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HPLC seminar

1. Introduction
High Performance Liquid Chromatography (HPLC) is one mode of chromatography, the most widely used analytical technique. Chromatographic processes can be defined as separation techniques involving mass-transfer between stationary and mobile phases. HPLC utilizes a liquid mobile phase to separate the components of a mixture. These components (or analytes) are first dissolved in a solvent, and then forced to flow through a chromatographic column under high pressure. In the column, the mixture is resolved into its components. The amount of resolution is important, and is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material in the column. The interaction of the solute with mobile and stationary phases can be manipulated through different choices of both solvents and stationary phases. As a result, HPLC acquires a high degree of versatility not found in other chromatographic systems and has the ability to easily separate a wide variety of chemical mixtures.

History of HPLC
Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During the 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and did not achive sufficiently high resolution to distinguish between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flowthrough time, thus reducing purification times of compounds being isolated by column chromatogaphy. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure was debated. (Analytical Chem. vol 62, no. 19, Oct 1, 1990). High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds. By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above those obtained using previous techniques. Computers and automation added to the convenience of HPLC. Additional column types giving better reproducibility were introduced and such terms as micro-column, affinity columns, and Fast HPLC began to immerge. The past decade has seen a vast undertaking in the development of micro-columns, and other specialized columns. The dimensions of the typical HPLC column are: XXX mm in length with an internal diameter between 3-5 mm. The usual diameter of micro-columns, or capillary columns, ranges from 3 m to 200 m. Fast HPLC utilizes a column that is shorter than the typical column. A Fast HPLC column is about 3 mm long and is packed with smaller particles. Currently, one has the option of selecting from a lot of columns for the separation of compounds, as well as a variety of detectors to interface with the HPLC in order to obtain optimal analysis of the compound. Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry,in actual fact these fields currently comprise only about 50% of HPLC users(Analytical Chem. vol 62, no.19, Oct 1, 1990). Currently HPLC is used in a variety of fields and industries including the cosmetics, energy, food, and environmental industries.

What is HPLC ?
1. Introduction

H P L C

: : : :

High Performance (Pressure) Liquid Chromatography

GC : Gas chromatography TLC : Thin layer chromatography IC : Ion chromatography

What is HPLC used for ?


1. Introduction

1. Separation of mixed components 2. Qualitative analysis / Quantitative analysis 3. Preparation of interest components Separation analysis and/or preparation of interest components

Separation and Analysis


1. Introduction

A A C B C A B C A C

A B C

Separation

B C

Qualitative analysis What are components A, B and C ? Quantitative analysis What is the concentration of components A, B and C ?

Results obtained by HPLC


1. Introduction
C A B

Chromatogram containing three peaks


Qualitative analysis (identification) and Quantitative analysis (determination) Can be performed using the information contained in the chromatogram

Chromatography : Method Chromatogram : Results Chromatograph : Instrument

Chromatogram
1. Introduction
Sample IN

Mobile phase IN column baseline

Sample IN F Mobile phase IN A C B A BC D E E D

Chromatogram

Identification
What is component A?
1. Introduction
C A B
Sample

Caffeine

Component A elutes the same time as a caffeine peak. Component A is identified as caffeine.

Determination
What is the concentration of component A?
1. Introduction
C A B

Caffeine (1mg/ml) 5ul injection (5ug)

Peak area (or height) is proportional to the concentration (or amount) of the component. The concentration of component A(caffeine) is determined by comparing the peak area with that of the standard caffeine peak.

Separation Mechanism
1. Introduction
Separation is determined by column (packing material) and mobile phase (solvent).

Mobile phase elutes components. Packing materials retain components in the column.
Mobile phase (solvent)


A B C C A B

Column time Packing material

C>B>A

Five modes in HPLC


1. Introduction
LC mode Normal phase chromatography Packing materials Silica gel Mobile phase n-Hexane/IPE MeOH/Water THF Buffer sol. Buffer sol. Interaction Adsorption Hydrophobic Gel permeation Ion exchange Affinity

Reversed phase chromatography Silica-C18(ODS) Size exclusion chromatography Ion exchange chromatography Affinity chromatography Porous polymer Ion exchange gel Packings with ligand

HPLC Basic Instrumentation


1. Introduction

Solvent Delivery Injector Column Separation Mobile phase Pump Sample Injection

Detector

Data Processor

HPLC Instrumentation
1. Introduction

Column oven

Data processor

Pump

Injector

Column

Detector

Drain

Gradient Elution Unit

Auto sampler

Reagent pump

Fraction collector

System Controller

The JASCO advanced technology team has again met the challenge and designed a new line of HPLC instruments, The LC-1500series more than satisfies in response to the growing demand for greatly expanded HPLC analyses in the fields of not only biochemistry, pharmaceutical and medical science, but also in the areas of among other organic and inorganic compounds, foods, agricultural sciences, polymeric and natural substances and pollution. The LC-1500 series comprises pumps, detectors, autosamplers, its own column oven and other units each having built-in intelligence and incorporating many features with much higher levels of operability and reliability in addition to multiple functions, higher performance and higher accuracy than before, making them the most advanced instruments available.

2. Parameters used in HPLC

2. Parameters used in HPLC

Parameters used in HPLC

Retention parameters Column efficiency parameters Peak symmetry parameters Condition for Separation Retention : When a component in a sample interacts with the stationary phase in the column and a delay in elution occurs. Column efficiency : Goodness of a column

2. Parameters used in HPLC

Retention parameters

tR : retention time (the time between the injection point and the maximum detector response for
correspondent compound)

vR : retention volume (tR x eluent flow rate) k : capacity factor t0 : the time required for the component not retained by the column to pass through the column

tR tR - t0 t0 k =

tR - t0 t0

2. Parameters used in HPLC

Column efficiency
The number of theoretical plates N is given by: 4 method FWHM method 5 method h x 0.044 h x 0.5 h

tR

W4 N = 16 ( tR / W4 )2

W1/2 2 N = 25 ( tR / W5 )2 N = 5.545 ( tR / W0.5) W5

The height of the theoretical plate H is given by: H=L/N L : Column length

2. Parameters used in HPLC

Peak symmetry
S : Symmetry factor ( T : Tailing factor )

h x 0.05 f W0.05 W0.05 S= 2f

S = 1 : The peak is completely symmetric. S > 1 : Tailing S < 1 : Leading

2. Parameters used in HPLC

Degree of separation

tR2 tR1 k1 k2 Separation factor : W1 W2 k2 = k1 Resolution : Rs = 2 x tR2 - tR1 W1 + W2

2. Parameters used in HPLC

Condition for good separation


A larger Rs value means a better separation. 1 4 - 1 k2 1 + k2 N

Rs = k2

1 + k2 - 1

: Capacity term increases the retention time : Selectivity term increases the time interval between peaks : Column efficiency term produce narrow peaks

2. Parameters used in HPLC

Parameters and selectivity


Longer retention time

Larger

Improved column efficiency

Review of Sections 1 and 2

What is HPLC ? What is HPLC used for ? What is Separation and Analysis ? Qualitative and Quantitative analysis from chromatogram HPLC Parameters

Review of Sections 1 and 2

What is HPLC ? What is HPLC used for ? What is Separation and Analysis ? H : High P : Performance (Pressure) Qualitative and Quantitative analysis from L chromatogram : Liquid C : Chromatography HPLC Parameters

Review of Sections 1 and 2

What is HPLC ? What is HPLC used for ? What is Separation and Analysis ? Qualitative and Quantitative analysis from 1. chromatogramSeparation of mixed components

2. Qualitative analysis / Quantitative analysis 3. Preparation of HPLC Parametersinterest components

Review of Sections 1 and 2

What is HPLC ? What is HPLC used for ? What is Separation and Analysis ? Qualitative and Quantitative analysis from chromatogram Qualitative analysis
What are components A, B and C ? HPLC Parameters Quantitative analysis What is the concentration of components A, B and C ?

Review of Section 1 and 2


Qualitative analysis (identification) and quantitative analysis (determination) can ? be performed using the information Contained in the chromatogram.

What is HPLC

What is HPLC used for ?

What is Separation and Analysis ? Qualitative and Quantitative analysis from chromatogram HPLC Parameters

Review of Sections 1 and 2

What is HPLC ? What is


Retention parameters HPLC used for ?efficiency parameters Column Peak symmetry parameters Condition for and Analysis What is Separation Separation

Qualitative and Quantitative analysis from chromatogram HPLC Parameters

3. Separation mode

Column and mobile phase solvent

3. Separation mode

Sample and Analytical method


What is the sample ? Concentration of the interested component Contaminant

In which materials ? In what concentration ? Which sample ? With which technique ?

Characteristics of the sample - Structure - Molecular weight - pKa - Solubility Analytical technique - Column - Mobile phase - Detector - Sample preparation

3. Separation mode

Sample information

Merck Index Great Chemical Dictionary Great BioChemical Dictionary Reports based on other measurement techniques

3. Separation mode

Method information

Society magazines Journal of Chromatography. Analytical Chemist Manufacturer JASCO Application data

3. Separation mode

HPLC separation mode

HPLC separation mode Normal phase chromatography (NP) Reversed phase chromatography (RP) Size exclusion chromatography (SEC) Ion exchange chromatography (IEX) Affinity chromatography

3. Separation mode

Separation modes and features


Mode
Normal phase chromatography Reversed phase chromatography

Stationary phase
Silica gel

Mobile phase
Organic solvent (n-Hexane/IPE) MeOH/Water

Interaction
Adsorption

Feature
Fat-soluble

Silica-ODS (Silica-C18)

Hydrophobic

Most widely used

Size exclusion Chromatography Non-aqueous (GPC) Porous Polymer Aqueous (GFC) Aqueous porous Polymer Ion exchange Chromatography Affinity Chromatography Ion exchange gel

Organic solvent (THF) Gel permeation Molecular weight distribution Buffer solution Gel permeation Protein Separation Buffer solution Ion exchange Separation of ionic substances Purification of enzymes and proteins

Packing with ligand

Buffer solution

Affinity

GPC : Gel Permeation Chromatography GFC : Gel Filtration Chromatography

3. Separation mode

Solvent used in HPLC and range of Application


Solvent Polarity 0.1 0.1 -0.2 1.9 2.4 2.4 2.8 2.7 3.1 3.9 4.0 4.0 4.4 3.9 4.1 4.7 4.8 5.1 4.3 6.0 5.8 6.4 7.2 5.1 10.2 E0 0.01 0.01 0.04 0.54 0.28 0.29 0.38 0.32 0.42 0.7 0.82 0.57 0.58 0.82 0.40 0.51 0.56 0.56 0.88 0.65 0.75 0.95 R.I. 1.389 1.372 1.423 1.398 1.365 1.494 1.350 1.498 1.421 1.397 1.385 1.405 1.370 1.384 1.443 1.376 1.420 1.356 1.359 1.370 1.341 1.428 1.477 1.326 1.333 b.p. 99 69 81 89 68 110 35 80 40 118 97 66 77 82 61 80 101 56 78 118 82 153 189 65 100 Viscosity UV cut off 0.47 0.30 0.90 0.36 0.38 0.55 0.24 0.60 0.41 2.6 1.9 0.46 0.43 1.9 0.53 0.38 1.2 0.30 1.08 1.1 0.34 0.80 2.00 0.54 0.89 197 190 200 220* 285 218 280 233 210 240 212* 256 205 245 329 215 330 210 190 268 268 205
200

UV transmittance
250 300 350

Isoctane LCn-Hexane Cyclohexane Triethylamine i-Proryl ether Toluene Ethyl ether Benzene Methylene chloride n-Butanol n-Propanol Tetrahydrofuran Ethyl acetate i-Propanol Chloroform Methylethyl ketone Dioxane Acetone Ethanol Acetic acid Acetonitrile Dimethylformamide Dinethylsulfoxide Methanol Water

3. Separation mode

Polar compounds
Polar compound

Non-polar compound

H H O H H C H H

Bonding electrons are not shared evenly. The end of the bond with electrons becomes partially negative. The end of the bondwithout electrons becomes partially positive.

Polar compounds are soluble in polar solvents. Non-polar compounds are soluble in non-polar solvents.

3. Separation mode

Normal Phase Chromatography


Interaction : Packing materials : Adsorption Polar ex. Silica gel Silica-NH2 Silica-CN Silica-OH ex. n-Hex/CH2CL2 iso-Oct/IPA iso-Oct/AcOEt

Mobile phase :

Non-polar

Sample :

Fat-soluble Different polarity

3. Separation mode

Normal Phase Chromatography


Packing material
The most popular packing material is silica gel. It is believed that silanol radicals ( -Si-OH ) on the surface of silica gel act as the active site and the sample is separated.

O H

O H

O H

O H

O H

Si

Si Si

the surface of silica gel

O H

3. Separation mode

Normal Phase Chromatography


Interaction
OH OH H2N O 2N

OH

H2N

NO2

OH

H2N

OH OH OH

O 2N

3. Separation mode

Normal Phase Chromatography


Mobile phase solvents n-Hexane iso-Octane Chloroform Dichloromethane Ethylacetate Isopropylalchol Tetrahydrofran Dioxane Acetonitrile Ethanol Methanol Amines Acids n-Hex iso-Oct CHCl3 CH2Cl2 AcOEt IPA THF CH3CN EtOH MeOH
High Low

Polarity

3. Separation mode

Normal Phase Chromatography


Retention behavior
Low A B C

n-Hex/AcOEt(60/40)
A Polarity of Mobile phase B C D

n-Hex/AcOEt(50/50)
A BC D

Polarity of sample components A

<

<

<

High

n-Hex/AcOEt(30/70)

3. Separation mode

Reversed Phase Chromatography


Interaction : Packing materials : Hydrophobic Non-polar ex. Silica-C18 Silica-C8 Polymer ex. MeOH/H2O CH3CN/H2O MeOH/Buffer sol.

Mobile phase :

Polar

Sample :

Having different length of carbon chain

3. Separation mode

Reversed Phase Chromatography


CH3 OSiCH2(CH2)16CH3

Silica-C18 Packing materials


Commonly used packing materials are hydrocarbons having 18 carbon atoms (called the Octadecyl radical) which are chemically bonded to silica gel (SilicaODS).Since the surface of the Silica-ODS is covered with hydrocarbon, the polarity of the packing material itself is very low.

Si

CH3 CH3 OSiCH2(CH2)16CH3 CH3 CH3 OSiCH3

Si

CH3 CH3 OSiCH2(CH2)16CH3 CH3

3. Separation mode

Reversed Phase Chromatography


Hydrophobic Interaction

CH3

CH2COOCH3

CH3

CH2COOCH3

Silica-C18 (ODS)

3. Separation mode

Reversed Phase Chromatography


Mobile phase solvents
Main solvent : MeOH - H2O CH3CN - H2O EtOH IPA THF DMF Acid Salt Ion-pairing agent

Sub solvent :

Additive :

3.Separation mode

Reversed Phase Chromatography


Retention behavior in reversed phase HPLC
CH3CN/H2O
(70/30) (60/40) (50/50)

A B C A B C A B C Carbon chain length of sample A < B < C A : p-Hydroxy ethyl benzoate B : p-Hydroxy propyl benzoate C : p-Hydroxy butyl benzoate
0 5 0 5 0 5 10 (min)

Low

Polarity of Mobile phase Column : Finepak SIL C18

High

3.Separation mode

Reversed Phase Chromatography


Length of packing materials carbon chains and retention time A
Finepak SIL C18 A B Finepak SIL C8 C B C

A
Mobile phaseCH3CN/H2O(40/60)

Finepak SIL C1 A : p-Hydroxy ethyl benzoate B : p-Hydroxy propyl benzoate C : p-Hydroxy butyl benzoate
0 5

B C

10

15

20

25

30 (min)

3.Separation mode

Reversed Phase and Normal Phase Chromatography


Comparison of Reversed Phase and Normal Phase

Normal phase Stationary phase Mobile phase Interaction Elution order


High polarity Low polarity Adsorption Low to High (Polarity)

Reversed phase
Low polarity High polarity Hydrophobic Short to Long (Length of Carbon chain)

3.Separation mode

Reversed Phase and Normal Phase Chromatography


Comparison of Reversed Phase and Normal Phase
Reversed Phase Reversed Phase Chromatography Chromatography
Finepak SIL C18 Finepak SIL C18 MeOH MeOH

VA VD

Normal Phase Normal Phase Chromatography Chromatography


n-Hexane/IPA(96/4) n-Hexane/IPA(96/4)
VE VD 10 (min) 0 10 20 (min)

Finepak SIL Finepak SIL

VA

3.Separation mode

Ion-exchange Chromatography
Ion-exchange Chromatography
Interaction : Ion-exchange

Stationary phase: Anion exchange gel Cation exchange gel Mobile phase : Sample : Buffer solution Ionic substances (Cations or Anions)

3.Separation mode

Ion-exchange Chromatography
Ion-exchange Gel

SO3- Na +

NR3

+ Cl -

SO3- Na + SO3- Na +

NR3+ Cl NR3+ Cl -

SO3- Na +

NR3+ Cl -

Cation exchange gel

Anion exchange gel

3.Separation mode

Ion-exchange Chromatography
Mobile phase solvents used for Ion-exchange

Buffer solution Salt concentration pH (Hydrogen ion concentration) Type of salt Additive (Organic solvent)

SO3 - Na + S+ SO3 Na
+

Na SO3 S
+

3.Separation mode

Ion-exchange Chromatography
Application data of Ion-exchange chromatography
Separation of polyphosphoric acid
1. 2E+ 05 uV

1 5.89

Column
2 9.49

Mobile phase

1. 0E+ 05

Reactor
4 17.79 3 13.58 5 21.65 6 24.65

8. 0E+ 04

7 26.95

Detection
8 28.83

: Finepak GEL SA-121 (6.0mmI.D. POLY_003.CH x 100mmL) : A= 0.1M KCl + 1% EDTA-4Na (pH 10.0 adjusted HCl) B= 1.0M KCl + 1% EDTA-4Na (pH 10.0 adjusted HCl) gradient : 1.8MH2 SO4(1L), (NH4o7)6MO24-4H2O (5g), Sand of zinc metal(0.6g) : 830nm

10 31.71

11 32.86 12 33.84 13 34.72 14 35.50 15 36.21 16 36.85 17 37.43 18 37.95 19 38.38 20 38.72

6. 0E+ 04

4. 0E+ 04

2. 0E+ 04

0. 0E+ 00

9 30.40

10. 0

20. 0

30. 00

40. 00

[m i n]

3.Separation mode

Ion Chromatography
Summary of Ion Chromatography
Purpose : Separation of inorganic ions, organic acids

Stationary phase: Anion exchange gel Cation exchange gel Mobile phase : Detection : Buffer solution Conductivity detector

3.Separation mode

Ion Chromatography
Suppressor and Non-suppressor

P
Mobile phase

pump

P
Mobile phase

pump

injector

injector

column

column

suppressor

Conductivity detector

Conductivity detector

3.Separation mode

Ion Chromatography
Cation measurement data
Sample Column Mobile phase Detector : Tap water : Shodex IC YK-421 : 5mM tartaric acid+2mM Gibicolin acid : Conductivity detector (CD-5)

Na+ 5.276ppm

Ca2+ 12.386ppm

K+ 0.785ppm

Mg2+ 1.829ppm

10

15

20(min)

3.Separation mode

Ion Chromatography
Anion measurement data
Sample : Tap water Column : Shodex IC I-524A Mobile phase : 2mM phthalic acid+1.84mM tris +300mM boric acid(pH4.0) Detector : Conductivity detector (CD-5)

Cl- 6.029ppm

F- 0.111ppm

SO42- 10.426ppm

NO3- 6.694ppm

10

15

20

25(min)

3.Separation mode

Size Exclusion Chromatography (SEC)


GPC and GFC
Non-aqueous SEC Interaction Packing Mobile phase Sample : : : : :

GPC (Gel Permeation Chromatography)


Gel permeation Cross-Linked porous Polystyrene Organic solvent (THF, CHCl3, DMF) Molecular weight distribution of polymer Synthetic Oligomer separation

Aqueous SEC Interaction Packing Mobile phase Sample

: : : : :

GFC (Gel Filtration Chromatography)


Gel permeation Hydrophilic silica gel / Hydrophilic porous polymer Buffer solution Separation of Water-soluble polymers (proteins, nucleic acid, sugar) oligomers

3.Separation mode

Size Exclusion Chromatography (SEC)


SEC Separation mechanism
B
Mobile phase

A D A D C D C

Small pore

Packing material

C B D

A+B

3.Separation mode

Size Exclusion Chromatography (SEC)


Gel permeation chromatography and calibration curve

1. 0E+ 05

uV

RI

Column : Shodex GPC KF-806Lx 2 Column Mobile phase : THF


PS-8420K PS-900K PS-110K
5. 00 10. 00 15. 00

6. 0E+ 04

4. 0E+ 04

2. 0E+ 04

0. 0E+ 00

PS-18.1K PS-2.98K PS-Oligomer


20. 00 25. 00

8. 0E+ 04

30. 00

35. 00 m n] [ i

3.Separation mode

Size Exclusion Chromatography (SEC)


Peak analysis of polymer to calculate molecular weight distribution
no 1 2 3 Vi 10.0 10.5 11.0 Hi 74 156 318

H1 V1

H2 H3
25.0 (min)

10.0

15.0

20.0

V2 V3

Retention time

3.Separation mode

Size Exclusion Chromatography (SEC)


Molecular weight calculation
N 1 2 3 Vi 11.12 11.37 11.62 Hi 74 387 1539 hi Mn = Mw = Mz = D Hi/ HiMi/ HiMi2/ Hi/Mi = 15.5104 Hi = 28.6104 HiMi = 46.5104 Mi 2094050 1734413 1432619 mi Hi/Mi Hi/Mi HiMi HiMi HiMi2 HiMi2

= Mw/Mn = 1.84

3.Separation mode

Size Exclusion Chromatography (SEC)


Column selection
Molecular weight of the sample : Exclusion limit molecular weight Ability to dissolve the sample Molecular weight distribution : Applicable to packing materials : Range of calibration curve

3.Separation mode

Size Exclusion Chromatography (SEC)


Column suited to the sample in terms of molecular weight
Shodex A-8012
EPIKOTE 3-8 1001 2 1 n=0

Shodex A-8032
2 1 3 4 5 6 7 8 n=0

EPIKOTE 828

15

20

25 min

30

40 min

Eluent : THF

Flow rate : 1.0ml/min

3.Separation mode

Size Exclusion Chromatography (SEC)


Solvent and column
Solvent
THF

Column
Finepak GEL 101F Shodex KF series Finepak GEL 101C Shodex K series Shodex KD series Shodex SB series Shodex KS series

CHCl3

DMF H2O, Buffer solution

3.Separation mode

Size Exclusion Chromatography (SEC)


Calibration curves for columns

Eluent : THF

3.Separation mode

Columns for exclusive use


Columns for Exclusive use
Amino acids : Aapak (Cation exchange) Organic acid : Shodex Ionpak KC-811 (ion exclusion and partition & adsorption) Sugar : Shodex Ionpak KS series (aqueous SEC) Shodex Sugar series (ligand exchange) Finepak SIL NH2-5 (Normal phase) Finepak GEL SA-121 (Strong anion exchange)

N-methyl carbamate : Carbamatepak (Reversed phase)

3.Separation mode

Columns for exclusive use


Amino Acid Analysis
Column : AApak Na II-H Mobile phase : Sodium citrate buffer Stepwise gradient Detection : OPA post label Ex 345nm Em 445nm Sample : Sake NH3 Leu Ala Gly ThrSer Pro Glu Val

Met

Tyr

Phe

Lys His

Asp

Ile

Arg

3.Separation mode

Columns for exclusive use


Organic Acid Analysis
Column : Shodex Ionpak KC811x2 Mobile phase : Detection : BTB post label UV 445nm Sample : Sake

citric pyrvic malic

succinic

lactic

pyroglutamic

acetic

3.Separation mode

Ion suppression method & Ion-pair chromatography


Ion suppression method & Ion-pair chromatography
Separation method to analyze ionic compounds by reversed-phase chromatography Ion suppression method Ion pair chromatography : Acidic ion components : Basic ion components / Acidic ion components

3.Separation mode

Ion suppression method & Ion-pair chromatography


Diagram of Ion suppression method
Add phosphoric acid
H+ H
+

HA

H+ H+ HA H+ H+ H+

A-

Silica-C18
H+ H+ A
-

Silica-C18

HA H+ H+

AA: Sample

+ H+

HA

H+ : Hydrogen ion
HA

: Sample

3.Separation mode

Ion suppression method & Ion-pair chromatography


Chromatogram when Ion suppression method is used
Benzoic acid p-Hydroxy ethyl benzoate

Finepak SIL C1 CH3CN/H2O (40/60)

Finepak SIL C1 CH3CN/0.2% H3PO4 (40/60)

propyl butyl

10 (min)

10 (min)

3.Separation mode

Ion suppression method & Ion-pair chromatography


Diagram of Ion-pair chromatography
SO3SO 3
+ R4 - N

+NR

Silica-C18 SO3-

SO3-

Silica-C18

+NR

Add Ion-pair reagent

SO - + 3 N R

SO3 +NR
- + NR 4

SO3SO3- + NR4
SO 3
-

Silica-C18

SO 3

SO - +
3

NR

SO3 -

3.Separation mode

Ion suppression method & Ion-pair chromatography


Chromatogram when Ion-pair chromatography is used
A A B B

Without Ion-pair reagent

With Ion-pair reagent

Typical ion reagents


Acidic ions : Tetra alkyl ammonium halide Basic ions : l-Alkyl sulfonate

3.Separation mode

Ion suppression method & Ion-pair chromatography


Ion-pair chromatography
Effects of basic additives
- Stable pH - Longer retention time - Ion pair reagent effect

3.Separation mode

Ion suppression method & Ion-pair chromatography


Acid and basic sample for Reversed phase LC
Method Ion suppression Sample
Weak acidic sample

Reversed phase
phosphoric acid acetic acid perchloric acid trifluoroacetic acid Tetra alkyl ammonium halide l-Alkykl surfonate (acetic acid) (trifluoroacetic acid) phosphate citrate

Ion pair

Acidic sample Basic sample

Addition of salt

Review of Section 3

4 separation modes Polarity of packing material and solvent Change of mobile phase and elution Ion suppression method and Ion pair method Salt effect

4. Gradient elution method

4. Gradient elution method


For separation of a sample containing many components

MeOH(100)

Mobile phase

Gradient MeOH/Water (50/50) 0.1M KH2PO4 Step wise 0.01M 0 5 10 Time(min) 15 20 25 0.5M

4. Gradient elution method


Advantage of gradient elution method
Isocratic elution method Gradient elution method

A
Finepak SIL C18 MeOH/1% AcOH(40/60)

B * * A
MeOH/1% AcOH(30/70) MeOH/1% AcOH 30/7045/55 Linear Gradient16min

A : Chlorogenic acid B : Rutin * : Impurity

4. Gradient elution method


Precautions in gradient elution method

- Can the gradient save time ? - Reproducibility - Baseline - Ghost peak - Salt

4. Gradient elution method


Effect of temperature on retention time
60*C 1 2 3 4 Finepak SIL C18 CH3CN/H2O(90/10) Sample 1. Benzene 2. Anthracene 3. Pyrene 4. Benz(a)pyrene 4

40 *C

20 *C

2 3 4

10

12 (min)

Review of Section 4

Gradient elution method Temperature effect

5. Detector

5. HPLC detectors
HPLC detectors
UV-VIS(Absorption) PDA (Absorption) Differential refractometer(Refractive index) Fluorometric (Florescence) Electrochemical (ECD) (Oxidation -reduction) Conductivity Mass Chiral (OR) Circular dichroism (CD)

5. HPLC detectors
UV/Vis detector
- Selective detection minimizing effects from other components - High sensitivity detection at maximum absorption wavelength

5. HPLC detectors
Improved selectivity
Traditional medicine berberine impurity berberine

impurity

7.385

Wavelength=260nm

Wavelength=340nm

7.395

5. HPLC detectors
Improved sensitivity
Saccharin (SAC) and sorbin acid (SOR)

230nm

265nm

SOR

0nm

SAC

12.250

SAC

SOR

10

20

10

Wavelength programming

Fixed wavelength at 265nm

12.467

3.575

3.608

20

5. HPLC detectors
UV spectrum measurement to find wavelength effective for wavelength programming
1.0 Diazepam (DZP) Absorbance

Nitrazepam (NZP) 0.5

Chronazepam (CZP) 210 250 300 Wavelength(nm) 350 0

5. HPLC detectors
Wavelength programming and fixed wavelength
Blood serum NZP : 420ng/ml CZP : 130ng/ml DZP : 440ng/ml

NZP

DZP

NZP 0 5 CZP DZP 10 Fixed wavelength

310nm 250nm

CZP

10

Wavelength programming

5. HPLC detectors
Optics of Multi-wavelength detector

I2 lamp D2 lamp

Grating UV/Vis detector Photo diode

lamp

Grating

Photo diode

Cell

Cell Photodiode array

5. HPLC detectors
Multi-wavelength detector 3D chromatogram

5. HPLC detectors
Multi-wavelength detector Cont. data

5. HPLC detectors
Features of Multi-wavelength detector
1. 2. 3. 4. Spectrum collection at any time Library search Purity check Quantitative analysis at 6 wavelengths

5. HPLC detectors
Principle of Fluorescence detector
(S1) Excited state(S2) (S3)

excitation

emission

H (fluorescence)

S Ground state 0 Mobile phase

5. HPLC detectors
Features of Fluorescence detector
1. Selective detection 2. Detection at any excitation or emission wavelength 3. High sensitivity

5. HPLC detectors
Wavelength programming by Fluorescence detector
Wavelength programming 0 6.6 10.0 min 450 525 nm VB2 Fixed wavelength Ex=275nm Em=400nm Fixed wavelength Ex=450nm Em=525nm

Ex 275 240 Em 400 350

VB2 Phosphate

VB2 Phosphate 10 20 min 0 10

VB6

VB6

VB1

10

20 min

20 min

Column : Finepak SIL C18S Mobil phase : MeOH/Phosphate Buffer Gradient

VB2

5. HPLC detectors
Selectivity of UV detector and Fluorescence detector
UV detector

Fluorescence detector

5. HPLC detectors
Principle of RI detection

light i i

light

r0

Solvent

Sample and solvent

r0>r

5. HPLC detectors
UV detector and RI detector
RI detector

UV detector

5. HPLC detectors
Considerations for IR detection
1. 2. 3. 4. Temperature change Replacement of solvent (reference cell and sample cell) Unstable when solvent mixed Replacement of solvent inside column

5. HPLC detectors
Detectors
UV Sensitivity Detection selectivity Temperature Influence ng selective Fluorescence pg highly selective RI g universal

small

small

large

Gradient elution possible

possible

impossible

5. HPLC detectors
Label method
Samples absorb less UV/Vis light . Samples do not fluoresce.

Improved sensitivity and selectivity required

Label method

5. HPLC detectors
Label method
Pre-label method
reagent sample (reaction) pump injector column

Post-label method
injector

column reactor

reagent

detector detector

5. HPLC detectors
Label method
Post-label method

Aminoacid

0PA ninhydrine guanidine brom thymol blue ethylenediamine THI NAD HSD

Fluorescence Absorption in Visible range Fluorescence Absorption in Visible range Fluorescence Fluorescence Fluorescence Fluorescence

Sugar Organic acid Catecholamine

Bile acid

5. HPLC detectors
Pre and Post column derivatization method

Pre-column LC system required Standard system

Post-column Reaction system is required

Reproducibility Operation Reagents Applicability

less than post-column good for all samples wide range spot only reagents limited routine

5. HPLC detectors
Pre-column derivatization method
Dabcyl-Cl
CH3 N CH3 N=N SO2 Cl

Amino acid
R

+
H2 N O

O H

pH8.170 12min R CH3 N CH3 N=N SO2 N H O O H

Dabcyl - Amino acid

5. HPLC detectors
Separation of Dabcyl - Amino acid
4.0E+04 uV

Wavelength : 465nm
3.5E+04

DABS-OH

NH3

40pmol each

15 16 8 14 3 56 17 9 7 13 12 11 10

1 2 3.0E+04

1.Asp 2.Glu 3.Ser 4.Thr 5.Gly 6.Ala 7.Arg 8.Pro 9.Val

10.Met 11.Ile 12.Leu 13.Phe 14.Cystine 15.Lys 16.His 17.Tyr

2.5E+04

2.0E+04

5.00

10.00

15.00

20.00

25.00 [min]

5. HPLC detectors
Post-column derivatization method
Amino acid
CHO

2-mercapto ethanol
+ HS CH2 CH2 OH + NH2 C

COOH R H

CHO

orthophthalaldehyde (OPA)
S CH2 H N C R CH2 OH

COOH

Derivative compound

5. HPLC detectors
Post column derivatization method
Ex : 345nm Em : 455nm

CysSO3H

Val

Asp Thr Ser Glu Pro

Gly Ala Cys

Met

Ile Leu

His

Phe

Tyr

Lys

20

40

60 (min)

Trp

Arg

Review of Section 5

Detectors Selectivity and sensitivity Pre-/Post- column derivatization methods

The JASCO advanced technology team has again met the challenge and designed a new line of HPLC instruments, The LC-1500series more than satisfies in response to the growing demand for greatly expanded HPLC analyses in the fields of not only biochemistry, pharmaceutical and medical science, but also in the areas of among other organic and inorganic compounds, foods, agricultural sciences, polymeric and natural substances and pollution. The LC-1500 series comprises pumps, detectors, autosamplers, its own column oven and other units each having built-in intelligence and incorporating many features with much higher levels of operability and reliability in addition to multiple functions, higher performance and higher accuracy than before, making them the most advanced instruments available.

6. Data processing

6. Data processing

Data processing in HPLC


1. Qualitative analysis 2. Quantitative analysis 3. Molecular weight distribution

6. Data processing

Qualitative analysis
1. 2. 3. Retention time Retention volume of the standard sample Sample components are collected after separation, and subjected to spectrometric analysis such as IR, NMR and MS.

6. Data processing

Identification from retention time


tR A B

Standard sample

Unknown sample

6. Data processing

Standard addition method

Standard addition

Target peak

6. Data processing Standard addition method


Retention time of standard sample is different from unknown sample

Standard sample

Unknown sample

Unknown sample and Standard sample

6. Data processing Identification using a different instruments after preparative analysis Identification from retention time

Limitation:
On flow UV spectrum On flow emission spectrum Multi-channel detector

Preparative analysis
Spectrum measurement using a different instrument

6. Data processing

Quantitative analysis
How much component A ?
The amount of a component can be calculated from the peak height and peak area of the chromatogram. Standard sample (1mg/ml)
A

Injection of 10g

Unknown sample
A

Injection of 10g

6. Data processing

Calibration method
External standard sample Internal standard sample

6. Data processing

External standard sample


Thiamiral in serum

Concentration(g/ml)
Finepak SIL C18T-5 CH3CN/10mM KH2PO4 aq. (50:50) UV 288nm

Peak area

Thiamiral

Thiamiral

6. Data processing

Internal standard sample


Anticonvulsants in serum
Standard sample Calibration curve
1PB 2DPH 3CBZ ISPhenacetin

Unknown sample

Peak area

concentration(g/ml) Concentration ratio


Finepak SIL C18T CH3CN/5mM KH2PO4 aq.

6. Data processing

Guide for selecting the internal standard sample


No overlapping peaks No Components included in unknown sample Chemical and physical stability High purity

6. Data processing

External standard and Internal standard samples


External standard injection volume impossible Internal standard volume to be added possible

Error Correction of Pre-treatment loss

6. Data processing

Caution when using an Integrator


One point calibration Integrator

True curve

error

large small

large

6. Data processing

Caution when using an Integrator


Two point calibration Integrator

True curve

error

large

small

large

6. Data processing

Baseline

6. Data processing Considerations when performing quantitative analysis


Standard sample Integrator Micro syringe Sample preparation Concentration change of standard sample Contamination

Review of Section 6
Identification 1. Retention time 2. Standard sample 3. After preparative analysis, measure spectrum using a different method Quantitative analysis 1. External standard sample 2. Internal standard sample 3. Items to consider when performing quantitative analysis

The JASCO advanced technology team has again met the challenge and designed a new line of HPLC instruments, The LC-1500series more than satisfies in response to the growing demand for greatly expanded HPLC analyses in the fields of not only biochemistry, pharmaceutical and medical science, but also in the areas of among other organic and inorganic compounds, foods, agricultural sciences, polymeric and natural substances and pollution. The LC-1500 series comprises pumps, detectors, autosamplers, its own column oven and other units each having built-in intelligence and incorporating many features with much higher levels of operability and reliability in addition to multiple functions, higher performance and higher accuracy than before, making them the most advanced instruments available.

7. Sample preparation

7. Sample preparation

Sample preparation
Cause Sample is not liquid. Concentration is too high. Concentration is too low. Contains foreign particles Problem not possible to inject over load for column / out of detection range cannot detect clogged up Countermeasures extraction / dissolving dilution concentration / derivative centrifugation / filtration solvent extraction /derivative solvent extraction /derivative pH adjustment

Includes components which damage column Includes interference for separation Solvent unsuitable quantitation error deterioration of column

7. Sample preparation

Sample preparation Method


Filtration Extraction Concentration 0.45um, 0.2um membrance filter Solvent extraction Solid phase extraction Evaporation Solid phase exraction (Bond Elut) Fused drying Organic acid

Deprotaination Homonization

Solid phase extraction


1. Activation Wash with MeOH Activate With H2O 2. Load sample
Sample Contaminant

7. Sample preparation
3. Wash
Wash with H2O orH2O/MeOH

4. Elute target compound


MeOH or Eluting solvent

Contaminant Vacuum

Target compound

Removing contaminants which have strong retention


1. Activate
Wash with MeOH Activate with proper solvent

7. Sample preparation
3. Elute a target compound

2. Load sample

Target sample Compound which has strong retention

Using vacuum or pressure

Compound which has strong retention

Target sample Vacuum

Concentration
1. Activate 2. Load and concentrate target sample
target sample

7. Sample preparation
3. Elute target sample

Wash with MeOH

Activate with H2O

Elute with MeOH

pump

Small amount of Target sample

Target compound is concentrated. Vacuum

7. Sample preparation

Considerations when preparing sample


Recovery rate Contamination

Review of Section 7

1. The most appropriate preparation method depends on various factors including the sample(target compound), the amount of target compound in the sample, and the kinds of contaminant. 2. Consider such factors as the sample state, amount, running cost, running time, and handling.

The JASCO advanced technology team has again met the challenge and designed a new line of HPLC instruments, The LC-1500series more than satisfies in response to the growing demand for greatly expanded HPLC analyses in the fields of not only biochemistry, pharmaceutical and medical science, but also in the areas of among other organic and inorganic compounds, foods, agricultural sciences, polymeric and natural substances and pollution. The LC-1500 series comprises pumps, detectors, autosamplers, its own column oven and other units each having built-in intelligence and incorporating many features with much higher levels of operability and reliability in addition to multiple functions, higher performance and higher accuracy than before, making them the most advanced instruments available.

8. Procedure for developing analytical conditions

7. Sample preparation

Procedure for developing analytical conditions


Step one : clear analytical purpose, and research the target compound. (1) Molecular weight Molecular structure Functional group (2) Solubility, stability UV, FP absorption (3) Amount of concentration, contaminant (4) Application data reference literature, magazines Development analytical conditions (trial and error) (1) When attempting to develop analytical conditions, use an appropriate concentration of standard solution (2) Check the detection limit and detection method (3) Prepare sample (4) Check contaminat and target compound peak separation

Step two :

7. Sample preparation

Procedure for developing analytical conditions


Step three : Establish analytical condition for routine analysis (1) Linearity of calibration curve (2) Reproducibility of analysis (3) Check for contaminants that retain strongly in the column (4) Check for Correlation with other methods. Routine quantitative analysis (1) Lifetime of column (2) Running cost (3) Develop analytical procedure (SOP) (4) Check HPLC and column performance.

Step Four