HPLC Flashcards
High Performance Liquid Chromatography (HPLC)
• HPLC is the liquid chromatography analog to GC.
• The principle of HPLC is based on diffusion control.
• The rate of distribution of sample/solutes between the stationary phase and the mobile phase is largely diffusion-controlled.
• Diffusion in liquids is extremely slow compared to that in gas
HPLC Components
✓Fine column packing is used, so these operate at high pressures (1000-3000psi).
✓The solvent reservoirs may contain solvents with a range of polarities for gradient elution.
✓Types of detectors for HPLC include refractometer, UV, diode array, fluorescence, and amperometric types
2 main type of HPLC
: liquid-liquid (partition chromatography) and solid-liquid (adsorption chromatography)
Stationary phase particles
: commonly microporous or diffusive particles which
are also permeable to solvents
HPLC Process
➢ The mobile phase will move around the particles of the stationary phase, and solutes diffuse into the stagnant mobile phase with the stationary phase.
➢Then, the solute goes out into the moving mobile phase.
➢The most non-polar bonded phases used for the reverse phase are C18 and C8.
➢However, between the two phases, the C18, also known as octadecylsilane (ODS) , non-polar compound is the most popular phase used in HPLC.
Instrument of HPLC
- Mobile-phase supply system - contains a high pressured pump to provide gradient elution.
- Sample injection system
- Column
- Detector-high sensitivity detectors are required
in HPLC
Pump
1) A pump forces the mobile phase through the column at a much greater velocity than gravity-flows columns
2) Pumps are designed in order to maintain a stable flow rate
Injector
1) Inject the liquid sample within the range of 0.1 - 100ml volume under high pressure
2) Produce minimum band broadening
3) Volume must be small (0.1 - 500 microL)
Columns
1) Smooth-bore stainless steel or Heavy-walled glass tubing
2) The most common material = Silica gel
Detector
1) HPLC detectors monitor the elute as it leaves the column
2) Produce an electronic signal proportional to the concentration of each separated components
Characteristics of detectors
1) High sensitivity
2) Fast response
3) Crucial in trace analysis
4) Insensitive to changes in type of solvent, flow rate, and temp
The most widely used detectors;
1) Spectrophotometers
2) Mass spectrometers
3) Refractive index detector
Normal Phase HPLC
1) Utilize polar adsorbent surface and non-polar eluent
2) Polar substances in the mixture sticks to polar adsorbent than non-polar
3) Non-polar will pass more quickly through the column
Reversed Phase HPLC
1) Utilize non-polar adsorbent surface and polar eluent
2) Polar molecules will travel through the column more quickly because there is strong attraction between polar solvent and polar molecules when passed through the column
3) Most commonly used form of HPLC
Retention Time
The time taken for a particular compound to travel through the column to the detector
Types of chromatic separation
1) Adsorption Chromatography
2) Ion-exchange Chromatography
3) Size Exclusion Chromatography
Adsorption Chromatography
1) Competition for adsorption sites occurs between the molecules of the mixture to be separated and the molecules of the mobile phase
2) Mobile phase can be either a single solvent or 2 or more depend on the analyte to be desorbed
3) Speed of migration of the component along the column dpend on adsorptive affinity
Ion-Exchange Chromatography
1) Molecules can be separated by their iconic charges in a process
2) Ion-exchange resins are used as the column packing materials
3) This method is used for separation of ionic species, such as amino acids
Size Exclusion Chromatography
1) Packing material with very small pore is used
2) Precisely controlled pore size materials in the column
3) Large molecules, such as polymers are physically prevented from passing through the column
Applications of HPLC
1) Chemistry and biochemistry research analyzing complex mixtures
2) Purifying chemical compunds
3) Quality control to ensure the purity of raw materials
4) Analyzing air and water pollutants
Disadvantages of HPLC
The disadvantage of this technique is generally related to the multiple step sample preparation that is needed before
chromatographic analysis
Applications of Chromatography in Halal Research/Industry
Cosmetics and personal care products.
• Muslim want to be assured that the ingredients, handling, processing, distribution, transportation, and types of cosmetic used are halal compliant.
• Halal cosmetic standard, halal certification, and the halal logo can be used as benchmarks for halal compliance.
• Two-dimensional gas chromatography hyphenated with time-of-flight mass spectrometry (GCxGC-TOF-MS) and gas chromatography mass spectrometry (GCMS) have been developed to detect non-halal substances such as gelatine, alcohol, fats, and oils in cosmetic products.
