Chapter 4Chapter 4 High Performance High Performance Liquid ChromatographyLiquid Chromatography（（ HPLCHPLC ））高效液相色谱高效液相色谱

19521952 ，， A.J.Martin and R.L.M.Synge reA.J.Martin and R.L.M.Synge received the Nobel Prize for the discoveceived the Nobel Prize for the discovery of partition chromatography.ry of partition chromatography. In 1941, they wrote:In 1941, they wrote: The mobile phase need not be a liquid but may be The mobile phase need not be a liquid but may be a a vapourvapour. We show below that the efficiency of co. We show below that the efficiency of contact between the phases is far greater in the chrntact between the phases is far greater in the chromatogram than in ordinary distillation or extracomatogram than in ordinary distillation or extraction columns. Very refined separation of volatile stion columns. Very refined separation of volatile substances should therefore be possible in a coluubstances should therefore be possible in a column in which a permanent gas is made to flow ovemn in which a permanent gas is made to flow over a r a gel impregnated with a nonvolatile solventgel impregnated with a nonvolatile solvent in in which the substances to be separated approximawhich the substances to be separated approximately obey Raoults’ law.tely obey Raoults’ law.

4.1 Introduction of HPLC4.1 Introduction of HPLC Development of HPLCDevelopment of HPLC

From LC to GCFrom LC to GC From LC to HPLCFrom LC to HPLC

Application of HPLCApplication of HPLC Special characters of HPLCSpecial characters of HPLC

High pressureHigh pressure High FlowHigh Flow

High performanceHigh performance OthersOthers Types of HPLCTypes of HPLC

Chemical-bond (distribution), Adsorption, Chemical-bond (distribution), Adsorption, Ion exchange*, ExclusionIon exchange*, Exclusion

4.2 Discussion of LC from the 4.2 Discussion of LC from the Rate TheoryRate Theory

H=A+B/u+CuH=A+B/u+Cu A=2A=2λλdpdp B→0B→0 C=?C=?

Mass transfer resistance Mass transfer resistance causing by the flowing mobile causing by the flowing mobile

phase phase

Mass transfer resistance causing Mass transfer resistance causing by the stagnatingby the stagnating （滞留） （滞留）

Equation of Rate-theory of HPLC Equation of Rate-theory of HPLC

涡流扩散项 纵向扩散 流动相传质 滞留区传质 固定相内传质涡流扩散项 纵向扩散 流动相传质 滞留区传质 固定相内传质

uDdC

DdC

DdC

uDCdH

s

fs

m

ps

m

pmmdp )(2

222

ConclusionConclusion

FineFine stationary phase stationary phase big big DDmm Low flowLow flow Extra-volume of the columnExtra-volume of the column TemperatureTemperature

Glossary Glossary HPLCHPLC Chemical-bond phase Chemical-bond phase

chromatographychromatography Ion exchange chromatographyIon exchange chromatography Exclusion chromatographyExclusion chromatography StagnateStagnate Isocratic and gradient elutionIsocratic and gradient elution

Typical HPLC chromatographs Typical HPLC chromatographs Pumping and Solvent SystemPumping and Solvent System Sample Injection SystemSample Injection System ColumnsColumns DetectorsDetectors

4.3 Instrument of HPLC4.3 Instrument of HPLC

4.3.1 Diagram of HPLC 4.3.1 Diagram of HPLC instrument instrument

Picture of HPLC instrumentPicture of HPLC instrument

4.3.2 Pumping and Solvent 4.3.2 Pumping and Solvent SystemSystem

Principle and Picture of reciprocating pump ( 往复泵）

Gradient elusion Gradient elusion （剃度洗脱）（剃度洗脱）

Reason for Reason for gradient elusiongradient elusion

Compared with Compared with GCGC

溶剂 A 溶剂 B

电磁阀 A 电磁阀 B

混合室

泵

溶剂 A 溶剂 B

高压泵 A 高压泵 B

混合室

Low-pressure mixing High-pressure mixing

Two types of gradient elusion

4.3.3 Injection system4.3.3 Injection system

Injection valve

4.3.44.3.4 Column Column

SizeSizePacking materialsPacking materialsPacking technologyPacking technology

4.3.5 Detectors 4.3.5 Detectors UV-detector ( 紫外检测器）Differential refractive index detector RI

（示差折光检测器）Fluorescence Detector ( 荧光检测器 )Electrical conductivity detector ( 电导检测器

)

UV detectorUV detector

Liquid flow cell for UV detection Liquid flow cell for UV detection in HPLCin HPLC

Single wavelength detector Single wavelength detector （固定波长的紫外检测器）（固定波长的紫外检测器）

Multiwavelength detector Multiwavelength detector （可变波长的紫外检测器）（可变波长的紫外检测器）

Photo-diode array detector Photo-diode array detector （光电二极管阵列检测器）（光电二极管阵列检测器）

Single wavelength detectorSingle wavelength detector

Multiwavelength detector Multiwavelength detector

DAD (DAD ( 二极管阵列二极管阵列 ) detector) detector

3-D figure of photodiode-array detect3-D figure of photodiode-array detector (or ( 二极管阵列检测器之三维图二极管阵列检测器之三维图 ))

Example of Selecting different wavelength

Characters of UV-detectorCharacters of UV-detector

Sample should be UV-Sample should be UV-absorption absorption

High sensitivityHigh sensitivity Large linear rangeLarge linear range Suitable for gradient elutionSuitable for gradient elution Most widely usedMost widely used

Refractive index detectorRefractive index detector(( 折光检测器折光检测器 ))

Properties of RI detectorProperties of RI detector

Non-specificNon-specific Low-sensitivityLow-sensitivity Easily affected by Easily affected by

temperaturetemperature Unsuitable for gradient Unsuitable for gradient

elutionelution

Fluorescence detectorFluorescence detector

Properties of Fluorescence Properties of Fluorescence detectordetector

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

High sensitivityHigh sensitivity

Electrochemical detector Electrochemical detector (( 电化学检测器电化学检测器 ))

Properties of Electrochemical Properties of Electrochemical detectordetector

Selective to electro-active Selective to electro-active subjectsubject

Easily affected by some surface-Easily affected by some surface-active substancesactive substances

Comparison of the detectors Comparison of the detectors   RI UV Flu. EC

selectivity General Special High Special

Special

Gradient elution no yes yes no

Linear range 104 105 103 104

Detection limit μg ng pg ngSensitive to temperature high low low middle

Property of mobile phase Unlimited limited limited limited

GlossaryGlossary PumpPump ValveValve LoopLoop Gradient elutionGradient elution Refractive indexRefractive index FluorescenceFluorescence CellCell DAD (photodiode array detector) DAD (photodiode array detector)

Chemically bonded phase HPLCChemically bonded phase HPLC Adsorption HPLCAdsorption HPLC Ion pair chromatographyIon pair chromatography Ion exchange and ion Ion exchange and ion

chromatographychromatography Size exclusion chromatography Size exclusion chromatography

4.4 Types of HPLC (HPLC 4.4 Types of HPLC (HPLC modules)modules)

4.4.1 Chemically bonded phase 4.4.1 Chemically bonded phase chromatography chromatography (( 化学键合相色谱法化学键合相色谱法 )) Liquid-liquid Partition chromatography Liquid-liquid Partition chromatography

(( 液液 -- 液分配色谱）液分配色谱） Normal phase liquid chromatographyNormal phase liquid chromatography ：： polapola

r stationary phase and less polar or nonpolar r stationary phase and less polar or nonpolar mobile phasemobile phase

Reverse phase liquid chromatography:Reverse phase liquid chromatography: Polar Polar mobile phase and less polar or nonpolar statimobile phase and less polar or nonpolar stationary phaseonary phase

Disadvantage of the partition chromatographDisadvantage of the partition chromatographyy

Solid phase of chemical-bonded phase Chro.Solid phase of chemical-bonded phase Chro.(1)(1) Reversed phases: Reversed phases: silica gel surface is modsilica gel surface is modified with alkyl group(non-polar, such as n-oified with alkyl group(non-polar, such as n-octadecylctadecyl ，， CC1818), becoming hydrophobic. ), becoming hydrophobic.

SiO

OH

SiO

OH

SiO

OH

固定相表面

C H Si Cl18 37 3

SiO

O

SiO

O

SiO

O

固定相表面

C H18 37Si

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

Mobile phase of HPLCMobile phase of HPLC①① General ruleGeneral rule Mobile phase play an important role on Mobile phase play an important role on kk SuitableSuitable k k is between 2 and 5is between 2 and 5②② Eluotropic seriesEluotropic seriesPolarity and polarity index of solventsPolarity and polarity index of solventsPolarity of solvent mixturesPolarity of solvent mixtures③③ Elution strengthElution strengthRelationship between polarity of solvent Relationship between polarity of solvent used as mobile phase and solid phase used as mobile phase and solid phaseElution behavior of reversed phase Elution behavior of reversed phase chro. and normal phase chro. chro. and normal phase chro.

④④ Other properties of solvent used as mobile pOther properties of solvent used as mobile phase:hase:chemical stablechemical stablecompatible to detectorcompatible to detectorlow viscosity (low viscosity ( 粘度粘度 ))others (boiling point, toxicity and expensothers (boiling point, toxicity and expense)e)⑤⑤ Strategy of choosing mobile phaseStrategy of choosing mobile phaseOptimizing Optimizing k k and and αα through experience or systematic procedure through experience or systematic procedure according to some rules (typical solvents taccording to some rules (typical solvents trial).rial).

⑥⑥ In reversed-phase Chro.:In reversed-phase Chro.: mixture of th mixture of the solvents methanol, acetonitrile and e solvents methanol, acetonitrile and tetrahydrofuran with water.tetrahydrofuran with water.In normal-phase chroIn normal-phase chro.. ：： mixture of dimixture of diethyl ether, methylene chloride and cethyl ether, methylene chloride and chloroform with n-hexanehloroform with n-hexane

⑦⑦ Isocratic elution and Gradient elutionIsocratic elution and Gradient elutionTroubles of gradient elusion (column, Troubles of gradient elusion (column, detector and pump)detector and pump)

4.4.2 Adsorption chromatography 4.4.2 Adsorption chromatography of HPLC (liquid-solid of HPLC (liquid-solid

chromatography)chromatography)

Mechanism of the separation Mechanism of the separation

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

Solid phaseSolid phase

Polar phasePolar phase ：： silica gelsilica gel ，， alumina etc.alumina etc.

non-polar phasenon-polar phase ：： active carbon, polymeactive carbon, polyme

r micro-beadr micro-bead

Mobile phase Mobile phase ε° of the solventε° of the solvent Mixture of the n-alkyl(Mixture of the n-alkyl( 正 构 烷正 构 烷

烃）烃） and and methylene chloridemethylene chloride Effect of water and other solventEffect of water and other solvent

Effecting of water to the adsorption Effecting of water to the adsorption HPLCHPLC

（（ 44 ）） Application of adsorption HPLCApplication of adsorption HPLC

Suitable to nonpolar substances which aSuitable to nonpolar substances which are difficult to dissolve in water, such as fre difficult to dissolve in water, such as fattiness and oilattiness and oil

Positional isomers or stereoisomersPositional isomers or stereoisomers

GlossaryGlossary Chemically bonded phase HPLCChemically bonded phase HPLC Reverse Phase HPLC (RP-HPLC)Reverse Phase HPLC (RP-HPLC) Normal phase HPLCNormal phase HPLC Liquid-liquid Partition HPLCLiquid-liquid Partition HPLC CC1818,C,C8 ,8 ,-NH-NH22, -CN, -CN Alkyl groupAlkyl group Elution strengthElution strength Methanol, acetonitrileMethanol, acetonitrile absorbent absorbent

4.3.3 Ion pair chromatography4.3.3 Ion pair chromatography(( 离子对色谱法离子对色谱法 )) （（ 11 ）） MechanismMechanism

If the analyte is ionIf the analyte is ion （（ AA++ ）） , which can not be ret, which can not be retained by the solid phase, an counter ion (Bained by the solid phase, an counter ion (B--) is ad) is added into the mobile phase to combine with the ided into the mobile phase to combine with the ionon （（ AA++ ）） ,forming an neutral molecule ,forming an neutral molecule （（ ion-pion-pair complex) and then retained by the solid phasair complex) and then retained by the solid phase.e.

( )

[ ] ,[ ][ ]

[ ] [ ][ ]

[ ]

W W O

OAB

W W

OAB W

W

AB W

A B A B

A BEA B

A BK E BA

E Bk

，分配系数

（（ 22 ）） Solid phase, moble phase and Solid phase, moble phase and ion-pair reagention-pair reagent Solid phaseSolid phase ：： CC1818,C,C88 Mobile phaseMobile phase ：： water solutionwater solution Ion-pair reagentIon-pair reagent ：： (C(C44HH99))44NN++(( 四丁基四丁基

铵正离子铵正离子 )) 、十六烷基三甲基铵正离子，、十六烷基三甲基铵正离子，ClOClO44-- ，十二烷基磺酸根等，十二烷基磺酸根等

4.4.4 Ion chromatography 4.4.4 Ion chromatography (( 离子色谱法离子色谱法 )) and ion exchange chromatographyand ion exchange chromatography(1) Mechanism(1) Mechanism Exchange equilibrium of the ion-exchangersExchange equilibrium of the ion-exchangers ：： cation exchangecation exchange ：： resin—SOresin—SO33--HH+++M+M+ + = resin—SO= resin—SO3-3-M M +++H +H

++

anion exchangeanion exchange ：： resin—NRresin—NR33++ClCl--+X+X- - = resin—NR= resin—NR3+3+X X --+Cl +Cl -- Retention differences depend on the tendency existinRetention differences depend on the tendency existin

g on the resin matrix, while the stronger the tendency g on the resin matrix, while the stronger the tendency the bigger tthe bigger tRR of the ion. of the ion.

The detection of ion chromatographyThe detection of ion chromatography Problems of detection of ICProblems of detection of IC Suppressor columnSuppressor column ：： Adding a suppressor to decrease tAdding a suppressor to decrease t

he background conductivity of the elution and increase he background conductivity of the elution and increase the signal-noise ratiothe signal-noise ratio 。。

ccanion: anion: R R—OH—OH--+H+H+ + CLCL--= R—Cl= R—Cl--+H+H22OO RR—OH—OH--+M+M+ + CLCL--= R—Cl= R—Cl--+MOH+MOH anion: anion: R R—H—H+ + + Na+ Na++OH-= R—NaOH-= R—Na+++H+H22OO RR—H—H+ + + Na+ Na++Br-= R—NaBr-= R—Na+++HBr+HBr Single columnSingle column ：： No need of the suppressor column, but No need of the suppressor column, but

with special ion-exchangers and eluents. with special ion-exchangers and eluents.

（（ 22 ）） Solid PhaseSolid Phase ion-exchanger:ion-exchanger: Coating on supporter Coating on supporter

or kind of ion-bonded phaseor kind of ion-bonded phase ，， the ion the ion exchangers are divided as cation exchaexchangers are divided as cation exchangers (strong/weak) and anion exchanngers (strong/weak) and anion exchangers (strong/weak). gers (strong/weak).

（（ 33 ）） Mobile phaseMobile phase Suppressor columnSuppressor column

Cation: HClCation: HCl ，， HNOHNO33 Anion: NaOH,NaHCOAnion: NaOH,NaHCO33/NaCO/NaCO33 。。

Single columnSingle column CationCation ： ： HClHCl ，， HNOHNO33(low concentration)(low concentration) ；； Anion: Salt of benzoic acid(Anion: Salt of benzoic acid( 苯甲酸苯甲酸 ), citric ac), citric acid(id( 柠檬酸柠檬酸 ) etc.) etc.

（（ 44 ）） Application of ICApplication of IC Inorganic anionInorganic anion Inorganic cation and organic acid, Inorganic cation and organic acid,

organic base, sugar etc. organic base, sugar etc. Name of the ion chromatography Name of the ion chromatography

and ion exchange and ion exchange chromatographychromatography

（（ 55 ）） Definition ofDefinition of ion chromatography and iion chromatography and ion exchange chromatograon exchange chromatographyphy

Example of applicationExample of application

4.4.5 Size-exclusion chromatograp4.4.5 Size-exclusion chromatographyhy（体积排阻色谱法）（体积排阻色谱法）（（ SECSEC ））

(1) Mechanism(1) Mechanism Special retention reason: the analyte is Special retention reason: the analyte is

trapped into the pore of stationary trapped into the pore of stationary phase.phase.Separation of molecules is due to their Separation of molecules is due to their different size --- excluded or retained different size --- excluded or retained according to the diameter of gel-pores. according to the diameter of gel-pores.

Separation of Size-exclusion Chromatography

Elution volume: VElution volume: VRR VVRR=V=V00+K+KDD·Vp·Vp

K=0, completely excludedK=0, completely excluded K=1, free permeation K=1, free permeation 0<K0<KDD<1.0<1.0

Sample with different size (more than Sample with different size (more than 10%).10%).

Water soluble: Gel filtration chromatographWater soluble: Gel filtration chromatography(y( 凝胶过滤色谱法）凝胶过滤色谱法）（（ GFCGFC ），）， analyzing proanalyzing protein.(tein.( 多肽、蛋白质、核酸、多糖多肽、蛋白质、核酸、多糖 ).).

Water insoluble: Gel permeation chromatogWater insoluble: Gel permeation chromatography (GPC) (raphy (GPC) ( 凝胶渗透色谱法凝胶渗透色谱法 )) ，， analyzing Manalyzing Mole Weight of high polymer (ole Weight of high polymer ( 高聚物高聚物 )) （如聚（如聚乙烯、聚氯乙烯等）乙烯、聚氯乙烯等） ..

（（ 22 ）） Solid phaseSolid phase Porous glass, silica particles, polyPorous glass, silica particles, poly

mers, polysaccharides(mers, polysaccharides( 多聚糖）多聚糖） Pore size of packing materials and Pore size of packing materials and

exclusion limitexclusion limit Being hydrophobic or hydrophilicBeing hydrophobic or hydrophilic Pressure limitPressure limit

（（ 33 ）） Mobile phaseMobile phase Sample-solubleSample-soluble Solid phase and detector compatibleSolid phase and detector compatible Buffers of water for GFCBuffers of water for GFC

Tetrahydrofuran for GPCTetrahydrofuran for GPC

（（ 44 ）） Application of SECApplication of SEC Separation and Analysis of big molecules Separation and Analysis of big molecules Distribution of Molecular Weight of Distribution of Molecular Weight of

polymer polymer

exampleexample

Polyethylene glycol Polyethylene glycol （（聚乙二醇）聚乙二醇）1.40000 2. 100001.40000 2. 100003. 3000 4. 1000 5.5003. 3000 4. 1000 5.500

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

water soluble---SEC, aqueous mobile phase MW ＞ 2000 water insoluble---SEC ， nonaqueous mobile phase water insoluble ---bonded phase (RP), adsorptionSample MW water soluble, nonionic---bonded phase (RP) ＜ 2000 acid---anion exchange/ion pair water soluble ， ionic basic---cation exchange/ion pair

Guide to liquid chromatography mode selection

HPLC chromatographic conditions Column Mobile phase Detectors and conditions Preparation of sample

GlossaryGlossary Ion pair chromatographyIon pair chromatography Counter-ionCounter-ion Ion pair reagent Ion pair reagent Ion chromatography Ion chromatography ion-exchange chromatographyion-exchange chromatography ResinResin CationCation AnionAnion Suppressor columnSuppressor column Size-exclusion chromatographySize-exclusion chromatography gel-filtration and gel permeationgel-filtration and gel permeation

Displacement pump Displacement pump 活塞泵活塞泵 diaphragm diaphragm 隔膜隔膜 Hydraulic Hydraulic 水力的 液压的水力的 液压的 Cartridge Cartridge 柱 桶柱 桶 Alkyl Alkyl 烷基烷基 Elutropic Elutropic 洗脱的洗脱的 Chiral Chiral 手性手性 isophthalic acid isophthalic acid 异酞酸异酞酸 Polysaccharid [Polysaccharid [ 生化生化 ]] 多醣多醣 ,, 聚糖聚糖 ,, 多聚糖多聚糖 Divinylbenzene [Divinylbenzene [ 化化 ]] 二乙烯基苯二乙烯基苯 Copolymerisate Copolymerisate 共聚物共聚物 Homolog Homolog 同系物同系物 Oligomer Oligomer 低聚物低聚物 dextrane [dextrane [ 化化 ][][ 药药 ] ] 右旋糖苷右旋糖苷 Phosphorescence Phosphorescence 磷光磷光

AliphaticAliphatic 〈化〉脂肪族的〈化〉脂肪族的 Cuvette Cuvette 小玻璃管小玻璃管 , , 透明小容器透明小容器 , , 试管试管 Phenanthrene [Phenanthrene [ 化化 ]] 菲菲