Basic High-Performance Liquid Basic High-Performance Liquid Chromatography (HPLC)Chromatography (HPLC)

辛耘企業（股）公司辛耘企業（股）公司

ContentsContents

Overview, Basic termsOverview, Basic termsHPLC ColumnsHPLC ColumnsMobile PhaseMobile PhaseHPLC EquipmentHPLC EquipmentHPLC ApplicationsHPLC Applications

Advantages of HPLCAdvantages of HPLC

Rapid and precise quantitative analysisRapid and precise quantitative analysis– Typical analysis time of 5-20 min, precision <0.5-1% RSDTypical analysis time of 5-20 min, precision <0.5-1% RSD

Automated analysisAutomated analysis– Using autosampler and data system for unattended analysis and report Using autosampler and data system for unattended analysis and report

generationgeneration

High sensitivity detectionHigh sensitivity detection– Detection limits of ng to pgDetection limits of ng to pg

Quantitative sample recoveryQuantitative sample recovery– Preparative technique from Preparative technique from g to kg quantitiesg to kg quantities

Amenable to diverse samplesAmenable to diverse samples– Can handle >60% of all existing compounds vs. 15% for GCCan handle >60% of all existing compounds vs. 15% for GC– Can analyze samples with little or minimal preparationCan analyze samples with little or minimal preparation

An HPLC ChromatogramAn HPLC Chromatogram

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00-50

125

250

375

550 TEST #1 20030402-STD-1 UV_VIS_1mAU

min

1 - 1.791 2 - 2.488 3 - 3.544

4 - toluene - 3.889

5 - 4.101

WVL:254 nm

HPLC: Basic TerminologyHPLC: Basic Terminology

Retention Time (tRetention Time (tRR))

Capacity Factor (k’)Capacity Factor (k’)Column EfficiencyColumn Efficiency– Plate number (n)Plate number (n)– Height Equivalent of a Theoretical Plate (HETHeight Equivalent of a Theoretical Plate (HET

P)P)

Selectivity (Selectivity ())Resolution (RResolution (Rss))

Retention Time (tRetention Time (tRR))

Retention time (RT)Retention time (RT)– RT is the time required to elute a peakRT is the time required to elute a peak– Expressed in terms of capacity factor (k’)Expressed in terms of capacity factor (k’)– RT is affected by flow rate, solvent strength, RT is affected by flow rate, solvent strength,

etc.etc.– RT increases with increase in column lengthRT increases with increase in column length

Capacity Factor (K’)Capacity Factor (K’)

k’ is a measure of peak retention or how mk’ is a measure of peak retention or how many times the peak is retained vs. an unretany times the peak is retained vs. an unretained peak (tained peak (t00))

k’=(tk’=(tRR-t-t00)/t)/t00=t=tRR’/t’/t00

Column EfficiencyColumn Efficiency

EfficiencyEfficiency– Measured in terms of theoretical plates (N)Measured in terms of theoretical plates (N)– Function ofFunction of

Column lengthColumn lengthParticle sizeParticle sizeFlow rateFlow rate

– Efficiency (N) doubles when particle size incEfficiency (N) doubles when particle size increases from e.g. 20 to 10 to 5 μmreases from e.g. 20 to 10 to 5 μm

Resolution (RResolution (Rss))

How well a mixture of components is sepaHow well a mixture of components is separatedratedControlling ResolutionControlling Resolution– Selectivity FactorSelectivity Factor

Change composition of mobile or stationary phaseChange composition of mobile or stationary phase– Efficiency FactorEfficiency Factor

Chang N by changing the flow rate, column length Chang N by changing the flow rate, column length or particle sizeor particle size

– Retention FactorRetention FactorChange eluent strengthChange eluent strength

ColumnColumn

Types of ColumnsTypes of Columns– MicroboreMicrobore

Diameter 1-2 mm; Length 7-30 cm; sample size 0.0Diameter 1-2 mm; Length 7-30 cm; sample size 0.01 mg; flow rate 0.1 mL/min1 mg; flow rate 0.1 mL/min

– Std. analyticalStd. analyticalDiameter 3-5 mm; Length 7-30 cm; sample size 0.1 Diameter 3-5 mm; Length 7-30 cm; sample size 0.1 mg; flow rate 1 mL/min mg; flow rate 1 mL/min

– PreparativePreparativeDiameter 5-20 mm; Length 25-50 cm; sample size Diameter 5-20 mm; Length 25-50 cm; sample size 10 mg; flow rate 10 mL/min10 mg; flow rate 10 mL/min

Column PackingColumn Packing

Particle sizeParticle size– Range from 3-20 μmRange from 3-20 μm– Smaller the particle, the higher the plate numbSmaller the particle, the higher the plate numb

er (N) and need higher pressure to move elueer (N) and need higher pressure to move eluent through the columnnt through the column

– Analytical application – particle size 3 -10 μmAnalytical application – particle size 3 -10 μm– Preparative application – particle size > 10μmPreparative application – particle size > 10μm

Column Packing MaterialsColumn Packing MaterialsSupport TypeSupport Type– Silica (or alumina) or polymer (cross-linked polystyrene)Silica (or alumina) or polymer (cross-linked polystyrene)

Bonded groupsBonded groups– C18, C8, C4, amino, cyano, phenylC18, C8, C4, amino, cyano, phenyl– Diethylaminoethyl (DEAE), sulfonate, quaternary ammoniumDiethylaminoethyl (DEAE), sulfonate, quaternary ammonium

Particle size (dParticle size (dpp): 3-, 5-, 7,- 10- or 20 μm): 3-, 5-, 7,- 10- or 20 μm– Efficiency is inversely proportional to dEfficiency is inversely proportional to dpp

– Column pressure is inversely proportional to (dColumn pressure is inversely proportional to (dpp))22

Pore size (dPore size (dporepore): 60-300A): 60-300A– Wide pore materials (300 A) are used for biomolecules or polymersWide pore materials (300 A) are used for biomolecules or polymers

Surface area: 90-400 mSurface area: 90-400 m22/g/g– High surface area maximizes solute interaction with bonded groupHigh surface area maximizes solute interaction with bonded group

ss

Column Connection Tubing and Column Connection Tubing and FittingsFittings

Connecting TubingConnecting Tubing– The i.d. and length of capillary tubing between The i.d. and length of capillary tubing between

the injector and the detector can affect band sthe injector and the detector can affect band spreadingpreading

– This problem of band spreading is pronounceThis problem of band spreading is pronounced when RT is small e.g. in microbore columnsd when RT is small e.g. in microbore columns

Column Length (L)Column Length (L)

Column efficiency (n) is proportional to LColumn efficiency (n) is proportional to LColumn can be connected together tp prodColumn can be connected together tp produce a longer column with higher efficiencyuce a longer column with higher efficiencyColumn back pressure is proportional to LColumn back pressure is proportional to LCommon column length is 10-25 cmCommon column length is 10-25 cmFast LC column are short column (3-10 cFast LC column are short column (3-10 cm) packed with small-particles (dp = 3 μm)m) packed with small-particles (dp = 3 μm)

Column Diameter (dColumn Diameter (dcc))

Sample capacity is proportional to (dSample capacity is proportional to (dcc))22

Flow rate is proportional to (dFlow rate is proportional to (dcc))22

Detection limits is inversely proportional (dDetection limits is inversely proportional (dcc))22

Column typeColumn type i.d.i.d.(mm)(mm)

Sample Sample capacity (mg)capacity (mg)

Flow rate (mFlow rate (mL/min)L/min)

Semi-preparativeSemi-preparative 8-208-20 10-5010-50 5-305-30

Stand AnalyticalStand Analytical 4.64.6 11 11

NarrowboreNarrowbore 2.02.0 0.20.2 0.20.2

MircoboreMircobore 1.01.0 0.050.05 0.050.05

Liquid Chromatographic ModesLiquid Chromatographic ModesNormal-phase or Liquid-solid (NP, LSC)Normal-phase or Liquid-solid (NP, LSC)– Separation based on adsorption/desorption of the analyte onto a Separation based on adsorption/desorption of the analyte onto a

polar surface (silica)polar surface (silica)

Reversed-phase (RPC)Reversed-phase (RPC)– Separation based on analytes’ partition coefficients between the Separation based on analytes’ partition coefficients between the

mobile phase and the bonded stationary phasemobile phase and the bonded stationary phase

Ion-exchange (IEC)Ion-exchange (IEC)– Separation based on ion-exchanging with the counter-ions and iSeparation based on ion-exchanging with the counter-ions and i

onic interaction with the bonded ionic grouponic interaction with the bonded ionic group

Size-exclusion (SEC or GPC)Size-exclusion (SEC or GPC)– Separation based on analyte’s molecular size and sieving action Separation based on analyte’s molecular size and sieving action

of the column packingof the column packing

Normal-phase or Liquid-solid Normal-phase or Liquid-solid Chromatography (LSC)Chromatography (LSC)

Normal phaseNormal phase– Used polar stationary phase packings and low polarity Used polar stationary phase packings and low polarity

solventssolvents– Non-polar compounds elute out first followed by increNon-polar compounds elute out first followed by incre

asingly polar compoundsasingly polar compounds– Eluents used – low to moderate polarity e.g. hexane, Eluents used – low to moderate polarity e.g. hexane,

CHCH22ClCl22, CHCl, CHCl33, etc., etc.ApplicationApplication– Isomers, compound class separations, etc.Isomers, compound class separations, etc.– Ideal for samples with low/moderate polarity, water inIdeal for samples with low/moderate polarity, water in

soluble and non-ionicsoluble and non-ionic

Reversed-phase Chromatography Reversed-phase Chromatography (RPC)(RPC)

Reversed phaseReversed phase– Most popular since it can handle the broadest variety Most popular since it can handle the broadest variety

of sample typesof sample types– Opposite of normal phase – stationary phase is non-pOpposite of normal phase – stationary phase is non-p

olar and mobile phase is polarolar and mobile phase is polar– C18 columns (e.g. octadecyl, -CC18 columns (e.g. octadecyl, -C1818HH3737))– Eluents used – aqueous solution of organic solvents e.Eluents used – aqueous solution of organic solvents e.

g. MeOH, ACN, THF, etc.g. MeOH, ACN, THF, etc.

ApplicationsApplications– Amino acids, homologs, herbicides, etc.Amino acids, homologs, herbicides, etc.

Mobile phaseMobile phase

HPLC mobile phases are usually a mixture of onHPLC mobile phases are usually a mixture of one or more solvents with these characteristicse or more solvents with these characteristics– Desirable Physical propertiesDesirable Physical properties

High purity, low cost, UV transparency, non-corrosive, low viHigh purity, low cost, UV transparency, non-corrosive, low viscosity, low toxicity, non-flammable, sample solubilityscosity, low toxicity, non-flammable, sample solubility

– StrengthStrengthStrength is related to polarity of solvent; Water is a strong solStrength is related to polarity of solvent; Water is a strong solvent in normal phase but a weak one in reversed-phasevent in normal phase but a weak one in reversed-phaseSolvent strengths under normal phase are characterized by Solvent strengths under normal phase are characterized by Hildebrand’s scale (EHildebrand’s scale (Eoo))

– SelectivitySelectivityDepends on dipole moment, induced dipole, H-bonding, and Depends on dipole moment, induced dipole, H-bonding, and dispersive characteristics of the solventsdispersive characteristics of the solvents

Mobile phaseMobile phase

ViscosityViscosity– Low viscosity solvents decreases the pressurLow viscosity solvents decreases the pressur

e to achieve a given flow ratee to achieve a given flow rate– Low pressure extends the lifetime of pumps aLow pressure extends the lifetime of pumps a

nd columnsnd columnsBoiling pointBoiling point– Low b.p. facilitate solvent removal from collectLow b.p. facilitate solvent removal from collect

ed fractionsed fractions– Some pumps have difficulty in pumping liquids Some pumps have difficulty in pumping liquids

with b.p. <40℃with b.p. <40℃

Mobile phaseMobile phaseFlammabilityFlammability– Many solvents used in LC are flammable\Many solvents used in LC are flammable\– LC equipment should be operated in well ventilated arLC equipment should be operated in well ventilated ar

ea away from sources of ignitionea away from sources of ignitionToxicityToxicity– Preferable to use low toxicity solvents for both safety Preferable to use low toxicity solvents for both safety

and disposal reasonsand disposal reasonsCostCost– Operation of LC – 1 mL/min for 8 hr/day for 5 day/weeOperation of LC – 1 mL/min for 8 hr/day for 5 day/wee

k uses 125 liters of solvent/yeark uses 125 liters of solvent/year– Prudent to consider cost of operation when selecting Prudent to consider cost of operation when selecting

solventsolvent

Common Solvents for HPLC Mobile Common Solvents for HPLC Mobile PhasePhase

SolventSolvent Solvent streSolvent strength (Ength (Eoo))

BpBp( )℃( )℃

Viscosity Viscosity (cP)(cP)

UV cut off UV cut off (nm)(nm)

Refractive Refractive indexindex

N-HexaneN-Hexane 0.010.01 6969 0.310.31 190190 1.371.37

TolueneToluene 0.290.29 7878 0.590.59 285285 1.491.49

Methylene Methylene chloridechloride

0.420.42 4040 0.440.44 233233 1.421.42

TetrahydroTetrahydrofuranfuran

0.570.57 6666 0.550.55 212212 1.411.41

AcetonitrileAcetonitrile 0.650.65 8282 0.300.30 190190 1.341.34

2-propanol2-propanol 0.820.82 8282 2.302.30 205205 1.381.38

MethanolMethanol 0.950.95 6565 0.540.54 205205 1.331.33

WaterWater LargeLarge 100100 1.001.00 <190<190 1.331.33

Common Mobile phase ModifiersCommon Mobile phase Modifiers

BuffersBuffers– To stabilize pHs of mobile phase under RPC or IEC (phosphate, To stabilize pHs of mobile phase under RPC or IEC (phosphate,

acetate, citrate)acetate, citrate)

AcidifiersAcidifiers– To suppress ionization of acidic analytes under RPC (phosphoric To suppress ionization of acidic analytes under RPC (phosphoric

acid, acetic acid)acid, acetic acid)

Ionic strengthIonic strength– To control elution of ionic analyte under IEC (i.e., NaCl)To control elution of ionic analyte under IEC (i.e., NaCl)

Ion-pair reagentsIon-pair reagents– For separation of ionic compounds under RPC (hexane sulfonatFor separation of ionic compounds under RPC (hexane sulfonat

e)e)

Amine modifiersAmine modifiers– To reduce tailing of basic analytes under RPC (triethylamine)To reduce tailing of basic analytes under RPC (triethylamine)

Basic Flow diagram of Basic Flow diagram of LLCCEluent

Pump

Column

Detector

Pumping Separation Detection Recording

F-

NO3- SO4

2-

Cl-

NO2-

Br-

HPO42-

Sample injection valve

Detection cell

Injection