Podcast
Questions and Answers
Why is analytical science important?
Why is analytical science important?
- To develop new theories about matter
- To explore the origins of matter
- To develop and use instruments and methods to identify matter (correct)
- To develop and use instruments and methods to create matter
What makes analytical chemistry a popular field of work?
What makes analytical chemistry a popular field of work?
- Its emphasis on kinetic theory
- Its focus on theoretical plate models
- Its application in gas chromatography
- Its instruments and methods for identifying matter (correct)
How do significant figures and decimal places help in measurements?
How do significant figures and decimal places help in measurements?
- They introduce errors in measurements
- They provide an exact measurement
- They indicate measurement certainty (correct)
- They change the nature of the measurement
What is the importance of providing units when measuring anything?
What is the importance of providing units when measuring anything?
What is the significance of a unit when reporting a measurement?
What is the significance of a unit when reporting a measurement?
Why should we pay attention to errors in measurements?
Why should we pay attention to errors in measurements?
What is the purpose of stating errors in measurements?
What is the purpose of stating errors in measurements?
How is precision related to random errors in measurements?
How is precision related to random errors in measurements?
What is the role of accuracy in measurements?
What is the role of accuracy in measurements?
How does chromatography differ from solvent extraction?
How does chromatography differ from solvent extraction?
What does a partition coefficient signify in chromatography?
What does a partition coefficient signify in chromatography?
How does precision relate to standard deviation in measurements?
How does precision relate to standard deviation in measurements?
What is the classification of chromatography based on the physical properties of the analyte?
What is the classification of chromatography based on the physical properties of the analyte?
In which type of chromatography does the competition occur between a liquid stationary phase and the mobile phase?
In which type of chromatography does the competition occur between a liquid stationary phase and the mobile phase?
What type of chromatography involves a competition between an ionic exchange resin stationary phase and liquid mobile phase?
What type of chromatography involves a competition between an ionic exchange resin stationary phase and liquid mobile phase?
Which type of chromatography is associated with the acronym LC/HPLC?
Which type of chromatography is associated with the acronym LC/HPLC?
In which type of chromatography does the mobile phase consist of gas?
In which type of chromatography does the mobile phase consist of gas?
What is the principle of competition in Permeation Chromatography?
What is the principle of competition in Permeation Chromatography?
What is the principle of chromatography?
What is the principle of chromatography?
In chromatography, what is the mobile phase?
In chromatography, what is the mobile phase?
Which type of chromatography involves large molecules being excluded?
Which type of chromatography involves large molecules being excluded?
What is the purpose of preparative chromatography?
What is the purpose of preparative chromatography?
Which category of chromatography involves molecules interacting differently with the mobile and stationary phase?
Which category of chromatography involves molecules interacting differently with the mobile and stationary phase?
What is the term for a substance that is going to be separated in chromatography?
What is the term for a substance that is going to be separated in chromatography?
In which type of chromatography is one kind of molecule in a mixture attached to a molecule covalently bound to the stationary phase?
In which type of chromatography is one kind of molecule in a mixture attached to a molecule covalently bound to the stationary phase?
What does ion-exchange chromatography primarily attract?
What does ion-exchange chromatography primarily attract?
What does size-exclusion chromatography exclude?
What does size-exclusion chromatography exclude?
What does an eluate refer to?
What does an eluate refer to?
According to the Theoretical Plate Model of Chromatography, what are the separate layers within the chromatographic column called?
According to the Theoretical Plate Model of Chromatography, what are the separate layers within the chromatographic column called?
Who proposed the Rate Theory of Chromatography in 1956?
Who proposed the Rate Theory of Chromatography in 1956?
What is the primary focus of the Kinetic Theory in chromatography?
What is the primary focus of the Kinetic Theory in chromatography?
In Gas Chromatography, what is the role of the mobile phase?
In Gas Chromatography, what is the role of the mobile phase?
What distinguishes Normal Phase Chromatography from Reverse Phase Chromatography?
What distinguishes Normal Phase Chromatography from Reverse Phase Chromatography?
Which theory of chromatography focuses on the concept that the chromatographic column contains multiple separate layers known as theoretical plates?
Which theory of chromatography focuses on the concept that the chromatographic column contains multiple separate layers known as theoretical plates?
How does an increase in the velocity of the mobile phase impact the band broadening in chromatography?
How does an increase in the velocity of the mobile phase impact the band broadening in chromatography?
According to the Van Deemter equation, what does the term 'u' represent?
According to the Van Deemter equation, what does the term 'u' represent?
What is one of the main causes of band spreading in chromatography?
What is one of the main causes of band spreading in chromatography?
How does the Rate Theory of Chromatography differ from the Plate Model?
How does the Rate Theory of Chromatography differ from the Plate Model?
In chromatography, which term measures the ratio at which a substance moves randomly from a region of high concentration to a region of lower concentration?
In chromatography, which term measures the ratio at which a substance moves randomly from a region of high concentration to a region of lower concentration?
Which factor contributes to band broadening in chromatography according to the Van Deemter equation?
Which factor contributes to band broadening in chromatography according to the Van Deemter equation?
Which factor dominates at low flow rates and is more significant in gas chromatography compared to liquid chromatography?
Which factor dominates at low flow rates and is more significant in gas chromatography compared to liquid chromatography?
What aspect of chromatographic peak shape is affected by the rate of elution according to the Rate Theory of Chromatography?
What aspect of chromatographic peak shape is affected by the rate of elution according to the Rate Theory of Chromatography?
In Van Deemter's model, what does term C represent?
In Van Deemter's model, what does term C represent?
In chromatography, what does the Van Deemter equation help us understand better?
In chromatography, what does the Van Deemter equation help us understand better?
What causes broadening of the solute band in chromatography according to the text?
What causes broadening of the solute band in chromatography according to the text?
What does Cs stand for in Van Deemter's model and what does it represent?
What does Cs stand for in Van Deemter's model and what does it represent?
What does the plate height (H) represent in the Theoretical Plate Model?
What does the plate height (H) represent in the Theoretical Plate Model?
In chromatography, what does the Distribution Constant (KC) denote?
In chromatography, what does the Distribution Constant (KC) denote?
What is the aim of varying solvent strength in chromatography?
What is the aim of varying solvent strength in chromatography?
What does the Separation Factor (α) describe in chromatography?
What does the Separation Factor (α) describe in chromatography?
What is the significance of Resolution (Rs) in chromatography?
What is the significance of Resolution (Rs) in chromatography?
How is the Number of Theoretical Plates (N) related to the plate height (H) in a chromatographic column?
How is the Number of Theoretical Plates (N) related to the plate height (H) in a chromatographic column?
What is a limitation of the van Deemter theory in terms of comparison between different chromatographic techniques?
What is a limitation of the van Deemter theory in terms of comparison between different chromatographic techniques?
Which factor is not considered by the van Deemter theory when comparing different types of columns?
Which factor is not considered by the van Deemter theory when comparing different types of columns?
How do systems operating under different pressures compare according to the limitations of the van Deemter theory?
How do systems operating under different pressures compare according to the limitations of the van Deemter theory?
What does the Kinetic theory fail to consider regarding system changes?
What does the Kinetic theory fail to consider regarding system changes?
In terms of efficiency, how does the Kinetic theory predict the change with mobile phase velocity?
In terms of efficiency, how does the Kinetic theory predict the change with mobile phase velocity?
What aspect does the Rate Theory by van Deemter focus on primarily?
What aspect does the Rate Theory by van Deemter focus on primarily?
What modification is recommended if the Rf values in silica gel TLC are too high?
What modification is recommended if the Rf values in silica gel TLC are too high?
In two-dimensional TLC, after the first run, what is the next step before re-running the plate?
In two-dimensional TLC, after the first run, what is the next step before re-running the plate?
What is the primary role of the layer in thin-layer chromatography (TLC)?
What is the primary role of the layer in thin-layer chromatography (TLC)?
What does multiple TLC (unidimensional multiple development) aim to achieve?
What does multiple TLC (unidimensional multiple development) aim to achieve?
How does varying the polarity of the mobile phase affect the separation in silica chromatography?
How does varying the polarity of the mobile phase affect the separation in silica chromatography?
Which method is NOT a detection and visualization technique used in Thin-Layer Chromatography?
Which method is NOT a detection and visualization technique used in Thin-Layer Chromatography?
In thin-layer chromatography, why is alumina considered more basic than silica?
In thin-layer chromatography, why is alumina considered more basic than silica?
In column chromatography, what pH range is typically used with silica as the stationary phase?
In column chromatography, what pH range is typically used with silica as the stationary phase?
What is a characteristic feature that distinguishes reverse-phase silica from normal-phase silica in TLC?
What is a characteristic feature that distinguishes reverse-phase silica from normal-phase silica in TLC?
What is the recommended particle diameter range when using silica in column chromatography?
What is the recommended particle diameter range when using silica in column chromatography?
Why is thin-layer chromatography (TLC) not recommended for preparative work involving large sample quantities?
Why is thin-layer chromatography (TLC) not recommended for preparative work involving large sample quantities?
What impact does an increase in mobile phase polarity have on the chromatographic strength in silica chromatography?
What impact does an increase in mobile phase polarity have on the chromatographic strength in silica chromatography?
What property of the eluted solute can be detected directly in HPLC using a detector with a range of 1-1000 pg?
What property of the eluted solute can be detected directly in HPLC using a detector with a range of 1-1000 pg?
Which chromatography technique requires the presence of a fluorophore for detection at concentrations between 1-10 pg?
Which chromatography technique requires the presence of a fluorophore for detection at concentrations between 1-10 pg?
What is the primary purpose of an electrochemical detector in HPLC?
What is the primary purpose of an electrochemical detector in HPLC?
Which type of detector in HPLC is suitable for detecting solutes in the range of 100-1000 ng based on changes in refractive index?
Which type of detector in HPLC is suitable for detecting solutes in the range of 100-1000 ng based on changes in refractive index?
At what concentration range can a UV/VIS detector operate effectively in HPLC for direct solute detection?
At what concentration range can a UV/VIS detector operate effectively in HPLC for direct solute detection?
What parameter is used to measure the adsorption energy per unit area of the solvent in chromatography?
What parameter is used to measure the adsorption energy per unit area of the solvent in chromatography?
In Thin-Layer Chromatography (TLC), why is it important for all solutes to be moderately soluble in the mobile phase?
In Thin-Layer Chromatography (TLC), why is it important for all solutes to be moderately soluble in the mobile phase?
What is the key factor that determines if a solvent has the correct strength for the mobile phase in Thin-Layer Chromatography?
What is the key factor that determines if a solvent has the correct strength for the mobile phase in Thin-Layer Chromatography?
What is the recommended pore size range for packing materials when the analyte has a molecular weight above 10,000 MW?
What is the recommended pore size range for packing materials when the analyte has a molecular weight above 10,000 MW?
What action should be taken if single solvents do not provide adequate resolution in Thin-Layer Chromatography?
What action should be taken if single solvents do not provide adequate resolution in Thin-Layer Chromatography?
In chromatography, what is the purpose of using larger particles (15-20 μm)?
In chromatography, what is the purpose of using larger particles (15-20 μm)?
What does the non-linear effect observed when mixing weak and strong solvents in chromatography highlight?
What does the non-linear effect observed when mixing weak and strong solvents in chromatography highlight?
What distinguishes gradient elution from isocratic elution in chromatography?
What distinguishes gradient elution from isocratic elution in chromatography?
When should a packing material with a pore size of 100 Ã… or less be used?
When should a packing material with a pore size of 100 Ã… or less be used?
What is the main advantage of using a 10 μm packing material in chromatography?
What is the main advantage of using a 10 μm packing material in chromatography?
What role does the molecular weight of an analyte play in choosing the appropriate pore size for packing material?
What role does the molecular weight of an analyte play in choosing the appropriate pore size for packing material?
What type of mobile phase is used in gas chromatography?
What type of mobile phase is used in gas chromatography?
In gas-liquid partition chromatography, what is the nature of the stationary phase?
In gas-liquid partition chromatography, what is the nature of the stationary phase?
What does the stationary phase in gas-solid adsorption chromatography directly interact with?
What does the stationary phase in gas-solid adsorption chromatography directly interact with?
What is the primary function of the carrier gas in gas chromatography?
What is the primary function of the carrier gas in gas chromatography?
What differentiates gas-liquid partition chromatography from gas-solid adsorption chromatography?
What differentiates gas-liquid partition chromatography from gas-solid adsorption chromatography?
What happens to the analyte in gas-solid adsorption chromatography?
What happens to the analyte in gas-solid adsorption chromatography?
In Size Exclusion Chromatography (SEC), how are molecules separated?
In Size Exclusion Chromatography (SEC), how are molecules separated?
What is another name for Size Exclusion Chromatography (SEC)?
What is another name for Size Exclusion Chromatography (SEC)?
Which type of chromatography involves small molecules entering the pores of particles?
Which type of chromatography involves small molecules entering the pores of particles?
What determines if a molecule will be attracted to an Anion-exchange resin in Ion-exchange Chromatography?
What determines if a molecule will be attracted to an Anion-exchange resin in Ion-exchange Chromatography?
What does Size Exclusion Chromatography (SEC) primarily aim to separate?
What does Size Exclusion Chromatography (SEC) primarily aim to separate?
Which method of chromatography involves mobile ions being held near cations covalently attached to the stationary phase?
Which method of chromatography involves mobile ions being held near cations covalently attached to the stationary phase?
What is the primary function of the stationary phase in size-exclusion chromatography?
What is the primary function of the stationary phase in size-exclusion chromatography?
What is the impact of having a larger particle size in a size-exclusion chromatography column?
What is the impact of having a larger particle size in a size-exclusion chromatography column?
What happens when particles with different pore sizes are mixed in a size-exclusion chromatography column?
What happens when particles with different pore sizes are mixed in a size-exclusion chromatography column?
What is the typical percentage of the total column volume occupied by the pores in size-exclusion chromatography?
What is the typical percentage of the total column volume occupied by the pores in size-exclusion chromatography?
How does the resolution of molecules in size-exclusion chromatography change as the particle size decreases?
How does the resolution of molecules in size-exclusion chromatography change as the particle size decreases?
Why does size-exclusion chromatography require long columns or multiple columns?
Why does size-exclusion chromatography require long columns or multiple columns?
What is the role of anion exchangers in ion-exchange resins?
What is the role of anion exchangers in ion-exchange resins?
In ion-exchange selectivity, the selectivity coefficient K is calculated using the concentrations of which ions?
In ion-exchange selectivity, the selectivity coefficient K is calculated using the concentrations of which ions?
What kind of ions do ion exchangers generally favor the binding of?
What kind of ions do ion exchangers generally favor the binding of?
What is the purpose of gradient elution in ion exchange processes?
What is the purpose of gradient elution in ion exchange processes?
What should be done to ensure the quality of the sample in size exclusion chromatography?
What should be done to ensure the quality of the sample in size exclusion chromatography?
What application is NOT typically associated with ion-exchange resins?
What application is NOT typically associated with ion-exchange resins?
How does increasing the concentration of anion C- impact the elution process in an ion exchange column?
How does increasing the concentration of anion C- impact the elution process in an ion exchange column?
How can a combination of packings with different separation ranges be achieved in size exclusion chromatography?
How can a combination of packings with different separation ranges be achieved in size exclusion chromatography?
What is crucial for the chemical nature of column packing in chromatography?
What is crucial for the chemical nature of column packing in chromatography?
In which type of chromatography is retention based on the attraction between solute ions and charged sites bound to the stationary phase?
In which type of chromatography is retention based on the attraction between solute ions and charged sites bound to the stationary phase?
What did Benoit show in 1967 regarding the calibration for polymers of different types?
What did Benoit show in 1967 regarding the calibration for polymers of different types?
What does the Universal Calibration Principle underestimate when analyzing polymers?
What does the Universal Calibration Principle underestimate when analyzing polymers?
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Study Notes
Module Overview
- Module CH3F2 consists of 24 lectures, 6 workshops, and a lab (33% of the module) and an exam (67% of the module)
- Module leader: Mark Barrow
Preparing for the Exam/Revision
- Lecture slides will be color-coded to indicate the importance of each topic:
- Green: Must-know topics (basic material)
- Yellow/Amber: Should-know topics (moderate material)
- Red: Useful but not necessary to know (advanced material)
Chromatography Overview
- Chromatography is a family of laboratory techniques used to separate, identify, and quantify the components of a mixture
- It operates on the principle of distribution of components between a mobile phase and a stationary phase over time
- Types of chromatography:
- Based on stationary phase support:
- Planar (thin layer chromatography, TLC)
- Column
- Based on the physical properties of the analyte:
- Volatility (gas chromatography, GC)
- Solubility (partition chromatography)
- Adsorptivity (adsorption chromatography)
- Charge (ion exchange chromatography)
- Size (size-exclusion chromatography)
- Based on the chromatographic principle:
- Adsorption
- Partition
- Ion exchange
- Permeation
- Based on stationary phase support:
Importance of Units and Significant Figures
- Units are essential to provide meaning to measurement results
- Significant figures and decimal places indicate the certainty of a measurement
- Errors and significant figures are important to understand the reliability of a measurement result
Precision and Accuracy
- Precision: how reproducible a measurement is
- Accuracy: how close to the true value a measurement is
- Errors can be classified as:
- Random (affecting precision)
- Systematic (affecting accuracy)
Chromatography Terminology
- Analyte: substance being separated
- Eluent: solvent used to move the analyte through the stationary phase
- Eluate: the mixture of analyte and solvent that emerges from the column
- Mobile phase: the phase that moves through the stationary phase
- Stationary phase: the phase that is held in place
- Chromatograph: the equipment used to perform chromatography
- Chromatogram: the detector signal vs. retention time or volume
Theoretical Plate Model
- Theoretical plate model: a concept used to measure column efficiency
- Plate height (H or HETP): the length of column required for each mobile phase/stationary phase equilibrium to be established
- Number of theoretical plates (N): the length of the column divided by the plate height
- Theoretical plate model limitations:
- Assumes infinitely fast equilibration
- Does not account for the dynamics of the separation
Rate Theory of Chromatography
- Proposed by van Deemter in 1956
- Accounts for the dynamics of the separation
- The Van Deemter equation for plate height (HETP) = A + B/u + Cu
- B term: longitudinal diffusion
- C term: resistance to mass transfer
- HETP: the smaller the better, indicating a more efficient column
Kinetic Theory of Chromatography
- Proposed by Giddings in 1965
- Widely used in the last 10 years
- Limitations of van Deemter's theory:
- No allowance for comparison between different chromatographic techniques
- No allowance for comparison between different types of columns
- No consideration of viscosity changes with pressure/temperature system changes
Thin-Layer Chromatography (TLC)
- Usually liquid/solid chromatography
- Stationary phase: alumina or silica gel coated onto a flat surface
- Mobile phase: solvents (usually fairly non-polar)
- Microscale, rapid (10-20 min)
- Not suitable for preparative work (10 mg max)
- Choice of mobile phase for silica chromatography:
- Vary polarity of mobile phase to increase or decrease time polar solutes spend on stationary phase
- Chromatographic strength of solvent: how fast it moves analytes
- Solvent strength parameter: εo = measure of adsorption energy / unit area of solvent### Thin-Layer Chromatography (TLC)
- Ethyl acetate is a good solvent for silica gel TLC
- To reduce polarity, add 5-10% hexane
- To increase polarity, add 5-10% methanol
- Adding a percentage of acetic acid or ammonia (or pyridine) can help maintain acidic or basic solutions and prevent tailing of zones
Two-Dimensional TLC
- Used for examining complex mixtures
- Plate is run in one direction, removed, dried, and then rotated 90° and run in another solvent
- The line of partially resolved components from the first run becomes the origin for the second development
Multiple TLC (Unidimensional Multiple Development)
- Increases resolution of components with Rf values below 0.5
- After a single run, the plate is removed, partially dried, and then placed again in the same solvent and run in the same direction
- Final Rf = 1 – (1 – Rf)n
Detection and Visualization
- Self-absorption in UV (254 nm)
- Fluorescence (UV excitation)
- Iodine vapor (1% alcoholic solution or iodine crystals in a jar)
- Charring reagents
- Geiger counter for radioactive materials
- Rhodamine B solution (50 mg/100 ml ethanol) for organic compounds
- Spraying with water for lipophilic solutes (e.g., steroids)
Column Chromatography
- Usually a preparative technique rather than analytical
- Silica or alumina used as stationary phase
- Particle diameter: 40-60 μm for silica, and 5 μm for alumina
Stationary Phase Pore Size
- Affects separation efficiency
- Choose pore size based on analyte molecular weight:
- <3,000 MW: 100 Ã… or less
- 3,000-10,000 MW: 100 Ã… - 130 Ã…
- >10,000 MW (peptides and proteins): 300 Ã…
Mobile Phase Solvents
- Normal phase: heptane, 1-chlorobutane, chloroform, methylene chloride, isopropyl ether, ethyl acetate
- Reverse phase: methanol, acetonitrile, acetone, ethanol, 2-propanol, THF
Mobile Phase Elution
- Isocratic: constant mobile phase composition
- Gradient: changing mobile phase composition during analysis
- Gradient elution: for mixtures with wide ranges of hydrophobicity
HPLC Detectors
- UV/VIS (0.1-1 ng)
- Fixed Variable (VWD)
- Photodiode array (DAD)
- FTIR (1mg)
- Refractive Index (100-1000 ng)
- Mass Spectrometry (1-1000 pg)
- Conductivity (500-1000 ng)
- ELSD (100-1000 pg)
- Electrochemical (10-1000 pg)
- Fluorescence (1-10 pg)
Gas Chromatography
- Gaseous analyte transported through the column by a gaseous mobile phase (carrier gas)
- Stationary phase: non-volatile liquid coated on the inside of the column or on a fine solid support
- Types: gas-liquid partition chromatography and gas-solid adsorption chromatography
Types of Chromatography
- Adsorption chromatography
- Partition chromatography
- Ion-exchange chromatography
- Size-exclusion chromatography
Size Exclusion Chromatography (SEC)
- Also called molecular exclusion chromatography, steric exclusion chromatography, gel filtration chromatography, or gel permeation chromatography
- Separation based on molecular size
- Stationary phase: cross-linked gels or porous silica
- Pore size determines separation range
- Columns: 7-8 mm diameter (analytical), 20-25 mm diameter (preparative)
Ion-Exchange Chromatography
- Retention based on attraction between solute ions and charged sites bound to the stationary phase
- Anion exchangers: positively charged groups on the SP attract solute anions
- Cation exchangers: negatively charged sites attract solute cations
- Ion-exchange resins: amorphous particles of organic material
Applications
- Converting a salt into another
- Pre-concentration of trace components of a solution
- Purification of water
- Separation of proteins
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