? ? ??? ?? ??? ????

CH3CN/H2O
-Si-O-Si-C18 --
non-polar
OH
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?? ?? ??? ??

• u-Bondapak C18, CN, Phenyl
• Resolve C8, C18, CN, Phenyl
• Lichrosorb RP-18, RP-8
• Deltapak C18, C8
• Symmetry C18, C8
• Symmetryshield C8

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C18 Column ????
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End-capping???
TMS-Cl
-Si-O-Si-C18
-Si-O-Si-C18
-Si-OH
non-polar
-Si-O-Si-(CH3)3
non-polar
-Si-O-Si-C18
-Si-O-Si-C18
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3. Ion Exchange(?? ?? ??) ????? ??? ?? ?? ??.
-
-
-

counter ion

sample
-

-
-


-
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??????? ??

• IC-pak Anion/Cation
• SAX(Strong Anion Exchenger)/SCX
• Protein-pak DEAE/SP HR

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D. Size Exclusion(?????)
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??? column? ??

• GPC Styragel HR, HT, HMW Series
• Aqueous GPC Ultrahydrogel Series
• Aqueous GFC Protein-pak Series

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????? ????
???
gt 2000
lt 2000
Y
Hanxane
Organic Sol.
Silica, Porasil
N
Y
N
Y
MeOH
CN, Ph, C8, C18
Water
N
Y
Y
THF
Styragel HR, HT, HMW
N
Ultrahydrogel Protein-pak
Y
IONIC
NR3, COO, SO3
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?? ?? ??(????)
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???(Resolution)

• R1/4(1-1/a )SQRT(N)k/(1k)
• a ??? ??
• N ???? (??? ??)
• k ????

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Detector??? ?? ??? ??? ?? ??? ??? ??? ???
????
1. Signal to noise ratio (S/N ratio) ? ?? ?
2. Noise , Drift ? ?? ?
3. Detection limit ? ?? ?
4. Flow rate , temperature , pressure ? ??? ?
5. Gradient ? ??? ?
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Detector? ??

• Optical Detector

1. UV/Visible Detector
2. Fluorescence Detector
3. Refractive Index Detector

• Electrochemical Detector

1. Conductivity Detector
2. Electrochemical Detector
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???? Detector? ??
Electro-
RI
UV/VIS
Fluorescence
Conductivity
chemical
Response
universal
selective
selective
selective
selective
Sensitivity
micro-
gram
nanogram
picogram
picogram
nanogram
( typical
)
Flow
YES
NO
NO
YES
YES
sensitivity
Temperature
YES
YES
YES
NO
NO
sensitivity
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???/??? ???
???? ?? ??? ?? ??? ??? ?? cell?? ??? ????
??? ????, ??? ??? ???? ??? , ??? ??? ???
?? ?? ?? ???? ??? ??? ???? ?? ??? ?????
???? ??. ? ? ???? ?? ?(A)?
- ???? ?? ??? ?? (C)? ???? ,
- ?? ??? , ??? ???? ?? spectrum( Molar
Absorptivity ) ,
- ?? ??? ???? ?? (path length , b ) ? ??? ??.
???
photodiode? ???? ????? ?? ?? , ??? ?? ??? ???
???? ??? ??.
A Ebc ( Lambert- Beer Law )
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• ??? ??

1) Fixed type
- ?? (Lamp)? ?? ??? ? ?? ???? ??? ? ?? -
Wavelength 214, 229, 254, 265, 280, 313, 340,
405, 436, 546 nm
2) Variable type
- 190-600 nm ??? ?? ????? ??? ??? ???? ????
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3) Photodiode Array Type
- 190 - 800 nm ? ? ??? ??? scan ??.
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?? ???
1) ??
Exitation filter? ??? ?? sample cell? ???? ?? ??
??
? excited state? ??? ?? , ground state? ?????
??? ?
??? ? ??? ?? ???? ??.
??? ????? ???? ??? ?? ???? ???? ? ??
??, ???? ???? ?? ?? ??? ??? ???? ????
?? ??? ??? ??? ??? ??? ??.
2) ?? ??
Benzene ?? ??? ? ???? -OH, -OCH3, -H2, -NH(CH3),
-N(CH3)2, -F, -CO?? ???
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3) ? ?
Photomultiplier Tube
Emition
Emission Slit
Grating
Mirror
Flow Cell
Collection
Mirror
Beam Splitter
Exitation
Exitation
Grating
Slit
Photo
Diode
Lamp
Focusing Mirror
Mirror
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?? ??

• HPLC grade solvent? ???? 18 mega ohm??.
• Low Viscosity
• ??? ???. (miscibility No. ?? 15?? )
• ???? ???? ????? ???.
• ???? ?? ??? ???? ??? ??.
• UV cutoff, refractive index? ?? ??.

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Buffer ??? ?? ??

• 1) ??? ???? -???? ? ?????
• 2) ?? ????? ?? ??,??? (pH??)
• 3) ??? ?? ??? ???? ??

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??? ???

• ??
• ?? ???? ??? ??? ???? ?? ?? ??? ??? ??????? ???
  ????
• ??
• PIC A / Low UV
• PIC B Series

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?????(I)

• ?? ??
• HPLC ? ??
• ???(???? 18mega ohm)
• ?? ? ?? ??
• ??? volume ?? ??? ??
• ?? ??? ?? ?? ,?? ?? ??
• ???? ??? ? ??.

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?????(II)

• Filter
• ??? FILTER(PVDF)
• WATER? ACTIVATION
• ??? FILTER(PTFE)
• MeOH/??? ??? ACTIVATION

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?????(III)

• ??
• ???? ??? air, oxygen, bubble??
• Vacuum Degassing(????)
• ?? ??
• Helium Sparging
• ?? ???? sparging
• Ultrasonication(??? ??)