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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:
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)
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