OBTAINING
SEPARATION
ADJUSTING SELECTIVITY
PRESENTED BY :
KASHIF
BASHIR
Selectivity
Selectivity is the ability of an HPLC method to separate two analytes
from each other. Selectivity usually is abbreviated with the Greek letter
α, and is calculated as:
α = k2 /
k1
If two compounds interact with the column or mobile phase in a
sufficiently different manner, we can separate them – if they interact
in the same manner, a single peak results
Some of the more important variables that affect selectivity are
1. The solvent strength and type
2. The temperature of the column
3. The buffer and other additives and
4. The type of column packing
After we obtain a reasonable retention time for
a separation we may need to adjust selectivity
Retention adjustment alone is insufficient to obtain a
satisfactory separation
Next step is adjusting the selectivity or relative peak
separation of our sample
In reverse phase HPLC, we control retention primarily by
adjusting the mobile phase strength.
Selectivity is a function of chemical interactions between
sample molecules, the mobile phase and stationary phase
For neutral samples adjusting the mobile phase organic
solvent is usually the best way to change selectivity
HPLC Chromatogram
Most Popular organic solvents used in
HPLC
Acetonitrile
Methanol
Tetrahydrofuran
Change to Methanol
Can be started with 90%
methanol
And to determine best
conditions for methanol
We can generalize
solvent strength
conversion although
specific retention
predictions are inaccurate
Solvent Nomograph
Rule of Three
This rule states that the retention factor,
k, changes approximately threefold for a
10% change in %B
%B = Change in organic solvent
45% methanol-water gave good
separation( Rs>1.5)
But retention time increased
55% methanol- water
Retention times are better but
resolution decreased to ~1.3.
Separation of peak 1& 2 also
decreases (bad)
Solvent Mixtures
1. We can try ternary system i.e., methanol ,
acetonitrile and water or
2. THF
Blending acetonitrile with methanol will result in
poor (worse) separation because of acetonitrile like
separation.
A Binary THF+ Water as mobile phase is simpler to
use than Ternary THF+ MeOH+ Water
Tetrahydrofuran
We need to determine starting
composition of THF-Water mobile
phase
We use nomograph
30% THF-Water average of two
conversions for 45 and 55%
Methanol-water system (Rs>1.7)
Decreasing retention further, the
peaks will become further
crowded
Tetrahydrofuran
5% decrease in organic solvent will further increase
retention with a run time of 20 min
Resolution has increased greater than 2 for all
peaks at a run time of approx. 25 min
Mixing methanol-THF will cause same problems like
acetonitrile-methanol
Separation using binary Tetrahydrofuran-water
phase is satisfactory
Summary
Strong acetonitrile + water was first used then diluted until
the best possible resolution was achieved in acetonitrile (35%)
Separation was unsatisfactory so we used solvent strength
conversion tool
Water-methanol as mobile phase that would yield approximate
retention with different selectivity
Retention estimates were inaccurate so we adjusted mobile
phase using Rule of Three as a guideline to produce best
methanol-water separation.
Much better than acetonitrile-water but still inadequate
So we switched to THF-Water as mobile phase which gave
satisfactory separation.
Summary
Rule of three enabled us to adjust the solvent strength in an
intelligent and time saving manner
Solvent Strength nomograph helped us to transfer from one
solvent to another
Both only gave us estimate and were inaccurate
But in spite of inaccuracies time was saved
Only solvent strength and type was changed, the same
principle applies for the adjusting
Mobile phase additives
pH and
Temperature