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Separation Reproducibility With the BioLogic Chromatography Systems

Kevin Thornton, PhD, Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA


Introduction
In chromatographic separations, sample component elution times are often used both for identification and to program automated chromatography steps like fraction collection. Therefore, the ability to obtain reproducible chromatographic separations is essential. In order to achieve reproducible sample runs, a chromatography system must maintain a precise flow rate, inject a consistent sample volume at a precise time, and detect sample components in a consistent manner. Additionally, the column used must maintain its function and flow characteristics over time. In this report we provide data that shows the highly reproducible separations that can be obtained with both BioLogic DuoFlow and BioLogic LP systems. These results were obtained on a medium-pressure BioLogic DuoFlow QuadTec basic system and a low-pressure BioLogic LP system equipped with a BioFrac fraction collector.


Methods
A test mixture that contained the proteins horse skeletal muscle myoglobin, chicken egg white conalbumin, and soybean trypsin inhibitor type II (all obtained from Sigma) in a ratio of 2:5:5 was dissolved in an appropriate volume of 20 mM Tris buffer, pH 8.1 (buffer A), filtered through a 0.22 m syringe filter, and kept on ice until injected onto a column.

The test mixture was purified in consecutive anion exchange runs using an UNO Q1 column or an Econo-Pac High Q cartridge connected to the DuoFlow or BioLogic LP system, respectively. Proteins were eluted with a linear gradient of 050% buffer B (buffer A + 1 M NaCl). All buffers were filtered and degassed prior to use. For each run on the DuoFlow system, a 50 l sample loop was overfilled with 150 l of the protein sample using an Econo gradient pump. For the BioLogic LP system, 1 ml of sample was loaded though port C of the BioLogic LP buffer selection valve. The UV detector and conductivity monitor supplied with each system were used to monitor the UV signal and conductivity, respectively. Protocol details are summarized in Tables 1 and 2 and the system components used are listed in Table 3.


Results
The BioLogic DuoFlow and BioLogic LP systems were used to successfully purify a protein test mixture with excellent reproducibility. A total of 80 ion exchange runs were performed on the BioLogic DuoFlow QuadTec system as a queue of alternating anion exchange and equilibration runs (160 runs total). As shown in Figure 1, the 80 separations were virtually indistinguishable. The observed protein retention times (Table 4) demonstrate consistent sample introduction and pump performance. The standard deviation associated with the gradient slope, 4.88 0.01 mS/cm/min, indicates excellent pump and mixer performance, which is critical for discrete protein resolution. These results confirm that the BioLogic DuoFlow chromatography system provides high run-to-run reproducibility.


On the BioLogic LP, 25 consecutive separations were performed using its Multirun feature. As shown in Figure 2, the separations were highly reproducible and exhibited extremely consistent protein elution times (Table 4). Gradient performance was also highly reproducible with a small standard deviation (Figure 2); the observed average gradient slope was 3.59 0.05 mS/cm/min. These results show that the BioLogic LP gives the consistent mixing and reliable pump performance required from a low-pressure chromatography system.


Conclusions
These results demonstrate that the BioLogic DuoFlow and BioLogic LP chromatography systems routinely deliver highly reproducible results. This high level of reproducibility is critical for obtaining consistent sample purity and for automating laboratory chromatography processes. Whether your separation requirements are analytical or preparative, the BioLogic DuoFlow and BioLogic LP chromatography systems provide high-performance, reliable results.


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