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AND RUN TIME <15mins!

Is resolution any better?

STEP 3 (G)

4.6mm Diameter

3.2mm Diameter

STEP 4 (G)

250mm Column

150mm Column

5%-100%B range over 17 mins

DIAMETER

GRADIENT

100mm Column

LENGTH

Use the produced scouting gradient to

estimate new %B range

Perform a run with new parameters.

Run a 'scouting gradient' using the same mobile phase composition as in isocratic, but vary composition from 5%B-100%B over 20 mins

No?

5um Particle Size

If the analytes elute more than 20% after the end of the gradient, use a stronger organic Solvent, or less retentive stationary phase, to allow better mobile phase retention

3.2um Particle Size

50%-100% B range, over 17 minutes

Column Volume

PARTICLE SIZE

C18

If resolution still unacceptable, alter chromatographic parameters such as Flow rate, column dimensions and gradient time as expressed by:

Altered by changing

column dimensions

Is DELTA tG < 0.25tG?

Most commonly used ligand type,

usually offers good separation, use

this if you don't know where to start

Yes

YES!

Is res>1.5?

Is K>15?

STEP 2

No?

Choose between Gradient

or Isocratic first run

Lucky, you're done!

Now just VALIDATE

(STEP 9)

Gradient Time (tg)

FLOW RATE

Yes?

Gradient Time

FLOW RATE

k*

YES!

k*

Has altering these parameters

given you Resolution >1.5?

STEP 1

Yes!

Select stationary Phase

OTHER

GREAT! Change the linear profile

of the run back to the isocratic

method, and finish up!

ISOCRATIC

Done!

Move to

validation

(step 9)

Depending on analyte properties,

you may chose a more specific

column to enhance separation

Yes!

Pillars of Creation

More specific choices of stationary phase are available to offer better separation of dipolar,

basic/acidic, or neutral analytes.

Is Resolution >1.5?

NO

Perform a first run with 100%B

Chose this option if specific analytes will have more optimized separation, or if the regular c18 column is not good enough

No...

That's to be expected

Is K>3

Chromatogram with PFP

Still not perfect?

STEP 3 (I)

No

Try altering the stationary

phase and starting again..

ALTER RETENTION

Chromatogram with Aqueous C18

That's alright, still

plenty to optimize

Alter retention factor (k) by

changing %B in accordance to

the K rule, until you get a k~10.

Chromatogram with IBD

Chromatogram with BiPhenyl

STEP 4 (I)

K RULE

ALTER SELECTIVITY

A change of %B by 10% will

produce an inversely proportional change

in retention factor, by a factor of 2-3

GOOD RESOLUTION?

HOW TO WORK IT OUT:

Change organic solvent type, use a

nomogram to determine isoelutropic %B

of different solvents

Calculate the retention factor of the previous run by taking the t0 as the beginning of the unresolved peak, and the rt of the final peak as the end of the unresolved peak. Now figure out how much %B should change to produce the desireable retention factor.

Perfect!

Nomogram

Res>1.5?

K>15 ?

Lucky, you're done!

Now just VALIDATE

(STEP 9)

No?

Chromatogram with 40% ACN

Keep going!

Good?

NGC 7293-God's Eye Nebula

Finished!

Move to

Validation

(STEP 9)

STEP 5 (I)

Further alter Selectivity

Selectivity can be further changed by

altering chemical parameters such as:

  • Temperature
  • %B (Mixture of solvents)
  • pH

Does not have a huge impact on

resolution as a 1C change in temp only

changes k by 1-2%. There is usually an

optimum temperature depending on analytes

This occurs as the increased temperature decreases the viscocity of the mobile phase and the exchange between mobile and stationary phase of the

analytes increases.

This also has the added effect of reducing

peak tailing as there is less mass transfer

(Efficiency increases)

TEMPERATURE

T=50 C

pH

Solvent Mixture

The more the anayltes in the sample are

unionized, the better their retention.

(As there is greater affinity to the

non-polar stationary phase)

pH=7

To select mixture, use solvent triangle:

Mixture = 40:20 (MeOH:ACN)

HPLC Method Development

Resolution>1.5?

K>15?

Yes

Validation

Time

(STEP 9)

No...

DONE!

Don't give up just yet

SYSTEM SUITABILITY

What:

According to the USP, system suitability is used to verify that the chromatographic system is adequate for intended analysis

Retention time: The peak should be well resolved from the void volume generally k>2.0

How:

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.

NGC 1976-Orion Nebula

You have successfully designed a robust and effective method! Go celebrate!

ROBUSTNESS

REPEATABILITY

What:

The analytical procedure's capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

How:

PLACKETT BURMAN

Acceptance criteria: Meet system suitability Rs and %RSD requirements for all experiments and samples (≤20% difference from the original method conditions).

What:

Expresses as the precision under the same operating conditions over a short interval of time.

How:

Minimum of 6 determinations at the 100% level

Acceptance criteria: RSD ≤2%

NGC 6543-Cat's Eye Nebula

INTERMEDIATE

PRECISION

Stefan Nebl

What:

Express within-laboratory variations.

How:

Studies should include varying days, analysts, equipment. Acceptance criteria: RSD≤2% on individual basis of 2% (RSD ≤% overall)

STEP 6 (I)

PRECISION

REPRODUCIBILITY

A function of all; repeatability,

intermediate precision and reproducibility

ALTER EFFICIENCY

What:

Ability reproduce data within the predefined precision at two different labs

How:

Acceptance criteria: RSD≤2% on individual basis of 2% (RSD ≤% overall)

By altering mechanical and equipment

parameters, the efficiency of the chromatogram

can be improved dramatically

ACCURACY

What:

Closeness of the test results obtained by the method to the true value.

Should be established across specified range of analytical procedure.

How:

Should be assessed using a minimum of 3 concentration levels, 50%,100%,150% each in triplicate (total of 9 determinations)

Should be reported as:

Percent recovery of known amount added

Acceptance criteria: Average recover 98-102%

“We are all connected; To each other, biologically. To the earth, chemically. To the rest of the universe atomically.”

― Neil deGrasse Tyson

LOQ

LOD

What:

To find the lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision.

How:

Continue diluting the 12.5% until :

Estimated by Signal to Noise Ratio of 10:1.

Change Flow rate

What:

Lowest amount of analyte in a sample that can be detected but not necessarily quantitated.

How:

Continue diluting the 12.5% until :

Estimated by Signal to Noise Ratio of 3:1.

Aim is to minimize Plate Height (HETP), thus maximizing the theoretical number of plates (N) to increase the value of k! This is tricky however as there is an optimum value achievable, must find experimentally. (Produce Van Deemter)

Improvement of efficiency minimally improves resoluton, mostly just for optimization.

Increased flow rate also decreases run time though!

NGC 4594-Sombrero Galaxy

Optimum!

Some stars shine forever

YEP!

Good, finish up

by validating

(STEP 9)

HETP

RANGE

What:

"The interval between the upper and lower concentrations of analyte in the sample that have been demonstrate to have a suitable level of precision, accuracy, and linearity.

Normally derived from Linearity studies.

Established by confirming that the method provides acceptable degree of linearity, accuracy, and precision."

How:

Acceptance Criteria: r2≥0.99

50% to 150% of test Concentration

No?

Chromatogram at multiple flow rates (3.5 ,3.0, 1.5, 0.5)

Go on to alter column dimensions..

STEP 7 (I)

LINEARITY

Further alter efficiency

through column dimensions

What:

Changing analyte concentration should produce a proportional and accurately predictable response.

How:

Produce tests with varying levels of analyte (150%, 100%, 50%, 25%, 12.5%) and plot the responses. The slope of the graph should fit within an r^2 value of 0.99

Changing column dimensions can affect both the resolution and the efficiency of the resultant chromatogram

Also occurs via optimization of HETP

SPECIFICITY

DIAMETER

What:

To ensure that the peak in question is due to the actual analyze and not "contaminated" with any impurities/degradation products

How:

Prove that degradation products don't elute in the same region as the peak in question. Produce this by forcing degradation of the analyte via: Heat, Humidity, Acid Hydrolysis, Base Hydrolysis, Oxidation, Light

DIAMETER

LONGITUDINAL DIFFUSION

BAND BROADENING

EFFICIENCY

Eta Carinae

SEPARATION

LENGTH

STEP 9

NGC 5139-Omega Centauri

LENGTH

METHOD VALIDATION

HETP

Resolution still not

good enough?

EFFICIENCY

Try altering particles

Necessary to demonstrate that the procedure you've developed is suitable and effective. Data that supports the use of your method must be gathered, as outlined in the following steps.

SEPARATION

Particle Size

and Shape

Size

HETP

Spherical particles preferable over

irregular as the more spherical, the

better the efficiency, column durability

and the lower the backpressure

YES!

EFFICIENCY

1,000,000 YEARS DUNGEON!

About time..

now for

Validation!

(STEP 9)

Try a gradient approach...

(step 2)

SEPARATION

NO!

STEP 8 (I)

Acceptable

Injection Volume

If it's still just below what

you need, try altering the

injection volume..

Is resolution finally

what you need?

This is just for optimization as the effect is minimal at best! When injection volumes exceed 20uL, peak width increases quite a bit, if need be, increase sample concentration, and decrease injection volume.

FINALLY!

Now just to

VALIDATE!

How's resolution now?

NGC 224-Andromeda Galaxy

pKa: 9.54 logP: -1.07

logS: -1.48 PSA: 58.2A

HBA: 2 HBD:2

Rotatable Bonds: 0

Solubility: 3600mg/L

MP: 338C

URACIL

STEP 0

SULINDAC

pKa: 4.7 logP: 3.42

logS: -4.2 PSA: 54.4A

HBA: 3 HBD: 1

Rotatable Bonds: 4

Solubility: 3000mg/L

MP: 183C

IDENTIFY COMPOUNDS

PIROXICAM

pKa: 6.3 logP: 3.06

logS: -4.16 PSA: 99.6A

HBA: 5 HBD: 2

Rotatable Bonds: 2

Solubility: 23mg/L

MP: 199C

Assess chemical properties such as: solubility, pKa, PSA, HBD/HBA etc.

KETOPROFEN

pKa: 4.45 logP: 3.12

logS: -3.7 PSA: 54.37A

HBA: 3 HBD: 1

Rotatable Bonds: 4

Solubility: 51mg/L

MP: 94C

NAPROXEN

pKa: 4.15 logP: 3.18

logS: -4.16 PSA: 46.5A

HBA: 3 HBD: 1

Rotatable Bonds: 3

Solubility: 15.9mg/L

MP: 153C

DICLOFENAC

PHENYLBUTAZONE

pKa: 4.15 logP: 4.51

logS: -4.8 PSA: 49.3A

HBA: 3 HBD: 2

Rotatable Bonds: 4

Solubility: 2.37mg/L

MP: 284C

pKa: 4.5 logP: 3.16

logS: -3.3 PSA: 40.6A

HBA: 2 HBD: 0

Rotatable Bonds: 5

Solubility: 47.5mg/L

MP: 105C

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