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First Edition: Nov. 2025 01-01027-EN
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A commercially available polystyrene molecular weight marker
(MW = 580, styrene oligomer, Figure 3 (left)) was used as a
simulated sample. This styrene oligomer contains multiple
compounds that have different number of repeating monomer
units. In this article, a fractionation of a specific molecule from the
styrene oligomer following the workflow described above is
introduced.
Shodex GPC K-2002, suitable for the molecular weight of the
target component, was used as the prep GPC column. The
chromatogram obtained without recycling conditions for target
compound confirmation is shown in Fig. 3 (right), and the
analytical conditions are listed in Table 1.
Separation of Styrene Oligomers
In recycling fractionation, if all peaks—including those other than
the target compounds—are recycled, the target peaks may
coelute with other lapped peaks in the previous cycle.
Therefore, it is appropriate to remove all peaks except the target
peaks intentionally during the initial elution. Consequently, after
confirming the elution retention times of the target peaks to be
fractionated, the timing for selecting the recycle valve position of
either “waste/collection” or “recycle” must be determined. This
ensures that only the target component peak and its nearby
contaminant peaks are recycled, making the separation achieved
by recycling visually apparent on the chromatogram.
An overview diagram of the recycle fractionation is shown in Fig.
4. At the timing when the target peak elutes during the initial
elution, the recycle valve is switched to the “recycle” side.
Subsequently, when good separation of the target peak from the
nearby contaminant peaks is confirmed through recycling, the
recycle valve is switched to the “waste/collection” side to remove
contaminant peaks and collect the target peak.
0.0 5.0 10.0 15.0 min
-50
0
50
100
150
200
250
mV
Fig. 3 (Left) Structural formula of polystyrene,
(Right) Chromatogram of styrene oligomer
Table 1 Analytical conditions
System
：
Nexera GPC + LC-40D recycle kit
Column
：
Shodex GPC K-2002
（
300 mm
×
20 mm I.D.
）
Flow rate
：
3.0 mL/min
Mobile phase
：
Chloroform
Column temp.
：
Room temperature
Injection vol.
：
100 µL
Vial
：
TORAST for LC 1.5 mL, Glass
Detection
：
UV (254 nm, conventional cell)
Automatic recycle fractionation can be performed using the time
program function equipped in LabSolutions. Here, a
chromatogram was obtained by recycling all peaks including
those other than the target compound as a preliminary trial. Then
the recycle valve time program was determined based on this
chromatogram. The red colored portion in Fig. 5 (Top) represents
the target compound.
During recycling fractionation (Fig. 5 (Bottom)), unwanted peaks
on both sides of target compound were roughly removed in the
first elution. The second elution and the third elution were
performed to improv the separation against the remained
unwanted peaks. The separated neighboring peaks were
removed in the fourth elution, and the appropriate interval
around the peak top of the target compound was fractionated.
The time program for this operation is shown in Table 2.
Combination with the analytical batch in LabSolutions provides
fully automated repeated recycle fractionation involving sample
injection, recycling, and collection.
Automated operation using time program
Time Unit Command Value Recycle valve position
1 0.01 Controller Event 0 Waste/Collection
2 14.70 Controller Event 1 Recycle
3 15.10 Controller Event 0 Waste/Collection
4 20.00 Controller Event 1 Recycle
5 52.00 Controller Event 0 Waste/Collection
6 59.40 Controller Event 1 Recycle
7 60.00 Controller Event 0 Waste/Collection
8 80.00 Controller Stop
0 10 20 30 40 50 60 70 min
-50
0
50
100
150
200
250
mV
0 10 20 30 40 50 60 70 min
-50
0
50
100
150
200
250
mV
Target compounds
Fig. 5 (Top) Chromatogram of recycling all peaks,
(Bottom) Chromatogram of recycling following valve switching time program
Table 2 Time program for recycle valve
: Flow out (Waste) : Recycle : Flow out (Collection)
This article introduced the principles and workflow of recycle
fractionation using the separation of styrene oligomers as a
simulated sample. Utilizing recycle fractionation improves the
separation of compounds that are difficult to separate using a
single column, resulting in high purity purification. Furthermore,
switching use of a single HPLC setup for both analysis and recycle
fractionation depending on the purpose can be performed by
adding a recycle fractionation unit to ordinary HPLC and GPC
systems, resulting in maximized instrument utilization.
Additionally, recycle fractionation can be automated using the
time program function and batch analysis function of
LabSolutions.
Conclusion
Fig. 4 Overview diagram of recycle fractionation
: Flow out
: Recycle
Target compounds
