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Analytical and Clinical > Week 3 - Small Molecule Chromatography > Flashcards

Week 3 - Small Molecule Chromatography Flashcards

1
Q

Small Molecules

A
  • compounds with Leo molecular weight (<1000Da)
  • amino acids, lipids, sugars, fatty acids, alkaloids, and others
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2
Q

Separation

A
  • similar to protein separation
  • small molecules are such smaller as they range between 10-1000 Da rather than 1.5 kDa-450kDa for protein
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3
Q

How can good separation be achieved?

A
  • achieving good separation between small molecule can often be more challanging
  • therefore, advanced chromatography is required for good seperation
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4
Q

Examples of Seperation

A

liquid chromatography (LC)

gas chromatography (GC)

thin-layer chromatography (TCL)

capillary electrophoresis (CE)

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5
Q

Examples of Detection Techniques

A

nuclear magnetic resonance spectroscopy (NMR) - robust but high cost

uncommon UV/Vis spectroscopy - simplest

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6
Q

The order of molecule elution depends on

A
  1. Stationary phase(SP) -MAIN FACTOR
  2. Mobile phase (MP) composition
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7
Q

Reverse phase of SP

A

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.

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8
Q

Normal phase of SP

A

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.

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9
Q

Hydrophilic Interaction LC (HILIC)

A

Similar to Normal phase, but aqueous layer is formed between polar stationary phase and organic mobile phase.

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10
Q

Which RP method is ideal for the separation of 3 cyclic compounds?

A

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

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11
Q

What is Gas Chromatography (GC) ideal for?

A

Ideal for the components of a volatile mixture.

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12
Q

What principle does Gas Chromatography (GC) operate on?

A

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).

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13
Q

Gas Chromatography (GC)

A

High Sensitivity and Specificity

Wide Range of Applications

Compatibility with Various Detectors

High Resolution and Efficiency

Quantitative Analysis

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14
Q

Gas Chromatography (GC) - High Sensitivity and Specificity

A

GC can detect and quantify very low concentrations of chemical substances, making it ideal for trace analysis.

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15
Q

Gas Chromatography (GC) - Wide Range of Applications

A

It is used in various fields such as environmental analysis, pharmaceuticals, petrochemical industry, food and flavor analysis, forensic science, and more.

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16
Q

Gas Chromatography (GC) - Compatibility with Various Detectors

A

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.

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17
Q

Gas Chromatography (GC) - High Resolution and Efficiency

A

GC is capable of separating complex mixtures into individual components due to the high efficiency of the columns used.

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18
Q

Gas Chromatography (GC) - Quantitative Analysis

A

It provides precise and accurate quantitative information about the components

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19
Q

Gas Chromatography (GC) - Steps

A
  1. Sample Injection and Vaporization
  2. Separation
  3. Detection
  4. Data Analysis
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20
Q

Gas Chromatography (GC) - Sample Injection and Vaporization

A

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.

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21
Q

Gas Chromatography (GC) - Separation

A

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.

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22
Q

Gas Chromatography (GC) - Detection

A

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.

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23
Q

Gas Chromatography (GC) - Data Analysis

A

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

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24
Q

What is a compound formed during coffee roasting?

A

Furan

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25
Q

Why is Furan in the watchlist?

A
  • 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.
26
Q

Supercritical Fluid Chromatography (SFC)
Principle of SFC

A
  • SF is a substance above critical point of T and P
  • Effuse through solids like a gas and dissolve materials like a liquid
27
Q

What is a SFC a hybrid of?

A

SFC is a hybrid of gas and liquid chromatography that combines some of the best features of each

28
Q

How is density controlled in SFC?

A

Density is controlled by controlling system pressure

29
Q

In SFC, mobile phase affinity for…?

A

… for the analyte is a function of mobile phase density

30
Q

What variation of the mobile phase composition in HPLC affect?

A

seperation

31
Q

In SFC when close to the critical point, what changes results into changes in density?

A

Close to the critical point, small changes in T or P result in large changes in density

32
Q

What type of samples is SFC useful for?

A

Useful for highly polar samples that are not easy to handle (high critical parameters & high reactivity)

33
Q

SFC Stationary Phases

A

Same as those for GC and LC, with some modifications.

Silica/Alumina

Bonded to provide less adsorptive packing material

Widely used polar S Phase

34
Q

Silica/Alumina

A

→ Useful for non-polar compounds

→ Leads to irreversible adsorption of some polar solutes

35
Q

Bonded to provide less adsorptive packing material

A
  • Octyl, octadecyl, cyanoalkyl, aminoalkyl, diolakyl
  • Need organic modifiers to elute analytes
36
Q

Widely used polar S Phase

A
  • 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
37
Q

SFC Steps

A
  1. Mobile phase (CO2)
  2. Pump
  3. Injection System
  4. Oven
  5. Column
  6. Detector
  7. Chromatogram/Computer
38
Q

SFC Advantages

A

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.

39
Q

Application in Chemical Synthesis

Purification of Products

A

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.

40
Q

Application in Chemical Synthesis
Monitoring Reaction Progress

A

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.

41
Q

Application in Chemical Synthesis
Characterization of Compounds

A

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.

42
Q

Why do we need a degassing unit?

A

liquids have lots of air bubbles which are not viable to the eye

43
Q

What determines how fast a molecule travels?

A

polarity and hydrophobic interactions

44
Q

When the stationary phase is hydrophobic, what happens if the molecules are more polar?

A

less interaction between compounds and stationary phase hence will travel faster

45
Q

When the stationary phase is hydrophobic, what happens if the molecules are more hydrophobic?

A

more interaction

will stick to columns and harder to travel
= takes longer

46
Q

What is reverse phase?

A

stationary phase is hydrophobic

no polar compounds

47
Q

What gas is used in gas chromatography?

A

nitrogen and CO2

48
Q

What is the stationary phase in gas chromatography?

A

silicone-based
hence hydrophobic

49
Q

Gas Chromatography - Basics

A
  • sample is evaporated and liquid is turned into gas
  • stationary phase is hydrophobic
  • seperation occurs
  • detector
50
Q

Why is CO2 used?

A

low critical temperature and pressure

51
Q

Polyphenols

A

antioxidant

found in beer

52
Q

How does small molecule detection - MS do to create a charge?

A

apply lots of electrical current to create charge

53
Q

What does preselection in small molecule detection allow?

A

allows increase in sensitivity of particular compound

54
Q

Triple quadrupole

A

expensive as they are highly specific/sensitive/accurate

55
Q

What happens in time of fly?

A

iron goes into system and flies around

it measures how long one molecule takes to travel from one place to another

56
Q

Orbit trap

A

small and uses a lot of electricity

57
Q

What is measured in orbit trap?

A

molecule occeslation - frequency moves around which differentiates different molecule

58
Q

is orbit trap of time of flight faster?

A

time of flight is faster

59
Q

What does GS441 do?

A

inhibit RNA amplification of RNA or virus

60
Q

Hormone Testing - Derivatisation

A

twist and change a bit so compound becomes more volitie
= easily turn gas to liquid and is ideal for Mass Spec

Analytical and Clinical (11 decks)

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