The document presents an overview of high performance liquid chromatography (HPLC). It discusses the key components of an HPLC system including the reservoir, pump, injector, separation column, and detector. It explains that compounds are separated on the column based on differences in how they partition between the mobile and stationary phases. The document also reviews different modes of HPLC, common applications, and advantages over gas chromatography.

1. Presented by: Mehdi Soleymani Goloujeh
Supervisor: Dr. S. Davaran

2. 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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3. 3
Ether
Chlorophyll
CaCO3
Chromatography
Colors
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4. 4
Water flow
Base
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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6. 6
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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8.  HPLC is a form of liquid chromatography used to separate compounds
that are dissolved in solution. HPLC instruments consist of a reservoir of
mobile phase, a pump, an injector, a separation column, and a detector.
 Compounds are separated by injecting a sample mixture onto the
column. The different component in the mixture pass through the
column at differentiates due to differences in their partition behavior
between the mobile phase and the stationary phase. The mobile phase
must be degassed to eliminate the formation of air bubbles.
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Isocratic system
Constant eluent composition
Gradient system
Varying eluent composition
 HPGE (High Pressure Gradient)
 LPGE (Low Pressure Gradient)
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Mixer
High-pressure gradient
Low-pressure
gradient unit
Mixer
Low-pressure gradient
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Principle Pattern An Example
Solvent Cabinet Vacuum Degasser
Binary Pump
Autosampler
Thermostatted
Column Compartment
Detector
Solvent Reservoirs
Controller
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13.  The column is one of the most
important components of the HPLC
chromatograph because the separation
of the sample components is achieved
when those components pass through
the column. The High performance
liquid chromatography apparatus is
made out of stainless steel tubes with a
diameter of 3 to 5mm and a length
ranging from 10 to 30cm.
 Normally, columns are filled with silica
gel because its particle shape, surface
properties, and pore structure help to get
a good separation. Silica is wetted by
nearly every potential mobile phase, is
inert to most compounds and has a high
surface activity which can be modified
easily with water and other agents. Silica
can be used to separate a wide variety of
chemical compounds, and its
chromatographic behavior is generally
predictable 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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16.  The function of the injector is to place the sample into the high-pressure flow
in as narrow volume as possible so that the sample enters the column as a
homogeneous, low-volume plug. To minimize spreading of the injected volume
during transport to the column, the shortest possible length of tubing should be
used from the injector to the column.
When an injection is started, an air actuator rotates the valve: solvent goes
directly to the column; and the injector needle is connected to the syringe. The
air pressure lifts the needle and the vial is moved into position beneath the
needle. Then, the needle is lowered to the vial.
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17. 17
Front View
Inject
Rear View
Load - Inject
Sample Loop
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18.  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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19. 19
Carbohydrates
1. 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
1
2
3
4
5
mAU
time
6
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20. Waste
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Injector
Detector
Column
Solvents
Mixer
Pumps
High Performance Liquid Chromatograph
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
High Performance Liquid Chromatograph
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Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Solvents
Mixer
Pumps
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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Injector
Detector
Column
Pumps
Solvents
Mixer
Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
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to
Injection
tR
mAU
tR
time
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 Constant
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40. Column Parameters
 Column Material
 Deactivation
 Stationary Phase
 Coating Material
Instrument Parameters
 Temperature
 Flow
 Signal
 Sample Sensitivity
 Detector
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41. Sample Parameters
 Concentration
 Matrix
 Solvent Effect
 Sample Effect
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42. Normal phase
Reverse phase
 Size exclusion
 Ion exchange
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43.  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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45. Chemical
Pharmaceuticals
Environmental
Bioscience
proteins
peptides
nucleotides
Consumer Products
Clinical
polystyrenes
dyes
phthalates
tetracyclines
corticosteroids
antidepressants
barbiturates
lipids
antioxidants
sugars
amino acids
vitamins
homocysteine
polyaromatic hydrocarbons
Inorganic ions
herbicides
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46. I need a quantitative separation of
carbohydrates in some of our
products as soon as possible.
I’ll need a separation technique.
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I’ll get on it!

47. 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/H
PLCINJ.HTM
 http://test-equipment.
globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytical_Instru
ments/Chromatographs/HPLC_Columns
 http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm