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Users can achieve a more efficient purification with reduced cost through peak recycling. This article explains when peak recycling is viable and what technical specification is required for this process.
简而言之，峰值回收利用多次使用柱床的长度来拉长色谱柱的分离长度。许多制备色谱法的用户都知道该术语，但是他们不知道该特殊解决方案在哪里有用。
At the time of peak recycling, the peaks are led back onto the column following elution, so that critical peak pairs running together can be further separated with each cycle. Since the eluent composition has to remain constant during multiple repetition cycles, an isocratic separation method is required for peak recycling.
Apreparative HPLC system由检测器，制备柱，准备泵，分级阀和注入系统组成。
Chromatography columns used for preparative applications are usually very expensive. Therefore, users strive to find the best compromise between the achieved separation efficiency and incurring costs. Thus, both the individual separation challenge and the purification requirements set the conditions. This is the reason why complex separation challenges can result in cost-ineffective solutions and high expenses.
In particular cases where small particle sizes or special phases are needed for an efficient purification, both the system requirements and the cost of the preparative column become a limiting factor. Peak recycling provides a simple solution to improve the separation performance without the requirement for larger investments.
Technical Requirements
Generally, the terms “recycling” and “recycling mode” are used to represent different types of recycling:
1. Time-based peak recycling
This is not an automated mode and is used with any chromatography software. However, it is more difficult to configure. Retention time changes within a single separation can produce variations in purity of the collected fractions.
2. Automated peak recycling:
使用此模式，该软件可以通过峰高和/或斜率控制回收利用。自动模式（例如OpenLab CD Ezchrom Edition）需要特定的软件包，以提供高度的安全性和舒适性。
3. Solvent recycling:
软件包（例如PurityChrom或ClarityChrom Prep）提供了溶剂回收，作为节省溶剂的额外选择。如果认为没有峰的色谱区域中存在的溶剂被认为是清洁的，则可以根据时间或检测器信号将其回收回干清库存。此模式可以大大降低系统的运营成本。
Just a T-fitting and a multi-position valve are needed to use the recycling mode.
T型拟合将多位置阀连接到通过端口的系统泵供应（在许多情况下，是阀门的第二个端口）。当系统从正常到回收模式跳过时，阀门会切换到该特定端口。然后，泵不是从库存中绘制洗脱液，而是将溶剂回收在整个系统中，如图1所示。
Figure 1.Preparative HPLC system with multi-position valve enabling peak recycling.
The samples are pumped in cycles until the valve is switched to either waste or fractionation position.
Application Example
使用峰值回收利用分离方法优化方法的一个实际示例是两个（+/-）-α-生育酚异构体的分离。
Compared to analytical HPLC, the potential for optimization is much more limited for preparative HPLC. For preparative applications in reversed phase mode, the option of column material is mostly limited to standard phases with minimum particle sizes of 10 µm. On the other hand, these preparative columns with inner diameters of 20 to 100 mm show a lower separation performance, yet they produce a much lower back pressure at high flow rates, compared to similar columns with small particle sizes.
Users who are dealing with a complex separation task like tocopherol, where the target substance elutes narrowly to the contaminant, will have limited options (Figure 2). The required high performance pumps, together with the costs for particular phases leading to better separation, often exceed the benefit. However, a compromise of typically impaired separation performance is not required. If the right technique is used, users can achieve excellent separation results, even with inexpensive large particle materials.
Vitamin E and Tocopherol
Vitamin E consists of a number of fat-soluble substances. It also contains eight variants of Tocopherol, of which only the α-Tocopherol is in active form. In the food industry, only the biologically active forms are relevant.
α-Tocopherol is mostly used as E307 in the food industry and as an ingredient in cosmetic products. The stereoisomers need to be chromatographically isolated from each other in order to assay their activities.
In general, users have to pay for higher yield with lower purity, or vice versa for the purification of (+/- )-α-Tocopherol at standard conditions. However, a baseline separation can be achieved via the elongation of the separation length by peak recycling after three repetition cycles (Figure 2, black). Therefore, fractionation can be performed without compromising the purity or yield.
Figure 2.Comparison of the separations of (+/- )-α-Tocopherol with more (5 µm, red) and less expensive column material (black) with the help of peak recycling.
Conclusion
The separation of the α-Tocopherol isomers can be enhanced through peak recycling, as shown in Figure 2, black. Both purity and yield are similar to the separation with more expensive column material (Figure 2, red).
标准HPLCsystem can be easily altered for the implementation of peak recycling.KNAUER HPLC systems具有模块化体系结构，使其适合这些技术的实现和优化。可以轻松地将多个组件（如多位置阀）进行更改，可以轻松地将其整合到系统中。
This information has been sourced, reviewed and adapted from materials provided by KNAUER Wissenschaftliche Geräte GmbH.
For more information on this source, please visitKNAUER Wissenschaftliche Geräte GmbH。