Loading presentation...

Present Remotely

Send the link below via email or IM


Present to your audience

Start remote presentation

  • Invited audience members will follow you as you navigate and present
  • People invited to a presentation do not need a Prezi account
  • This link expires 10 minutes after you close the presentation
  • A maximum of 30 users can follow your presentation
  • Learn more about this feature in our knowledge base article

Do you really want to delete this prezi?

Neither you, nor the coeditors you shared it with will be able to recover it again.


Make your likes visible on Facebook?

Connect your Facebook account to Prezi and let your likes appear on your timeline.
You can change this under Settings & Account at any time.

No, thanks

HPLC Method Development Flow Chart

No description

Christopher Hendy

on 2 June 2015

Comments (0)

Please log in to add your comment.

Report abuse

Transcript of HPLC Method Development Flow Chart

HPLC Method Development Flow Chart
Be aware of the physicochemical properties of the analytes
Select a column
Select the type of run
make sure backpressure does not exceed 4000
implement scouting gradient
implement 100% organic solvent
is delta Tg < 0.25 x Tg?
calculate the % B at half delta TG and conduct an isocratic run at this %B
adjust organic range to begin at the % B of first eluted peak
is resolution > 1.5?
is resolution > 1.5?
adjust the gradient time Tg
is resolution > 1.5?
adjust the organic range
is resolution > 1.5?
reduce the length of the column
is resolution > 1.5?
change organic solvent
is resolution > 1.5?
reduce the sample injection volume
is resolution > 1.5?
re-develop the method:

-change the column

-change to isocratic conditions

-change the solvent
very close;
within 0.1 of Rs = 1.5
is K< 3?
compound is not suitable for analysis by reverse-phase HPLC
is resolution > 1.5?
reduce % B by 10% increments
is K less than 15 and resolution > 1.5?
change solvent but keep solvent strength constant with the use of a nomogram
is K less than 15 and resolution > 1.5?
is there observable/appreciable separation of all peaks after using two different mobile phases?
with the aid of a nomogram, choose an isoleluotropic solvent mixture that yields close to the target K
is K less than 15 and resolution > 1.5?
is resolution almost at target (>1.4) and run time acceptable?
Start editing efficiency.
Change flow rate. Start at the maximum flow rate, then lower in increments.
Increase column length in increments.
is resolution 1.5 or greater?

run time acceptable?
Decrease particle size
Decrease injection volume
is resolution > 1.5?
Increase temperature.
is K less than 15 and resolution > 1.5?
Adjust pH
is K less than 15 and resolution > 1.5?
re-develop the method:

-change the solvent type

-change the column

if neither of these things work,
-try a gradient method
is resolution 1.5 or greater?

run time acceptable?
is resolution 1.5 or greater?

run time acceptable?
method validation
For compound quantification, an external standard of (at least) 3 known concentration are injected and a calibration curve is derived from the AUC of each injection
Being able to prove that any peak is caused by a lone compound without interference from degradation products or impurities.
Can be confirmed by analysing varying concentrations of a compound (over 5 concentrations, 12.5% - 150%) and plotting the results.

Analysing different concentration should yield predictable results with an R squared value of 0.99 or greater.
Below this is unacceptable.
Limit of Detection
The lowest concentration of analyte within a sample that can be detected but not necessarily quantitated

Acceptance criteria: Signal:Noise = 3:1
Limit of Quantitation
The lowest concentration of analyte within a sample that can be quantified with suitable accuracy and precision

Acceptance criteria: Signal:Noise = 10:1
System Suitability
Used to verify that the chromatographic system is adequate for the intended analysis
Accuracy Tests
A measure of closeness of results yielded by a specific method to a known true value.

This is assessed using 3 concentration levels (minimum) (e.g. 50%, 100% and 150%), each in triplicate.

Reported as 'percentage recovery of known amount added'

Acceptance criteria: average recover 98-102%
A measure of the capacity of the analytical procedure to remain unaffected by small, deliberate variations in method parameters. This provides an indication of reliability during normal usage.

Plackett Burman acceptance criteria:
Meet system suitability and %RSD requirements for all experiments and samples (<20% difference from the original method conditions)
This is the interval between the lowest and highest concentrations of analyte sample that were used in the linearity experiments to confirm that the method has acceptable levels of linearity, accuracy and precision.

Acceptance criteria: R squared > 0.99

50% to 150% of test concentration.
Precision Testing

Defined by concordance of test results when the method is applied to multiple samplings of a heterogenous sample.

Measured by RSD (Relative Standard Deviation)
Acceptability Criteria:

Resolution between API and nearest impurity > 2.5

tR ~ the peak should be well-resolved from the void volume ( k > 2 )

Overall standard precision/repeatability (n>5) for AUC, tR and RSD (<1%)

Tailing factor < 2

Plate count (N) > 10,000

Calibration curve R squared of 0.995
= yes, proceed to next step
= no, change a condition
= directional arrow; shows you next step of the method or directs you to a required decision

-this arrow can be followed when there is an alternative to 'yes' or 'no'
= an adjustment or decision to be made
= something you need to pay attention to
= criteria required to advance to next step
it is important that we are aware of the polarity and pKa's of the analytes in order to a) know if reverse-phase analysis is suitable and b) assist in choosing a suitable column
This will damage the machine.

Start with a low flow rate and gradually increase.

Flow rate and back
pressure depend on
the column (and particle) dimensions.
A scouting gradient is a run with a gradient time of ~20mins over the organic range 5-100%

They help to determine if an isocratic run is possible, and if so at what %B
Handy Equations
the run time can be significantly reduced by reducing the gradient time, although peak separation must not be compromised
This does not have to be done in a linear fashion. This method is very much trial and error.
Altering the length of the column changes linear velocity and plate count

Varying column length can help to optimise linear velocity and increases the plate count at the expense of increased backpressure and tR
Every 10% decrease in %B equates to an approximate 2-3 fold increase in K. Using this knowledge, a %B can be estimated with reasonable accuracy to increase K within method limits (<15)
Doing so decreases backpressure through viscosity reduction. This will allow for a higher flow rate implementation reducing the run time and potentially optimising Vm
Through this parameter the ionisation states of the compounds can be controlled and varied.

pH can only be between 2.5 and 8. Values outside of this destroy the beads/ligand
Efficiency method modifications are implemented when resolution is very close to acceptable but still not quite there (within 0.1 of Rs = 1.5). No major changes to resolution occur here.
Altering flow rate changes linear velocity.

By optimisation of FR the theoretical plate height can be reduced, increasing the plate count and henceforth efficiency.
Doing so alters Vm and increases the plate count.

Care must be taken to adjust flow rate with length increase, for an increase in column length corresponds to an increase in backpressure.
retention factor (k)
resolution (Rs)
selectivity (a)
theoretical plate height
This is the ability to reproduce data within the predefined precision across two (or more) different laboratories
Acceptance criteria: RSD<2% on individual basis of 2% (RSD<2% overall)
Precision under the same operating conditions over a short interval of time

A minimum of 6 determinations at the 100% level
Acceptance criteria: RSD<2%
Intermediate Precision
Precision under variations within the same laboratory (days, analysts, equipment)
Acceptance criteria: RSD<2% on individual basis of 2% (RSD<2% overall)
This can be done by instigating degradation of analytes and spiking the sample with suspect impurities. Resolved identifiable peaks account for acceptable specificity
resolution > 1.5?

run time < 15 minutes?

sensitivity > 1?

backpressure < 4000 psi?
(<9000 if using UPLC)

The nomogram depicts conversions between solvent types whilst retaining the same solvent strength. For example, runs conducted with 50% ACN and 60% ACN should have approximately similar overall run times, whilst the selectivity of the two runs is likely to change.
This is due to different compounds engaging in different types and extents of interactions with different solvents based on their chemical properties.
For example, using a more acidic solvent is likely to decrease the retention time of a basic analyte due to ionisation.
Increasing temperature alters the pKa of the analytes, and to different extents.

The degree of ionisation is dependent on the chemical properties of the compound
decreasing particle size reduces eddy diffusion and band broadening by limiting the variety of paths a compound can take within the column.

doing this also increases the theoretical plate height.
this technique aims to limit longitudinal diffusion, and consequently reduce band broadening
= explanation / elaboration
changing the organic modifier allows for new interactions (and extents of) to occur between the analytes and the mobile phase; resulting in potentially major changes in selectivity
this technique aims to limit longitudinal diffusion, and consequently reduce band broadening
Successful method.
Tg denotes the time taken to get from 5%B (0 minutes) to 100% (20 minutes)
Full transcript