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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
CHANGE
NO
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
Acceptance criteria: Meet system suitability Rs and %RSD requirements for all experiments and samples (≤20% difference from the original method conditions).
How efficient was the HPLC method is separating the compounds of interest with achieving the set goals. (i.e Resolution)
Through continuous dilution of original sample. Conduct tests using a at least 5 concentration levels for accuracy (150%, 100%, 50%, 25%, 12.5%)
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:
Acceptance Criteria r2≥0.99
50%-150% of [test]
Flow rate: 0.75 ml/min
- evident peak tailing
- low efficiency
Conditions
pH: 3
B type: mix
%B: 2040
Column: C18
Column Dimensions: 150x4.6x5
[sample]: 0.1 mg/ml
injection Volume: 20ul
Flow rate 1.5 ml/min
Flow rate
LOD (limit of detection)
Changes in Linear velocity affects the amount of time in which the compounds spend in the detector.
When column diameter and flow rate change disproportionally, the retention time of the compounds will also change. (Linear Velocity)
Note: Increasing Flow rate, increases back pressure of the HPLC, keep flow rate at a value that maintains bp < 4000 psi
50% ACN
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
65% MeOH
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
LOQ (limit of quantification)
COLUMN DIAMETER: 4.6 mm
COLUMN DIAMETER: 3.2 mm
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
- 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
- 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.
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
Peak with band broadening
Disproportional changes in diameter, and flow rate will affect Linear Velocity.
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 without band broadening
Efficiency enhances resolution through changes in column parameters and theoretical plate number.
%B
How close actual result from the method is to expected result, the true value.
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
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
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
LIGAND
Particle size: 3 um
- High efficiency
USE FIRST
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
B
A
Particle size 5 um
- lower efficiency
- loss of sensitivty in peaks
Length: 150mm
Diameter: 4.6mm
particle size: 5μm
ACCEPTANCE CRITERIA: Average recover 98-102%
Less convoluted route of analytes in the column, incresases efficiency.
Influences the path of the analyte in the statiotnary phase as it travels with the mobile phase flow.
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
Column Length: 100 mm
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: 150 mm
Column length directly affects efficiency (N) of analysis, through the theoretical plate number of a column.
longer column = greater N
IMPROVING RETENTION 2 <K< 20
Flow rate should be chosen according to diameter and length.
Standard value: 1.5 ml/min
Acceptance Criteria: RSD ≤2%
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
RUN TIME > 20 mins
Acceptance Criteria: RSD ≤ 2% on individual basis of 2% ( RSD ≤ Overall)
Organic range: 5-100%
- wider organic range
- Run time is longer
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 50-100%
- shorter organic range
- analytes elute earlier
- run time is decreased
Conditions:
Mobile Phase: ACN & Water
Organic range: 5-100%
Run time: 20 mins
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
Organic Range
Look out for changes in Peak tailing!
Δtg < 0.25 x tg
Acceptance Criteria: RSD ≤ 2% on individual basis of 2% ( RSD ≤ Overall)
Δtg < 0.25 x tg
ISOCRATIC ANALYSIS IS POSSIBLE
ti = elution time of the initial peak
tf= elution time of final peak
Flow rate: 1.5 ml/min
- t0 is earlier
- shorter run time
- increased selectivity
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 ml/min
- t0
- longer run time
- increased efficiency
-decreased selectivity
Flow rate
- 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%
Improved Sensitivity?
tg: 15 mins
- longer run time
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: 10 mins
- shorter run time
- t0 did not change
- change in K
- selectivity is constant
Rs > 1.5
improve run time and sensitivity?
Gradient time (tg)
Column length: 150 mm
- Run time approx 14 mins
- t0: ~2.5mins
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: 250 mm
- Run time approx 15 mins
- t0: ~3.2 mins
- improved selectivity between peak 4 &5.
Column void volume (Vm)
LEGEND
Further improved
important information
isocratic analysis
Gradient analysis