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FOOD30008 > (9) Principles of Chromatography > Flashcards

(9) Principles of Chromatography Flashcards

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1
Q

principles of chromatography

A
  1. involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between 2 different phases
  2. one phase is the mobile phase the other is the stationary phase
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2
Q

Distribution coefficient

A
  • ratio of how much retained in solid phase to how much migrated with the mobile phase
  • different affinity of components to stationary phase causes the separation
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3
Q

Types of chromatography system

A
  1. Thin layer chromatography (TLC)
  2. Low pressure liquid chromatography
  3. High pressure chromatography
  4. Gas chromatography
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4
Q

Mechanism of TLC

A
  • consists of a stationary phase (silica gel) which thinly coats a glass or plastic plate and a solvent (mobile)
  • the sample (either liquid or dissolved in a volatile solvent) is deposited as a spot on the stationary phase
  • one edge of the plate is then placed in a solvent reservoir and the solvent moves up the plate via capillary action
  • when the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir
  • the separated spots are visualized with UV light or by placing the plate in iodine vapour
  • the different components in the mixture move up the plate at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase
  • the constituents of a sample can be identified by simultaneously running standards with the unknown
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5
Q

How to quantitate TLC?

A

retention factor

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6
Q

Mechanism of LC

A
  • useful for separating ions/molecules dissolved in a solvent
  • Components within a mixture are separated in a column based on each component’s affinity for the mobile phase; varies due to difference in adsorption, ion exchange partitioning or size
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7
Q

Describe components of an LC system

A
  • column with fritted bottom that holds a stationary phase in equilibrium with a solvent

LC stationary phases and their interaction with solutes:

  1. Solids (Adsorption)
  2. Liquids on an inert solid support (partitioning)
  3. ionic groups on a resin (ion-exchange)
  4. Porous inert particles (Size exclusion)
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8
Q

LC procedure

A
  • mixture is loaded onto the top of the column followed by more solvent
  • different components in the mixture pass through the column at different rates due to the difference in their partitioning behaviour between the mobile liquid phase and the stationary phase
  • the compounds are separated by collecting aliquots of the column effluents as a function of time
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9
Q

Five types of liquid chromatography

A
  1. liquid/solid chromatography (adsorption)
    - normal phase (m: hydrophobic; s: hydrophilic)
    - reverse phase (m: hydrophilic; s: hydrophobic)

*Separation is based on the competition of the sample for the active sites on an absorbent such as silica gel (eg. hydrophillic solutes will adhere more to a hydrophilic stationary phase so hydrophobic components will be eluted quicker)

  1. Liquid/liquid chromatography (partition)
    - normal and reverse phase
  • The stationary solid surface is coated with a 2nd liquid (stationary) which is immiscible in the solvent (mobile) phase
  • Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation
  1. Size exclusion chromatography
    - Mechanical sorting of molecules based on the size of the molecules in solution
    - small molecules are able to permeate more pores and are therefore retained longer than large molecules
  • smaller molecules can enter more volume of the stationary phase and hence travel through the column slowly (get into small spaces) . Larger molecules will be swept away in the mobile phase, therefore having a smaller retention time.
  • The stationary phase is a gel that consists of spherical beads which contain pores of a specific size distribution. Consequently, molecules are eluted in order of decreasing MW.
  1. ion exchange chromatography
    - involves the separation of ionizable molecules based on their total charge.
  • This enables the separation of similar types of molecules that would be difficult to separate by other techniques because the charge carried by the molecule of interest can be readily manipulated by changing buffer pH.
  • The stationary phase is either an anion exchange resin or a cation exchange resin, depending on which ion is intended to be captured (if ion has a net positive charge, a negatively charge cation exchange resin is chosen and vice versa).
  1. separation by hydrophobicity
    - protein often contain a variable surface patches of hydrophobicity; changing the concentration of salt can help molecules interact with each other (by removing the thin layer of water around it so both hydrophobic molecules can interact with each other)
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10
Q

LC application

A
  1. conventional LC used in preparative scale work to purify and isolate some components of a mixture
  2. in ultra trace separation where small disposable columns are used once and discarded eg pesticides
  3. Analytical separation of solutions for detection or quantification typically use HPLC
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11
Q

What is supercritical fluid chromatography?

A
  • A supercritical fluid is one that is above its critical pressure and temperature
  • can be formed from a conventional gas by increasing pressure or conventional liquid by raising temperature
  • SFC can be performed using packed column or capillaries
  • SFC has been used for non-polar compounds (Fats and oils)
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12
Q

Components of HPLC

A

HPLC separates compounds that are dissolved in solution

  1. reservoir for mobile phase
  2. Pump
  3. injector
  4. separation column (guard and analytical)
  5. detectors which rely on a change in:
    - refractive index (RI)
    - UV-Vis absorption OR
    - fluorescence (after excitation with a suitable wavelength)
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13
Q

HPLC mechanism

A
  • Compounds are separated by injecting a small volume of the sample mixture onto the column
  • the different components in the mixture passes through the column at different rates due to difference in their partitioning behaviour between the mobile liquid phase and the stationary phase
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14
Q

Methods to improve HPLC performance

A
  1. Setup
    - Select proper stationary and mobile phase (eg hydrophobic/philic)
  2. Column
    - Increase column length
    decrease column diameter (longer and smaller column provides better resolution)
    - Uniformly pack column
  3. Operational
    - decreased flow rate
    - proper pressure used
    - gradient elution used (use 2/3 different mobile phases in different proportions several times)
    - decrease sample size
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15
Q

Principles of GC

A
  • the partitioning of molecules between the gas (mobile phase) and the liquid (stationary phase)
  • suited for the analysis of thermally stable volatile substances (vaporize without decomposition)
  • it requires sample prep before injection
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16
Q

How to increase volatility before GC analysis

A

Triacylglycerols and phospholipids are typically saponified (to fatty acid + alcohol) and fatty acids are esterified to form fatty acid methyl ester (FAME)

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17
Q

GC Method

A
  • Involves a sample being vapourised and injected onto the head of the chromatographic column
  • the injection port is maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture (to ensure all sample changed to gas; if not condensation will take place and give false result); increase temperature over time to elute the high BP components
  • the sample is transported through the column by the flow of inert gaseous mobile phase (generally inert gas such as helium, argon or nitrogen)
  • the column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid
  • less volatile molecules interact with the stationary phase and move slowly while the more volatile molecules move with the mobile phase and move fast down the column to the detector
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18
Q

Types of GC columns

A

Packed (not really used anymore

  • filled with stationary phase
  • for concentrated samples

Capillary (longer and smaller d the column has, the better the separation)

  • stationary phase coated on the inner surface
  • for analytical work
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19
Q

Types of detectors: GLC

A

Vary in terms of sensitivity and cost
1. Thermal conductivity

Measures the changes of thermal conductivity due to the sample
Samples can be recovered
- sensitivity: µg

  1. Flame ionisation detector
    - has 3 inlets: carrier gas from column, hydrogen and oxygen gas
    - the ignitor ignites hydrogen and oxygen to produce a flame; when sample molecules reaches the flame, it gets ionised and electrons are released
    - across the flame there are 2 electrodes, each with a positive and negative charge. The electrodes detects the electrons in the form of current which is amplified and detected by software which plots a peak with respect to retention time (current vs RT)
    - area under the peak gives info about the concentration of the sample; if conc is less, area is small
  • sensitivity: ng
  1. Electron capture
    sensitivity: pg
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20
Q

Characteristic of a detector

A
  1. Sensitivity
  2. Stability
  3. reasonable life-time
  4. A linear response characteristics over a range of sample concentration
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21
Q

Sample derivatization and their issues

A

sample derivative is a non-volatile compound made volatile (derivation of glucose with trimethylchlorosilane (TMS))

Issues

  • Time consuming
  • side reactions
  • loss of samples
22
Q

Features of GLC

A
  • often good separation
  • short analysis time
  • small sample needed- µl
  • good detection system
  • quantitatively analysed

> material has to be volatized (bc separation based on BP)
material has to be volatized at 250C without decomposition
destruction of samples with FID

23
Q

Discuss qualitative analysis for chromatography

A
  1. Comparison of retention time of unknown and standards
  2. the collection of each peak as it emerges from the detector and subsequent characterisation using chemical and instrumental tests (eg direct the effluent into an infrared or mass spec)
24
Q

Discuss retention time

A

Time required for the sample to travel from the injection port through the column into the detector

Tentative identification of unknowns by retention time
- test for similar retention time compared to standard compounds

25
Q

Discuss normalisation

A

The percent composition is determined by measuring the area of each peak and dividing the individual areas by the total area

26
Q

Discuss absolute calibration

A
  • calibration curve of either peak area/peak height vs concentration of the sample is prepared
  • unknown is then chromatographed, its peak and height determined and the concentration of the unknown is obtained from the calibration curve
27
Q

What are the fundamental concepts of GC?

A
  1. Distribution value = distribution coefficient
    - different affinity of components to stationary phase causes the separation
    - sample components that partition strongly to the stationary phase spend a greater amount of time in the column
  2. Resolution
  3. Selectivity
  4. Column efficiency (Plates)
28
Q

Discuss resolution of GC

A

resolution is a measure of the separation of two peaks of different retention time t in a chromatogram

  • the resolution is affected by:
    1. compactness of peaks and distance between peak centres
    2. distribution coefficient, selectivity and the number of theoretical plates
  • If the distribution coefficient is small, then the selectivity is also small
29
Q

Discuss selectivity of GC

A

The measure of the preference a stationary phase reveals towards one solute over another and is expressed as a ratio

30
Q

Discuss column efficiency (plates)

A

Efficiency in chromatography can be expressed quantitatively by the number of theoretical plates, N
or the height equivalent to a theoretical plate, H
H = L/N
L= column length

  • larger N, better the resolution/separation of different fatty acids
  • small H is GOOD
31
Q

Discuss plate height

A

A measure of column efficiency

small plate height = narrow peaks = better separation

32
Q

How to maximize resolution

A
  1. Increasing plate
    - an increase in plate number by lengthening the column leads to an increase in retention time and increase in band broadening (may not be desirable)
    Instead, to increase N, the height equivalent can be reduced by reducing the size of the stationary phase particles
2. Selectivity and retention factor
to improve separation, change
- mobile phase composition
- column temperature
- composition of stationary phase
  1. Optimising flow rate
    the flow rate of the mobile phase can also affect analyte resolution, by affecting the plate height
    - higher flow rate reduces retention time but must play around with flow rate to ensure the base/width of the peak is still acceptable
33
Q

Discuss capillary electrophoresis

A

CE is used for complex hydrophilic macromolecules and highly charged solutes
-implement separation by analysing mass:charge ratio

It is a hybrid of column chromatography and electrophoresis
protein applications includes:
- capillary isoelectric focusing
- SDS electrophoresis

ADV: higher resolution compared to standard chromatography

DISADV: Generally limited to analytical work (not for purification and not as widely applicable as HPLC)

34
Q

Mech. of CE

A

The capillary interior shows molecules of different size and charge separating

  • free solution capillary electrophoresis
  • the positive molecules migrate towards the negative electrode while the electro osmotic flow carries the neutral and and negatively charged molecules in the same direction
  • narrow bore fused silica capillaries to separate a complex array of large and small molecules
  • high voltages are used to separate molecules based on differences in size, charge and hydrophobicity
  • injection into the capillary is accomplished by immersing the end of the capillary into a sample vial and applying pressure, vacuum or voltage
  • depending on the types of capillary and buffers, CE can be segmented into several separation techniques

** molecules are separated based on their mass:charge ratio; will all move in the same direction due to capillary action

35
Q

Discuss capillary zone electrophoresis

A
  • free solution CE
  • simplest form of CE
  • separation based on differences in the charge to mass ratio of the analytes
  • fundamental to CZE are homogeneity of the buffer solution and constant field strength through the length of the capillary
  • the separation relies principally on the pH- controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute
36
Q

TLC

A

*useful for separating organic compounds
simple and rapid method to monitor the extent of a reaction/check the purity of organic compounds
- mobile phase: solvent
- stationary phase: solid adsorbent on a flat support (silica gel/paper)

37
Q

LC

A

used in separating ions or molecules that are dissolved in solvent
stationary phase: solid/ion exchange resin

38
Q

HPLC

A
  • a form of liquid chromatography that separates compounds that are dissolved in solution
  • variation of LPLC that utilises high pressure pumps to increase the efficiency of separation

mobile phases:
1. Polar/hydrophilic solvents:
Water>methanol>acetonitrile> Ethanol> Oxydipropionitrile

  1. Non-Polar/hydrophobic solvents:
    N-Decane> N-Hexane> N-Pentane> Cyclohexane
39
Q

Main differences between HPLC and GC

A
  • mobile phase (liquid VS gas)

- hplc at room temperature, gc at high temp

40
Q

Supercritical fluid chromatography (SFC)

A
  • at ultra high pressure

supercritical fluid is one that is above both it’s critical pressure and temperature

41
Q

GC

A

applied to volatile organic compounds

  • mobile: gas
  • stationary: usually liquid on a solid support/ sometimes a solid adsorbent
42
Q

what is an adsorbent?

A

porous solids which bind liquid or gaseous molecules to their surface

43
Q

LC equipment: analytical and guard column

A

Analytical is longer
Guard protects analytical from being contaminated with sample; traps/seals any contaminant in solvent before going into A
- guard column significantly cheaper, extends the shelf life of the analytical column

44
Q

Difference between HPLC and LC

A

HPLC uses a pump to increase pressure, and increase the rate in which solvent travels through the column, reducing the time of separation

45
Q

temperature and separation for GC

A
  • partitioning behavior is dependent on temperature so, the separation column is usually contained in a thermostat controlled oven
  • separating components with a wide range of boiling point is accomplished by starting at a low oven temperature and increasing the temperature over time to elute the high-boiling point components (Temp gradient)
46
Q

GC detectors: advantage of thermal conductivity

A
  • can collect the solute and subject to other analyses; in FID, sample will be subjected to very high temperatures and disintegrate, cant collect for further analysis (sample will be hydrolysed and broken up)
47
Q

can we use GC to analyse carbs?

A

Yes, but need to apply special derivation bc with GC analysis, key factor to ensure that the solute analysed is heat resistant

48
Q

the theoretical plate model

A

N can be defined as the length of column in which the solute undergoes one complete equilibration between the two phases

  • the plate model supposes that the chromatographic column contains a large number of separate layers called theoretical plates
  • separate equilibrations of the sample between the stationary and mobile phase occur in these plates
  • the analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next
  • plates are used to measure column efficiency by:
    > measuring the number of theoretical plates in a column, N
    > stating the plate height (height equivalent to a theoretical plate (smaller better)) –> sharper peaks
49
Q

how to improve column quality in order to improve peak resolution and separation?

A

based on equation h=l/n, can increase L and consequently separate peaks from each other BUT base of peak becomes very long and wide which is not good in terms of chromatography separation
- better alternative is to increase N without changing L by changing type of resin used (change size of particles in resin/use diff column with smaller resin)
> smaller the size of the particle, the larger the SA so larger the number of theoretical plates

50
Q

maillard reaction

A
  • when reducing sugars (glucose, fructose, lactose) and amino compounds (free AA, peptides or proteins) are present together in a system (eg food), browning/maillard reactions may occur
  • not catalysed by enzymes; affected by temp, concentration of reactants, pH or preservatives
  • interaction of amino compound with monosaccharides initially involves addition of a carbonyl group to a primary amino group of an AA, peptide or protein followed by water elimination
  • leads to intermediary imine which cyclizes to a glycosylamine
51
Q

GC: peaks overlapping

A
  • this issue is related to very bad resolution which is directly linked to low number of theoretical plates
  • the larger the n value, the larger the number of theoretical plates and hence the better the separation

Can be solved in different ways:

  1. try to change the mobile phase, flow rate, temperature
  2. if none successful, look into how we can increase n
  3. based on equation can increase L (longer column) but the disadvantage we will have is a very wide base peak (less compact and sharp; we want sharp and narrow peaks)
  4. so we can increase n by decreasing height of column; choose smaller resin particle size (larger SA)
52
Q

Internal vs external standard

A
  1. Internal:
    - An internal standard in chromatography is a chemical substance that is added in a constant amount to samples.
    - This substance can be used for calibration by plotting the ratio of the preak area of sample to standard as a function of the analyte standard concentration. This is done to correct analyte losses during sample preparation.
  2. External:
    An external standard is like the internal standard (known behaviour), but is not added to the unknown. Rather it is run alone, as a sample, and usually at different concentrations, so you can generate a standard curve. Can also establish retention times and peak area for each peak to be compared with those of the sample.

FOOD30008 (9 decks)

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