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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

Choose one

CHANGE

Particle Size

ISOCRATIC

NO

GRADIENT

HPLC METHOD DEVELOPMENT FLOW CHART

No?

CHANGE

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

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).

SPECIFICITY

How efficient was the HPLC method is separating the compounds of interest with achieving the set goals. (i.e Resolution)

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

- Assuming values,

K, run time, selectivity and backpressure

are ideal.

- Resolution is the only goal left.

- jump to changing 'efficiency' to achieve improved resolution.

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

ALMOST!

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

No?

Flow rate 1.5 ml/min

  • peak tailing and low efficiecny remains
  • no change in dimensions.
  • decreased tR
  • change in LV
  • HETP

LOD & LOQ

Flow rate

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

Changes in Linear velocity affects the amount of time in which the compounds spend in the detector.

  • Width (AUC)
  • longitudinal diffusion

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

CHANGE

ALL GOOD!

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)

  • 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

DONE

COLUMN DIAMETER: 4.6 mm

COLUMN DIAMETER: 3.2 mm

ORGANIC SOLVENT

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

Another option to alter solvent strength is to use a mixed solvent system. Therefore combining two organic modifiers as %B.

e.g MeOH:ACN

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

Exploring retention factor (K)

- 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.

At this point the only response that needs perfecting is RESOLUTION. (Rs >1.5)

Column 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

- No evident retention (no analysis) for these 7 compounds

- Non-polar compounds eluted with void volume t0

( compare back to reference on the separation of brown and green peak, swap of blue and pink peak's position)

Peak with band broadening

Disproportional changes in diameter, and flow rate will affect Linear Velocity.

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.

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

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.

ACCURACY

Efficiency enhances resolution through changes in column parameters and theoretical plate number.

ALL GOOD!

%B

GOALS

No?

REFERENCE

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)

ALL GOOD!

2 < K< 20

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

GOALS

Method Validation

Rs >1.5

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

2 < K< 20

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

Dimensions

USE FIRST

Selectivity > 1

SELECTIVITY

EFFICIENCY

CHANGE

  • 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

Rs >1.5

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

Best to alter one parameter, before combining two changes to see their individual effect

  • Long C18 hydrocarbon tail as the bonded phase (increased hydrophobic interactions)
  • Expected to interact with non-polar compounds
  • C18 hydrocarbon chain, minus a methyl group with the addition of a polar group.
  • increased Dipolar and acidic property of ligand

B

A

Run time < 15 mins

Particle size 5 um

- lower efficiency

- loss of sensitivty in peaks

Length: 150mm

Diameter: 4.6mm

particle size: 5μm

  • 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)

Selectivity > 1

DONE

ACCEPTANCE CRITERIA: Average recover 98-102%

Backpressure < 4000psi

  • Highly electronegative aromatic ring as the bonded phase
  • 5 Fluorine groups increase the bascity of the ligand.
  • C-F bonds increase Dipolar property
  • Two aromatic rings
  • Hydrophobic effect
  • Delocalised electrons in the continuous Pi cloud increases Dipolar interactions.

pH

Run time < 15 mins

The 2pH rule is used to explain the ionisation state of analytes 2 pH units above and below their pKa.

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.

Backpressure < 4000psi

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.

Ambient to 50 degrees celcius

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

  • 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

PRECISION

TEMPERATURE

INCREASE

CHOOSE STATIONARY PHASE LIGAND

Temp should remain below the boiling point of the mobile phase liquid

Do not exceed the column's max temp.

Change in temp alters viscosity of MP and decreases interaction time (run time).

- 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

How?

Column Length: 100 mm

DONE

  • lower plate number
  • lower efficiency
  • shorter run time
  • low selectivity

" The measure of the degree of agreement among test results when the method is applied repeatedly to multiple samplings of a homogeneous sample"

- USP

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

- Multiplying factor effect [(xK= KB+10% /KB) is between 2 and 3 ]

- Decrease in %B by 50% has increased retention (accordian effect)

  • Higher plate number
  • Higher efficiency
  • improved selectivity
  • dissapearance of one peak?

No?

Column Length

Column length directly affects efficiency (N) of analysis, through the theoretical plate number of a column.

longer column = greater N

GOALS

Effect on Back pressure:

- Decreasing %B and increasing %A will increase viscosity.

-Increase in backpressure

2 < K< 20

ALL GOOD!

CHANGE

Rs >1.5

Selectivity > 1

Run time < 15 mins

%B

Backpressure < 4000psi

DONE

100%B

IMPROVING RETENTION 2 <K< 20

Flow rate should be chosen according to diameter and length.

Standard value: 1.5 ml/min

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]

SET FLOW RATE

Acceptance Criteria: RSD ≤2%

ALL GOOD!

SET CONDITIONS (mechanical parameters)

- Flow rate

- Column stationary phase/dimensions

- Detector (UV-vis 254nm)

- Temperature

- Elution type

DONE

INTERMEDIATE PRECISION

GOALS

IDENTIFY

COMPOUNDS

  • 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)
  • Using the scouting gradient, if compounds elute after 20 mins a change in column is required.
  • A less retentive column would be required

2 <K< 20

Rs> 1.5

Backpressure <4000

tR (run time) < 15 mins

ALMOST!

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)

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

CHOOSE DETECTOR

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

SCOUTING GRADIENT

  • Starting point for ISOCRATIC or GRADIENT HPLC analysis.

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

REPRODUCIBILITY

Organic Range

Look out for changes in Peak tailing!

  • Precision in achieving the same results using different labs

Δtg < 0.25 x tg

Acceptance Criteria: RSD ≤ 2% on individual basis of 2% ( RSD ≤ Overall)

ELUTION TYPE

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

GOALS

Flow rate

2 < K< 20

- 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%

Rs >1.5

ALL GOOD!

Selectivity > 1

Improved Sensitivity?

Run time < 15 mins

Backpressure < 4000psi

tg: 15 mins

- longer run time

SET TEMPERATURE

DONE

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

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

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