introduction to high performance liquid chromatography-hplc

Presented by: Mehdi Soleymani Goloujeh

Supervisor: Dr. S. Davaran

Outlook:

History

Introduction

The components of the high performance liquid chromatograph

What affects system

The separation process

The chromatogram

The most common modes of HPLC

HPLC Applications

The differences between HPLC and GC

Advantages of High Performance Liquid Chromatography

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Medical Nanotechnology Department

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Ether

CaCO3

Chlorophyll

Chromatography

Colors

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Base

Water flow

Light leaf

Heavy stone

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Chromatography in which the mobile phase is a liquid. The liquid used as the mobile phase is called the “eluent”.

The stationary phase is usually a solid or a liquid.

In general, it is possible to analyze any substance that can be stably dissolved in the mobile phase.

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Chromatography: Analytical technique

Chromatograph: Instrument

Chromatogram: Obtained “picture”

Chromatographer: Person

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Higher degree of separation! Refinement of packing material (3 to 10 µm)

Reduction of analysis time! Delivery of eluent by pump Demand for special equipment that can withstand high pressures

The arrival of high performance liquid chromatography!

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HPLC is a form of liquid chromatography used to separate compoundsthat are dissolved in solution. HPLC instruments consist of a reservoir ofmobile phase, a pump, an injector, a separation column, and a detector.

Compounds are separated by injecting a sample mixture onto thecolumn. The different component in the mixture pass through thecolumn at differentiates due to differences in their partition behaviorbetween the mobile phase and the stationary phase. The mobile phasemust be degassed to eliminate the formation of air bubbles.

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Isocratic systemConstant eluent composition

Gradient systemVarying eluent composition

HPGE (High Pressure Gradient)

LPGE (Low Pressure Gradient)

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High-pressure gradient

Mixer

Low-pressure

gradient unit

Low-pressure gradient

Mixer

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Principle Pattern An Example

Detector

Thermostatted

Column Compartment

Autosampler

Binary Pump

Vacuum DegasserSolvent Cabinet

Solvent Reservoirs

Controller

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The column is one of the mostimportant components of the HPLCchromatograph because the separationof the sample components is achievedwhen those components pass throughthe column. The High performanceliquid chromatography apparatus ismade out of stainless steel tubes with adiameter of 3 to 5mm and a lengthranging from 10 to 30cm.

Normally, columns are filled with silicagel because its particle shape, surfaceproperties, and pore structure help to geta good separation. Silica is wetted bynearly every potential mobile phase, isinert to most compounds and has a highsurface activity which can be modifiedeasily with water and other agents. Silicacan be used to separate a wide variety ofchemical compounds, and itschromatographic behavior is generallypredictable and reproducible.

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Problems caused by dissolved air in the eluent Unstable delivery by pump

More noise and large baseline drift in detector cell

In order to avoid these problems, the eluent must be degassed.

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The function of the injector is to place the sample into the high-pressure flowin as narrow volume as possible so that the sample enters the column as ahomogeneous, low-volume plug. To minimize spreading of the injected volumeduring transport to the column, the shortest possible length of tubing should beused from the injector to the column.

When an injection is started, an air actuator rotates the valve: solvent goesdirectly to the column; and the injector needle is connected to the syringe. Theair pressure lifts the needle and the vial is moved into position beneath theneedle. Then, the needle is lowered to the vial.

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Front View

Inject

Rear View

Load - Inject

Sample Loop

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Absorbance (UV with Filters, UV with Monochromators)

IR Absorbance

Fluorescence

Refractive-Index

Evaporative Light Scattering Detector (ELSD)

Electrochemical

Mass-Spectrometric

Photo-Diode Array

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Carbohydrates1. fructose

2. Glucose

3. Saccharose

4. Palatinose

5. Trehalulose

6. isomaltose

Zorbax NH2 (4.6 x 250 mm)

70/30 Acetonitrile/Water

1 mL/min Detect=Refractive Index

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5

mAU

time

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Injector

Detector

Column

Solvents

Mixer

Pumps

High Performance Liquid Chromatograph

Waste

Separation in based upon differential

migration between the stationary and

mobile phases.

Stationary Phase - the phase which

remains fixed in the column, e.g. C18,

Silica

Mobile Phase - carries the sample

through the stationary phase as it

moves through the column.

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Injector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time


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Injector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time


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Injector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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Injection

to

tR

mAU

time

tR

to - elution time of unretained peak

tR- retention time - determines sample identity

Area or height is proportional

to the quantity of analyte.

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Mobile Phases

Flow Rate

Composition

Injection Volume

Column

Oven Temperature

Wavelength

Time ConstantMehdi Soleymani


Column Parameters

Column Material

Deactivation

Stationary Phase

Coating Material

Instrument Parameters

Temperature

Flow

Signal

Sample Sensitivity

Detector

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Sample Parameters

Concentration

Matrix

Solvent Effect

Sample Effect

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Normal phase

Reverse phase

Size exclusion

Ion exchange

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Normal Phase.- Polar stationary phase and non-polar solvent.

• Reverse Phase.

- Non-polar stationary phase and a polar

solvent.

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Types of Compounds Mode Stationary

Phase

Mobile Phase

Neutrals

Weak Acids

Weak Bases

Reversed

Phase

C18, C8, C4

cyano, amino

Water/Organic

Modifiers

Ionics, Bases, Acids Ion

Pair

C-18, C-8 Water/Organic

Ion-Pair Reagent

Compounds not

soluble in water

Normal

Phase

Silica, Amino,

Cyano, Diol

Organics

Ionics Inorganic Ions Ion

Exchange

Anion or Cation

Exchange

Resin

Aqueous/Buffer

Counter Ion

High Molecular Weight

Compounds

Polymers

Size

Exclusion

Polystyrene

Silica

Gel Filtration-

Aqueous

Gel Permeation-

Organic

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Chemical

Environmental

Pharmaceuticals

Consumer Products

Clinical

polystyrenes

dyes

phthalates

tetracyclines

corticosteroids

antidepressants

barbiturates

amino acids

vitamins

homocysteine

Bioscience

proteins

peptides

nucleotides

lipids

antioxidants

sugars

polyaromatic hydrocarbons

Inorganic ions

herbicides

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I need a quantitative separation of

carbohydrates in some of our

products as soon as possible.

I’ll need a separation technique.

I’ll get on it!

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I have two separation techniques in my lab,

High Performance Liquid Chromatography

and Gas Chromatography. Which one should I use?

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High separation capacity, enabling the batch analysis of multiple components

Superior quantitative capability and reproducibility

Moderate analytical conditions Unlike GC, the sample does not need to be vaporized.

Generally high sensitivity

Low sample consumption

Easy preparative separation and purification of samples

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http://192.215.107.101/ebn/942/tech/techfocus/1071main.html

http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.html

Skoog, Holler, and Neiman. Principles of Instrumental Analysis. 5th ed. Orlando: Harcourt Brace & Co., 1998.

http://weather.nmsu.edu

http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm

http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html

http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLCHP1090/HPLCINJ.HTM

http://test-equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytical_Instruments/Chromatographs/HPLC_Columns

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm

http://192.215.107.101/ebn/942/tech/techfocus/1071main.html

http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.html

http://weather.nmsu.edu/

http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm

http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html

http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLCHP1090/HPLCINJ.HTM

http://test-equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytical_Instruments/Chromatographs/HPLC_Columns

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm

introduction to high performance liquid chromatography-hplc

Science

mobile phase

components of chromatography

principles of chromatography

technique chromatography

river mouth

separation tube

separation column

stationary riverbed