HPLC_PART II
High
Performance
Liquid
Chromatography
Pumps:
• A high-pressure pump [solvent delivery system or solvent manager] is used to generate and
meter a specified flow rate of mobile phase. Pumps are required to deliver a constant flow of
mobile phase at pressures ranging from 1 to 550 bar (14.6 to 8000psi).
Commonly used pumps in HPLC are-
1. Reciprocating piston pump:
2. Syringe pump and
3. Constant pressure pump
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Injector:
• The solute mixture is introduced into the chromatographic column by means of a suitable
injection device.
• The sample solution is injected through a self sealing rubber septum or Teflon disk using a
micro-liter syringes.
• Typical sample volumes for manual injector are 5-20 μL.
Manual injection device Microliter syringe
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Automatic injectors (autosamplers)
• Continuously Inject variable volumes of sample ranging from 1–100 µL.
• Precision and accuracy are much more than manual injectors but more expensive initially.
• Modern sophisticated autosamplers not only do sample injection but also can do a number of
other functions such as :
1. Diluting,
2. Performing derivatization by mixing with different reagents,
3. Adding internal standard and
4. Transferring reagents and sample to an empty vial
for pretreatment etc.
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Autosampler
Elution Techniques in HPLC:
Elution through HPLC are of two types
1. Isocratic elution
2. Gradient elution
Isocratic Elution:
If a solvent or mixture of solvent, having fixed composition and fixed polarity is pumped through out
the overall analytical procedure, then it is called isocratic elution.
Gradient Elution:
In this type of elution, polarity of solvent is changed gradually.
Low pressure gradient elution
High pressure gradient elution
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Isocratic
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Low Pressure Gradient
(quaternary pumps)
Atmospheric pressure High pressure
Mixing on low
pressure side
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High Pressure Gradient
(binary pumps)
Mixing on high pressure side
Atmospheric pressure High pressure
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mixing
Low pressure mixing High pressure mixing
Both systems have their own advantages and it cannot be claimed that one is
superior to the other.
• Due to heat of mixing, composition can vary on mixing but present day
software provides correction features.
• Use of more than one pump in high-pressure pump means increased cost and
greater maintenance.
• High-pressure systems are usually limited to 2 solvents
• High-pressure mixing provides precise control and reproducibility of mobile
phase composition up to 0.1% 9
• Low-pressure mixing can be more useful in development work as operation up
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Explain the figure….
Based on mode of separation:
Normal Phase vs. Reverse Phase
• If the stationary phase is more polar than the mobile phase, the separation is deemed
normal phase.
• If the stationary phase is less polar than the mobile phase, the separation is reverse phase.
• Typical columns for normal phase separation are packed with alumina or silica.
• Alkyl, aliphatic or phenyl bonded phases are typically used for reverse phase
separation.
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Preparation of Stationary phase for HPLC.
The stationary phase of NP-HPLC usually involves silica (O=Si=O).
SiO2
O=Si=O
Si OH
Polar Group
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Stationary Phase for NP-HPLC
Si Si-OH Unmodified Silica (USP-L3)
Si Si-CH2-CH2-CH2-NH2 Amino (USP-L11)
Si Si-CH2-CH2-CH2-CN Cyano (USP-L10)
OH
Si Si-CH2-CH2-CH2-OCHCH2 Diol (USP-L)
OH
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Explain the figure….
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Mobile Phase for NP-HPLC
Primary solvents (non-polar)
– Hydrocarbons (Pentane, Hexane, Heptane, Octane)
– Aromatic Hydrocarbons (Benzene, Toluene, Xylene)
– Pet. Ether
– Methylene chloride
– Chloroform
– Carbon tetrachloride
Secondary solvents
– Methyl-t-butyl ether (MTBE), Diethyl ether, THF, Dioxane, Pyridine,
Ethyl acetate, Acetonitrile, Acetone, 2-propaol, ethanol, methanol
A primary solvent is used as mobile phase. Addition of secondary solvents
is to adjust retention time.
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