Ch 12 : Basic principles of chromatography Flashcards
What is meant by extraction?
Transfer of a solute from one liquid phase to another.
FYI for UROP
How does batch extraction work?
A solvent containing a solute is mixed with a second immiscible solvent, and the mixture is shaken. The solution is left to stand and come to an equilibrium, and layer containing solute is removed.
FYI for UROP
What is the equation for partition coefficient?
K = concentration of solute in phase 1 / concentration of solute in phase 2
FYI for UROP
When is continous extraction carried out and how does it work?
Continuous extraction is carried out when organic substances dissolve more in water than in organic solvents or in a solid phase and are less soluble in organic solvents.
- organic solvent extracts lipophilic compounds when run through solids / aqueous mixture, and organic solvent is recycled to recover more lipophilic compounds with less solvent
FYI for UROP
How does countercurrent extraction work?
2 or more solutes with different partition coefficients are separated from each other by a series of partitions between 2 immiscible liquid phases. There is a stationary phase which is fixed, and mobile phase which moves along the stationary phase.
FYI for UROP
The ____ the partition coefficients, the better the separation.
Bigger
Chromatography
What is the definition of chromatography?
A general term applied to a wide variety of separation techniques based on partitioning / distribution of solute sample between mobile phase and stationary phase
Chromatography
What is the idea behind chromatography?
Chromatograpy separates different components/compounds in a sample based on their polarity
Chromatography
What state is the mobile phase usually in?
Liquid / gas
Chromatography
What state is the stationary phase usually in?
Solid / liquid
Liquid Chromatography
What state is the stationary and mobile phase in paper chromatography?
Stationary phase : water (supported with cellulose paper, trapped between cellulose fibres of paper and doesnt move along w sample)
Mobile phase : liquid
Liquid Chromatography
Briefly explain how paper chromatography works.
- Dissolved sample applied as a asmall spot at origin line (1.5cm away from strip)
- Strip is allowed to dry
- Strip is suspended in a close container / chamber
- Mobile phase (developing agent) moves up the paper strip, allowing chromatogram to be developed
- After developing agent travelled the length of the paper, strip is removed and separated zones are detected
Liquid Chromatography
What are components in the sample charactised by in paper chromatography / thin layer chromatography?
Their relative mobility in solvent (Rf values)
Liquid Chromatography
What does Rf value stand for? What is the equation?
Retention factor. It is the distance moved by component / distance moved by solvent
Liquid Chromatography
The procedure between thin layer chromatography and paper chromatography are similar. What is the main difference between these 2 methods?
The stationary phase.
- In paper chromatography, stationary phase is water entrapped between cellulose fibres of paper
- In thin layer chromatography, silicia gel / aluminium oxide is coated on glass plates
Liquid Chromatography
What is the advantage of thin layer chromatography over paper chromatography?
Silica gel has many polar –OH groups, and particulates are way smaller. This provides higher resolution (better separation of components in sample)
Liquid Chromatography
In thin-layer chromatography, what kind of compounds are eluted first?
Non-polar compounds, as polar compounds are strongly adsorbed to the –OH groups on the statinary phase
Adsoprtion Chromatography
What kind of chromatography is adsorption? (state of stationary and mobile phase)
What kinds of compounds does adsorption separate?
solid-liquid chromatography
Adsorption is mainly used for fat-soluble vitamins / aromatic / aliphatic **non-polar **compounds
- TLDR : non-polar, lipophilic compounds
Adsoprtion Chromatography
What is the idea behind adsoprtion chromatography?
Solvent (liquid) and solute (sample which is dissolved in solvent) molecules are competing for active site in the stationary phase.
To elute solute, a solvent with suitable solvent strength (higher strength than solute) must be chosen, so that the solvent molecules bind to the stationary phase while solute elutes out
Adsoprtion Chromatography
Adsorption coefficient always remains constant. True or False?
Adsorption coefficient – a measure of how quickly surfactant molecules are adsorbed at a surface
False. adsorption depends on concentration. The higher the concentration and the more solutes are added, additional solutes may take more time to bound to adsorption sites.
Adsoprtion Chromatography
In adsorption chromatography, what kind of stationary phase is used?
Polar (solid)
Partition Chromatography
What is the state of stationary phase and mobile phase in partition chromatography?
Partition chromatography : liquid-liquid
- Stationary phase is a liquid that is being held by inert solid material
- Mobile phase is an immiscible solvent (opposite polarity from stationaryphase)
Partition Chromatography
What is the idea behind partition chromatography?
As the mobile phase flows through the statonary phase, there is close contact between 2 phases. thus, the solutes will partition between 2 liquid phases according to their partition coefficient
Partition Chromatography
What is the polarity of the stationary and mobile phase in:
(i) Normal phase systems
(ii) Reverse phase systems
(i) Normal phase systems
- Stationary phase : polar
- Mobile phase : non-polar
- Thus normal phase systems separate polar hydrophilic substances by retaining them in the column
(ii) Reverse phase systems
- Stationary phase : non-polar
- Mobile phase : polar
- Thus reverse phase systems separate non-polar lipophilic substances by retaining them in the column
Partition Chromatography
Supposing you did a normal phase partition chromatography, where you wanted to separate and identify polar compounds by retaining them in a column. How can you elute the polar compounds out if you want to remove them from the column?
- Choose a Mobile Phase, where solvent / solvent mixture is more polar than the stationary phase.
- Prepare a Gradient: If the compounds have different polarities or if you want to elute them at different times, you can prepare a gradient elution. This involves starting with a less polar mobile phase and gradually increasing the polarity over time.
Hydrophobic Interaction chromatography
What is the idea behind hydrophobic interaction chromatography?
Hydrophobic portions of large biomolecules such as proteins from hydrophobic interactions with ligands binded to the stationary phase, thus allowing separation of proteins.
Hydrophobic Interaction chromatography
What is the stationary phase in Hydrophobic Interaction chromatography?
It involves hydrophobic ligands binded to a hydrophilic support (solid), such as cellulose, chitosan etc
Hydrophobic Interaction chromatography
Why can’t the solid support material that is used to hold the hydrophobic ligands be hydrophobic too? Why must it be hydrophilic?
This may cause the hydrophobic parts of proteins to strongly bind to the support material too, causing denaturation
Hydrophobic Interaction chromatography
What other characteristics must the support material possess?
polymer that has high degree of crosslinking to provide rigidity and high surface area
Hydrophobic Interaction chromatography
what kind of susbtances are the hydrophobic ligands usually?
Linear chain alkane that is chemically bonded to the support material (but other groups with aromatic groups can be used too)
Ion-exchange chromatography
Ion-exchange chromatography involves separating ions of different types. What are the 3 categories of ion types that can be separated?
- Cation & anion (batch extraction)
- ionic & non-ionic (batch extraction)
- separating mixtures of similarly charged species (chromatography)
Hydrophobic Interaction chromatography
What charactersitic does the mobile phase have, that drives hydrophobic interactions between proteins and ligands?
High salt (ion concentration)
- The salts reduce the solubility of hydrophobic regions of proteins, causing them to bind to the hydrophobic ligands on the stationary phase.
Ion-exchange chromatography
In ion exchange chromatography, what is the stationary phase?
Cations / anions that are covalently attached and fixed to solid support material
Ion-exchange chromatography
In cation exchanger, what is the charge of the stationary phase and mobile phase?
Cation exchanger: to bind and separate cations from mixture
- stationary phase : negatively charged
- mobile phase : positively charged (cation)
Ion-exchange chromatography
In anion exchanger, what is the charge of the stationary phase and mobile phase?
anion exchanger: to bind and separate anion from mixture
- stationary phase : positively charged
- mobile phase : negatively charged (anion)
Ion-exchange chromatography
What is the main diiference between strong ion exchangers and weak ion exchangers?
- Strong ion exchangers are fully ionized over a wide pH range. This means that they maintain a consistent charge (positive for strong cation exchangers, negative for strong anion exchangers) regardless of the pH of the solution.
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- However, weak ion exchangers are only partially ionized depending on the pH of the solution. Their charge (positive for weak cation exchangers, negative for weak anion exchangers) can vary with pH.This means that binding capacity changes with change with pH
Ion-exchange chromatography
How can one separate compounds with similar adsorption coefficient in weak ion exchangers?
Make fine-tuned adjustments by adjusting pH (not salt conc!)
- weak exchanger = binding capacity changes w pH
- note: if proteins are present and if you want to separate proteins, make sure to avoid isoelectric point of protein if not they will just get eluted
12.4.5. Affinity chromatography
What is the idea behind affinity chromatography?
Affinity chromatography separates components based on a specific, reversible interaction between a solute and ligand molecule
12.4.5. Affinity chromatography
What makes up the stationary phase in chromatography?
Ligands (e.g. enzyme inhibitors, antibodies / molecules that selectively and reversibly bind to complementary analyte molecules)
which are binded to a support material
- porous, stable, high surface area material that does not adsorb anything
12.4.5. Affinity chromatography
What are the 2 steps in immobilising ligands on support material in affinity chromatography?
- Activation - A reagent reacts with functional groups on the support to produce activated matrix
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- Coupling
- Removal of excess reagent will cause ligand to be coupled to activated matrix
12.4.5. Affinity chromatography
What is meant by specific and general ligands?
Specific – only binds to 1 particular solute
General – bind to certain classes of solutes
- bound solutes can then be separated as a group or individually, depending on elution technique used
12.4.5. Affinity chromatography
What is meant by nonspecific elution technique in affinity chromatography? (Check ans agn)
Ligand-analyte binding is (not??) disrupted by changing mobile phase pH / ionic strength / temperature etc
12.4.5. Affinity chromatography
What is meant by (bio)specific elution technique in affinity chromatography?
Free ligands, which may be identical or different from immbolised ligands), may be added to mobile phase and compete for binding sites in analyte
12.4.6. Size-Exclusion Chromatography (SEC) – easiest
What is the principle behind Size-Exclusion chromatography (SEC)?
Molecules are separated based on size, with no interaction between solute and stationary phase.
12.4.5. Affinity chromatography
In (bio)specific elution, should the free ligands or immbolised ligand have a higher affinity for the analyte of interest?
Free ligand, as the primary goal of (bio)specific elution is to displace the analyte from the immobilized ligand on the stationary phase. (want to elute out the specific compound)
12.4.6. Size-Exclusion Chromatography (SEC) – easiest
How does SEC work?
- Column packing material contains pores that are comparable in size to molecules of interest to be fractionated
- Solutes that are too big pass out and gets eluted since they cannot enter the interstitial space in the column, while solutes of interest are within the spaces in the column
12.4.6. Size-Exclusion Chromatography (SEC) – easiest
What is meant by
1. Void volume, V0?
2. Total permeation volume, Vt?
Void volume, Vo : Volume of mobile phase that occupies the space outside the porous beads within the chromatography column
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Total permeation volume, Vt = V0 + internal pore volume (volume of liquid in pores)
12.4.6. Size-Exclusion Chromatography (SEC) – easiest
____ molecules have free access to all available pore volume, also known as ____
Small, total permeation volume
Chromatography analysis
In Chromatography analysis, what does 1 chromatographic peak represent?
It represents 1 component found in the sample
Chromatography analysis
Choosing a separation mechanism for chromatography is important. What are the main characteristics of :
1. Isocaratic elution
2. Gradient elution
1) Isocratic elution - solvent composition and elution rate remains constant
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2) Gradient elution – mobile change composition is varied (by changing pH) or flow rate is varied.
What 3 parameters define the chromatographic peak of a component in the sample?
- Retention time
- Peak width
- Peak height
What is the significance of retention time in a chromatographic peak?
Retention time allows for identification of compounds as each compounds has a unique retention time
What is the significance of peak width in a chromatographic peak? [2]
Significance:
1) Resolution: Narrower peaks indicate better separation and resolution between components. Wider peaks can suggest poor separation or interactions within the column.
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2) Efficiency: Peak width is related to column efficiency; narrower peaks usually indicate higher efficiency and better performance of the chromatographic system.
What is the significance of peak area / peak height in a chromatographic peak? [2]
Peak Area (A) or Peak Height (H)
Significance:
1) Quantification: The peak area is directly proportional to the amount of the component in the sample, allowing for quantitative analysis. Peak height can also be used for quantification but is less reliable than peak area, especially for peaks with varying shapes.
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2) Calibration: By comparing the peak area (or height) of the sample with those of known standards, the concentration of the component in the sample can be determined.
What is meant by resolution and how does a graph of high resolution and low resolution look like?
Resolution refers to the degree of separation between two adjacent peaks in a chromatogram
- High resolution : two components in the mixture are well-separated and appear as distinct peaks
- Low resolution : peaks overlap / not clearly distinguishable
What is meant by retention factor and its significance? [2]
- Definition: Retention factor (also known as capacity factor) is a dimensionless number that describes the retention of a component relative to the mobile phase.
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Significance:
1) Behavior: It helps in understanding the interaction of the component with the stationary phase relative to the mobile phase.
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2) Optimization: It is used in method development and optimization to achieve better separation.
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3) Allows for identification of components when compared to standard (mote: rf values can differ based on certain factors!)
What are the 4 factors affecting resolution (separation)?
- Temperature
- Flow rate
- Stationary phase used
- Mobile phase used
What is meant by the plate theory in column efficiency? (Height equivalent to theoretical Plate, HETP)
The concept of theoretical plates is used tod escribe the efficiency of a column. HETP is the height of each imaginary segment of a column, and the shorter the height, the better the efficiency of separation (components travels less before it gets separated)
From the graph, how can one tell that there is high column efficiency?
Sharp narrow peaks indicate that there are more theoretical plates and thus better separation of components
What is dead volume in column selectivity?
volume in the column that is not available for the separation of compounds, i.e. volume of mobile phase that is not retained under separation conditions
What is column selectivity represented by in the graph?
distance/relative separation between peaks in chromatogram
What is meant by column capacity factor, K’?
amount of time a solute spends in/on the stationary phase relative to the mobile phase
How can one tell that there is high column selectivity from the chromatogram?
Peaks are well separated
How can we conduct quantitative analysis (conc of a compound) with chromatography?
Usage of data analysis software that can recognise start, maximum and end of each chromatographic peak, even when not fully resolved from one another
- Start, maximum and end of each peak is used by software to calculate retention times, peak height and peak area
- This numerical data is compared to known compounds in a database with known concentrations and thus find out the identity + concentration of the component of interest
Peak area can be compared with external or internal standards. What is the procedure of comparing peak area with external standards? (and also what is plotted on standard curve)
Chromatogram of standard and unknown sample are carried out separately.
- Sample Analysis: An identical volume of the sample is chromatographed, and the peak area of the sample’s compound(s) is compared to the calibration curve to determine the sample’s concentration.
- ratio of analyte singnal to internal standard signal is plotted n standard curve
Peak area can be compared with external or internal standards. What is the procedure of comparing peak area with internal standards? (and also what is plotted on standard curve)
- Samples (e.g. 1M, 2M, 3M….)
- Inject known standards alone into the chromatography (e.g. 1M, 2M, 3M….)
Standards = analyte of interest + internal standard - measure peak area of each known standard, and calculate ratio of peak area of analyte to internal standard
- Plot calibration curve of ratio against conc
- Inject unknown sample into chromatograph, from peak area of analyte and compound of interest → calculate ratio → find conc
