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Copy of Method Development Flow Chart
2
Transcript
Method Development
pH affects the degree of ionisation of ionic analytes
Changing
solvent type
Solvent strength
- Methanol
- ACN
- Buffer
Changing mobile phase
Percentage of Organic phase (B)
An increase of 10%B reduces k
by 2-3 fold
one
two
Column type
Isocratic elution method
k= (tR-t0)/t0
(α)
Selectivity
Retention factor (k)
αa= k2/k1=(tR2-t0)/(tR1-t0)
∆tg < 0.25xtg
Optimisation of k ~5
2<k<20
Sample Classification
tg=k(1.15∆ФVm)/F
Length (L)
Changing temperature
Initial Conditions
Resolution
Scouting Gradient
Rs=1/4√N x (αa-1)/aα x k/(k+1)
An increase in column T by 1oC,
k by 1-2%
Increase
- Column type
- Flow rate
- Mobile phase
- Temperature
Conditions: ACN 5 – 100 %
with water in 20 minutes
Run Time
Analyse the sample
- Chemical structure (functionality)
- Molecular weights
- pKa values
%B=(∆%Bf)/tg x (ti+tf)/2+%Bi
%Bi=%B at first peak
%Bf = % B last peak
Particle diameter (dp)
Decrease
three
Gradient elution method
Back Pressure
Number of Theoretical
plate (N)
∆tg > 0.25xtg
Reduce
Optimisation of N
Viscosity (n)
Column diameter (dc)
Velocity (u)
Changing
flow rate
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