Waters Advanced Purification Glass Columns - Manual (Page 9)

[ Care and Use ManUal ]
Waters Advanced Purification Columns 9
This section describes three equations to adjust the sample load
and flow rate. Use Equation l to calculate the sample load and
Equation 2 to calculate the flow rate. Alternately, use Equation 3 to
calculate a scale factor, which you may use to obtain the preparative
value. In all three equations, if you use preparative columns and
analytical columns of the same length, both sample mass and flow
rate are proportional to the square of the column cross section.
In the following equations:
p = preparative column
a = analytical column
m = mass injected
I = column length
d = column diameter
F = flow rate
v = column volume
sf = scale factor
t = times in gradient table
V
d
= delay volume of the instrument
Equation 1
To increase sample load in proportion to the volume of the packed bed:
Equation 2
To increase flow rate in proportion to the volume of the packed bed:
Equation 3
To calculate the scale factor:
Multiply the analytical sample mass and/or flow rate by the scale
factor to obtain the preparative value. You should obtain similar
retention times and peak widths for both the analytical and the
preparative column and thus, similar resolution.
c. Adjusting Gradient Parameters
If you have scaled the flow rate in proportion to the column volume, the
gradient time profile should remain constant. However, due to pressure
limitations on the preparative instrument or column, you may have to reduce
the flow and adjust the gradient time table. Use the following formula to
• Keep a constant ratio between the gradient volume and column volume
• Adjust the gradient table
d. Adjusting Gradient Delay Volume
Before the gradient reaches the inlet of the column, there is an isocratic
period caused by the delay volume between the point of mixing of the gradi-
ent and the column inlet. The isocratic period exists until the delay volume is
purged.
In small columns, the delay volume results in a longer isocratic period than
in larger preparative columns. To maintain the same separation for analytical
and preparative columns, you must delay the gradient for the preparative
column by the some number of column volumes. Use the following equation
to calculate the additional gradient delay time for the preparative column: