HPLC

Chromatography is used to separate mixtures of substances into their components All forms of chromatography work on the same principle

They all have a stationary phase (a solid or a liquid supported on a solid) and a mobile phase (a liquid or a gas)

The mobile phase flows through the stationary phase and carries the components of the mixture with it Different components travel at different rates

TLC-Thin layer chromatography is done exactly as it says - using a thin uniform layer of silica gel or alumina coated onto a piece of glass metal or rigid plastic

The silica gel (or the alumina) is the stationary phase The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later The mobile phase is a suitable liquid solvent or mixture of solvents

The diagram shows the plate after the solvent has moved about half way up itThe solvent is allowed to rise until it almost reaches the top of the plate That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase

What separates the compounds as a chromatogram develops

What separates the compounds as a chromatogram develops

As the solvent begins to soak up the plate it first dissolves the compounds in the spot that you have put on the base line The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards

How fast the compounds get carried up the plate depends on two thingsbullHow soluble the compound is in the solvent This will depend on how much attraction there is between the molecules of the compound and those of the solventbullHow much the compound sticks to the stationary phase - the silica gel for example This will depend on how much attraction there is between the molecules of the compound and the silica gel

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC

High performance liquid chromatography is basically a highly improved form of column chromatography Instead of a solvent being allowed to drip through a column under gravity it is forced through under high pressures of up to 400 atmospheres That makes it much faster

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

bull No air bubbles (Priming)bull Use degassed buffers

Injector Module

Column Inlet

Detector

FractionCollector

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Chromatography is used to separate mixtures of substances into their components All forms of chromatography work on the same principle

They all have a stationary phase (a solid or a liquid supported on a solid) and a mobile phase (a liquid or a gas)

The mobile phase flows through the stationary phase and carries the components of the mixture with it Different components travel at different rates

TLC-Thin layer chromatography is done exactly as it says - using a thin uniform layer of silica gel or alumina coated onto a piece of glass metal or rigid plastic

The silica gel (or the alumina) is the stationary phase The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later The mobile phase is a suitable liquid solvent or mixture of solvents

The diagram shows the plate after the solvent has moved about half way up itThe solvent is allowed to rise until it almost reaches the top of the plate That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase

What separates the compounds as a chromatogram develops

What separates the compounds as a chromatogram develops

As the solvent begins to soak up the plate it first dissolves the compounds in the spot that you have put on the base line The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards

How fast the compounds get carried up the plate depends on two thingsbullHow soluble the compound is in the solvent This will depend on how much attraction there is between the molecules of the compound and those of the solventbullHow much the compound sticks to the stationary phase - the silica gel for example This will depend on how much attraction there is between the molecules of the compound and the silica gel

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC

High performance liquid chromatography is basically a highly improved form of column chromatography Instead of a solvent being allowed to drip through a column under gravity it is forced through under high pressures of up to 400 atmospheres That makes it much faster

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

bull No air bubbles (Priming)bull Use degassed buffers

Injector Module

Column Inlet

Detector

FractionCollector

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

TLC-Thin layer chromatography is done exactly as it says - using a thin uniform layer of silica gel or alumina coated onto a piece of glass metal or rigid plastic

The silica gel (or the alumina) is the stationary phase The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - for reasons you will see later The mobile phase is a suitable liquid solvent or mixture of solvents

The diagram shows the plate after the solvent has moved about half way up itThe solvent is allowed to rise until it almost reaches the top of the plate That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase

What separates the compounds as a chromatogram develops

What separates the compounds as a chromatogram develops

As the solvent begins to soak up the plate it first dissolves the compounds in the spot that you have put on the base line The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards

How fast the compounds get carried up the plate depends on two thingsbullHow soluble the compound is in the solvent This will depend on how much attraction there is between the molecules of the compound and those of the solventbullHow much the compound sticks to the stationary phase - the silica gel for example This will depend on how much attraction there is between the molecules of the compound and the silica gel

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC

High performance liquid chromatography is basically a highly improved form of column chromatography Instead of a solvent being allowed to drip through a column under gravity it is forced through under high pressures of up to 400 atmospheres That makes it much faster

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

bull No air bubbles (Priming)bull Use degassed buffers

Injector Module

Column Inlet

Detector

FractionCollector

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

The diagram shows the plate after the solvent has moved about half way up itThe solvent is allowed to rise until it almost reaches the top of the plate That will give the maximum separation of the dye components for this particular combination of solvent and stationary phase

What separates the compounds as a chromatogram develops

What separates the compounds as a chromatogram develops

As the solvent begins to soak up the plate it first dissolves the compounds in the spot that you have put on the base line The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards

How fast the compounds get carried up the plate depends on two thingsbullHow soluble the compound is in the solvent This will depend on how much attraction there is between the molecules of the compound and those of the solventbullHow much the compound sticks to the stationary phase - the silica gel for example This will depend on how much attraction there is between the molecules of the compound and the silica gel

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC

High performance liquid chromatography is basically a highly improved form of column chromatography Instead of a solvent being allowed to drip through a column under gravity it is forced through under high pressures of up to 400 atmospheres That makes it much faster

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

bull No air bubbles (Priming)bull Use degassed buffers

Injector Module

Column Inlet

Detector

FractionCollector

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

What separates the compounds as a chromatogram develops

What separates the compounds as a chromatogram develops

As the solvent begins to soak up the plate it first dissolves the compounds in the spot that you have put on the base line The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards

How fast the compounds get carried up the plate depends on two thingsbullHow soluble the compound is in the solvent This will depend on how much attraction there is between the molecules of the compound and those of the solventbullHow much the compound sticks to the stationary phase - the silica gel for example This will depend on how much attraction there is between the molecules of the compound and the silica gel

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC

High performance liquid chromatography is basically a highly improved form of column chromatography Instead of a solvent being allowed to drip through a column under gravity it is forced through under high pressures of up to 400 atmospheres That makes it much faster

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

bull No air bubbles (Priming)bull Use degassed buffers

Injector Module

Column Inlet

Detector

FractionCollector

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

What separates the compounds as a chromatogram develops

As the solvent begins to soak up the plate it first dissolves the compounds in the spot that you have put on the base line The compounds present will then tend to get carried up the chromatography plate as the solvent continues to move upwards

How fast the compounds get carried up the plate depends on two thingsbullHow soluble the compound is in the solvent This will depend on how much attraction there is between the molecules of the compound and those of the solventbullHow much the compound sticks to the stationary phase - the silica gel for example This will depend on how much attraction there is between the molecules of the compound and the silica gel

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC

High performance liquid chromatography is basically a highly improved form of column chromatography Instead of a solvent being allowed to drip through a column under gravity it is forced through under high pressures of up to 400 atmospheres That makes it much faster

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

bull No air bubbles (Priming)bull Use degassed buffers

Injector Module

Column Inlet

Detector

FractionCollector

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY - HPLC

High performance liquid chromatography is basically a highly improved form of column chromatography Instead of a solvent being allowed to drip through a column under gravity it is forced through under high pressures of up to 400 atmospheres That makes it much faster

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

bull No air bubbles (Priming)bull Use degassed buffers

Injector Module

Column Inlet

Detector

FractionCollector

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Fast Protein Liquid Chromatograph (FPLC)

1

2

3

5

4

bull No air bubbles (Priming)bull Use degassed buffers

Injector Module

Column Inlet

Detector

FractionCollector

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

HPLC is a separation technique that involves

bullthe injection of a small volume of liquid sample

bullinto a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

bullwhere individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Definitions1048729 Stationary phase-- common name for the column packing material in any typeof chromatography-- those samples which have stronger interactions with thestationary phase than with the mobile phase will elute fromthe column less quickly and thus have a longer retentiontime while the reverse is also true

1048729 Mobile phase-- liquid media that continuously flows through the columnand carries the analytes a carrier for the sample solution-- normally use mixtures of solvents as mobile phase

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

HPLC system

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

FOUR TYPES OF LIQUID CHROMATOGRAPHY

bull Partition chromatography

bull Adsorption or liquid-solid chromatography

bull Ion exchange chromatography

bull Size exclusion or gel chromatography

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

WHAT AFFECTS SYSTEM Column Parameters

bull Column Materialbull Deactivationbull Stationary Phasebull Coating Material

Instrument Parameters

bull Temperaturebull Flowbull Signalbull Sample Sensitivitybull Detector

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

WHAT AFFECTS SYSTEM

Sample Parameters

bull Concentrationbull Matrixbull Solvent Effectbull Sample Effect

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Several column types(can be classified as )

bullNormal phase

bullReverse phase

bullSize exclusion

bullIon exchange

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Normal phase

bullIn this column type the retention is governed by the interaction of the polar parts of the stationary phase and solute For retention to occur in normal phase the packing must be more polar than the mobile phase with respect to the sample

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Reverse phase bullIn this column the packing material is

relatively nonpolar and the solvent is polar with respect to the sample Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase Typical stationary phases are nonpolar hydrocarbons waxy liquids or bonded hydrocarbons (such as C18 C8 etc) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Size exclusion

bullIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores The large molecules elute before the smaller molecules

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Ion exchange bullIn this column type the sample

components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase Separations are made between a polar mobile liquid usually water containing salts or small amounts of alcohols and a stationary phase containing either acidic or basic fixed sites

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors

Types of Detectorsbull Absorbance (UV with

Filters UV with Monochromators)

bull IR Absorbance

bull Fluorescence

bull Refractive-Index

bull Evaporative Light Scattering Detector (ELSD)

bull Electrochemical

bull Mass-Spectrometric

bull Photo-Diode Array

• HPLC
• Slide 2
• Slide 3
• Slide 4
• Slide 5
• Slide 6
• Slide 7
• Slide 8
• Slide 9
• Slide 10
• HPLC system
• FOUR TYPES OF LIQUID CHROMATOGRAPHY
• WHAT AFFECTS SYSTEM
• WHAT AFFECTS SYSTEM (2)
• Several column types (can be classified as )
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
• Types of Detectors