Stable and efficient nanoliter two-dimensional separation technology——online dual-phase reversed-phase chromatography

For trace and complex samples, such as proteomics samples, residual host cell protein (HCP) in protein drugs, etc., not only a highly sensitive nanoscale liquid phase is required, but also a more sufficient separation. On-line 2D NanoLC separation technology (on-line 2D NanoLC) came into being, and has become an indispensable separation method for the analysis of small and complex samples.

Traditional nanoliter online two-dimensional technology generally uses strong cation exchange (SCX) as the first dimension, and reversed-phase chromatography (RP) as the second dimension separation method. This method is based on the orthogonal relationship between the ionic characteristics and the hydrophobicity of the sample in the salt solution. However, SCX-RP technology has many difficulties in the separation and upgrading of nano. The difficulty comes mainly from the SCX separation dimension. In SCX separation, it is necessary to use a higher concentration salt solution as the mobile phase, but the salt-containing mobile phase is prone to salting out or causes the sample to precipitate in the pipeline, and the internal diameter of the nanoliter liquid phase pipeline is very small (25-100 Micron). Therefore, when the SCX-RP separation is actually used, the pipeline is often blocked and the experiment fails.

To this end, in addition to providing traditional SCX-RP separation technology, Waters creatively developed a two-phase two-phase separation method. (RP-RP). This RP-RP technology does not need to use high-concentration salt solution as the mobile phase, avoiding the problem of pipeline blockage easily caused by ion exchange separation, thereby greatly improving the system stability and practicality of the nanoliter two-dimensional liquid phase. What is more exciting is that Jarrod A. Marto of Harvard Medical School conducted a comprehensive experimental comparison and found that the liquid quality analysis results obtained by using RP-RP separation technology are better than SCX-RP method (Figure 1) [1] RP -RP double reversed two-dimensional method can help scientists get more protein analysis results.

This is because: 1. The salt buffer used by the SCX method is prone to ionic noise background, which affects the quality of mass spectrometry data; 2. The separation effect of SCX depends on the number of charges carried by the polypeptide, and the number of types of charge carried by the polypeptide is limited, so Dimensional SCX resolution is poor, resulting in low quality of liquid quality data information.

Figure 1

R PR P dual reversed-phase separation technology uses reversed-phase chromatography in both the first and second dimensions, so how does it achieve the orthogonality of the separation properties necessary for two-dimensional separation? It turned out that after research, it was found that at different pH values Under the environment, the reverse-phase retention behavior of peptides is different (Figure 2) [2]. Based on this property, Waters scientists have developed a unique RP-RP nanoliter online two-dimensional system-nanoACQUITY UPLC® System with 2D-LC. The separation column of this system uses UPLC's consistent sub-2 micron particle packing, so it has the advantages of UPLC's ultra-high resolution. In addition, it can achieve precise nanoliter flow rate without splitting, which can save the laboratory a huge purchase cost of high-purity mobile phase and waste liquid treatment costs, and is more environmentally friendly. The advantages of nanoACQUITY UPLC System with 2D-LC dual reversed two-dimensional system are summarized as follows:

â–  Compared with SCX-RP technology, more protein identification results can be obtained by using RP-RP system.

â–  RP-RP system is more stable and durable than SCX-RP system.

â–  Compared with nano HPLC, nanoACQUITY UPLC has superior separation effect of UPLC.

â–  Accurate delivery without splitting

Figure II

The structure and analysis flow of the nanoACQUITY UPLC System with 2D-LC dual reversed online two-dimensional system is shown in Figure 3, which includes three chromatographic columns: high pH reversed column, capture column, and low pH reversed column. In this system, the first dimension chromatography column is a high pH chromatography column. After the sample enters the first-dimension chromatography column, the first-dimension gradient pump can automatically increase the organic proportion in steps according to user requirements, so as to elute different hydrophobic peptides in the sample in batches. The peptide eluted from the high pH reversed phase column will be captured by the enrichment column. Each batch of enriched peptides will enter the low pH reversed-phase analysis column in the linear gradient mode of the second dimension pump, where after sufficient separation, the sample will reach the ion source and enter the mass spectrometer.

The lower left picture is a schematic diagram of the structure. Step â‘ : After the sample is collected by the autosampler, it is pushed into the high pH chromatography column by the first dimension gradient pump. Step â‘¡: Under the action of the first-dimension pump step gradient, the sample flushes out a part of the peptide and is enriched by the capture column. The second-dimension gradient pump dilutes the solvent into a system suitable for trapping column enrichment by applying 9 times the water-phase mobile phase of the first-dimension pump. Step â‘¢: After the six-way valve is switched, the second-dimensional pump fully separates the peptide samples through a linear gradient and sends them to mass spectrometry for analysis. After performing step â‘ , step â‘¡ and step â‘¢ are alternately performed until the required analysis is completed. The nanoACQUIT Y UP LC System with 2D-LC has been widely used in the analysis of peptides and liquids, and has helped researchers achieve many valuable research results.

Figure 3. NanoACQUITY UPLC System with 2D-LC system structure and analysis flow chart.

references

(1) Zhou F, Cardoza JD, Ficarro SB, Adelmant GO, Lazaro JB, Marto JA. Online Nanoflow RP-RP-MS Reveals Dynamics of Multicomponent Ku Complex in Response to DNA Damage. J Proteome

Res. 2010, 9, 6242-6255.

(2) Gilar M, Olivova P, Daly AE, Gebler JC. Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. J. Sep. Sci. 2005, 28, 1694–1703 .

About Waters Corporation () For more than 50 years, Waters Corporation (NYSE: WAT) has made significant advancements in the fields of medical services, environmental management, food safety, and global water quality monitoring by providing practical and sustainable innovations. Laboratory-related institutions have created business advantages. As the pioneer of a series of separation science, laboratory information management, mass spectrometry and thermal analysis technologies, Waters ’major breakthroughs and laboratory solutions have created a lasting platform for customers’ success. With a revenue of US $ 1.85 billion in 2011, Waters will continue to lead customers around the world to explore science and achieve outstanding achievements.

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