HPLC Basics- Principle and Scope

HPLC – Principle | Scope | Diagram | Separation Mechanism | Separation Modes | Detectors for HPLC |

• Concept and scope of HPLC
• Separation Mechanisms 
• Instrumentation
• Detectors for HPLC 

Basic Terms :

• High Performance Liquid Chromatography (HPLC)
• Chromatography: Method of analysis
• Chromatograph: Instrument
• Chromatogram: Resulting graph
• Chromatographer: Person performing analysis

Concept of Chromatography :

• Chromatography is an analytical method that the compounds are physically separated prior to measurement
• The main purpose of chromatography is to separate and quantify the target sample in the matrix

Why use HPLC ?

• Simultaneous Analysis
• High Resolution
• High Sensitivity (ppm-ppb)
• Good repeatability
• Small sample size
• Moderate analysis condition
  • no need to vaporize the sample like GC
• Easy to fractionate the sample and purify
• No destructive for many detectors

Scope of HPLC :

Field	Typical mixtures
Pharmaceuticals	Antibiotics, sedatives, steroids, analgesics, crude drugs, cosmetics
Biochemical	Amino acids, proteins, peptides, carbohydrates, lipids, enzymes, medicines, hormone
Food products	Mycotoxins, additives, saccharides, amino acids, vitamins, fatty acid, coloring agents, antibacterials
Industrial chemicals	Condensed aromatics, surfactants, propellants, dyes, polymers, plasticizers
Forensic chemistry	Drugs, poisons, blood alcohol, narcotics
Environmental field	Inorganic ions, organic acids, agricultural chemicals, pesticides, herbicides, phenols,
Clinical medicine	Bile acids, drug metabolites, urine extracts, estrogens

Flow Diagram of HPLC :

Chromatogram :

• t_R  : Retention time
• A   : Area
• h   : Height

Some Important Terms :

• Chromatogram: A plot of detector signal output versus time or elution volume.
• Mobile phase: The liquid that moves the solute through the column.
• Stationary phase: The packing material of the column, which is the immobile phase involved in the chromatographic process.
• Peak: The visual representation on the chromatogram based on the detector’s electrical response due to the presence of a sample component inside the flow cell.
• Retention time: The time taken by the analyte peak to reach the detector after sample injection.
• Qualitation: An analysis process which is designed to identify the components of a substance or mixture.
• Quantitation: An analysis process which is designed to determine the amounts or proportion of the components of a substance.

Separation Mechanism :

Compounds are separated because the molecules move at different rates in the column.

Due to different interaction between stationary phase and different sample, the molecules move at different rate, therefore separation can be done.

Separation Modes :

• Normal phase chromatography
• Reversed phase chromatography
• Ion chromatography
• Size exclusion chromatography
• Affinity chromatography

	Stationary phase	Mobile phase
Normal phase	High polarity (hydrophilic)	Low polarity (hydrophobic)
Reversed phase	Low polarity (hydrophobic)	High polarity (hydrophilic)

• Normal-Phase/Absorption Chromatography:
  • Stationary phase: High polarity (hydrophilic) : Silica gel or polar functional group which is chemically bonded on the surface of silica gel.
  • Mobile phase: Low polarity (hydrophobic) : Non-polar solvent such as hexane.

• Reversed-Phase Chromatography:
  • Stationary phase: Low polarity: Octadecyl group bonded silica gel (called “ODS” or “C18”)
  • Mobile phase: High polarity: Water, methanol, acetonitrile, etc.

• Stationary Phase in Reversed Phase Column
  • C18 (ODS) type
  • C8 (octyl) type
  • C4 (butyl) type
  • Phenyl type
  • TMS type
  • Cyano type

Mobile Phase for Reversed Phase HPLC :

• Water / buffer + Organic solvent
  • Organic solvents:
    • Methanol
    • Acetonitrile
    • THF
  • Buffer:
    • Phosphate buffer
    • Acetate buffer
• Ratio of aqueous and organic solvents is important

• Isocratic and Gradient:
  • Isocratic elution : fixed composition.
  • Gradient elution : if the mobile phase composition is gradually changed during analysis.

Guard Column and Pre-Column:

Pre-column: Traps impurities and substances that interfere with analysis in the “mobile phase”.

Guard column: Traps contaminants and insoluble substances in the “sample”

Detectors for HPLC :

• UV-VIS – Ultraviolet / Visible detector
• PDA – Photodiode Array detector
• RF – Fluorescence detector
• CDD – Conductivity detector  
• RID – Refractive Index detector
• ECD – Electrochemical detector
• ELSD – Evaporative light scattering detector
• MS – Mass spectrometer detector

1. UV-VIS – Ultraviolet / Visible detector:

• Advantage:
  • Sensitivity is high
  • Relative robust to temperature and flow rate change
  • Compatible with gradient elution
• Disadvantage
  • •Only compounds with UV or visible absorption could be detected.

2. PDA – Photodiode Array detector:

• Advantages
  • PDA Detector could analyze a sample simultaneously at many different wavelengths.
  • UV Visible spectra are useful for compound identification, checking peak purity, as well as finding the optimum absorbance for the compounds.
  • UV Visible spectra of many compounds could be stored in the spectrum libraries, which are useful for compound identification.
  • Relatively robust to temperature and flow rate fluctuations
  • Compatible with gradient elution.
• Disadvantages
  • Slightly less sensitive than UV-Visible detector.

3. RF – Fluorescence detector:

Fluorescence is a type of luminescence in which the light energy is released in the form of a photon in nanoseconds to microseconds

• Advantage
  • Sensitivity is higher than UV-Vis detector
  • Selectivity is high because relatively few compounds fluorescence
  • Compatible with gradient elution
• Disadvanage
  • Difficult to predict fluorescence
  • Greatly affected by environment
    • Solvent
    • pH
    • Temperature
    • Viscosity
    • Ionic strength
    • Dissolved gas

4. CDD – Conductivity detector :

• Advantages
  • Respond to ionic compounds and suitable for ion chromatography.
  • High sensitivity for low concentration range
• Disadvantages
  • Sensitive to the fluctuations in the solvent flow and mobile phase composition
  • Not compatible with gradient elution

5. RID – Refractive Index detector:

• Advantage
  • Responds to nearly all solutes
  • Unaffected by flow rate
• Disadvantage
  • Not as sensitive as most other types of detectors
  • Could not be used with gradient elution

6. ECD – Electrochemical detector :

Electrochemical detector responds to compounds that can be oxidized or reduced, such as phenols, aromatic amines, ketones, aldehydes.

• Advantages
  • Selective as relatively few compounds are electro-active.
  • Excellent sensitivity for low concentration range.
• Disadvantages
  • Sensitive to temperature and flow rate fluctuations
  • Not compatible with gradient elution.
  • Aqueous or other polar solvents containing dissolved electrolytes are required and they must be rigorously free from oxygen.

7. ELSD – Evaporative light scattering detector :

• Detection Pinciple
  • Nebulization
  • Evaporation
  • Detection

ELSD responds to compound that is less volatile than that of the mobile phase

Applications of ELSD :

• Advantages
  • •Most compounds can be detected (universal detector)
  • •Compatible with gradient elution
• Disadvantages
  • •Mobile phase must be volatile.
  • Nebulizing gas is required

8. MS – Mass spectrometer detector:

• Ion Detector
  • Electron Multiplier
    • 1. A series of dynodes maintained at ever-increasing potentials
    • 2. Ions strike the dynode surface, resulting in the emission of electrons.
    • 3. these secondary electron are then attracted to the next dynode where more secondary electrons are generated
    • 4. ultimately resulting in a cascade of electrons

• Ionization of Compounds in MS Detector
  • ESI
    • drugs and their metabolites
    • peptides
    • proteins
    • many kinds of natural product – (-OH, -NH₂,-COOH, SO₂, PO₃ etc.)
  • APCI
    • pesticides
    • steroids
    • drugs

Selection of Detectors

Detectors	Type of compounds can be detected
UV-Vis & PDA	Compounds with chromophores, such as aromatic rings or multiple alternating double bonds.
RF	Fluorescent compounds, usually with fused rings or highly conjugated planar system.
CDD	Charged compounds, such as inorganic ions and organic acid.
ECD	For easily oxidized compounds like quinones or amines.
RID & ELSD	For compounds that do not show characteristics usable by the other detectors, eg. polymers, sccharides.

Reference : Shimadzu

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