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HPLC Parameter Influence Concept Map FINAL SUBMISSION

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Christopher Hendy

on 25 June 2015

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Transcript of HPLC Parameter Influence Concept Map FINAL SUBMISSION

Resolution
Run Time
Back Pressure
Sensitivity
Peak Tailing
Column Length
Column Diameter
Particle Size
Solvent Type
% B
Pore Size
Temperature
Injection Volume
Sample Concentration
Flow Rate
Wavelength
pH
Ligand Type
column
equipment
sample
mobile phase
Legend
= primary controllable parameter
= Royal Family
= responses
=Chemical implication
= Mechanical implication
= other/both
= partially controllable parameter
= calculated responses
selectivity
retention
efficiency
linear velocity
viscosity
gradient retention
AUC
plate number


tR
peak height
ionisation state of compound
UPLC
end capping
carbon load
metal ion content
practical plate height
Van Deemter equation
eddy diffusion (A term)
mass transfer
(C term)
t0
peak width
longitudinal diffusion
(B term)
gradient time
HPLC Parameter Influence Concept Map
Dictated by the retention time of the final eluting peak

HPLC analysis aims to optimise
resolution in the minimum amount of time.

Run times less than 15 minutes are ideal, up to 30 minutes is acceptable
Defined by the mass/concentration of solute analysed by a detector which yields signal:noise = 2:1
Is a measure of how effectively the analytes are
separated by the column/chromatographic conditions.

An Rs value of 1.5 or greater indicates total separation of peaks; therefore each peak can be accurately and individually assessed.

Resolution is directly affected by efficiency, selectivity and retention - and indirectly affected by all parameters which influence the aforementioned.
'the king of HPLC analysis'
Is a description of peak shape deviation with respect to a symmetrical peak.

Peak tailing is associated with poor resolution and inaccurate quantitation of peaks.

The 'A' and 'B' terms in the peak tailing equation denote the front and back half-widths at 10% of the peak height.
Pressure created by the high-pressure hydraulic pumps force the mobile phase and analytes through the tightly-packed stationary phase.

HPLC backpressure should not exceed 4000 psi
UPLC, 9000 psi

Pressures in excess of these values risk machine failure
overcomes
this is an indication of the migration rate of an analyte under specific chromatographic conditions

ideally k < 15
a measure of a chromatographic system/method's ability to identify all components present in the sample.

selectivity values of (a) < 1 are not acceptable
a measure of the dispersion of the analyte band as it progresses through the column.

High plate numbers increase efficiency by reducing peak width and consequently the potential for peak overlap

Plate counts (N) of 10,000 or greater are acceptable
the ionisation state of a compound is dependent on the pH of the solvent and its pKa

this information can be used to manipulate retention and enforces why being aware of the properties of the analytes is useful

ionised molecules are not retained
t0 represents the 'dead time'; the amount of time an unretained compound takes to elute from the column
tR represents retention time; how long it takes for an analyte to elute from a HPLC column
The area under the curve is directly proportional to the concentration of analyte in the injected sample
viscosity is 'force per unit area resisting uniform flow'

increases in viscosity result in an increase in backpressure
This is the speed at which the mobile phase travels through the column
decreases the amount of silanol groups therefore decreasing peak tailing
metal ions within silica are responsible for peak tailing
a result of the many paths a molecule can travel through the random arrangement of stationary phase particles, with each path varying in distance
this is observed when the band of analyte molecules disperse in random directions due to the concentration gradient being lower at the band perimeter, resulting in a broader and less concentrated band
all molecules will interact with the stationary phase in a unique fashion

If i compound is susceptible to pore penetration, rentention is increased and the band is broadened
decreasing particle size = decreased eddy diffusion and consequently reduces HETP
increasing temperature results in a decreased tR

kinetic energy is increased and interactions occur more rapidly and briefly between analytes and the stationary phase

increasing temperature also corresponds to a decrease in viscosity and backpressure as the velocity of the solvent molecules increase and less intermolecular interactions occur
the longer the column, the longer a compound will take to elute

Increases in column length correspond to increases in both tR and t0
Mobile phase velocity is inversely proportional to AUC.
As the speed of the mobile phase increases, molecules bypass the detector resulting in an inaccurately low AUC
different ligands will interact with different compounds variably and hence tR is dependent on the analyte's affinity for the ligand
AUC is at a maximum when the lamp wavelength matches the maximum absorbance of the analyte

When using photo-diode array (PDA), all analytes are subject to their max absorption wavelength and will yield the max potential AUC

When conducting analysis at a fixed wavelength, a maximum AUC will not be obtained unless the analyte has a maximum absorption at the delivered wavelength.
greater carbon load = more potential interactions to be made (between a retained compound and the stationary phase) and hence increased tR
decreasing %B corresponds to an increase in viscosity and consequently backpressure (aqueous solvent is more viscous than organic and requires more pressure to push through the column)
= explanation/justification
mini with text
Different molecules interact with different solvents in unique ways. This can modify tR in an unpredicatable manner
Increasing the speed of the mobile phase decreases the time spent by compounds in the column (t0 decreases)
Increasing the speed of the mobile phase decreases the time spent by compounds in the column (tR decreases)
Dominates changes in retention time.

As %B decreases, the mobile phase becomes less polar and hydrophobic analytes will begin to interact with the non-polar stationary phase to a greater extent, increasing retention time and separation of compounds
comes about due to resistance of the mobile phase to travels through the incredibly small and tightly-packed spaces between mobile phase beads
= Mechanical Parameter
= Chemical Parameter
% B range
(at a thick white line junction)
= Main Link
A 10% decrease in %B corresponds to 2-3 x increase in retention factor
area under the curve increases proportionally (linearly) with an increase in injection volume
when particle size is decreased, backpressure is increased as a result of the solvent being being more difficult to force through
Retention time of analytes are reduced at higher flow rates (ref being 1.5mL/min) as the compounds are being pushed through the column faster, resulting in faster elution
a decrease in column diameter (reference diameter = 4.5) corresponds to a reduced retention time as a result of an increased linear velocity and faster elution of compounds
Increasing flow rate (which increases LV) decreases the area under the curve as the detector begins to 'miss' molecules due to the speed of the mobile phase (molecules passing through before they can be detected
the green line with the circles represents retention time of a compound in a 250mm column (ref = 150). Retention time increases proportionally to the increase in column length as the analytes/mobile phase have a longer distance to travel, therefore eluting later. The extra time spent in the column also allows for more compound/stationary phase interactions to occur
The green line with circles represents peak 7 when run through a UPLC. The length of the column and the diameter are lower than the standard reference HPLC therefore compounds reach the detector faster
An increase in linear velocity corresponds to an increased HEPT and a decreased N via mass transfer effects
different solvent types exhibit varying degrees of intermolecular interactions and as a result, their viscosities vary
As pKa is temperature dependent, the extent of ionisation of compounds will vary with changes in temperature.

As a result acid/base and ulterior interactions between compounds themselves and between compounds and the mobile phase will vary, modifying the extent of retention
larger column diameters result in an increase in backpressure as greater mobile phase volumes within a column require more pressure to transport through
longer column lengths result in an increase in backpressure as greater mobile phase volumes within a column require more pressure to transport through the column
Higher flow rates result in a magnified backpressure for it requires more pressure to push a larger volume of mobile phase through the column in the same amount of time
increasing %B results in a linear increase in peak height and a logarithmic decrease in peak width
delta phi
column diameter increases band broadening as the compound band is less restricted to dispersion
Solvent type, solvent composition, solvent pH, temperature and ligand type all affect selectivity through tR
= Important Link
=3
20uL
60-90
amb - 50 degrees
254 nm
1.5mL/min
C18
5uM
4.6 mm
150 mm
0.05 g/mL
MeOH/ACN/H2O
15 min
2-15 mins
approx. 1 min
B/A < 2
acceptable sensitivity = signal:noise = 10:1
>10,000
>50,000
0.1-0.8
Full transcript