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HPLC Method Development
Step
- Leanne Kwok -
Decision
Path
Know your compounds:
pKa, acidic and basic functional groups
Select a column type:
What stationary phase do you want to have?
Choose a method:
Isocratic
(ISO)
100%B run
Gradient
(GRA)
Scouting gradient
Advantage of scouting gradient over isocratic 100%B:
- gives more information (ideal %B) in only one run compared to isocratic which may need two runs to get the same information
Scounting gradient
100%B
Organic range usually 5-100%B
tg usually 20 min
To ensure that all compounds are eluted with in 1-3 min
A 'potato' peak around this time
?
YES to
all
Run time < 20 min
K < 20
Rs > 1.5
NEARLY
All but Rs:
1.4 < Rs < 1.5
NEARLY
Run time < 20 min
K < 20
1.4 < Rs < 1.5
NO
to
all
Method change?
Decrease %B
ISO or GRA
To change to isocratic method a condition must be met: delta tg > 0.25xtg
Decreasing %B will increase run time and hopefully separate the 'potato' peak (from 100%B).
Can decrease B by 10% less (i.e. 100 - 10 = 90%)
This will help in calculation of multiplying factor, so we can then use the K Rule to help achieve goal of run time < 20 min and K < 20
Calculation:
RUN TIME and K GOAL
?
delta tg > 0.25xtg?
YES to
all
Run time < 20 min
K < 20
Backpressure < 4000 psi
Rs > 1.5
NEARLY
All but Rs:
1.4 < Rs < 1.5
NO
to
all
Calculate %B
(start at 5-10% less than %B calculated)
Scouting gradient allows you to calculate a %B that can best be used to start isocratic method
Calculate %B as the tR for the first peak after t0 peak
Calculate multiplying factor
Multiplying factor (MF) = k2/k1 (shown in table -> (v) column 5, row 2)
Calculate %B for desired k value:
Example calculations:
slope: (organic range / tg)
= (100%-5%/20)
= 95%/20
= 4.75% per min
tR (first peak after t0) - t0
= 19.16 - 2 = 17.16 min
tR x t0 + (%B organic range start at)
= (17.16 min x 4.75% per min)+5%
= 81.51%+5%
= 86.51%B
(start at 5-10% less than %B calculated)
Run isocratic run at new %B
Run at newly calculated %B
?
Change organic range to start at new %B and end at 100%
Run at new organic range
NO
to
all
Run time < 20 min
K < 20
Rs > 1.5
NEARLY
All but Rs:
1.4 < Rs < 1.5
YES to
all
?
YES to
all
Run time < 20 min
K < 20
Backpressure < 4000 psi
Rs > 1.5
RESOLUTION GOAL
NEARLY
All but Rs:
1.4 < Rs < 1.5
NO
to
all
Change selectivity
There are a few approaches to changing selectivity:
50%ACN
-> Changing solvent:
ACN, MeOH
Mix (preferably done after the two solvents have been tried separately)
NOTE: remember to consult nomogram
65%MeOH
Changing selectivity
There are many approaches to changing selectivity:
Mix
Organic range, tg, flow rate, l
SELECTIVITY
-> Changing temperature:
from ambient to 50 deg C
SELECTIVITY
50 deg C
-> pH:
can separate two close peaks by their pka
pH 7
?
YES to
all
Run time < 20 min
K < 20
Backpressure < 4000 psi
Rs > 1.5
?
YES to
all
Run time < 20 min
K < 20
Backpressure < 4000 psi
Rs > 1.5
NEARLY
All but Rs:
1.4 < Rs < 1.5
NO
to
all
NEARLY
All but Rs:
1.4 < Rs < 1.5
NO
to
all
Change stationary phase
(Step 0 Part 2)
Change efficiency
There are many approaches to changing efficiency: flow rate, l, d, ps, pore size
Flow rate
When changing flow rate, may change %B as well to ensure run time and K are within limits
Change column:
l (250 mm, 150 mm, 100 mm)
d (4.6 um, 3.2 um)
ps (5 microns, 3 microns)
pore size (coreshell)
UPLC for smaller l, d and ps
EFFICIENCY
?
YES to
all
Run time < 20 min
K < 20
Backpressure < 4000 psi
Rs > 1.5
NO
to
all
Change stationary phase
(Step 0 Part 2)
Method Validation
The purpose of completing a method validation is to ensure that the HPLC method was suitable.
METHOD VALIDATION
Precision
Accuracy
Limit of Detection
Limit of Quantitation
Specificity
Linearity
Range
Robustness
YES
NO
to
all
?
YES to
all
Run time < 20 min
K < 20
Rs > 1.5
NEARLY
Run time < 20 min
K < 20
1.4 < Rs < 1.5
NO
to
all
NO
YES to
all