Yours Chromatographically, Kaushik Zala

Sample goes from detector into                  Sample goes from detector into
waste                                                            fraction collector
Goal: Quantification and/or                        Goal: Isolation and/or
identification of compounds                        purification of compounds







In analytical chromatography the sample amounts applied
to the column are typically in the μg range but can be
lower. The mass ratio of compound to stationary phase
on the column is less than 1:100000. The applied sample
volume is also usually much smaller than the column
volume (< 1:100). Under these conditions good separations
with sharp and symmetrical peaks can be achieved. The
biggest difference in preparative HPLC is the much higher
amount of sample applied to the stationary phase. The
impact on the chromatography, the methods of injecting
larger amounts of sample and the scale up of an analytical
method are described in the next chapters.


ADSORPTION ISOTHERM



The goal of analytical HPLC is the quantitative and/or
qualitative determination of a compound. Important
chromatographic parameters to achieve reliable and accurate
results are resolution, peak width and peak symmetry.
If more and more sample amount is applied to the column,
the peak height and peak area increases but the peak
symmetry and the capacity factor remain unchanged as
shown in figure 1.









In analytical HPLC the optimal peak shape resembles
a Gaussian curve. The peak’s standard deviation σV
describes its symmetry and how well it resembles a
Gaussian curve. The capacity factor (k’) is the retention
time (t) relative to the retention time of a non-retained
compound (t0). If more than a certain amount of sample
is injected onto the column the adsorption isotherm
becomes non-linear. This means the peak becomes unsymmetrical,
shows strong tailing and the capacity factor
decreases as shown in figure 2. In preparative HPLC this
effect is called concentration overloading. In some cases,
depending on the compound, the capacity factor increases
with increasing overloading, which leads to a strongly
fronting peak. Since the adsorption isotherm is dependant
on the compounds the chromatographic system column
loadability has to be determined for each preparative
HPLC experiment.







Column loading and
overloading



For purification of large sample amounts two methods are
possible: Scale-up of the analytical system or column overloading.
Scale-up of the analytical system would mean 



increasing the sample volume with the column length
and sample concentration remaining constant. The peaks
would then remain sharp and symmetrical. The disadvantage
of this method is that large columns and high solvent
volumes are required to separate rather small amounts of
compound, hence, the method would not be economical.
Therefore, column overloading, that is, increasing the
applied sample amount under the same analytical conditions,
is usually the method of choice. Using column overloading
allows to separate samples in the milligram range
even on analytical columns. For larger amounts of sample
an additional scale-up of the system is necessary. Column
overloading can be done in two ways – concentration or
volume overloading. In concentration overloading the
concentration of the sample is increased but the sample
volume injected remains the same. The capacity factor, k',
decreases and the peak shape changes from a Gaussian
curve to a triangle as shown in figure 3. Concentration
overloading is only possible when the sample compound
has good solubility in the mobile phase.







If the compound has poor solubility, concentration overloading
cannot be used and more sample volume must
be injected. This technique is called volume overloading.
Beyond a certain injection volume the peak height does not
increase and the peaks become broader and rectangular.
In preparative HPLC concentration overloading is favored
over volume overloading because the sample amount,
which can be separated, is higher. Since the solubility of
compounds is usually a limiting factor both overloading
techniques are used in combination. Table 3 gives an
overview of the overloading techniques.



1. Optimization of the analytical method regarding
resolution
2. Column overloading on the analytical column
3. Scale-up to the preparative column







The two parameters that must be scaled up when going
from a column with smaller i.d. to a column with larger
i.d. are the flow rate and the sample amount applied to the
column. To scale up the flow rate the upper equation in







figure 5 is used, in the shown example the flow of
0.6 mL/min on a 3 mm i.d. column is scaled up to a
21.2 mm i.d. column. For scaling up the amount of
compound loaded onto the column the lower equation
is used, it makes no difference if it is used to scale up
the concentration or the injection volume. The factor CL
equals 1 if two columns of the same length are used.
After the scale-up calculations and the first preparative
run, it is not uncommon to further optimize the parameters
to achieve the best separation results.



The three important parameters used to judge the result
of a preparative run are purity of the product, yield and
throughput. Since the parameters are dependent on each
other it is not possible to optimize a preparative HPLC
method with respect to all three parameters (figure 6).







Chromatogram 1 shows a preparative HPLC run capable
of very high throughput but the separation of the two
compounds is poor. It might be possible to obtain some
fractions with high purity for each compound but the
recovery, that is, the yield is rather low. In chromatogram 2
the peaks are well separated, therefore, it is possible
to get both compounds in high purity and yield but the
throughput is very low. Chromatogram 3 would be an
optimized preparative HPLC run with a compromise to all
three parameters. The peaks are almost baseline-separated,
which leads to high purity and yield and throughput is
as high as possible. The most important parameter for
which the separation has to be optimized depends on the
application. If, for example, a compound has to be isolated
for activity or toxicity testing, it is necessary to obtain
this compound in high purity. Throughput and the yield
are less important. If a synthesis intermediate has to be
purified the purity is not of highest importance as long as
it is sufficient for the next synthesis step. However,
throughput is an issue here because it is usually necessary
to complete the entire synthesis as fast as possible. The
yield is also important because the compound is valuable
and the loss of compound should be minimized.







A purification run
can never be optimized
with respect to the three
parameters: purity,
recovery and
throughput!


keep reading kaushikzala on blog spot..........chapter 2 coming soon