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Questions and Answers
What principle underlies the separation in reversed phase HPLC?
What principle underlies the separation in reversed phase HPLC?
Which type of column packing material has the highest hydrophobicity for reverse phase chromatography?
Which type of column packing material has the highest hydrophobicity for reverse phase chromatography?
In normal phase HPLC, what type of interactions primarily facilitate the separation process?
In normal phase HPLC, what type of interactions primarily facilitate the separation process?
Which solvent is considered the least polar in reversed phase chromatography?
Which solvent is considered the least polar in reversed phase chromatography?
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What characterizes the stationary phase in reversed phase chromatography?
What characterizes the stationary phase in reversed phase chromatography?
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Which of the following materials is considered the most polar in normal phase chromatography?
Which of the following materials is considered the most polar in normal phase chromatography?
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What is the primary function of Ion-Exchange chromatography?
What is the primary function of Ion-Exchange chromatography?
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Which solvent is considered the least polar in the context of normal phase chromatography?
Which solvent is considered the least polar in the context of normal phase chromatography?
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In Gel-Permeation chromatography, what primarily determines the separation of molecules?
In Gel-Permeation chromatography, what primarily determines the separation of molecules?
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Which type of ion-exchange packing material is classified as acidic?
Which type of ion-exchange packing material is classified as acidic?
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What is a major trade-off when optimizing chromatographic resolution?
What is a major trade-off when optimizing chromatographic resolution?
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Which mode of HPLC interactions is specifically designed for separating molecules based on size?
Which mode of HPLC interactions is specifically designed for separating molecules based on size?
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In HPLC, a pressurized polar solvent is used to carry a sample through what type of structure?
In HPLC, a pressurized polar solvent is used to carry a sample through what type of structure?
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What type of chromatography would use a high-velocity mobile phase?
What type of chromatography would use a high-velocity mobile phase?
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What characteristic property of a molecule is primarily identified in HPLC?
What characteristic property of a molecule is primarily identified in HPLC?
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Which of the following is NOT a mode of HPLC interactions?
Which of the following is NOT a mode of HPLC interactions?
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What dual capability does HPLC provide in its analysis?
What dual capability does HPLC provide in its analysis?
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Which type of differential distribution primarily involves different chemical states?
Which type of differential distribution primarily involves different chemical states?
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What is generally compromised when increasing the length of a chromatographic column?
What is generally compromised when increasing the length of a chromatographic column?
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What does the term 'Eddy Diffusion' refer to in chromatography?
What does the term 'Eddy Diffusion' refer to in chromatography?
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In the context of chromatography, what does HETP stand for?
In the context of chromatography, what does HETP stand for?
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Which of the following terms is used to describe the retention factor in chromatography?
Which of the following terms is used to describe the retention factor in chromatography?
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What is the primary factor affecting molecular diffusion in a chromatography column?
What is the primary factor affecting molecular diffusion in a chromatography column?
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Which separation technique utilizes different rates of migration in an electric field?
Which separation technique utilizes different rates of migration in an electric field?
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In chromatography, the relationship expressed by H=Au1/3 + B/u + Cu indicates what?
In chromatography, the relationship expressed by H=Au1/3 + B/u + Cu indicates what?
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Which technique is specifically used for extracting analytes using a supercritical fluid?
Which technique is specifically used for extracting analytes using a supercritical fluid?
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What materials are used for the packing of columns in GPC?
What materials are used for the packing of columns in GPC?
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Which of the following solvents is suitable for bio-polymers in GPC?
Which of the following solvents is suitable for bio-polymers in GPC?
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What type of pump is commonly used in HPLC systems?
What type of pump is commonly used in HPLC systems?
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What is the typical diameter range for analytical columns in HPLC?
What is the typical diameter range for analytical columns in HPLC?
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Which feature is NOT common in analytical chromatograph designs?
Which feature is NOT common in analytical chromatograph designs?
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Which type of column uses hydrophobic solvent for sample adhesion?
Which type of column uses hydrophobic solvent for sample adhesion?
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What is the primary purpose of a pulse dampener in HPLC pumps?
What is the primary purpose of a pulse dampener in HPLC pumps?
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Which of the following statements about SDVB beads is true?
Which of the following statements about SDVB beads is true?
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What is the primary purpose of using reverse phase columns in HPLC?
What is the primary purpose of using reverse phase columns in HPLC?
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Which of the following solvents is commonly used in normal phase HPLC?
Which of the following solvents is commonly used in normal phase HPLC?
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What is the main role of the differential refractive index detector in HPLC?
What is the main role of the differential refractive index detector in HPLC?
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What effect does increasing solvent flow rate have on chromatographic peaks?
What effect does increasing solvent flow rate have on chromatographic peaks?
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Which detector is best suited for detecting drugs at parts per trillion (PPT) levels?
Which detector is best suited for detecting drugs at parts per trillion (PPT) levels?
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Which pressure range is typical for reverse phase HPLC systems?
Which pressure range is typical for reverse phase HPLC systems?
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What is a characteristic of using more polar solvents in chromatographic applications?
What is a characteristic of using more polar solvents in chromatographic applications?
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Which type of solvent selection is crucial for protecting PEEKK polymer surfaces?
Which type of solvent selection is crucial for protecting PEEKK polymer surfaces?
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Study Notes
Basic Principles of Chromatography (FDSC 624)
- Chromatography is a separation technique based on differential distribution
- Different methods exist, categorized by the type of differential distribution.
Types of Differential Distribution
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Differential Distribution by Different Chemical States of the Same Matter:
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Distillation
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Sublimation
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Crystallization
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Zone melting
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Precipitation
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Differential Distribution by Different Chemical Phases:
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Absorption (partition)
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Adsorption
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Liquid-liquid extraction (LLE)
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Solid-phase extraction (SPE)
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Supercritical fluid extraction (SFE)
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Solid-phase microextraction (SPME)
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Differential Distribution by Different Chemical Environments or Locations:
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Separation based on different migration rates under a field
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Electric field: electrophoresis, and field-flow fractionation
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Gravitational field: centrifugation and filtration
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Thermal field: Thermal diffusion
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Membrane (semi-permeable): dialysis, osmosis, and ultra-filtration
Chromatographic Techniques
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Classical Column Chromatography
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Uses a packed column for separation
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Separation occurs based on differences in affinity to stationary/mobile phase
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Thin Layer Chromatography (TLC)/Planar Chromatography
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Uses a planar stationary phase
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Separation occurs based on differences in affinity to stationary/mobile phase
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High Performance Liquid Chromatography (HPLC)
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Uses a packed column for separation
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Separation occurs based on differences in affinity to stationary/mobile phase and size
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Gas Chromatography (GC)
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Uses a packed column for separation
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Separation occurs based on differences in boiling point
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Supercritical Fluid Chromatography (SFC)
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Uses a supercritical fluid as the mobile phase
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Separation occurs based on differences in interaction with the stationary/mobile phase
Chromatographic Definitions
- Migration time: time it takes for the non-retained species to travel through the column
- Retention time:
- Time for analyte A
- Time for analyte B
- Adjusted retention time: time corrected to exclude the migration time of the nonretained species
- Peak Width (WA, WB): measure of band spreading
- Column Length: length of the column packing material
- Flow rate: volume of mobile phase passing through the column per unit time.
- Volume of stationary phase: Volume of the stationary phase in the column
- Concentration of analyte: concentration of analyte in mobile and stationary phase
Chromatographic Relationships
- Linear mobile-phase velocity: speed of the mobile phase
- Volume of mobile phase: the amount of mobile phase used.
- Retention factor: measure of the analyte's affinity to the stationary phase
- Distribution constant: measure of the analyte's distribution between the two phases.
- Selectivity factor: measure of the selectivity of the separation.
- Resolution: measure of the separation between two peaks.
- Number of Plates: measure of column efficiency.
- Plate height: measure of the height per theoretical plate in the column.
Rate Theory
- Plate height (H): describes the efficiency of a column; minimize
- Eddy diffusion (A): term showing how mobile phase spreads out
- Molecular diffusion (B): term showing how the analyte molecules move through the column
- Mass transfer (C): term showing how the movement of analyte between the mobile and stationary phases
- Velocity of mobile phase (u): measures how fast the mobile phase moves.
Eddy Diffusion
- Packing factor (λ)
- Diameter of packed particles (dp)
Molecular Diffusion
- Obstruction factor (Ψ)
- Diffusion coefficient (DM)
Mass Transfer
- Retention factor (k)
- Diffusion coefficient (Ds)
- Average film thickness (df)
Chromatography: Peak Resolution
- Poor resolution (a)
- More separation (b)
- Less band spread (c)
Quantitative Analysis
- Peak areas
- Peak heights
- Calibration and standards
- Internal standard method
Liquid Chromatography (HPLC)
- Uses a pressurized polar solvent to carry a sample through a tubular column filled with non-polar packing.
- Allows separation of organic species.
- Qualitative and quantitative analysis supported by various detectors
- Different modes of separation categorized:
- Adsorption Chromatography
- Partition Chromatography
- Ion-exchange Chromatography
- Size-exclusion Chromatography
Adsorption Chromatography
- Stationary phase is a solid
- Separations occur through adsorption/desorption steps.
- Common stationary phases: silica and alumina.
Partition Chromatography
- Separation based on solute partitioning between two liquid phases
- Separation based on relative solubility.
- Normal phase: polar stationary phase, non-polar solvent.
- Reverse phase: non-polar stationary phase, polar solvent.
Ion-exchange Chromatography
- Stationary phase has a positively or negatively charged surface.
- Separation based on charged solute molecules in mobile phase binding opposingly charged surfaces on stationary phase
- Commonly using weak exchange resins.
Size-exclusion Chromatography
- Separation based on molecular size.
- Using gels with controlled pore size.
- Larger molecules elute first (shorter distance to travel).
Basic HPLC Theory
- All organic molecules have a characteristic retention time.
- Different modes of operation: normal phase, reverse phase, ion-exchange and gel-permeation
- Chromatographs have several common components:
- Solvent/pump (mobile phase)
- Injection port/valve
- Columns (stationary phase)
- Detectors (up to four)
- Output (analog or digital)
HPLC System Design
- Components: pump, filter, injector, column, detector, data recorder, waste solvent reservoir.
HPLC Pump
- Reciprocating, cam-driven, single-piston pumps. Uses a simple pulse dampener to create a consistent solvent flow.
Injections Valve
- High-pressure, fluoropolymer material to accommodate different sample loops, syringes and automated sampler ports.
Tubing and Fittings
- Conventional stainless steel; high pressure and solvent-resistance; for GPC/SEC work, and NPC.
- Fluoropolymer (PEEK); reduced ionic contamination for bio-assays & RPC.
Columns & Fittings
- Packed columns with 3-10 micron silica-based stationary phases to provide maximum data quality and minimum time using metal or polymer high-pressure fittings and seals.
Column
- Normal or reverse phases
- Gel permeation, ion-exchange
Normal Phase Columns
- Uses hydrophilic stationary phase and non-polar solvent.
- Useful for low polarity and petrochemical samples, tests for molecular weight determinations.
Reverse Phase Columns
- Uses hydrophobic stationary phase and polar solvent.
- Useful for high polarity and hydrophilic samples. Most popular form of HPLC.
Solvent Selection
- Normal phase: hexane, methylene chloride, THF, DMF, DMSO
- Reverse phase: 90% water, methanol & acetonitrile; some add small amounts of aprotic solvents (THF).
HPLC Detectors
- UV/Vis (fixed & variable wavelengths)
- Refractive Index
- Fluorescence
- Evaporative Light Scattering
- Electro-Chemical
LC Detectors: Differential Refractive Index
- Looks at the difference between a static reference cell and a sample flow cell
- Specialized for sugars and carbohydrates, polymer (molecular weight) determinations.
Filter Fluorescence
- Use excitation and emission filters to allow analysis of drugs, natural products, vitamins, and specialized chemicals at a PPT level
- Used for poly-aromatic analyses and environmental work
HPLC: Important Features
- Solvent pump rate (micro-LC, analytical, semi-prep)
- System pressure rating (normal phase, reversed phase)
Chromatographic Controls
- Higher flow rate = closer peaks
- More polar solvent = faster separation
- Higher pressure = closer peaks
- Lower flow, polarity, pressure = better resolution
- Controlled flow = peaks with minimum resolution loss
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Description
Test your knowledge on various chromatography techniques including reversed phase, normal phase, ion-exchange, and gel-permeation chromatography. This quiz covers principles, materials, and solvent interactions essential for successful chromatographic separation. Perfect for students and professionals in chemistry and biochemistry fields.
