Monolith columns for liquid chromatographic separations of intact proteins: A review of recent advances and applications.

In this article we survey 256 references (with an emphasis on the papers published in the past decade) on monolithic columns for intact protein separation. Protein enrichment and purification are included in the broadly defined separation. After a brief introduction, we describe the types of monolithic columns and modes of chromatographic separations employed for protein separations. While the majority of the work is still in the research and development phase, papers have been published toward utilizing monolithic columns for practical applications. We survey these papers as well in this review. Characteristics of selected methods along with their pros and cons will also be discussed.

Narrow, Open, Tubular Column for Ultrahigh-Efficiency Liquid-Chromatographic Separation under Elution Pressure of Less than 50 bar.

We report that we can achieve extremely high separation efficiencies using a narrow, open, tubular (NOT) column for liquid-chromatographic separations, and we can carry out these separations under an elution pressure of no more than 50 bar. To improve the separation efficiency in packed-column liquid chromatography, one of the most effective approaches is to reduce the monodispersed-particle sizes. A direct consequence of reduced particle size is an increased elution pressure. High efficiencies have been obtained in ultrahigh-performance liquid chromatography (UPLC) using 1-2 µm or even submicron particles, and high elution pressures (greater than 1000 bar) are commonly used to carry out these separations. Open, tubular (OT) columns have been predicted to be the most efficient columns for high-efficiency liquid-chromatographic separations, as long as the column diameter is sufficiently small (1-2 µm). However, high efficiencies have not yet been publically reported, possibly because of the challenges (such as picoliter-volume detection, nanocapillary-column preparation, low sample loadability, etc.) of utilizing 1-2 µm diameter capillaries. In this paper, we show how we overcame these problems and achieved extremely high separation efficiencies using a 2 µm inner diameter capillary. We see 200+ apparent peaks with a peak capacity of 810 within 54 min when separating a sample from trypsin-digested cytochrome C, and we count 440 apparent peaks with a peak capacity of 1640 within 172 min when separating a sample from pepsin/trypsin-digested Escherichia coli cell lysate.

Two-dimensional liquid chromatography consisting of twelve second-dimension columns for comprehensive analysis of intact proteins.

A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 µm i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second-dimension columns, while the second-dimension separations were carried out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances.