Evert-Jan Sneekes, Laurent Rieux and Remco Swart
Thermo Fisher Scientific, Amsterdam, The Netherlands
Summary
Liquid chromatography (LC) miniaturization is usually done to increase sensitivity, which is an inevitable requirement for proteomics to detect low abundance peptides and proteins. Other advantages of miniaturization include increased efficiency in connection to mass spectrometry and reduced solvent consumption. Today, miniaturized liquid chromatography is easier to use, and its instrumentation and connection are the same as in conventional LC systems. This offers more opportunities for miniaturized liquid chromatography, especially for capillary and microfluidic LC applications, in new workflows and applications.
Key words
RSLCnano , nanoliter liquid chromatography, capillary liquid chromatography, microfluidic liquid chromatography, mass spectrometry, nanoViper
introduction
The development of miniaturized liquid chromatography began about thirty years ago, and in 1987 the first commercial products appeared. Over time, nanoliter liquid chromatography has become an indispensable tool for bioanalytical research, especially in the field of proteomics that requires the highest level of sensitivity. However, with the daily application of nanoliter liquid chromatography in proteomics laboratories, people sometimes forget the principle of improved sensitivity. Understanding these principles and their actual impact can help people see how miniaturized liquid chromatography can benefit proteomics, bioanalysis, and more.
Sensitivity- size factor
In order to understand the sensitivity of a column with a smaller inner diameter, it is necessary to first determine the difference between the sample concentration and the sample amount. This can be summarized as follows:
• Sample injection of liquid chromatography refers to the injection of a certain volume of a certain sample.
- This is a fixed amount: for example 1 μL 1 pmol/μL = 1 pmol
• When the sample is injected, the fixed amount of this sample is diluted by the liquid chromatography system.
- This will get a new concentration
• Smaller volumes of liquid chromatography will produce higher concentrations at the same injection volume
- Columns with smaller internal diameters are more sensitive than large-diameter columns
Figure 1. The same injection volume is different in the LC system.
Figure 1 shows the injection of the same injection volume (four “peptidesâ€) on a column with internal diameters of 4.6 mm and 75 μm, respectively, which is clearly higher. The relative increase in sensitivity can be calculated using Equation 1.
Optical detectors (such as UV detectors) and electrospray mass spectrometers are concentration-sensitive instruments. Their signal is proportional to the concentration of the sample in the peak eluted from the column.
Figure 2 plots the relative sensitivity of a column of different diameters to a 4.6 mm diameter column (calculated by Equation 1). As can be seen from the figure, the sensitivity of the column with the smallest inner diameter (50 μm and 75 μm) can be theoretically increased by several thousand times. However, the inset shows that the sensitivity of the column has changed significantly from 4.6 mm to 1 mm (approximately 20 times). While some do not require absolute sensitivity, but miniaturization can provide other benefits, such as easy connection to mass spectrometry, reduced solvent usage (see calculation examples), and waste disposal and cost reduction, these column internal diameter ranges are perfectly suited for practical applications. .
Reduce solvent usage:
Imagine analysis on a column with an internal diameter of 4.6 mm using the following parameters:
- Flow rate is 1.0 mL / min
- 70% mobile phase A
- 2 liter solvent bottle
All mobile phase A will be depleted in two days.
The application was run at a flow rate of 50 μL/min using microfluidic liquid chromatography and the 500 mL bottle was maintained for one week.
Figure 2. Relative increase in sensitivity for a series of id columns compared to 4.6 mm id.
Ultra-high performance liquid chromatography (UHPLC) for nanoscale, capillary and microflow liquid chromatography
The calculated sensitivity increase is actually a scaling factor between conventional and miniaturized liquid chromatography applications, and flow rate is the best example. Typically, the flow rate (inner diameter 4.6 mm) in conventional liquid chromatography is 1000-1200 μL/min, and the nanoliter liquid chromatography (inner diameter 75 μm) is 0.250-0.300 μL/min. The ratio of 4000 reflects the scale factor between the 4.6 mm internal column and the nanoliter LC column and will result in the same (linearly) linear velocity of the two columns.
The Thermo Scientific TM Dionex TM UltiMate TM 3000 RSLCnano system (Figure 3) is designed to provide the very low flow rates and very high pressures required for modern nanoliter liquid chromatography. The UltiMate 3000 RSLCnano system delivers flow rates as low as 20 nL/min* and as high as 50 μL/min under UHPLC pressure, making it the perfect instrument for nanoscale, capillary and microflow LC analysis. The 1 module has a built-in column oven that supports a range of applications while minimizing the physical distance between the various components. This allows the connecting tube to be as short as possible.
Another important aspect of the connecting tube is the inner diameter of the tube. The inner diameter of the nanotube liquid chromatography (20 μm) is approximately 10 times smaller than the inner diameter of the conventional pipe. Typically, capillary-stage and micro-flow liquid chromatography are used with connecting tubes with internal diameters of 50 μm and 75 μm, respectively.
The best results are obtained if the proper piping is properly connected. Thermo Scientific TM Dionex TM nanoViper TM connector system provides factory system, manpower UHPLC linker, zero dead volume. The nanoViper fittings are integrated into the tubing and column and can be assembled without tools and without the risk of introducing dead volume or capillary breakage during attachment.
* Minimum flow rate is required when required
Figure 3. UltiMate 3000 RSLCnano system, internal view, and nanoViper connector
Miniaturized Liquid Chromatography - Application
Miniaturized liquid chromatography can be divided into three or three flow rates: nanoscale, capillary and microflow liquid chromatography. Nanoscale liquid chromatography refers to applications with flow rates below 1000 nL/min, capillary-grade liquid chromatography with low flow rates (1–10 μL/min), and micro-flow liquid chromatography covers flow rates over 10 μL/min Applications.
The following are typical application examples for each flow rate range. The first and most common example, nanoscale liquid chromatography is used in the proteomics discovery workflow. Here, the limitation of the amount of sample is the main reason for using nano-upgraded liquid chromatography. In the second example, capillary-level liquid chromatography was applied to the quantitative analysis of target peptides in complex matrices. Samples may be easier to obtain, but throughput is also critical, and capillary-grade LC can do both. For the third example, microfluidic liquid chromatography was used to increase throughput in the analysis of monoclonal antibody (MAb) digests. Sample size is no longer a limiting factor here, but increased sensitivity and system durability are valuable. Finally, an example of non-biological analysis is given.
In proteomics research, researchers face a double challenge. The protein concentration to be studied is low and the sample volume is small, resulting in an extremely low content. In addition, the samples are very complicated. These factors have led to the development of a cashier upgraded LC system and columns with very high separation capabilities to ensure special applications, as shown in Figure 4. A similar experiment was performed by Köcher et al., who averaged 2,516 proteins in trypsin-digested HeLa cells by performing an 8-hour gradient elution on a 50 cm column. 2
Figure 4. High-resolution isolated E. coli digest after 10 hours gradient elution on a 75 μm (inside diameter) × 50 cm nanoliter LC column
The next example is the application of capillary-grade liquid chromatography in a targeted quantitative workflow. In this case, capillary-grade liquid chromatography was chosen to increase throughput, increase durability and ease of use while maintaining high sensitivity. Yeast digests with isotopically labeled peptides were separated on a column with an internal diameter of 300 μm. The sample was separated at a flow rate of 4 μL/min over 30 minutes and connected to a Thermo Scientific TM TSQ Vantage TM triple quadrupole mass spectrometer using a standard HESI-II interface for detection in SRM mode with a detection limit of 10 amol (Figure 5). See Thermo Scientific's Application Report 583 for more details. 3
Figure 5. Extraction chromatogram of peptide SAAGAFGPELSR added to 500 ng yeast digestate matrix at different concentrations
The following microflow examples are particularly useful for high efficiency and high throughput peptide mapping matching applications in the biopharmaceutical industry. In this particular example, the monoclonal antibody digest was first isolated by a 30 minute gradient elution at a flow rate of 6 μL/min. In two successive steps, the flow rate was increased to 15 μL/min while the gradient was shortened. Figure 6 shows how the analysis time is reduced to 1/3 without degrading the data quality. Increasing the flow rate on a standard particle size column in a similar manner may complicate the connection of the chromatographic system to the mass spectrometer and require two mobile phases to be changed daily.
Figure 6. Accelerated analysis of monoclonal antibody Lys C digest on a Thermo Scientific TM Acclaim TM PepMap TM RSLC analytical column at 300 μm (inside diameter) × 15 cm
Finally, Xiang He et al. demonstrated the promising results of micro-flow liquid chromatography in the fields of forensic toxicology. 4 Established a simple, highly sensitive method for quantifying the analysis of cannabinoids in biological samples to meet the needs of forensic toxicology. This method is similar in design and cycle time to typical standard particle size filler applications, but with higher sensitivity for the analysis of further diluted samples. Forensic toxicologists can apply this versatile workflow to other common illegal/abuse drug quantitative analyses. Reagent consumption is also significantly reduced by more than 90% and the maintenance frequency of the mass spectrometer is reduced.
in conclusion
Miniaturization of liquid chromatographs is generally only considered to be associated with nanoscale liquid chromatography that is required by proteomics research to provide higher sensitivity. However, capillary-grade and micro-flow liquid chromatography have greatly improved sensitivity. In addition, UHPLC capabilities are guaranteed over the entire flow rate range (from nanoscale to traditional), greatly simplifying the scale of the analysis without compromising performance.
Although proteomics remains the primary focus of nanoscale LC applications, there is also a lot of potential for biopharmaceutical and forensic/toxicology applications. As mentioned earlier, the user's use of miniaturized liquid chromatography not only improves sensitivity, but also makes it easier to connect to mass spectrometry, while also significantly reducing sample and solvent consumption. This not only compensates for extended operating time, but also reduces operating costs. Switching to capillary-grade liquid chromatography or micro-flow liquid chromatography means a perfect balance between sensitivity and durability, throughput and ease of use.
So go back to the question in the title: "The miniaturization of the liquid chromatograph; why do we do this?", because there are many benefits!
references
1. Rieux, L.; Sneekes, EJ.; Swart, R. Nano LC: Principles, Evolution, and State-of-the-Art of the Technique. LCGC NA2011, 29 (10), 926–934, [Online] Http:// com/lcgc/ Column%3A+Innovations+in+HPLC/Nano-LC-Principles-Evolution-and-State-of-the-Art
/ArticleStandard/Article/detail/745381 (accessed August 7, 2013).
2. Köcher, T.; Swart, R.; Mechtler, K. Anal. Chem. 2011, 83, 2699–2704.
3. Kiyonami, R.; Swart, R; Zabrouskov, V.; Huhmer, A. Increased Robustness and Throughput for Targeted Protein Quantification Using Capillary Flow and a Conventional ESI Probe, Thermo Scientific Application Note 583, [Online] http:// ource
resourceId=102334&storeId=11152&from=search# (accessed August 7, 2013).
4. He, X.; Kozak, M.; Nimkar, S. Anal. Chem. 2012, 84 (18), 7643–7647.
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