Principles and Practical Aspects of Preparative Liquid Chromatography

Analytical Semi-preparative Preparative Pilot
Purification range [mg] 1–15 7–70 30–300 64–640 180–1800 400–4000 700–7000 600–16000 2800–28000
4.6 mm
0.8–2.0
mL/min
9.4 mm
(0.5 inch)
21.2 mm
(1 inch)
4–10
mL/min
18–42
mL/min
Column inside diameter [mm]
30 mm
50 mm
(2 inch)
75 mm
(3 inch)
100 mm
(4 inch)
150 mm
(6 inch)
200 mm
(8 inch)
34–85
mL/min
94–236
mL/min
212–931
mL/min
378–945
mL/min
800–2100
mL/min
1100–3375
mL/min
Figure 2.1 Overview of recommended flow rates (in mL/min)
and sample amounts (in mg) for typical column sizes.
Typically, the amount of crude sample can vary from 0.1 to 1.0 % based on
the weight of the sorbent. Recommended values are based on reversedphase
sorbent with a density of 0.6 g/mL and for a column or bed length of
150 mm. A 21.2 by 150 mm column contains about 32 g of sorbent.
Let us consider this example of a typical scientist’s requirement;
“I would like to purify 100 mg of crude material per injection.
Which column dimensions and what flow rate do you recommend?“
The recommendations in Figure 2.1 suggest that this demand can be
met using a 21.2-mm id column with particle sizes between 5 and 10
microns, which deliver good results for the majority of purification tasks.
The available column lengths from 50 to 250 mm need to be matched
with the requested injection amounts. A simple rule to select a suitable
length of a 21.2-mm id column is: 50 mm for 50 mg; 250 mm for 250 mg.
If separation becomes too difficult, the amount of injected crude material
needs to be decreased or the amount of stationary phase needs to be
increased. When decreasing the injected amount, always first reduce
the injection volume, then the concentration. Increasing the amount of
stationary phase can be realized by increasing the column diameter or
the column length, or both.
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