Chromatography Flashcards
What is chromatography?
a technique used to separate and analyze the
components of a mixture based on their physical and chemical properties.
Can be used for separating and detecting (if paired with a detection method like MS) multiple pesticides for example.
What does chromatography involve?
The technique involves passing a mixture through a stationary phase, such as a solid or liquid, which interacts differently with the various components of the mixture, causing them to move at different rates through the stationary phase.
List the different types of chromatography.
Define: stationary phase
- the immobile phase that is used to separate the components of a mixture.
- solid or liquid material that is packed into a column or coated onto a surface and provides the surface for the analyte
Define: mobile phase.
- the fluid that carries the mixture being analyzed through the stationary phase.
- liquid or gas that flows through the stationary phase and transports the analyte along the
column or surface.
Compare stationary phase and mobile phase.
What is a column?
- The ‘column’: vessel containing the stationary phase; allows passage of mobile phase
What are methods of separation?
What is absorption?
With regard to chromatography.
- Partitioning
- Analyte in partition in two immiscible liquids
- Molecules of the mobile phase are dissolved into the stationary phase.
What is adsorption?
With regard to chromatography
- Analyte attaches to a surface
- Analytes will adsorb out of solution onto the solid phase (in liquid chromatography)
- As the mobile phase changes during elution, the adsorbed solutes will partition back inot the solution (be absorbed into the mobile phase)
What is liquid chromatography?
- Separation of a liquid chemical mixture into a subset of components (solutes)
- Separation is based on chemical or physical properties of the solutes
- Solutes differentially distribute as they flow around/over the separating medium
Describe the general process of chromatography.
- Mobile phase containing solutes are added to the column
- Solutes travel through the column by:
- Adsorbing onto stationary phase
- Absorbing back into the mobile phase
- Constant on/off process
- There will always be some solutes in/on both phases
- Depending on how much the solutes interact with the column, they move faster/slower relative to each other
A hydrophobic stationary phase interacts more strongly with more hydrophobic (nonpolar) molecules.
Describe silica as a stationary phase material.
- Crosslinked tetraethyl orthosilicate
- Highly customizable pore size and volume
- Can easily functionalize silanol groups
- Si-OH group
- Modify to have specific properties (cationic/anionic; hydrophobic/hydrophilic)
However, not all Si-OH may be modified, and it is only stable at pH 2-8.
What are the downsides of silica as a stationary phase material?
- Not all Si-OH may be modified
- Unintended sample binding/not binding enough
- Only stable pH 2 – 8
- Acidic mobile phase may hydrolyze functional groups
- Basic mobile phase may solubilize silica
- Can clog column
What are the benefits of using a polymeric resin for a stationary phase? [2]
- Stable across pH 1 - 13 (depends on the polymer)
Resists hydrolysis - longer column life
What are the downsides of using a polymeric resin for a stationary phase? [3]
- Not as strong as silica
- Cannot withstand high pressure
- Can sometimes swell depending on the organic solvent
Give two examples of polymeric resins used as stationary phase materials.
- Polymethacrylate (A)
- Polyvinyl alcohols (B)
Define: mobile phase [4]
- Solvent used to perform experiments
- Some LC methods use two mobile phases
- Must be compatible with the sample and column
- More details in section for each type of LC
Hexane
Describe: paper chromatography
- Stationary phase: a piece of paper or a cellulose-based material that acts as an absorbent; a polar and hydrophilic material
- Mobile phase: liquid solvent
- The solvent moves through the paper by capillary action
- The retention factor (Rƒ)
- The ratio of the distance travelled by the solute to the distance travelled by the solvent.
- Rf value is constant for a given substance
under given conditions.
What is the retention factor?
- The retention factor (Rƒ)
- The ratio of the distance travelled by the solute to the distance travelled by the solvent.
- Rf value is constant for a given substance under given conditions.
Paper chromatography
What are the benefits of paper chromatography?
- Simple and inexpensive
Widely used in the separation and analysis of small molecules, such as amino acids, sugars, and dyes
Describe thin-layer chromatography.
- Stationary phase: is a layer of adsorbent
(usually silica gel, or aluminium oxide) on substrate such as glass, plastic, or aluminium foil - Mobile phase: liquid solvent
Basically the same as paper chromatography.
What are the benefits of TLC? [4]
Simplicity, relative low cost, high
sensitivity, and speed of separation
More accurate than paper chromatography.
For a successful separation, one must have knowledge of the chemical properties of the […]
- Analyties of interest
- Stationary phase
- Mobile phase
List 5 types of chromatography (excluding paper & TLC).
- Adsorption(Liquid-Solid)Chromatography
- Partition (Liquid-Liquid)) chromatography
- Ion-exchange chromatography
- Size exclusion chromatography
- Affinity chromatography
Briefly describe adsorption chromatography.
Liquid-solid chromatrography
Briefly describe partition chromatography.
Liquid liquid chromatography
Briefly describe ion-exchange chromatography.
Briefly describe size exclusion chromatography.
Briefly describe affinity chromatography.
What is the stationary phase in adsorption (liquid-liquid) chromatography?
- Stationary phase (adsorbent)
- Finely divided solid (max. surface area)
- Permit differential interaction with components to be separated
- Commonly used: silica (acidic); alumina (basic)
What is the mechanism of adsorption chromatography, and what are the intermolecular forces that occur?
- solvent and solutes compete for binding sites on the surface
- changing the strength of the mobile phase will alter solute-surface interactions, e.g., increasing polarity of the mobile phase to elute the
solute from a polar surface
What is the mechanism of partition chromatography?
- Column of finely divided inert support material was used to hold one liquid phase (stationary phase), while the second liquid, an immiscible solvent (mobile phase), flowed over it, thus providing intimate contact between the two phases.
- Solutes partitioned between the two liquid phases according to their partition coefficients
Adsorption and partition chromatography may be reverse phase or normal phase.
True or False?
True.
Adsorption and partition chromatography may be only reverse phase or normal phase.
True or False?
False.
Adsorption and partition chromatography may be reverse phase or normal phase.
Define: normal phase chromatography.
Stationary phase is polar and mobile phase is non-polar
Define reverse phase chromatography.
Stationary phase is non-polar and mobile phase is polar.
Define: ion-exchange chromatography.
A separation technique that separates molecules based on their affinity for an oppositely charged stationary phase.
Note: The analytes can be eluted from the stationary phase by changing the conditions of the mobile phase, such as pH, salt concentration, or buffer composition.
What are the two types of ion-exchange chromatography?
- Cation - the stationary phase contains negatively charged functional groups, and positively charged analytes are retained on the column.
- Anion - the stationary phase contains positively charged functional groups, and negatively charged analytes are retained on the column.
Note: The analytes can be eluted from the stationary phase by changing the conditions of the mobile phase, such as pH, salt concentration, or buffer composition.
-
Cation exchange chromatography - the stationary phase contains negatively charged functional groups, and positively charged analytes are retained on the column.
True or False?
True.
-
Cation exchange chromatography - the stationary phase contains positively charged functional groups, and negatively charged analytes are retained on the column.
True or False?
False.
* Cation exchange chromatography - the stationary phase contains negatively charged functional groups, and positively charged analytes are retained on the column.
-
Anion exchange chromatography - the stationary phase contains positively charged functional groups, and negatively charged analytes are retained on the column.
True or False?
True.
-
Anion exchange chromatography - the stationary phase contains negatively charged functional groups, and positively charged analytes are retained on the column.
True or False?
False.
* Anion exchange chromatography - the stationary phase contains positively charged functional groups, and negatively charged analytes are retained on the column.
What are applications of ion-exchange chromatography?
Widely used in the purification and separation of biomolecules such as proteins, nucleic acids, and carbohydrates, as well as in the analysis of small ions and organic molecules.
Define: size exclusion chromatography.
A chromatographic separation procedure that
separated analyte molecules according to their size or geometry and some cases by their molecular weight.
What is size exclusion chromatography used for?
Separation of macromolecules like proteins, enzymes, antibodies, nucleic acids (DNA and RNA), and industrial polymers.
Describe the column used in size exclusion chromatography.
- column contains a porous matrix of spherical
particles or beads. - columns are composed of dextran polymers
(Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P)
Define: affinity chromatography.
A chromatographic technique used to separate and purify molecules based on their specific interactions with a ligand immobilized on the stationary phase.
* The specificity of the interaction between the ligand and the analyte allows for high selectivity and high resolution separations.
Ligand can be a variety of molecules, such as antibodies,
enzymes, lectins, nucleic acids, or small molecules that
have a high affinity for the target molecule.
What are controllable sample properties in chromatography? [5]
- pH
- Volume
- Buffer/salts/ions
- Solvent
- Particulate matter
Why do we need to control sample properties like pH, volume, etc. in chromatographic analysis?
- Can influence outcome of analysis
- Impact peak shape/separation, retention times, quantitation limits, compound selectivity
- Can damage column
- Particulate matter, high/low pH
- Columns are expensive
In sample preparation, why is particular matter important to control?
- Columns have small pore sizes and tight packing
- Particulates can clog column (can’t always clean and columns are expensive!)
In sample preparation, what are solutions to particular matter?
- Centrifugation at high speed to remove non-soluble matter (>40,000 g)
- Syringe filter (0.22 µm; ensure sample has low binding to filter material)
What pH considerations are necessary in sample preparation for chromatographic analysis?
- Be aware of column limitations
- Could dissolve/hydrolyze column contents
- Hydrolyze ligands
- Loss of resolution, binding capacity, etc.
- Controlled pH may be required
- E.g., Ion-exchange
- If pH too low/high, may not be in an ionized state (low affinity), or proper ionized state (too much affinity)
What are solvent considerations that are necessary in sample preparation for chromatographic analysis?
- Be aware of column limitations
- Some solvents may damage either the stationary phase, or the column housing itself
- Can impact separation and/or resolution
- Make sure they are compatible with the sample
- Polar sample –> probably want polar solvent
- For proteins: make sure they don’t induce unfolding
Describe the output from a chromatography run.
Contains:
* Analyte peaks
* User information
* When sample was added (injection time)
* System settings (flow rate, pressure)
* Changes in solvent proportions
* pH
* Electrical conductivity
* Absorbance (if analytes allow)
* And more
What are t0, tR1, and t’R1 in a chromatogram?
- Note, you can use time and volume interchangeably (many prefer volume, or ‘column volumes’ because they are more well-defined than time) - V0, VR1, and V’R1.
Define: resolution
The ability to distinguish peaks in the resolution.
What is resolution affected by ? [3]
Fundamental column parameters:
1. Selectivity or separation factor (alpha)
2. Capacity factor (k’)
3. Efficiency or theoretical plates (N)
What is selectivity?
- The ratio of retentions of analytes
- Indicates how long one solute is retained in the stationary phase in the relation to another
- Is affected by the chemistry of the entire system
- For separation to occur:
- 𝛼 > 1 (otherwise peaks overlap)
What is capacity factor?
- Capacity factor
- Is an indication of how long a compound can be retained by the stationary phase
- Impacted by the:
- Solute’s relative affinity for the mobile and stationary phases
- Packing of solid phase
- For good separation to occur:
- 𝑘′ ≥ 1 ; 2 – 10 is good (otherwise peaks overlap)
For good separation to occur, 𝑘′ ≥ 1.
True or False?
True.
Otherwise peaks overlap
Capacity factor.
For good separation to occur, 𝑘′ < 1.
True or False?
False.
For good separation to occur, 𝑘′ ≥ 1.
Capacity factor
For good separation, 𝛼 > 1.
True or False?
True.
Otherwise, peaks overlap
Selectivity
For good separation, 𝛼 ≤ 1.
True or False?
False.
For good separation, 𝛼 > 1.
Otherwise, peaks overlap.
Selectivity
What is efficiency?
- Also referred to as ‘Number of theoretical plates’
- Measures the degree of band broadening
- Informally, how well the analytes interact with the system as a whole
A lower number of theoretical plates means a less intense peak.
True or False?
True.
A higher number of theoretical plates means a less intense peak.
True or False?
False.
A lower number of theoretical plates means a less intense peak.
What factors influence the number of theoretical plates?
Many aspects of the system to varying degrees.
If k’ is increased, the resolution is better, but the peaks become broader.
True or False?
True.
If k’ is decreased, the resolution is better, and the peaks become narrower.
True or False?
False.
If k’ is increased, the resolution is better, but the peaks become broader.
If N is decreased the resolution is worse because of the broadening of the peak widths.
True or False?
True.
Decreasing α will increase resolution.
True or False?
False.
Increasing α will increase resolution.
If N is decreased the resolution is better because of the broadening of the peak widths.
True or False?
False.
If N is increased the resolution is better because of the narrowing of the peak widths.
If k’ is increased, the resolution is worse, but the peaks become narrower.
True or False?
False.
If k’ is increased, the resolution is better, but the peaks become broader.
If N is increased the resolution is better because of the narrowing of the peak widths.
True or False?
True.
Increasing α will increase resolution.
True or False?
True.
[…] is the most powerful parameter for increasing resolution in chromatographic analysis.
α - selectivity/separation factor
How may quantitative analysis be performed in chromatographic methods?
Can be performed by:
* Comparing peak areas
* Comparing peak heights
* Using standards to identify components in a mixture
* Internal standard method
* Add a known compound that is similar to the analytes
* You know the concentration and retention time
* Can estimate the concentration of other unknowns using the peak area ratio
In quantitative chromatographic analysis, compare the external and internal standard methods.
- External method: area of analyte
- Internal method: ratio of analyte and internal standard area
Why is an internal standard added to both standard and unknown?
It ‘compensates’ for losses during sample preparation or variability during the analytical determination.
List relevant factors for selecting an internal standard. [6]
- should be similar to analyte chemically and physically.
- should elute near to main analyte and well resolved.
- should not present in Orginal Sample matrix.
- should be unreactive to sample matrix.
- should be available in high pure form
- The concentration of Internal standard should be about to half (either in height or response) to the main analyte (this is to distinguish two peaks instantly in case of RT shifting).
Describe the general liquid chromatography system design.
Describe the pump used in LC.
- Responsible for delivering the mobile phase (solvent) at a constant flow rate to the column.
- The solvent pump used in HPLC is typically a high-pressure reciprocating piston pump or a high-pressure gradient pump.
What does the solvent pump rate depend on? [2]
Column size and type of chromatography
Compare system pressure rating of FPLC, HPLC, and UHPLC.
Each column (and system) will have a recommended maximum flow rate and pressure. What happens if these recommendations are not followed?
- If not followed, system or column can be damaged
- System: $75k minimum
- Columns: Highly variable. Range from <$100 to >$1000s
Describe the injection valve of HPLC.
- Must withstand high pressure (if HPLC/UHPLC)
- Ports for sample loops, liquid syringes, auto-sampler ports
Describe detectors used in liquid chromatography. [5]
- UV/Vis (fixed & variable wavelengths)
- Refractive Index
- Fluorescence
- Evaporative light scattering
- LC output has solvent evaporated; scattering of light by dry molecules is quantified
- Electrochemical
- Measures current induced by oxidation or reduction of molecules
What does evaporative light scattering measure?
- LC output has solvent evaporated; scattering of light by dry molecules is quantified.
What does electrochemical LC detection entail?
- Measures current induced by oxidation or reduction of molecules.
Describe UV-VIS LC detectors. [3]
- Useful for most general purpose analyses
- Can detect wide range of analytes
- Fixed, variable wavelength systems
Describe fluorescence LC detectors. [2]
- Use excitation and emission filters allow drugs, natural products, vitamins, and other specialized chemicals to be determined at parts-per-trillion level
- Very sensitive
- Use of excitation wavelength to generate emission signal gives rise to the high sensitivity of
fluorescence-based analyses (in general)
List some LC applications.
What happens if you increase k’?
If k’ is increased, the resolution is better, but the peaks become broader
What happens if you increase N?
If N is increased the resolution is better because of the narrowing of the peak widths.
What happens if you increase alpha?
- Increasing alpha will increase resolution
- Alpha is the most powerful parameter for increasing resolution.
