Instrumental requirements for nanoscale liquid chromatography
J P Chervet1, M Ursem, J P Salzmann
1LC Packings, Baarsjesweg 154, 1057 HM Amsterdam, The Netherlands.
Analytical Chemistry
|May 31, 2011
Summary
Miniaturized nanoscale liquid chromatography (nano-LC) offers high sensitivity for protein analysis. This technique achieves excellent reproducibility and performance, making it ideal for microcharacterizing valuable protein samples.
Area of Science:
- Analytical Chemistry
- Chromatography
- Biochemistry
Background:
- Conventional High-Performance Liquid Chromatography (HPLC) faces limitations in sample volume and sensitivity for analyzing minute biological samples.
- Miniaturization of HPLC systems into nanoscale liquid chromatography (nano-LC) offers a solution for analyzing small quantities of complex mixtures.
Purpose of the Study:
- To detail the principles and practical implementation of down-scaling conventional HPLC to nano-LC systems.
- To evaluate the performance characteristics of nano-LC, including reproducibility, efficiency, and sensitivity.
- To highlight the suitability of nano-LC for microcharacterization of proteinaceous samples.
Main Methods:
- Down-scaling of HPLC components using a down-scaling factor (ratio of column diameters squared).
- Utilizing specialized micro-components: microflow processor, longitudinal nanoflow cell, microinjection valve, and low-dispersion tubing.
- Performance evaluation through retention time reproducibility measurements and plate count determination.
Main Results:
- A down-scaling factor of approximately 3800 was validated for system components.
- Excellent retention time reproducibility was achieved (±0.1% isocratic, ±0.2% gradient).
- High system efficiency demonstrated with plate counts exceeding 100,000/m.
- Exceptional mass sensitivity enabling detection in the low femtomole and subfemtomole ranges (e.g., 520 amol for BSA).
Conclusions:
- Nano-LC is a practical and effective miniaturized version of HPLC.
- The system exhibits high performance in terms of reproducibility, efficiency, and sensitivity.
- Nano-LC is highly attractive for the microcharacterization of valuable and minute protein samples.
- Coupling nano-LC with mass spectrometry is a promising future direction.
Related Concept Videos
00:57High-Performance Liquid Chromatography: Instrumentation
High-performance liquid chromatography, or HPLC, is an analytical technique that separates liquid samples under high pressures. An HPLC instrument consists of glass bottles for storing solvents called mobile phase reservoirs. HPLC-grade solvents are used to maintain high purity, and the dissolved gases are removed using a degasser, such as a vacuum pumping system or sparging with helium. The solvents are then pumped into the analytical column using a screw-driven syringe or reciprocating pumps.
01:11High-Performance Liquid Chromatography: Introduction
High-performance liquid chromatography(HPLC), formerly referred to as High-pressure liquid chromatography, is a powerful technique used to separate, identify, and quantify components in complex mixtures. The term "high pressure" refers to using high pressure to push the liquid mobile phase through the tightly packed columns.
In HPLC, two phases play a critical role in the separation process:
In HPLC, two phases play a critical role in the separation process:
01:05High-Performance Liquid Chromatography: Elution Process
In High-Performance Liquid Chromatography (HPLC), the elution process is critical to the separation of analytes and the quality of chromatographic results. Elution describes how compounds move through the column and separate based on their interactions with the mobile and stationary phases. This process determines the resolution, peak shape, and retention times in the chromatogram, which are essential for identifying and quantifying components in complex mixtures. Understanding the elution...
01:15High-Performance Liquid Chromatography: Types of Detectors
The role of the detectors in High-Performance Liquid Chromatography (HPLC) is to analyze the solutes as they exit from the chromatographic column. The detector recognizes the solute's property and generates corresponding electrical signals, which are converted into a readable graph of the detector's response versus elution time called a chromatogram at the computer. There are several types of HPLC detectors, each with its own advantages and limitations, depending on the analyte properties and...