High-performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC) is a well-known and widely used separation technique in the field of chemistry and research. The authors of this article explore and perform a method of using microchip HPLC to illustrate potential improved and more efficient separations of peptides and proteins.

Different advantages to using improved systems of HPLC were discussed. For example, the typical HPLC with 1 mL and 4.6 mm capacity and internal diameter is at a disadvantage to microbore and capillary-based HPLC columns that have 100 um to 2 mm internal diameters. A microchip system also provides an option for analyzing solvents at low volumes and different separations. Recent attempts at microchip HPLC consisted of controlling pump pressure and sample injections into microsystems. A micro device that uses on-chip injection, separation, and detection was used. The authors used this injection method with a miniaturized LIF detector. This detector was used to detect eluted molecules that were fluorescently tagged and to test repeatability and linearity. The microchip was made of silica substrates using photolithography, wet etch, and bonding techniques.

They coated the inside surfaces of the chip with fluoroalkyl coating to recude the surface friction coefficient.

A microwave injector, made by laser-polymerization, served as a valve seat between the buffer channel and the sample channel and was positioned perpendicular to the separation monolith. They attached the separation media by incubating the microchip in a mixture of glacial acetic acid, DI water, and 3-(trimethoxysilyl) propyl acrylate for 30 minutes and then rinsing with acetonitrile. Contact lithography was used for separation. It allowed for polymerization of the monolith separation medium before the chip was rinsed again with acetonitrile. Pressure pulses, by use of manually operated syringe pumps, were sent to the sample line for injections. The buffer line pressure was held constant at 150 psi below the pressure in the sample channel at a maximum of a 1600 ms interval in order to switch the valve and inject the sample. The authors concluded that controlling the interval of pressure pulses was successfully performed for separations.

The injections showed reproducible data with a 3.9% RSD for peak area and 3.6% RSD for retention time. The peak areas for the injection of peptides using the on-chip RP-HPLC separations were repeatable with 3.8% RSD and 0.03% RSD retention time. As for the issue of switching samples and sample carryover the authors tested, there was a switching limit of ~0.2 Hz that illustrates the picoliter-injection HPLC system can successfully rapidly switch samples. The authors also concluded that their system could be connected to a nanospray mess spectrometer for analysis of different analytes.

Future work was proposed and suggestions of different uses for the microchip HPLC were given such as environmental monitoring. Unfortunately, the article was extremely complex and in what it seemed to be an attempt to be concise writers, the authors butchered the flow of explaining their technique and how the system actually worked better than less advanced ones.

Reichmuth, D. S.; Shepodd, T. J.; Kirby, B. J. Anal. Chem. 2005. 77, 2997-3000.