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APPLIED ANALYTICAL METHODS

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by

Dimuthu Wickramasinghe

on 26 April 2015

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Transcript of APPLIED ANALYTICAL METHODS

APPLIED ANALYTICAL METHODS
CONCEPT MAP

MOBILE PHASE
SAMPLE
COLUMN
SOLVENT COMPOSITION
SOLVENT TYPE
SOLVENT pH
EQUIPMENT
The solvent is comprised of an aqueous phase (%A) and an organic phase (%B).
Changing the proportions of each phase will inherently change the overall polarity of the solvent, as well as the overall viscosity of the solvent.
This will have an impact on the interactions between the
SAMPLE
and the
MOBILE PHASE
, which affects retention time, retention factor and selectivity
The change in viscosity has an impact on the flow rate of the
MOBILE PHASE
, which also affects retention time.
SELECTIVITY
RETENTION TIME
RETENTION FACTOR
(FACTOR FROM FUTURE CYCLE)
Similar to Solvent composition, changing the type of organic or aqueous phase, will likely influence many factors in the experiment
Changes to solvent type can possibly change; pH, polarity, charge of sample, viscosity.
This will have an impact on the interactions between the
SAMPLE
and the
MOBILE PHASE
, which affects retention time, retention factor and selectivity
FLOW RATE
Similar to Solvent composition, changing the type of organic or aqueous phase, will likely influence many factors in the experiment
Changes to solvent type can possibly change; pH, polarity, charge of sample, viscosity.
This will have an impact on the interactions between the
SAMPLE
and the
MOBILE PHASE
, which affects retention time, retention factor and selectivity
COMPOUND
The compound itself inherently affects a variety of factors in HPLC
Depending on it's own properties, it could have different effects with the
MOBILE PHASE
and
COLUMN
Properties such as; polarity, ionisation, aromaticity and hydrophobicity impact the number and strength of interactions with both the
MOBILE PHASE
and
COLUMN
.
These interactions influence; retention time, retention factor and selectivity.
INJECTION VOLUME
The main factor that injection volume affects is sensitivity, increasing the amount of sample injected increases the peak height and area
This is because the
detector
is better able to distinguish component peaks from any background noise.
SELECTIVITY
RETENTION TIME
RETENTION FACTOR
(FACTOR FROM FUTURE CYCLE)
(FACTOR FROM FUTURE CYCLE)
LIGAND
The Ligand is the most important part of the HPLC column, they are the beads.
These beads are tightly packed together within the column, the ligand "arms" "wave" about to interact with incoming sample particles traveling through the column
The type of ligand use affects the run time by either retaining the sample depending on it's affinity for it.
Making changes to the type of ligand used in the column will affect; Retention time, retention factor, selectivity.
COLUMN DIAMETER
COLUMN LENGTH
Changing the diameter of the column has a similar effect to that of changing the width of a hose.
This change will likely result in a change of flow rate.
In addition to flow rate, back pressure will be changed for example, decreasing the diameter of the column will result in a larger back pressure due to the same amount of eluent is pumped through a smaller surface area. In order to achieve this, higher velocity is required which increases back pressure.
Changing the length of the column does not necessarily affect flow rate, however it will inherently affect run time.
This affects Retention time, but not retention factor, back pressure is also affected.
SELECTIVITY
RETENTION TIME
RETENTION FACTOR
FLOW RATE
BACK
PRESSURE
PARTICLE SIZE
The size of the particles within the column have a direct impact on the HPLC run and resulting chromatogram
Reducing particle size reduces the effect of Eddy Diffusion this is because the smaller the particles create smaller alterations to the path and therefore less band broadening.
EDDY DIFFUSION
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