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HPLC METHOD DEVELOPMENT FLOW CHART

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Iasmin Inocencio

on 1 June 2015

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Transcript of HPLC METHOD DEVELOPMENT FLOW CHART

START

SCOUTING GRADIENT
ISOCRATIC
GRADIENT
IDENTIFY
COMPOUNDS
SET CONDITIONS (mechanical parameters)
- Flow rate
- Column stationary phase/dimensions
- Detector (UV-vis 254nm)
- Temperature
- Elution type
CHOOSE STATIONARY PHASE LIGAND
Dimensions
Length: 150mm
Diameter: 4.6mm
particle size: 5μm
SET FLOW RATE
CHOOSE DETECTOR
SET TEMPERATURE
Note:
- Higher temps reduce viscosity. This lowers backpressure allowing higher flow rates to be used.
- Reduction in viscosity, means less %B is required in MP.
- Temps must be lower than boiling point of mobile phase
- check column instructions for maximum temperature

100%B
ELUTION TYPE
GOALS
2 <K< 20
Rs> 1.5
Backpressure <4000
tR (run time) < 15 mins

2 < K< 20
Rs >1.5
Backpressure < 4000psi
Run time < 15 mins
%B
DONE
Effect on Back pressure:

- Decreasing %B and increasing %A will increase viscosity.
-Increase in backpressure

IMPROVING RETENTION 2 <K< 20
- No evident retention (no analysis) for these 7 compounds
- Non-polar compounds eluted with void volume t0
Exploring retention factor (K)
Flow rate should be chosen according to diameter and length.

Standard value: 1.5 ml/min
Further improved
CHANGE
Selectivity > 1
GOALS
2 < K< 20
Rs >1.5
Backpressure < 4000psi
Run time < 15 mins
Selectivity > 1
GOALS
SELECTIVITY
- Multiplying factor effect [(xK= KB+10% /KB) is between 2 and 3 ]
- Decrease in %B by 50% has increased retention (accordian effect)
ORGANIC SOLVENT
pH
TEMPERATURE
INCREASE
Compare to reference
pH 3-7 resulted in change of peak order (2&3, 6&7 swapped)(ionisation of two peaks causing them to elute sooner than before)
The separation between peaks 3&4 did not improve
Ambient to 50 degrees celcius
Decrease in run time ( ~ 7mins down to ~5.5mins)
Evident change in selectivity of a few peaks, caused overlap.
Reference chromatogram was at an ambient temperature

How?
Choose one
Best to alter one parameter, before combining two changes to see their individual effect
Changing
organic solvent
can change the solvent strength and hence elution strength of the mobile phase. This could also alter interaction with analytes and enhance selectivity.

e.g use MeOH instead of ACN
Another option to alter solvent strength is to use a
mixed solvent
system. Therefore combining two organic modifiers as %B.

e.g MeOH:ACN
pH of the mobile phase controls the ionization state of the compounds in the column, may it be on the stationary phase or the analytes.
Therefore it is important to check the suitable pH values of the column in use.


The 2pH rule is used to explain the ionisation state of analytes 2 pH units above and below their pKa.
Do not exceed the column's max temp.

Change in temp alters viscosity of MP and decreases interaction time (run time).
Temp should remain below the boiling point of the mobile phase liquid
2 < K< 20
Rs >1.5
Backpressure < 4000psi
Run time < 15 mins
Selectivity > 1
GOALS
EFFICIENCY
Column Length
Particle Size
Column Diameter
ALL GOOD!
No?
- Assuming values,
K, run time, selectivity and backpressure
are ideal.
- Resolution is the only goal left.
- jump to changing 'efficiency' to achieve improved resolution.
- Assuming values,
K, run time, selectivity and backpressure
are ideal.
- Resolution is the only goal left.
- jump to changing 'efficiency' to achieve improved resolution.
Temp also changes pKa of
compunds in the column, which changes ionisation state leading to a decrease or increase in run time
Detector chosen must be based on the analyte. Inability for detector to absorb the analyte at interest, results in no peak and therefore no analysis.
( 254nm; UV-vis)
2 < K< 20
Rs >1.5
Backpressure < 4000psi
Run time < 15 mins
Selectivity > 1
GOALS
DONE
Increasing the LV, will decrease band broadening of the mobile phase within the column. Therefore there is less dispersion of the analytes in the column and they are able to elute at a higher concentration.
(Longitudinal diffusion)
Peak with band broadening
Peak without band broadening
Method Validation
RUN TIME > 20 mins
Starting point for ISOCRATIC or GRADIENT HPLC analysis.

Physiochemical properties
PSA, LogP, LogD, solubility, pKa

It is important to determine whether the analytes of interest are acidic or basic. Therefore the correct mobile pH can be applied to keep the analytes in the favourable ionisation state.
For reverse HPLC, UNIONISED is most favourable due to non-poalr stationary pahse interactions.

For acidic analytes pH 3 is good (below 2 is not ideal for the bonded phase)

For basic analytes pH 7 is good (above pH 8 destroys stationary phase and potentially column)

Remember: stronger the acid, smaller the pKa
stronger the base, the larger the pKa
Using the scouting gradient, if compounds elute after 20 mins a change in column is required.
A less retentive column would be required

Conditions:

Mobile Phase: ACN & Water
Organic range: 5-100%
Run time: 20 mins


Δtg < 0.25 x tg
ti = elution time of the initial peak
tf= elution time of final peak
Δtg < 0.25 x tg


ISOCRATIC ANALYSIS IS POSSIBLE
NO
Flow rate
LIGAND
A
B
- Performing a gradient run, start organic range at the %B in which the first peak from the scouting gradient analysis eluted.

e.g In the chromatorgram to the left, it is 60%
When performing an isocratic run, the %B of the mobile phase is estimated from from the scouting gradient.

Use the %B that corresponds to tf(average)= Δtg/2




%B
Example of a 55%B 45%A analysis, using a %B composition of 40:20 (MeOH:ACN)

- MeOH is decreases the solvent strength of B
- postivie effect on selectivity.
Nomogram was used to calculate a %B composition that mainted the isoelutropic quality of the analysis. This allows a mobile phase of the same strength to be used so that previously achieved results remain the same (K), only altering the interactions of the analytes to change selectivity.
( compare back to reference on the separation of brown and green peak, swap of blue and pink peak's position)
REFERENCE
Organic Range
Flow rate
Gradient time (tg)
Column void volume (Vm)
Look out for changes in Peak tailing!
Rs > 1.5
improve run time and sensitivity?
CONDITIONS
Column dimensions: 100x4.6x5
Temp: 20 degrees celsius
wavelength: 254 nm
%B: 20:40 ACN:MeOH
Flow rate: 1.5 ml/min
pH: 3
Injection Volume: 20ul
[sample]: 0.1 mg/ml
tg: 10mins
Organic range: 5-100%
- wider organic range
- Run time is longer

Organic range 50-100%
- shorter organic range
- analytes elute earlier
- run time is decreased
At this point the only response that needs perfecting is RESOLUTION. (Rs >1.5)


Efficiency enhances resolution through changes in column parameters and theoretical plate number.
CONDITIONS
pH: 3
Injection volume: 20ul
[sample]: 0.1 mg/ml
temp: 50 degress C
Column dimensions: 150x4.6x5
tg: 10
Mobile phase: MeOH:ACN:H20 (40:20:40)
orgainc range: 5-100%
Temp: 50 Degrees C
Flow rate: 1.5 ml/min
- t0 is earlier
- shorter run time
- increased selectivity

Flow rate: 1 ml/min
- t0
- longer run time
- increased efficiency
-decreased selectivity
CONDITIONS
pH: 3
Injection volume: 20ul
[sample]: 0.1 mg/ml
temp: 50 degress C
Column dimensions: 150x4.6x5
Flow rate: 1.5 ml/mim
Mobile phase: MeOH:ACN:H20 (40:20:40)
Orgainc range: 5-100%
tg: 15 mins

- longer run time

tg: 10 mins

- shorter run time
- t0 did not change
- change in K
- selectivity is constant

CONDITIONS
pH: 3
Mobile phase: MeOH:ACN:Water (40:20:40)
Flow rate: 1.0 ml/min
Injection Volume: 20 ul
[sample]: 0.1 mg/ml
Column diamter: 4.6 mm
Particle size: 5
tg: 10 mins
organic range: 5-100%
Temp: amb
Column length: 150 mm

- Run time approx 14 mins
- t0: ~2.5mins
2 < K< 20
Rs >1.5
Backpressure < 4000psi
Run time < 15 mins
Selectivity > 1
GOALS
Improved Sensitivity?
important information
isocratic analysis
Gradient analysis
LEGEND
ALL GOOD!
65% MeOH
CONDITIONS
pH: 3
Temp: AMB
Mobile phase: 50:50 (ACN:Water)
Injection volume: 20ul
Flow rate: 1.5 ml/min
Column dimensions: 150x4.6.5
[sample] : 0.1 ug/l
50% ACN
CONDITIONS
pH: 3
Temp: AMB
Mobile phase: 65:35 (ACN:Water)
Injection volume: 20ul
Flow rate: 1.5 ml/min
Column dimensions: 150x4.6.5
[sample] : 0.1 ug/l
Conditions
pH: 3
Mobile phase: 50:50 (ACN:Water)
Temp: AMB
Column: C18
Dimensions: 150x4.6x5
Detector: 254nm UV-vis
Flow rate: 1.5 ml/min
injection volume: 20ul
[sample]: 0.1 ug/l
Conditions
pH: 7
Mobile phase: 50:50 (ACN:Water)
Temp: AMB
Column: C18
Dimensions: 150x4.6x5
Detector: 254nm UV-vis
Flow rate: 1.5 ml/min
injection volume: 20ul
[sample]: 0.1 ug/l
Conditions
pH: 3
Mobile phase: 50:50 (ACN:Water)
Temp: 50 Degrees C
Column: C18
Dimensions: 150x4.6x5
Detector: 254nm UV-vis
Flow rate: 1.5 ml/min
injection volume: 20ul
[sample]: 0.1 ug/l
Conditions
pH: 3
Mobile phase: 40:20:40 (MeOH:ACN:Water)
Temp: AMB
Column: C18
Dimensions: 150x4.6x5
Detector: 254nm UV-vis
Flow rate: 1.5 ml/min
injection volume: 20ul
[sample]: 0.1 ug/l
Conditions
pH: 3
B type: mix
%B: 2040
Column: C18
Column Dimensions: 150x4.6x5
[sample]: 0.1 mg/ml
injection Volume: 20ul


Flow rate: 0.75 ml/min
- evident peak tailing
- low efficiency
Flow rate 1.5 ml/min
peak tailing and low efficiecny remains
no change in dimensions.
decreased tR
change in LV
HETP


When column diameter and flow rate change disproportionally, the retention time of the compounds will also change. (Linear Velocity)
Changes in Linear velocity affects the amount of time in which the compounds spend in the detector.
Width (AUC)
longitudinal diffusion
Note: Increasing Flow rate, increases back pressure of the HPLC, keep flow rate at a value that maintains bp < 4000 psi
Column Length: 250 mm

- Run time approx 15 mins
- t0: ~3.2 mins
- improved selectivity between peak 4 &5.
CONDITIONS
pH: 3
Mobile phase: 40:20 MeoH:ACN
Temp: AMB
Flow rate: 3 ml/min
Column Length: 100mm
Particle size: 5
CONDITIONS
pH: 3
Mobile phase: 40:20 MeoH:ACN
Temp: AMB
Flow rate: 2.0 ml/min
Particle size: 5
Column Length: 100mm
Disproportional changes in diameter, and flow rate will affect Linear Velocity.
COLUMN DIAMETER: 3.2 mm
COLUMN DIAMETER: 4.6 mm
- Shorter run time
- LV must be too high as it appears to have merged three different analytes when passing the detector.
low efficiency
- solution would be to perhaps decrease the flow rate with this small diameter.
- longer run time
- With a larger column diameter and 1 ml/min higher flow rate, the middle peaks have improved seperation.
- peak widths remain broad, efficiency still low
Influences the path of the analyte in the statiotnary phase as it travels with the mobile phase flow.


Less convoluted route of analytes in the column, incresases efficiency.
Conditions
pH: 3
Mobile Phase: 40:20 (MeOH:ACN)
Temp: AMB
Flow rate: 0.5 ml/min
injection volume: 20ul
[sample]: 0.1 mg/ml
Column length: 150 mm
Column diameter: 4.6 mm
Particle size: 3 um
- High efficiency
Particle size 5 um
- lower efficiency
- loss of sensitivty in peaks
CHANGE
CONDITIONS:
pH: 3
Mobile Phase: 40:20 (MeOH:ACN)
Flow rate: 1.5 ml/min
Temp: AMB
[sample]: 0.1 mg/ml
Injection volume: 20 ul
Column Diameter: 4.6 mm
Particle Size: 5 um

Column Length: 100 mm
Column Length: 150 mm
lower plate number
lower efficiency
shorter run time
low selectivity
Higher plate number
Higher efficiency
improved selectivity
dissapearance of one peak?
Column length directly affects efficiency (N) of analysis, through the theoretical plate number of a column.

longer column = greater N
An approach that determines the suitablity and credibility of the analytical procedure. This is achieved via a planned and systematic collection of data to prove the worth of the analytical procedure outlined
SPECIFICITY
How efficient was the HPLC method is separating the compounds of interest with achieving the set goals. (i.e Resolution)
USE FIRST
DONE
ALL GOOD!
DONE
ALL GOOD!
DONE
ALL GOOD!
CHANGE
No?
CHANGE
No?
Based on the knowledge of your compounds of interest, let that be the beginning of your HPLC METHOD DEVELOPMENT ADVENTURE
Choose initial conditions to create a reference for your method.
- column (ligand and dimensions)
- Mobile phase (type, %B, pH)
- flow rate
- Temperature
- Detector wavelength
- injection volume
- sample concentration
Long C18 hydrocarbon tail as the bonded phase (increased hydrophobic interactions)
Expected to interact with non-polar compounds
Highly electronegative aromatic ring as the bonded phase
5 Fluorine groups increase the bascity of the ligand.
C-F bonds increase Dipolar property
C18 hydrocarbon chain, minus a methyl group with the addition of a polar group.
increased Dipolar and acidic property of ligand
Ultra IBD contains a Polar group bonded mid-hydrocarbon chain.
Polar group is more accessible to analytes in the column higher up the chain
large increase in acidic capabilities due to hydrophillic quality.
ability to form H bonds with other compounds (water in mobile phase)
Two aromatic rings
Hydrophobic effect
Delocalised electrons in the continuous Pi cloud increases Dipolar interactions.
LINEARITY
Determining the proportional changes of analyte concentraiton and AUC.
Linear realtionship (y = mx + b )
Through continuous dilution of original sample. Conduct tests using a at least 5 concentration levels for accuracy (150%, 100%, 50%, 25%, 12.5%)
acceptance criteria r2≥0.99
RANGE
An analysis derived from linearity studies, which investigates the interval between the upper and lower concentrations of the sample analyte which demonstrate a suitable level of:
Precision
accuracy
linearity
Acceptance Criteria r2≥0.99
50%-150% of [test]
LOD & LOQ
LOD (limit of detection)
lowest amount of analyte detected in sample.
not always quantifiable
continue dilutions from linearity tests (from 12.5 %)
stop dilution when Signal to Noise ratio is 3:1
LOQ (limit of quantification)
lowest quantifiable amount of analyte detected in sample.
suitable accuracy and precsion
continue dilutions from 12.5%
stop dilutions when Signal to Noise ratio is 10:1
ACCURACY
How close actual result from the method is to expected result, the true value.
Established upon a stated range of analytical approaches
Assessed using a minimum of 3 concentration levels (50%, 100%, 150%) X 3 ( 9 results)
Data should be measured and reported as the percent difference between mean value and true value with confidence intervals. (e.g +- 1 SD)
ACCEPTANCE CRITERIA: Average recover 98-102%
PRECISION
" The measure of the degree of agreement among test results when the method is applied repeatedly to multiple samplings of a homogeneous sample"
- USP
REPEATABILITY
Precision of method to achieve the same results over a short time interval.
identical conditions.
analysis involves minimum of 6 determinations at 100% of test [target]
Acceptance Criteria: RSD ≤2%

INTERMEDIATE PRECISION
Investigates the precision of the method when conducted under non-lab related conditions.
Test performance varies day, analyts and equipment.
Experimental design should be utilized (PB&Anova)
Acceptance Criteria: RSD ≤ 2% on individual basis of 2% ( RSD ≤ Overall)
REPRODUCIBILITY
Precision in achieving the same results using different labs
Acceptance Criteria: RSD ≤ 2% on individual basis of 2% ( RSD ≤ Overall)
ROBUSTNESS
Analytical procedure to measure the methods resistance to minor changes in parameters
Extent of effect (if any, ideally none)
PB
Acceptance criteria: Meet system suitability Rs and %RSD requirements for all experiments and samples (≤20% difference from the original method conditions).

SYSTEM SUITABILITY
Information provided by USP to determine the chromatographic system is adequate for intended analysis.

Retention time: The peak should be well resolved from the void volume generally k>2.0
Repeatability: RSD Area and tR≤ 1% for n ≥ 5 is desirable
Tailing Factor: T ≤ 2
Theoretical plates: N > 10,000
Calibration curve: R2 of 0.995
DONE
ALL GOOD!
CHANGE
No?
HPLC METHOD DEVELOPMENT FLOW CHART
ALMOST!
ALMOST!
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