1

Part I – Basic Principles of HPLCPart I – Basic Principles of HPLC

Chromatography and HPLCVarious HPLC modes and applications

Normal phaseReversed phaseReversed phase ion pairing Ion exchange (IC)SEC (GPC/GFC)Chiral separation

HPLC system modes Isocratic elution Gradient elution

2

What is chromatography?What is chromatography?

Chromatography is one of the separation technique.

The main purpose of chromatography is

to separateseparate and quantifyquantify the target sample in the matrix.

3Why mixed sample Why mixed sample can be separated? can be separated?

river bed

direction of flow

4

What is the difference?What is the difference?

Interaction is different by gravityStrong

Weak

5How can the separation How can the separation be carried out?be carried out?

Separation can be carried out by the column.

packing material, 3-5um

column

6Interaction between packing Interaction between packing

material and samplematerial and sample

Due to difference interaction between packing material and sample, separation can be done.

packing material

sample A

sample B

7

colum

n

mixed sample

Separation can be done Separation can be done by the columnby the column

8

What kind of chromatography?What kind of chromatography?

High Performance Liquid chromatography (HPLC)

Gas Chromatography (GC) Thin-Layer Chromatography (TLC) Capillary Electrophoresis(CE)

9

Advantage of ChromatographyAdvantage of Chromatography

Simultaneous Analysis

High Resolution

High Sensitivity (ppm-ppb)

Small Injection Volume (1-100uL)

10

Advantage of HPLCAdvantage of HPLC

Moderate analysis condition - no need to vaporize the sample like

GC

Easy to fractionate the sample and purify

Good repeatability (C.V. < 1%)

11

Flow Diagram of HPLCFlow Diagram of HPLC

pumpinjector

column

ovendetector

12

Normal Phase ModeNormal Phase Mode

Petroleum etherPetroleum ether

CaCOCaCO33

Chlorophyll'sChlorophyll's

ChromatoChromatograph

ColorsColors

13

Effect of stationary phaseEffect of stationary phase

C18 (ODS)

Strong

C8

sample

sample

sample

C4

Medium

Weak

14

Elution orderElution order

SECColumn

15Relationship between Relationship between

MW and RTMW and RT

Molecu

lar Weigh

t (LogM

W)

Exclusion limit

Permeation limit

Time

16

Isocratic Elution SystemIsocratic Elution System

pump injector

column

ovendetector

Single SolventSingle Solvent

17

Gradient Elution SystemGradient Elution System

pumpinjector

column

ovendetector

pump

A

B

Time

B con

centration

18

Isocratic Elution ModeIsocratic Elution Mode

Long Time AnalysisLong Time Analysis

Bad SeparationBad Separation

MeOH / H2O = 6 / 4

MeOH / H2O = 8 / 2

( column : ODS type )

19

Gradient Elution ModeGradient Elution Mode

95%

30% MeO

H con

centration

20

Part II Instrumentation of HPLCPart II Instrumentation of HPLC

Solvent delivery pump

Sample injector

Column oven

Detector

21

Flow Diagram of HPLCFlow Diagram of HPLC

pumpinjector

column

ovendetector

22

Desirable Pump PerformanceDesirable Pump Performance

High Pressure Resistance

Precise Flow Rate

Low Pressure Fluctuation

23

Plunger reciprocating pumpPlunger reciprocating pump

motor and cam

plungerplunger seal

check valve

pump head

10 -100uL

24

AdvantageLow pressure fluctuationVery easy to replace the another

solvent

DisadvantageChange the plunger seal

Plunger reciprocating pumpPlunger reciprocating pump

25Dual plunger Dual plunger

with tandem flow linewith tandem flow line

check valve

Main plunger Sub plunger

Low pressure fluctuation UV / PDA detectorFluorescence detector

The number of maintenance parts is less. So this design is suitable for routine analysis.

26Dual plunger Dual plunger

with parallel flow line with parallel flow line

check valve

plunger head

Very low pressure fluctuation Refractive index detector Conductivity detectorElectrochemical detectorMS detector

The number of maintenance parts is more.

27

Single plunger Single plunger

check valve

High pressure-fluctuation Low sensitivity analysis using UV / PDA and Fluorescence detector

The number of maintenance parts is minimized.

28

HPLC InjectorsHPLC Injectors

Manual injector

Auto injector

29

6 port valve system6 port valve system

column

load position inject position

pump pump column

sample loop

30

Manual InjectorManual Injector

Rheodyne Manual InjectorRheodyne Manual Injector

31

Manual InjectorManual Injector

[LOAD][LOAD] [INJECT][INJECT]

Column

Pump

Column

Pump

32

Injection MethodInjection Method

Partial Injection MethodPartial Injection Methodbetter to inject less than half volume of

sample loop

Loop Injection MethodLoop Injection Methodbetter to inject more than 3 times

volume of sample loop

33

Dispersion and Dilution EffectDispersion and Dilution Effect

loop

pump

column

drain

drainpump

column

loop

drain

drain

Sample ZoneDilution ZoneDispersion Zone

Less than half volume Less than half volume More than half volume More than half volume

34

Dispersion and Dilution EffectDispersion and Dilution Effect

drain

drain

pump

column

loop

Sample ZoneDilution Zone

Less than 3 timesLess than 3 times More than 3 timesMore than 3 times

drain

drain

pump

column

loop

35

INJECT / LOAD positionINJECT / LOAD position

[INJECT ][INJECT ] position position

[LOAD][LOAD] position position

36

CautionCaution

Do not use pointed or beveled needle tip. Must use square end type.

Do not use more than pH 10 solution.Must change rotor seal.

37Column Temperature Column Temperature

Control DevicesControl Devices

Column Oven (heat /or cool) Heated Column Jacket

Aluminum block

Insulated Column Jacket Water bath

Column temperature control devices are functioning to keep the column temperature constant. The temperature fluctuation of column will influence retention time reproducibility.

38

Detectors for DetectorsDetectors for Detectors

Ultraviolet / Visible detector (UV/VIS)Photodiode Array detector (PDA)Fluorescence detector (RF)Conductivity detector (CDD)Refractive Index detector (RID)Mass spectrometer detector (MS)

39

Ultraviolet / Visible detectorUltraviolet / Visible detector

Ein Eout

A= εCl = - log (Eout / Ein)

l

C : concentration

Lambert-Beer's law

(A : absorbance)

CellCell

40

Ultraviolet / Visible detectorUltraviolet / Visible detector

Sample Cell

Reference Cell

Photodiode

Photodiode

Ein

EinEin

Grating

D2 / W lamp

λ Eout

41

Lambert-Beer's lawLambert-Beer's law

A= εCl = - log (Eout / Ein)

Absorb

ance

Concentration

linear range2.5

42

SpectrumSpectrum

275 nm275 nm

208 nm208 nm275 nm275 nm

208 nm208 nm

43

Additional FunctionsAdditional Functions

Dual Wavelength modeRatio Plot modeWavelength Time Program modeWavelength Scan mode

44

Sample Cell

512 Elements Photodiode Array

Grating

D2 / W lamp

Photodiode Array detectorPhotodiode Array detector

One element can detect one absorbance at one wavelength.

45

Photodiode Array detectorPhotodiode Array detector

TimeW

avelength

Ab

sorban

ce

Chromatogram

Spectrum

46

Photodiode Array detectorPhotodiode Array detector

UV spectra of peaks are obtained, which are supportive for identification.

By checking peak purity, one can know if any impurity is present.

Identification by SpectrumIdentification by SpectrumElution sequence

Peak 1

Peak 2

Peak 3

Acetonitrile30% 15%

Azelaic acidAzelaic acid

Benzoic acidBenzoic acid

Nitrobenzoic acidNitrobenzoic acid

48

Comparison by 3 Point SpectrumComparison by 3 Point Spectrum

Acetyl Salicylic Acid

down slope

up slope, peak top

49Cell design Cell design

of Fluorescence detectorof Fluorescence detector

50

Conductivity detectorConductivity detector

I

V

L

A A

K (conductivity) = I [A] / E [V] =A [cm2] / L [cm] * k (k : specific conductivity)

k= (I/E)*(L/A)k= (I/E)*(L/A)

electrode

51

Conductivity detectorConductivity detector

Conductivity is very affected to temperature. Must keep the cell in the temperature control devise.

52

Type of Ion ChromatographyType of Ion Chromatography

Non-Suppresser typesimple system (easy maintenance)low conductivity mobile phase

Suppresser Typelow back grand currentdifficult maintenance

53

Non-Suppresser TypeNon-Suppresser Type

extremely low ion exchanger

Low conductivity mobile phase

54

Suppresser TypeSuppresser Type

55

Conductivity and Peak ResponseConductivity and Peak Response

56Chromatogram of Cations Chromatogram of Cations

in Tap waterin Tap water

Analytical Conditions Column : Shim-pack IC-C3 Mobile phase : 2.0 mM Oxalic Acid Flow rate : 1.0 mL/min Temperature : 40 C Injection volume : 100 uL

Peaks 1. Na (8.25 ppm) 2. NH4 (0.01 ppm) 3. K (1.66 ppm) 4. Mg (2.22 ppm) 5. Ca (11.85 ppm)

57LCMSLCMS

Atmospheric Pressure IonizationAtmospheric Pressure Ionization

Pneumatically Assisted ElectroSpray(ESI)

Atmospheric Pressure Chemical Ionization(APCI)

High Voltage

3) Coulon Exclusion

Ion Evaporation

2) Evaporation

of Solvent

Liquid Samples

+ - + -+-

+-

+ -+

+

+

-

-+-++

+-+-+-+++-+-

++

++

+

+

Liquid Samples HeaterC oron a D ischarge

Nee dle

Ion molecular reaction

Nebulizing gas

Nebulizing gas

58

Design of LCMSDesign of LCMS

API Probe

Atmosphere High Vacuum

RP TMP1 TMP2(Vacuum pump system)

MSdetector

59What kind of compounds What kind of compounds

can be analyzed ?can be analyzed ?

ESI drugs and their metabolites peptides proteins many kinds of natural product (-OH, -NH2,-COOH, SO2, PO3 etc.)

APCI pesticides steroids drugs

60What kind of benefits What kind of benefits LC/MS users can get ?LC/MS users can get ?

Powerful qualitative capabilityPowerful qualitative capability Selective quantitative capability

M/Z

M/Z

M/Z