Week 3 - Small Molecule Chromatography Flashcards
Small Molecules
- compounds with Leo molecular weight (<1000Da)
- amino acids, lipids, sugars, fatty acids, alkaloids, and others
Separation
- similar to protein separation
- small molecules are such smaller as they range between 10-1000 Da rather than 1.5 kDa-450kDa for protein
How can good separation be achieved?
- achieving good separation between small molecule can often be more challanging
- therefore, advanced chromatography is required for good seperation
Examples of Seperation
liquid chromatography (LC)
gas chromatography (GC)
thin-layer chromatography (TCL)
capillary electrophoresis (CE)
Examples of Detection Techniques
nuclear magnetic resonance spectroscopy (NMR) - robust but high cost
uncommon UV/Vis spectroscopy - simplest
The order of molecule elution depends on
- Stationary phase(SP) -MAIN FACTOR
- Mobile phase (MP) composition
Reverse phase of SP
SP is normally hydrocarbons (e.g. C8, C18) attached to a silica- based backbone, therefore in hydrophobic nature.
Hydrophilic compounds will elute first, then hydrophobic compounds will elute last.
Normal phase of SP
SP is polar compounds (e.g. NH2, APS) attached, therefore in hydrophilic nature.
Hydrophobic compounds will elute first, and then hydrophilic compounds will elute last.
Hydrophilic Interaction LC (HILIC)
Similar to Normal phase, but aqueous layer is formed between polar stationary phase and organic mobile phase.
Which RP method is ideal for the separation of 3 cyclic compounds?
Method 1
– Shortest run, but no clear separation between Benzoic acid and Benzylamine.
Method 2
– Increase in organic composition. Good separation between all.
Method 3
– Increase in pH changed the elution order and run time.
Overall, method 2 because of good separations, yet it has a short run time.
Change in pH and Temp. also affects elution order
What is Gas Chromatography (GC) ideal for?
Ideal for the components of a volatile mixture.
What principle does Gas Chromatography (GC) operate on?
Operates on the principle of partitioning between a mobile phase (carrier gas) and a stationary phase (a liquid or solid coated onto the inside surface of a capillary column or packed in a column).
Gas Chromatography (GC)
High Sensitivity and Specificity
Wide Range of Applications
Compatibility with Various Detectors
High Resolution and Efficiency
Quantitative Analysis
Gas Chromatography (GC) - High Sensitivity and Specificity
GC can detect and quantify very low concentrations of chemical substances, making it ideal for trace analysis.
Gas Chromatography (GC) - Wide Range of Applications
It is used in various fields such as environmental analysis, pharmaceuticals, petrochemical industry, food and flavor analysis, forensic science, and more.
Gas Chromatography (GC) - Compatibility with Various Detectors
GC can be coupled with different types of detectors (e.g., Flame Ionization Detector [FID], Mass Spectrometer [MS], Electron Capture Detector [ECD]), enhancing its applicability to analyze a wide range of compounds.
Gas Chromatography (GC) - High Resolution and Efficiency
GC is capable of separating complex mixtures into individual components due to the high efficiency of the columns used.
Gas Chromatography (GC) - Quantitative Analysis
It provides precise and accurate quantitative information about the components
Gas Chromatography (GC) - Steps
- Sample Injection and Vaporization
- Separation
- Detection
- Data Analysis
Gas Chromatography (GC) - Sample Injection and Vaporization
A small volume of the liquid sample is injected into the injection port where it is vaporized to form a gas.
This gas is then carried into the column by an inert carrier gas (mobile phase), such as helium or nitrogen.
Gas Chromatography (GC) - Separation
The vaporized sample is transported through the column by the carrier gas.
The column contains a stationary phase, which can be a liquid or solid.
Components of the vaporized sample distribute themselves between the mobile phase and the stationary phase, based on their relative affinity for the stationary phase.
Components with less affinity for the stationary phase travel faster and elute (exit) the column earlier than those with greater affinity.
Gas Chromatography (GC) - Detection
As each component exits the column, it passes through a detector specific to the type of analysis being conducted.
The detector generates a signal proportional to the amount of the component present, producing a peak on a chromatogram.
Gas Chromatography (GC) - Data Analysis
The chromatogram displays the separation of the components as distinct peaks.
Each peak’s retention time (the time taken for a component to pass through the column) helps in identifying the component, while the peak area or height can be used to quantify the concentration
What is a compound formed during coffee roasting?
Furan
Why is Furan in the watchlist?
- These are on the watchlist due to their potential toxicity effect on the body.
- GC-MS is a popular method to detect and quantify furan and its derivates in food.
Supercritical Fluid Chromatography (SFC)
Principle of SFC
- SF is a substance above critical point of T and P
- Effuse through solids like a gas and dissolve materials like a liquid
What is a SFC a hybrid of?
SFC is a hybrid of gas and liquid chromatography that combines some of the best features of each
How is density controlled in SFC?
Density is controlled by controlling system pressure
In SFC, mobile phase affinity for…?
… for the analyte is a function of mobile phase density
What variation of the mobile phase composition in HPLC affect?
seperation
In SFC when close to the critical point, what changes results into changes in density?
Close to the critical point, small changes in T or P result in large changes in density
What type of samples is SFC useful for?
Useful for highly polar samples that are not easy to handle (high critical parameters & high reactivity)
SFC Stationary Phases
Same as those for GC and LC, with some modifications.
Silica/Alumina
Bonded to provide less adsorptive packing material
Widely used polar S Phase
Silica/Alumina
→ Useful for non-polar compounds
→ Leads to irreversible adsorption of some polar solutes
Bonded to provide less adsorptive packing material
- Octyl, octadecyl, cyanoalkyl, aminoalkyl, diolakyl
- Need organic modifiers to elute analytes
Widely used polar S Phase
- Polysiloxanes– stable, flexible Si–O bond lead to good diffusion.
- Substituted with chemical groups for selective interaction with analyte
- Polymethylsiloxanes–increase efficiency in separating closely related polar analytes
- Cyanopropyl polysiloxanes-useful for compounds with –COOH
SFC Steps
- Mobile phase (CO2)
- Pump
- Injection System
- Oven
- Column
- Detector
- Chromatogram/Computer
SFC Advantages
Advantage 1: High penetration
- With higher penetration ability than water, it can easily pass through coffee beans
- Supercritical carbon dioxide can penetrate the interior of coffee beans without having to grind the beans to increase their surface area.
Advantage 2: High solubility.
- Substances that contact supercritical carbon dioxide are quickly dissolved and extracted from the coffee bean.
Advantage 3: Evaporation at ambient temperatures/pressures.
- CO2 residues are unlikely to remain in coffee beans.
Advantage 4: Low toxicity.
- Even if residues remained in coffee beans, they would not be toxic.
- In this way, the properties of supercritical carbon dioxide are used beneficially for manufacturing decaffeinated coffee.
Application in Chemical Synthesis
Purification of Products
After a chemical synthesis reaction, the reaction mixture often contains the desired product and unreacted starting materials, by-products, and catalysts.
Chromatography is used to separate and purify the desired product. For instance, column chromatography, including flash chromatography, is a common technique for purifying small organic molecules.
The choice of stationary phase (e.g., silica gel) and mobile phase (solvent system) depends on the properties of the compounds in the mixture.
Application in Chemical Synthesis
Monitoring Reaction Progress
Analytical chromatography techniques, such as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), are used to monitor the progress of a reaction.
By comparing the chromatograms of samples taken at different times during the reaction, chemists can determine when the reactants have been consumed and the reaction is complete.
Application in Chemical Synthesis
Characterization of Compounds
After purification, it is crucial to confirm the structure and purity of the synthesized compounds.
Techniques like HPLC coupled with Mass Spectrometry (HPLC-MS) or Gas Chromatography-Mass Spectrometry (GC-MS) provide detailed information about the molecular weight and structure of the compounds.
This information is vital for verifying that the correct product has been synthesized.
Why do we need a degassing unit?
liquids have lots of air bubbles which are not viable to the eye
What determines how fast a molecule travels?
polarity and hydrophobic interactions
When the stationary phase is hydrophobic, what happens if the molecules are more polar?
less interaction between compounds and stationary phase hence will travel faster
When the stationary phase is hydrophobic, what happens if the molecules are more hydrophobic?
more interaction
will stick to columns and harder to travel
= takes longer
What is reverse phase?
stationary phase is hydrophobic
no polar compounds
What gas is used in gas chromatography?
nitrogen and CO2
What is the stationary phase in gas chromatography?
silicone-based
hence hydrophobic
Gas Chromatography - Basics
- sample is evaporated and liquid is turned into gas
- stationary phase is hydrophobic
- seperation occurs
- detector
Why is CO2 used?
low critical temperature and pressure
Polyphenols
antioxidant
found in beer
How does small molecule detection - MS do to create a charge?
apply lots of electrical current to create charge
What does preselection in small molecule detection allow?
allows increase in sensitivity of particular compound
Triple quadrupole
expensive as they are highly specific/sensitive/accurate
What happens in time of fly?
iron goes into system and flies around
it measures how long one molecule takes to travel from one place to another
Orbit trap
small and uses a lot of electricity
What is measured in orbit trap?
molecule occeslation - frequency moves around which differentiates different molecule
is orbit trap of time of flight faster?
time of flight is faster
What does GS441 do?
inhibit RNA amplification of RNA or virus
Hormone Testing - Derivatisation
twist and change a bit so compound becomes more volitie
= easily turn gas to liquid and is ideal for Mass Spec
