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HPLC - concept map FINAL COPY

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on 25 June 2015

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Transcript of HPLC - concept map FINAL COPY

HPLC - concept map
Resolution
Resolution is calculated by the 2 closest peaks and it is influenced by many factors.
The equation for resolution is;

Sample
Equipment
Column
Mobile phase
Back pressure
Run time
Run time is not a calculated response, but it is the time for all compounds to elute out.
Run time shouldn't be too long, as you don't want to spend hours in the lab just waiting for the last peak

Sensitivity
Map Legend
= Chemical parameter
tR
t0
AUC
Width
Height
Ligand type
Sample solvent
Sample conc.
pH
type of B
Injection vol
Length
Diameter
Particle size
Wavelength
Temperature
HETP
Linear velocity
Pore size
= Mechanical parameter
= Both chemical and mechanical parameter
Peak tailing/fronting
Peak tailing and fronting should not be present in an ideal chromatogram - it increases the chance of peak overlap
Viscosity
Back pressure is not a calculated response - however, exceeding back pressure will turn off the HPLC machine, so back pressure must never exceed the maximum limit
(4000psi)

Efficiency (N)
Efficiency can be calculated using the length of the column and HETP, or using tR and width of a peak
N = length/HETP
N = 16・(tR/width)^2
Selectivity (alpha)
Selectivity is calculated by relative distance of two peaks
α = tR2/tR1
Retention factor (K)
Retention factor is calculated by the retention time of molecule and uracil
K = (tR - t0)/t0
AUC
Width
Height
HETP
tR
t0
By every 10% change in B, each compound K will be multiplied by a factor in between 2 ~ 3. All compounds have different multiplying factor. In fact, the same compound has different multiplying factor between different %B
%B
Incrased flow rate using same column diameter = increased linear velocity
Particles move faster in the column, so the run time is shorter
Since tR is affected, the tR of the last peak changes the run time.

K does not change because tR and t0 changes proportionately
Flow rate, and tR and t0
%B and retention factor (K)
%B and tR (but not t0)
As %B increases, retention time decreases since the mobile phase polarity ("non-polarity") gets close to that of the stationary phase. The analyte molecules have less preference towards the stationary phase over the mobile phase, as there is less difference in polarity between them. More detail on the flow chart.

x-axis = %B
y-axis = time
Flow rate and back pressure
As flow rate increases, resistance in the column also increases.
Back pressure goes up as a consequence
Wavelength and AUC
If UV-Vis detector is used, changing the wavelength of the detector will change AUC.
This is because different particles absorb light at different wavelength.
The chromatogram seen on the computer screen is absorbance against time. If the wavelength is set so that one compound doesn't absorb light at all, the detector will not "detect" that compound.
Injection volume and width
As injection volume increases, width also increases because the more you inject, the more longituginal diffusion that occurs.
Injection volume and HETP
As injection volume increases, longitudinal diffusion also increases (band broadening)
= Minor link. This parameter is not used to change this response.
= Link between a parameter and a response. Although they may cause changes in the response, they are not used as the main source.
= Main link. The parameter is varied to change this parameter.
Particle size and back pressure
Smaller particle size increases back pressure.

A tube filled with sand will be much harder to push liquid through than a tube filled with marbles.
Pore size and HETP
Pore size affects mass transfer.

Mass transfer is when analyte molecules move in to the pores of the particles and interact with the stationary phase ligands
Bigger pore size = some molecules move in deep, so more retained but some only interact with the surface, so less retained = band broadening.
Column length and back pressure
Increased column length increases back pressure.

It's easy to blow out a piece of paper stuck in a 10cm straw than in a 25cm straw.
Sample conc. and AUC
Effectively, AUC corresponds to the amount of molecules detected by the detector.

Increased sample concentration increases the amount of molecules in the sample, so the AUC goes up.
pH and ligand type
Some ligands can be destroyed by change in pH.
For example, usual C18 column can only work in pH 2~8 because at pH<2, bonded ligands can be hydrolysed, and at pH>8, the silica particles can dissolve.
pH, and tR (and K and selectivity)
pH will change the ionisation state of some molecules.

Ionised molecules are less likely to interact with the stationary phase, so they will have shorter retention times. Unionised molecules are more likely to interact with the stationary phase, so they will have longer retention times.

pH is a chemical parameter, so it will not change t0. This means that K is changed.

K will change disproportionately for different molecules within the sample, so selectivity also changes.
Temperature and selectivity
Temperature changes pKa of acidic/basic molecules.

Refer to
pH and selectivity
for relationship between ionisation state and retention times.
injection volume and AUC
Effectively, AUC corresponds to the amount of molecules detected by the detector (at a given wavelength).

Increased injection volume increases the amount of molecules in the sample, so the AUC goes up.
Flow rate and linear velocity
At a given column diametee, higher flow rate results in higher linear velocity.

At a higher flow rate, more molecules are being pushed into the column, so the molecules must travel faster to accomodate the number of molecules being pushed.
Linear velocity, and tR and t0
At a higher linear velocity, the molecules are moving faster, so their tR and t0 will decrease.

K is not affected by LV since both tR and t0 change proportionately. Also, K is a result of change in chemical parameter, but LV is a mechanical parameter.
Linear velocity and back pressure
Increase in LV increases back pressure because resistance in the column increases as LV increases.
Temperature and ligand type
Bonded phase on a ligand can be hydrolysed.
This hydrolysis process can be sped up by increased temperature.
Solvent type, and efficiency
Solvent miscibility is an important factor.
Immiscible solvents can produce precipitation, immmiscible emulsions or cavitation.
Cavitation will cause gas bubbles to go into the column, destroying silica particles.
This gives poor effiency and/or peak fronting.
Width and efficiency
As effiency can be calculated by N = 16・(tR/width)^2, increase in width (but no change in tR) will decrease efficiency.
Linear velocity, and HETP
Increased LV will give less HETP until a point. After that, increased LV continuously gives increased HETP.
LV and longitudinal diffusion
Longitudinal diffusion occurs as a result of analyte molecules diffusing in the column.

The faster they travel, the less time they have to diffuse so there will be less band broadening.
LV and mass transfer
Mass transfer occurs as a result of molecules moving into the pores of the startionary phase particles and interacting with the bonded phase within the pores.
Some molecules will move deep into the pores but some will only interact at the surface.
Higher LV means that these molecules that only interact at the surface have moved further down the column by the time the molecules that were deep in the pores come out to elute.
Diameter and linear velocity
At a given flow rate, decreaseing the diameter will give increased LV (faster speed) since more molecules must "fit" in a narrower column.
Particle size and HETP
Particle size affects Eddy diffusion.

Eddy diffusion is band broadening as a result of analyte molecules taking different "paths" to elute out of the column.

Smaller particle size minimises Eddy diffusion.
Pore size and retention time
Increasing the pore size means decreased surface area (of the silica particles). This decreased surface area leads to decreased carbon density.
Dereased carbon density gives decreased retention time as there is less bonded phase for molecules to interact with.
“large-pore column can be one way to reduce retention” – John Dolan [1]
tR
= Primary response. This is the reponse that you see on the chromatogram.

They are the walls of the castle
= Secondary response. They are calculated from the primary response.

They are the knights that protect the royal family.
Resolution
Resolution is calculated by the 2 closest peaks and it is influenced by many factors.
The equation for resolution is;

= The main reponse. The size indicates the importance of the responses.

They are the royal family; King, Queen, Prince, Princess, and the baby.
Viscosity and back pressure
As the viscosity of the solvent goes up, the back pressure also goes up since more "power" needs to be applied to push solvent that is more resistant to flow.
Efficiency (N)
Efficiency can be calculated using the length of the column and HETP, or using tR and width of a peak
N = length/HETP
N = 16・(tR/width)^2
Selectivity (alpha)
Selectivity is calculated by relative distance of two peaks
α = K2/K1
Retention factor (K)
Retention factor is calculated by the retention time of molecule and uracil
K = (tR - t0)/t0
Retention factor (K)
Retention factor is calculated by the retention time of molecule and uracil
K = (tR - t0)/t0
Type of B, and tR (and retention factor and selectivity)
Changing the organic solvent will change the tR of peaks in the chromatogram by changing the type and extent of interactions that occur between the analyte molecules, and stationary and mobile phases. t0 is not affected by type of B, so retention factor changes.

All molecules within the sample are affected differently which gives rise to the difference in selectivity.
http://www.shimadzu.com/an/hplc/support/faq/acetonitrile/2-1.html
HEPT and efficiency
HETP (Height equivalent to one theoretical plate) is inversely related to the efficiency of the column. As HETP increases, the number of theoretical plate (N) that can fit in a column, which is effectively the efficiency of the column, decreases.
Ligand type and retention time (and retention factor and selectivity)
There are different types of column ligands available: the most common ligand type used is the C18 column, but there are also biphenyl, C18 aqueous, PFP, etc.

These different ligands possess different functional groups on the bonded phase (ligand arms) which differ in the type/extent of interactions that they can offer to the analyte molecules.

Ligand type will only change tR, so K and subsequently selectivity are also changed.
%B and ligand type
Highly aqueous mobile phase will cause the bonded phase on the ligand particles to collapse onto themselves.
There are no bonded phase arms for analyte molecules to interact with.
Another effect is that at a very low %B (highly aqueous solvent), the arms collapse onto themselves. This will decrease the retention time of everything as there are no more bonded phase for analyte molecules to interact with.
%B and viscosity
All solvents have different viscosity. Unless the solvent has the same visocity as water, changing the composition of organic/water will change viscosity.
%B and selectivity
Every molecule has different multiplying factor so they will all change their K differently (disproportionately).
However, this is a very minor effect.
Gradient retention factor (K')
Gradient retention factor is calculated by
Gradient time (tg)
Gradient retention factor (K')
Gradient retention factor is calculated by
= Does not fall into primary/secondary/main response.

They are like a lake near the castle. It's something nice to have, but not necessary.
K' of 5 should be aimed for average separation. Use the euqation to calculate the tg and delta phi that gives K' around 5
Gradient time and run time
Longer gradient time gives longer run time.

Highlighted red is the gradient time
Temperature and viscosity
Viscosity decreases with increased temperature
Efficiency and resolution
Larger N value improves resolution
Selectivity and resolution
According to the eqation, larger alpha improves resolution. Small alpha makes the middle part of the equation close to 1, making it less siginificant.
Retention factor and resolution
Large K is required to give high resolution. As K increases, the Retention part of the equation gets closer to 1.
Retention time and retention factor
As retention factor relies on tR (and t0), anything that changes tR only or tR ant t0 disproportionately will change K
Ligand type and peak tailing
Free silanols, found on C18 columns can cause peak tailing of bases. These silanols are acidic form of silicon and attract bases however, most of the silicon has the hydrophobic ligands attached. Because only a small proportion of silanols are available, only a small portion of the bases are attracted an elute a little bit later than the other molecules. 

End-capping the free acidic silanols will provide "protection" against base retention.
Column length, and tR and t0
Longer column length means that the molecules must travel a longer distance to reach the detector, hence longer tR and t0
Column length and efficiency
Using the equation N = length/HETP, longer length gives higher efficiency. This is because a longer column can fit more plates (given that HETP stays constant)
Column length and K'
Column diameter and K'
pH and peak tailing
Peak tailing occurs as a result of bases being attracted to acids. Changing pH will change the ionisation state of acids/bases.
Ionised acids will be more likely to attract bases and ionised bases are more likely to interact with acids.

Using pH that makes both acids and bases be ionised will create the worst peak tailing.
tR and efficiency
Efficiency can be calculated by 16・(tR/width)^2
If tR increases by a greater magnitude than width, it will increase efficiency.
tR and efficiency
Efficiency can be calculated by 16・(tR/width)^2
If tR increases by a greater magnitude than width, it will increase efficiency.
tR and run time
It is tR of the last peak that defines the run time
tR and run time
It is tR of the last peak that defines the run time
Retention time and retention factor
As retention factor relies on tR (and t0), anything that changes tR only or tR ant t0 disproportionately will change K
Linear velocity and sensitivity
As LV increases and molecules pass the detectors quickly, less molecules are detected. The detector needs time to precisely detect molecules.
K and selectivity
Selectivity is a measure of relative distances between two peaks (measured through K). If K of two peaks change disproportionately, selectivity is changed.
Efficiency and resolution
Larger N value improves resolution
Selectivity and resolution
According to the eqation, larger alpha improves resolution. Smaller alpha makes the middle part of the equation closer to 0.
Retention factor and resolution
Large K is required to give high resolution. As K increases, the Retention part of the equation gets closer to 1.
Width and efficiency
As effiency can be calculated by N = 16・(tR/width)^2, increase in width (but no change in tR) will decrease efficiency.
HEPT and efficiency
HETP (Height equivalent to one theoretical plate) is inversely related to the efficiency of the column. As HETP increases, the number of theoretical plate (N) that can fit in a column, which is effectively the efficiency of the column, decreases.
Gradient time and K'
Flow rate and K'
Sensitivity is defined as the smallest amount/concentration that gives signal:noise of
2:1
Height and sensitivity
Height of the peak must be at least twice (usually three times) of the noise for it to be detected (LOD)

For a peak to be quantifiable, the peak must be more than 10 times the noise (LOQ)
Flow rate and AUC
Decreased flow rate increases AUC because molecules travelling at a lower rate will be more detected than molecules travelling with a higher rate.
Solvent type and peak fronting
When a sample dissolved in a solvent stronger than the mobile phase gets injected, the molecles will preferentially stay in the sample solvent (as it is more hydrophobic than the mobile phase). However, as it travels through the column, the "end" of the sample solvent is diluted by the mobile phase, decreasing its' solvent strength. With a weaker solvent, the molecules will prefer to partition into the stationary phase.
The molecules at the "front" of the sample solvent will elute out before others, giving peak fronting.
Injection volume and peak fronting
Increased injection volume will increase the amount of analyte molecules pasing through the column. As molecules partition into the stationary phase, some molecules are "left out" and cannot interact with the stationary phase as other molecules are occupying the bonded phases. These molecules will then travel forward in the column to find free bonded phase to interact with.
Extra colum volume
Extra colum volume and width
Extra column volume is the volume between the injector and the column, and column and the dector ie volume of the tubing which analytes travel through.

Bigger the extra column volume, the more chance the analytes will diffuse (longitudinal diffusion).
Therefore to avoid band broadening, extra column volume should be reduced as much as possible [2].
References
http://www.chromacademy.com/hplc-training.html
Most pictures taken from CHROMacademy unless otherwise stated

All Excel graphs taken from/produced using values on 2015 Practicals POS table.

[1] http://www.sepscience.com/Techniques/LC/Articles/702-/HPLC-Solutions-10-Pore-Size-vs-Particle-Size

[2] http://www.chromacademy.com/chromatography-HPLC-extra-column-volumes.html

Injection volume needs to be carefully determined to avoid this effect.
http://www.crawfordscientific.com/Chromatography-Technical-Tips-Sample-Diluent-Effects-in-HPLC.html
Type of B and viscosity
Different solvents have different viscosity
Height and sensitivity
Height of the peak is a visual representation of sensitivity
Ayame Saito 25133381
Flow rate
Changing pH will only have effect on ionisable molecules
Resolution should be greater than 1.5
Run time should be as short as possible without compromising the resolution.
Retention factor should be between 1~10 or 2~20 (depending on literature)
Selectivity should be more than 1 to ensure good separation between two peaks
Efficiency should be over 4,000. Around 10,000 is best
K and selectivity
Selectivity is a measure of relative distances between two peaks (measured through K). If K of two peaks change disproportionately, selectivity is changed.
UPLC
Ultra performance liquid chromatography - able to withstand very high pressure
= The machine "kills" the response - with UPLC, back pressure does not need to be taken into consideration when changing other parameter.s
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