Chapter 4 High Performance Liquid Chromatography (HPLC)

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Chapter 4 High Performance Liquid
Chromatography(HPLC)

• ??????

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1952,A.J.Martin and R.L.M.Synge received the
Nobel Prize for the discovery of partition
chromatography.

• In 1941, they wrote
• The mobile phase need not be a liquid but may
  be a vapour. We show below that the efficiency of
  contact between the phases is far greater in the
  chromatogram than in ordinary distillation or
  extraction columns. Very refined separation of
  volatile substances should therefore be possible
  in a column in which a permanent gas is made to
  flow over a gel impregnated with a nonvolatile
  solvent in which the substances to be separated
  approximately obey Raoults law.

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4.1 Introduction of HPLC

• Development of HPLCFrom LC to GC
• From LC to HPLCApplication of HPLC
• Special characters of HPLCHigh pressure
• High FlowHigh performance
• Others
• Types of HPLCChemical-bond (distribution),
  Adsorption, Ion exchange, Exclusion

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4.2 Discussion of LC from the Rate Theory

• HAB/uCu
• A2?dp
• B?0
• C?

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Mass transfer resistance causing by the flowing
mobile phase
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Mass transfer resistance causing by the
stagnating(??)
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• Equation of Rate-theory of HPLC
• ????? ???? ????? ????? ??????

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Conclusion

• Fine stationary phase
• big Dm
• Low flow
• Extra-volume of the column
• Temperature

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Glossary

• HPLC
• Chemical-bond phase chromatography
• Ion exchange chromatography
• Exclusion chromatography
• Stagnate
• Isocratic and gradient elution

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4.3 Instrument of HPLC

• Typical HPLC chromatographs
• Pumping and Solvent System
• Sample Injection System
• Columns
• Detectors

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4.3.1 Diagram of HPLC instrument
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Picture of HPLC instrument
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4.3.2 Pumping and Solvent System
Principle and Picture of reciprocating pump (???)
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Gradient elusion (????)

• Reason for gradient elusion
• Compared with GC

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• Two types of gradient elusion

Low-pressure mixing
High-pressure mixing
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4.3.3 Injection system
Injection valve
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4.3.4 Column

• Size
• Packing materials
• Packing technology

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4.3.5 Detectors

• UV-detector (?????)
• Differential refractive index detector RI
  (???????)
• Fluorescence Detector (?????)
• Electrical conductivity detector (?????)

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UV detector
Liquid flow cell for UV detection in HPLC
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• Single wavelength detector (??????????)
• Multiwavelength detector (??????????)
• Photo-diode array detector (??????????)

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Single wavelength detector
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Multiwavelength detector
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DAD (?????) detector
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3-D figure of photodiode-array detector
(????????????)
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Example of Selecting different wavelength
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Characters of UV-detector

• Sample should be UV-absorption
• High sensitivity
• Large linear range
• Suitable for gradient elution
• Most widely used

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Refractive index detector(?????)
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Properties of RI detector

• Non-specific
• Low-sensitivity
• Easily affected by temperature
• Unsuitable for gradient elution

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Fluorescence detector
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Properties of Fluorescence detector

• High selectivity ( for protein, drug)
• High sensitivity

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Electrochemical detector (??????)
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Properties of Electrochemical detector

• Selective to electro-active subject
• Easily affected by some surface-active substances

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Comparison of the detectors
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Glossary

• Pump
• Valve
• Loop
• Gradient elution
• Refractive index
• Fluorescence
• Cell
• DAD (photodiode array detector)

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4.4 Types of HPLC (HPLC modules)

• Chemically bonded phase HPLC
• Adsorption HPLC
• Ion pair chromatography
• Ion exchange and ion chromatography
• Size exclusion chromatography

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4.4.1 Chemically bonded phase chromatography
(????????)

• Liquid-liquid Partition chromatography (?-?????)
• Normal phase liquid chromatographypolar
  stationary phase and less polar or nonpolar
  mobile phase
• Reverse phase liquid chromatography Polar mobile
  phase and less polar or nonpolar stationary phase
• Disadvantage of the partition chromatography

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• Solid phase of chemical-bonded phase Chro.(1)
  Reversed phases silica gel surface is modified
  with alkyl group(non-polar, such as
  n-octadecyl,C18), becoming hydrophobic.

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• (2) More about the solid phase of RP-HPLC
• Principle of the retaining of the analyte
• Alkyl group and other groups
• Stability of the solid phase
• (3) Normal phase polar functional groups
  chemically modified on the surface of silica
  gelThe group could be diol, cyano, amino, etc.

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Mobile phase of HPLC

1. General rule Mobile phase play an important role
   on k Suitable k is between 2 and 5
2. Eluotropic seriesPolarity and polarity index of
   solventsPolarity of solvent mixtures
3. Elution strengthRelationship between polarity of
   solvent used as mobile phase and solid
   phaseElution behavior of reversed phase
   chro. and normal phase chro.

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• Other properties of solvent used as mobile
  phasechemical stablecompatible to detectorlow
  viscosity (??)others (boiling point, toxicity
  and expense)
• Strategy of choosing mobile phaseOptimizing k
  and a
• through experience or systematic procedure
  according to some rules (typical solvents trial).

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1. In reversed-phase Chro. mixture of the solvents
   methanol, acetonitrile and tetrahydrofuran with
   water.In normal-phase chro.mixture of diethyl
   ether, methylene chloride and chloroform with
   n-hexane
2. Isocratic elution and Gradient elutionTroubles
   of gradient elusion (column, detector and pump)
   

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4.4.2 Adsorption chromatography of HPLC
(liquid-solid chromatography)

• Mechanism of the separation
• By using solid absorbent as stationary phase
  and solvent as mobile phase, the solute of being
  analyzed could be absorbed by the active centers
  on the surface of the stationary phase and then
  desorbed by the solvent. Difference of this
  procession could cause the separation of
  different analyte.

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• Solid phase
• Polar phasesilica gel,alumina etc.
• non-polar phaseactive carbon, polymer micro-bead

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• Mobile phase
• e of the solvent
• Mixture of the n-alkyl(????)and methylene
  chloride
• Effect of water and other solvent

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Effecting of water to the adsorption HPLC
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(4)Application of adsorption HPLC

• Suitable to nonpolar substances which are
  difficult to dissolve in water, such as fattiness
  and oil
• Positional isomers or stereoisomers

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Glossary

• Chemically bonded phase HPLC
• Reverse Phase HPLC (RP-HPLC)
• Normal phase HPLC
• Liquid-liquid Partition HPLC
• C18,C8 ,-NH2, -CN
• Alkyl group
• Elution strength
• Methanol, acetonitrile
• absorbent

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4.3.3 Ion pair chromatography(??????)

• (1)Mechanism
• If the analyte is ion(A), which can not be
  retained by the solid phase, an counter ion (B-)
  is added into the mobile phase to combine with
  the ion(A),forming an neutral molecule (ion-pair
  complex) and then retained by the solid phase.

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(2)Solid phase, moble phase and ion-pair reagent

• Solid phaseC18,C8
• Mobile phasewater solution
• Ion-pair reagent(C4H9)4N(???????)????????????,Cl
  O4-,????????

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4.4.4 Ion chromatography (?????) and ion
exchange chromatography

• (1) Mechanism
• Exchange equilibrium of the ion-exchangers
• cation exchangeresinSO3-HM resinSO3-M
  H
• anion exchangeresinNR3Cl-X- resinNR3X
  -Cl -
• Retention differences depend on the tendency
  existing on the resin matrix, while the stronger
  the tendency the bigger tR of the ion.

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• The detection of ion chromatography
• Problems of detection of IC
• Suppressor columnAdding a suppressor to
  decrease the background conductivity of the
  elution and increase the signal-noise ratio?
• canion ROH-H CL- RCl-H2O
• ROH-M CL- RCl-MOH
• anion RH NaOH- RNaH2O
• RH NaBr- RNaHBr
• Single columnNo need of the suppressor
  column, but with special ion-exchangers and
  eluents.

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• (2)Solid Phase
• ion-exchanger Coating on supporter or kind of
  ion-bonded phase,the ion exchangers are divided
  as cation exchangers (strong/weak) and anion
  exchangers (strong/weak).

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• (3)Mobile phase
• Suppressor column Cation HCl,HNO3 Anion
  NaOH,NaHCO3/NaCO3?
• Single column Cation HCl,HNO3(low
  concentration) Anion Salt of benzoic
  acid(???), citric acid(???) etc.

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  (4)Application of IC

• Inorganic anion
• Inorganic cation and organic acid, organic base,
  sugar etc.
• Name of the ion chromatography and ion exchange
  chromatography

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  (5)Definition of ion chromatography and
ion exchange chromatography
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Example of application
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4.4.5 Size-exclusion chromatography(???????)(SEC
)

• (1) Mechanism
• Special retention reason the analyte is trapped
  into the pore of stationary phase.Separation of
  molecules is due to their different size ---
  excluded or retained according to the diameter of
  gel-pores.

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Separation of Size-exclusion Chromatography
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• Elution volume VR VRV0KDVp
• K0, completely excluded K1, free permeation
  0ltKDlt1.0
• Sample with different size (more than 10).

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• Water soluble Gel filtration chromatography(?????
  ??)(GFC),analyzing protein.(????????????).
• Water insoluble Gel permeation chromatography
  (GPC) (???????),analyzing Mole Weight of high
  polymer (???)(??????????).

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(2)Solid phase

• Porous glass, silica particles, polymers,
  polysaccharides(???)
• Pore size of packing materials and exclusion
  limit
• Being hydrophobic or hydrophilic
• Pressure limit

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  (3)Mobile phase

• Sample-soluble
• Solid phase and detector compatible
• Buffers of water for GFCTetrahydrofuran for GPC

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(4)Application of SEC

• Separation and Analysis of big molecules
• Distribution of Molecular Weight of polymer

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example

• Polyethylene glycol (????)
• 1.40000 2. 10000
• 3. 3000 4. 1000 5.500

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4.4.6 Guide to liquid chromatography mode
selection

• According to the analytical task
• Concentration
• interference (composition)
• Information of the analyte
• structure
• molecular weight
• acidity
• solubility

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• Guide to liquid chromatography mode selection

water
soluble---SEC, aqueous mobile phase
MW gt2000 water insoluble---SEC,nona
queous mobile phase

water insoluble ---bonded
phase (RP), adsorption Sample


MW water soluble,
nonionic---bonded phase (RP) lt2000


acid---anion exchange/ion pair
water soluble,ionic

basic---cation exchange/ion pair
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• HPLC chromatographic conditions
• Column
• Mobile phase
• Detectors and conditions
• Preparation of sample

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Glossary

• Ion pair chromatography
• Counter-ion
• Ion pair reagent
• Ion chromatography
• ion-exchange chromatography
• Resin
• Cation
• Anion
• Suppressor column
• Size-exclusion chromatography
• gel-filtration and gel permeation

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• Displacement pump ???
• diaphragm ??
• Hydraulic ??? ???
• Cartridge ? ?
• Alkyl ??
• Elutropic ???
• Chiral ??
• isophthalic acid ???
• Polysaccharid ????,??,???
• Divinylbenzene ??????
• Copolymerisate ???
• Homolog ???
• Oligomer ???
• dextrane ?? ????
• Phosphorescence ??

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• Aliphaticlt?gt????
• Cuvette ????, ?????, ??
• Phenanthrene ??