Post on 15-Jul-2015
RP-HPLC – BASICSS.Karthikumar., M.Sc.,M.Phil.,M.Tech.,(Ph.D)
Assistant Professor, Department of Biotechnology
Kamaraj College of Engineering and Technology
Virudhunagar-626001, Tamilnadu, INDIA
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Invention of Chromatography by M.Tswett
Ether
CaCO3
Chlorophyll
Chromatography
Colors
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High Pressure Liquid Chromatography
High Performance Liquid Chromatography
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Advantages of High Performance Liquid Chromatography
• High separation capacity,
• 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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Fields in Which High Performance Liquid Chromatography Is Used
• Biogenic substances• Sugars, lipids, nucleic acids,
amino acids, proteins, peptides, steroids, amines, etc.
• Medical products• Drugs, antibiotics, etc.
• Food products• Vitamins, food additives, sugars, organic
acids, amino acids, etc.
• Environmental samples• Inorganic ions
• Hazardous organic substances, etc.
• Organic industrial products• Synthetic polymers, additives, surfactants,
etc.
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Interaction Between Solutes, Stationary Phase, and Mobile Phase
• Differences in the interactions between the solutes and stationary and mobile phases enable separation.
Solute
Stationary
phaseMobile phase
Degree of adsorption,
solubility, ionicity, etc.
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Stationary Phase and Mobile Phase Used in Normal Phase Mode
• Stationary Phase• Silica gel: -Si-OH
• Cyano type: -Si-CH2CH2CH2CN
• Amino type: -Si-CH2CH2CH2NH2
• Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
• Mobile Phase• Basic solvents: Aliphatic hydrocarbons, aromatic hydrocarbons, etc.
• Additional solvents: Alcohols, ethers, etc.
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Relationship between Hydrogen Bonding and Retention Time in Normal Phase Mode
OH
HO
SiOH
SiOHStrong
Weak
Steric hindrance
Very weak
?
• Polar Functional Groups• -COOH
• Carboxyl groups
• -NH2
• Amino groups
• -OH• Hydroxyl groups
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Reversed Phase Chromatography• Stationary phase: Low polarity
• Octadecyl group-bonded silicalgel (ODS)
• Mobile phase: High polarity• Water, methanol, acetonitrile
• Salt is sometimes added.
Stationary
phaseMobile phase
Normal
phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed
phase
Low polarity
(hydrophobic)
High polarity
(hydrophilic)
• Nonpolar Functional Groups• -(CH2)nCH3
• Alkyl groups
• -C6H5
• Phenyl groups
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Separation Column for Reversed Phase Chromatography
• C18 (ODS) type
• C8 (octyl) type
• C4 (butyl) type
• Phenyl type
• TMS type
• Cyano type
Si -O-Si
C18 (ODS)
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
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Hydrophobic Interaction
H2O
H2OH2O
H2O
H2OH2O
H2O
Network of hydrogen bonds
H2O
H2OH2O
H2O
H2O H2O
H2O
Nonpolar solute
If a nonpolar
substance is added...
…the network is broken and...
H2OH2O H2O
H2OH2O H2O
H2O
Nonpolar solute
Nonpolar stationary phase
…the nonpolar substance
is pushed to a nonpolar
location.
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Effect of Chain Length of Stationary Phase
C18 (ODS)
Strong
C8
C4
Medium
Weak
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Relationship Between Retention Time and Polarity
C18 (ODS)
CH3
StrongWeak
OH
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Comparison of Normal Phase and Reversed Phase
• Normal Phase• Effective for separation of
structural isomers
• Offers separation selectivity not available with reversed phase
• Stabilizes slowly and is prone to fluctuations in retention time
• Eluents are expensive
• Reversed Phase• Wide range of applications
• Effective for separation of homologs
• Stationary phase has long service life
• Stabilizes quickly
• Eluents are inexpensive and easy to use
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HPLC - Hardware
Solvent Delivery System, Degasser, Sample
Injection Unit, Column Oven
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Pump
Sample injection unit
(injector)
Column
Column oven
(thermostatic
column chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
Degasser
Flow Channel Diagram for High Performance Liquid Chromatograph
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Solvent Delivery Pump
• Performance Requirements• Capacity to withstand high load pressures.
• Flow rate does not fluctuate.
• Solvent replacement is easy.
• The flow rate setting range is wide and the flow rate is accurate.
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Solvent Delivery Pump:Schematic Diagram of Plunger Pump
Motor and cam
Plunger
Plunger seal
Check
valves
Pump head
10 -100µL
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Pump – Mixing & Flow Rate System
• Isocratic system• Constant eluent composition
• Gradient system• Varying eluent composition
• HPGE (High Pressure Gradient)
• LPGE (Low Pressure Gradient)
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Difference in the Chromatogram
• In isocratic mode
Long analysis time!!
Poor separation!!
CH3OH / H2O = 6 / 4
CH3OH / H2O = 8 / 2
• If the eluent composition is changed gradually during analysis... Gradient system
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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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Degasser Heated stirring is used to reduce the dissolved airin the solution in the mobile phase bottle to thesaturated solubility level.
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Mixing, Filtration, and Offline Degassing of the Eluent
Decompression
by aspirator
Ultrasonic
cleaning unit
Decompression
by aspirator
Membrane filter with pore
size of approx. 0.45 µm
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Online Degasser
Gas-liquid separation membrane methodHelium purge method
Helium
cylinder
To draft
To pump
Eluent container
Regulator
Drain valve
To pump
Eluent container
Polymeric film tubeVacuum chamber
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Preparing pH Buffer Solution
• Use a weak acid with a pKa value close to the desired pH value.
• Example: Preparing a buffer solution for a pH value of around 4.8. Use acetic acid, which has a pKa value of 4.8.
• Make the concentrations of HA and A- roughly equal. Mix an acid with its salt.
• Example: Mix acetic acid and sodium acetate so that they have the same molar concentration.
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Buffer Solutions Used for HPLC Eluent
• Requirements• High buffering power at
prescribed pH.
• Does not adversely affect detection.
• Does not damage column or equipment.
• Inexpensive.
• Commonly Used Acids• Phosphoric acid
• pKa 2.1, 7.2, 12.3
• Acetic acid• pKa 4.8
• Citric acid• pKa 3.1, 4.8, 6.4
• Concentration• If only to adjust pH, 10
mmol/L is sufficient.
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pH of Eluent and Retention of Ionic Solutes
COOH
COO
H
pH of eluent
Acidic
Alkaline
+
Increased
hydrophobicity
Increased
hydrophilicity
High RT
Low RT
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Replacement of Eluent
• Mutually insoluble solvents must not be exchanged directly.
• Aqueous solutions containing salt and organic solvents must not be exchanged directly.
Water
Hexane
2-Propanol
Buffer solution
Water-soluble
organic solvent
Water
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Sample Injection Unit (Injector)
INJECT position
LOAD position
From pump
To column
From pump
To column
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Column Oven
• Air circulation heating type
• Block heating type• Aluminum block heater
• Insulated column jacket type• Water bath
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HPLC Detectors
• UV-VIS absorbance detector
• Photodiode array-type UV-VIS absorbance detector
• Fluorescence detector
• Refractive index detector
• Evaporative light scattering detector
• Electrical conductivity detector
• Electrochemical detector
• Mass spectrometer
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UV-VIS Absorbance Detector
A = e·C·l = –log (Eout / Ein)
l
C: Concentration
(A: absorbance, E: absorption coefficient)
Detection cell
Ein Eout
A
C
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Comparison of Detectors
Note: The above table indicates general characteristics. There are exceptions.
Selectivity SensitivityPossibility of
Gradient System
AbsorbanceLight-absorbing
substancesng Possible
Fluorescence Fluorescent substances pg Possible
Differential
refractive indexNone µg Impossible
Evaporative light
scatteringNonvolatile substances µg Possible
Electrical
conductivityIonic substances ng Partially possible
ElectrochemicalOxidizing / reducing
substancespg Partially possible
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Chromatogram
tR
t0
Inte
nsity o
f d
ete
cto
r sig
nal
Time
PeaktR : Retention time
h
A
t0 : Non-retention time
A : Peak area
h : Peak height
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Retention Factor, k
tR
t0
Str
ength
of
dete
cto
r sig
nal
Time
tR: Retention time
t0: Non-retention time
0
0R
t
ttk
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Theoretical Plate Number, N
W
W1/2
H1/2
H
2
.21
R
R
/
R
W
t
W
2
2
2
545
16
Area
Ht
tN
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