Introduction into analytical separation techniques Flashcards
LO
- Understand the importance of separation techniques e.g., GC, LC, CE in analytical science
- Know how and why they work
- Be aware of the various modes of separation and which might be best suited for your application
Why do we separate mixtures?
- Purification of target molecules
- To find out what’s in it (qualitative analysis)
- To determine the concentration of specific components (quantitative analysis)- determination of an analyte
- To understand a system, e.g., to ensure that manufactured products are safe for human consumption or that environments do not contain harmful pollutants
- The analyte is what we are trying to find out what it is, and the matrix is what the analyte is in i.e., drug analytes are in a urine matrix
What is the SP?
What forms can it beand the type of chromatography that form is found in?
o Can be solid or a liquid coated onto a solid support
o The SP may be coated onto a flat surface (PLANAR chromatography e.g, TLC)
o Or packed into a column (column chromatography e.g.,HPLC, GC)
o Or coated into the walls of a very narrow column(**open tubular chromatography **e.g., capillary GC)
What is the MP and the terminology associated with it at each stage of its flow
o Can be a liquid or a gas (fluid)
o The MP is often called the **eluent **
o When the eluent emerges from the end of a column, it is called the eluate
o The process of passing liquid or gas through the column is called elution
When the SP is packed into a column what are the formats the column can be?
Particles
Monolith
When as a monolith form, what is this and what are the different scales or bore can it be?
Column can be any length with internal bore
Three scales or bore: standard bore 4mm,micro bore 2mm and capillary bore which is small and around 50-500µm
What are the two types of open tubular forms of chromatography?
PLOT
WCOT
When it is a columnar scale column (GC) tell me some of the features of the SP
- SP is a liquid coat on inside wall of capillary and the mobile phase is a heated gas flowing through the middle of it
- The liquid SP can have several thicknesses
- The liquid SP can absorb analyte
Define the following involved in analytical separations:
* Chromatogram
* Peak
* Injection
* Elution
* Isocratic elution
* Gradient elution
*** Chromatogram **
o This is the output received from the detector as a single response versus time
o For electrically driven separations these are terms electropherograms
* Peak
o A peak in a chromatogram is a signal indicating that an analyte is eluting from the SP
*** Injection **
o The sample is introduced into the flow of bulk BP as a discrete volume of plug which is swept onto the column
* Elution
o The process of removing solutes from the column/ stationary phase using an “eluent”
*** Isocratic elution **
o On MP/ eluent
*** Gradient elution **
o Two or more re-proportioned overtime
What is the retention time and how does it relate to the chromatographic process?
- During elution, solutes continually distribute themselves between the SP and the MP
- While in the MP, they are carried forward with it, but they remain at a standstill in the SP
- Different molecules have different affinities for the SP- those with the greatest affinity for the SP stay here the longest
- Movement from the time of injection is timed and called the retention time
- The interaction of B with the SP is lower; therefore, it will not be “retained” as long as A, therefore separation occurs.
- A solute C that has no interaction passes through “unretained”
- Order of solute C<B<A
Peaks associating with different retention times of different solutes A,B and C
What are the different separation modes of chromatography?
And what type of chromatography is each one related to?
Adsorption: Normal phase and reversed phase
Partition: PDMS in open tubular GC, RP
**Ion-exchange **: IEX and IIC
Molecular exclusion: gel permeation
Affinity: IMAC, immunoaffinity
What happens during the adsorption separation mode?
o Analyte binds to a surface
o In NP and RP
o Solid SP and liquid/gaseous MP
o Solute is adsorbed on the surface of solid particles
o Interaction occurs via H-bonding, London dispersive and dipolar/induced dipole forces(van der Waals)
o Degree of adsorption depends on the relative polarities of the MP and SP
o E.g., adsorption to C8 or C18 chains in RPC
What happens during the partition separation mode?
o Analytes interact within SP
o DMS in GC
o C18 in RP
o A liquid SP forms a thin film on the surface of a solid support
o Movement of the analyte is determined by its relative solubility in the two phases
o Interaction based on hydrophobic and H-binding forces e.g., GC
What happens during the ion exchange separation mode?
o Charged analyte with oppositely charged surface ions
o Carboxylate or sulphonate group for cations or quaternary ammonium group for anions
o Similarly charged species to analyte is added to eluent to displace bound analyte on the surface leading to an ion exchange mechanism
o SP contains fixed charged groups e.g., R-SO32- or -R-(CH3)3N+ and + mobile counter ions (e.g.,H+ or OH-) which exchange with an analyte bearing an opposite charge
What happens during the molecular exclusion separation mode?
o Exclusion of species from porous SP based on their size
o Small molecules work through large pores in SP and therefore have a longer route to travel
o Analytes remain in the MP
o Separation arises because of variations in diffusion among the pores of an inert solid- molecules are separated based on size
What happens during the affinity separation mode?
o A complex molecule interacts with a covalently or electrostatically attached molecules on the SP surface
o E.g., histidine-tagged proteins (as analytes) and their interaction with metals on a stationary surface (immobilised metal affinity chromatography)
For efficient separation, generally only 1 mode is used for 1 column. However what is the exception to this and what is an example of where this happens
o the exception is partition and adsorption where high affinity is still maintained
o NOTE: when more than one of these are used at the same time, there is termed a “multi-modal” separation- e.g., hydrophilic interaction chromatography **(HILIC) **uses both partition and adsorption to achieve this separation
What is retention time and the equation to define it?
Define the following and how to calculate them:
* Retention time
* Void time
* Adjusted retention time
* Retention factor
* Resolution
What are the different chromatogram peak shapes?
- Good peak shape= Gaussian- evenly distributed around a
mean central point- this is the tr marker -
Non-gaussian: poor peak shape: termed **tailed, fronted,
or split ** - Asymmetry, As= y/x
- Should be shaped like a bell curve with mean distribution
Around mean apex which is used to find retention time
What kind of chromatogram peak is preferred?
Narrow and sharp peak as this represents efficiency as means that more sample can be tested at any time as opposed to wide peaks
How do you characterise symmetry of a peak and when is it considered asymmetric?
o If you want to characterise the symmetry of a peak; a division of y (min)/x (min) is performed.
**>1 is asymmetry. Symmetric peaks are rare. **
How can you tell if peaks are fronted, tailed or split?
If shallow or sloped to front of the peak, then it is fronted
If shallow or sloped to rear of the peak, then it is** tailed **
Occasionally, **split **peaks may be observed. These are not the same as two species which have separated but usually when separating a mixture. It is generally due to a problem with the chromatogram.
Whats the equation to define retention factor
What is retention factor used for?
What are the expected values?
What units are used within the equation?
- Used to compare separation of same mixture on different columns of different dimensions
- Normally retention to t0
- Retention factor is the same as capacity factor which it is sometimes referred to as. But always use retention factor, expressed as ‘k’
*** K values of 1-5 **are preferred by sometimes with large screening samples of 30 compounds or more 0-20 can be seen
- If K falls outside the general 1-5 range, then certain things can be done to help speed up or slow down the separation; increase mobile phase flow rate, change composition of MP solvent etc.
- K unitless as min cancel out
- Retention time is not in mins or seconds but **decimal minutes **
e.g., peak at 3 mins 15 seconds –> 3.25 decimal minutes (calculations won’t work using minutes and seconds)
What is the selectivity factor, alpha ?
Described the selectivity between two different solutes which may be separated in a chromatogram
What is the equation to define the selectivity factor?
How else may the selectivity factor be calculated?
What is a theoretical plate?
- The height equivalent to a theoretical plate (H) is a hypothetical distance along a SP for one complete band equilibration to occur (as shown in green)
- Number of discrete lengths where the solutes interact with the SP above in green
- This is not a fixed quantity in practice because the width of plugs broadens across the column but can still be used to assess and compare separation between systems
How is plate height defined for a given column length?
How can the number of theoretical plates, N be calculated?
NB: Make sure read if width at baseline or width at half height so you know which equation to use in an exam
Peaks are generally in a curved form, so how is W and W0.5 calculated?
Triangulation
Example of triangulation
Peak 4 used in this example
With the same chromatogram as above you can see that the retention time for peak 4 is roughly less than 2 but to accurately measure you would follow these steps…
What does resolution measure?
What does it take into consideration and what is the equation to calculate it?
Resolutions in practice…
What is a useful equation that is readily derived which is related to the resolution of a column to N, K’ and alpha?
What does it mean under the following circumstances:
If alpha is close to 1…
If N is small…
What is Rs proportional to?
Compare the selectivity factor and resolution
What is the Van Deemter equation?
The van Deemter curve
How is the A term defined and what does it consider?
In the A term what is channelling and Eddy diffusion?
Describe and explain the B term
Describe and explain the C term
Equations to know
Main topics to know
What causes the chromatographic peak and what term is this associated with?
C term
The MP and SP both contribute to what and how is this defined?
What is the rate of migration proportioal to and what term is this associated with?
B term
