Agilent Technologies Preparative separation User Manual
Preparative separation of a binary
compound mixture – recovery of pure
compounds and solvent consumption
Abstract
This Application Note describes the separation of two compounds from
a binary compound mixture using the Agilent 1100 Series purification
system. The parameters purity, recovery, analysis run time, solvent con-
sumption and liquid phase composition are monitored and their interre-
lation is explained. Further, we discuss how the analysis can be opti-
mized with regard to these parameters.
Application
Udo Huber
Introduction
Separation of discrete compounds
from a mixture is a typical task in
preparative liquid chromatography. The simplest task is isolating
two compounds from a binary
mixture, however, it is also possible to isolate pure compounds
from more complex compound
mixtures, such as natural product
extracts 1, for example. The compounds to isolate are typically isomers or enantiomers
2,3
.
In this Application Note we
demonstrate the separation of two
compounds from a binary compound mixture using the Agilent
1100 Series purification system.
The method developed on an analytical column was scaled up to
preparative scale and the compounds were separated in milligram quantities. The separation
was done repeatedly, varying the
composition of the liquid phase to
achieve a separation with good
recovery and purity with minimum
solvent consumption.
Equipment
The system included two Agilent
1100 Series preparative pumps, an
Agilent 1100 Series diode array
detector, an Agilent 1100 Series
column organizer and an Agilent
220 micro plate sampler modified
for higher flow rates. The system
was controlled using the Agilent
ChemStation (revision A.08.04)
and the micro plate sampling software (revision A.03.02).
Results and Discussion
Overloading of the analytical column
The separation of the binary compound mixture was first done on
an analytical column. Separation
was achieved isocratically with a
water/ acetonitrile mixture
(figure 1).
Figure 1
Analytical separation of binary compound mixtures
Time [min]
24681012
Absorbance
[mAU]
0
500
1000
1500
2000
2500
30 % B
20 % B
15 % B
10 % B
Mobile phase: water = A
acetonitrile = B
Isocratic: between 10 and 30 % B
Stop time: 12 min
Column: Zorbax SB-C18
3 x 150 mm, 5 µm
Flow: 0.6 ml/min
Injection: 5 µl
Column temperature: ambient
UV detector: DAD 270 nm/16
(reference 360 nm/100)
Standard cell
(10 mm pathlength)
Since the crude product was very
soluble concentration overloading
was possible. Figure 2 shows that
the analytical column could be
loaded with up to 500–1000 µg of
each compound
Scale up to preparative scale
The scale-up from the analytical to
the preparative column was calculated using the formulae shown in
figure 3. After the first preparative
run the flow rate was changed to
25 ml/min to achieve comparable
retention times.
0.025 µg
0.25 µg
2.5 µg
25 µg
250 µg
Isocratic: 10 % B
Time [min]
2 4 6 8 10 12 14
Absorbance
[mAU]
0
1000
2000
3000
4000
5000
500 µg
1000 µg
Analytical column
Zorbax SB-C18
3 x 150 mm, 5 µm
Preparative column
Zorbax SB-C18
21.2 x 150 mm, 5 µm
Flow: 0.6 ml/min
Flow ~ 30 ml/min
Volume: 15 µl/injection
Volume: 750 µl/injection
x
1
=
r
2
1
×π
x1 = max. volume column 1
r1 = radius column 1
x2 = max. volume column 2
r2 = radius column 2
CL= ratio lengths of columns
= 15 µl
= 1.5 mm
= ?
= 10.6 mm
= 1
x
2
r
2
2
×π
×
1
C
L
V
1
V
2
r
1
r
2
2
2
=
.
.
Figure 2
Overloading of the analytical column
Figure 3
Scale up from analytical preparative column
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