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