Microbore columns utilizing polypropylene capillary-channeled polymer (C-CP) fibers as the stationary phase in high-performance liquid chromatography (HPLC) have been investigated. The polypropylene C-CP fiber diameter is approximately 50 microm, with eight channels along the periphery of the fiber ranging in diameter from approximately 12 to 35 microm. The polypropylene C-CP fibers were packed into fluorinated ethylene propylene (FEP) tubing, 1.3 mm inner diameter, with lengths of 500, 750, and 1,000 mm, to examine the effects of increased column length with regards to plate height, resolution and analysis time. The low backpressures characteristic of the C-CP fiber stationary phases allow the length of the column to be increased without significantly decreasing the specific permeability. The high specific permeability (approximately 5x10(-8) cm2) of the C-CP packed microbore columns yields a relatively low backpressure of 2.35 MPa at the highest flow rate of 17 microL/s (54 mm/s) for a 1,000 mm column. Radial compression of the soft-walled FEP tubing is accomplished by pulling the 1.7 mm o.d. column through a 1.4 mm diameter orifice. Reducing the inner diameter of the column from 1.3 to 1.0 mm lowered the interstitial fraction from 47% to 42%, decreased the A-term contributions to band broadening, resulted in a significant decrease in average plate height (approximately 30%), and increased resolution (approximately 36%) at identical linear velocities. Although the lower void volume of the radially compressed column increased the backpressure from 0.57 to 2.11 MPa at a linear velocity of approximately 20 mm/s, the specific permeability only decreased from approximately 7x10(-8) to 4x10(-8) cm2.

Capillary-channeled polymer (C-CP) fibers as a stationary phase in microbore high-performance liquid chromatography columns

The performance of microbore columns with polypropylene (PP) capillary-channeled polymer (C-CP) fibers as the support/stationary phase for separation of macromolecules has been investigated. Polypropylene C-CP fibers (40 μm diameter) were packed in fluorinated ethylene propylene (FEP) tubing of inner diameter 0.8 mm and lengths of 40, 60, 80, and 110 cm. The performance of PP fiber packed microbore columns (peak width, peak capacity, and resolution) was evaluated for separation of a three-protein mixture of ribonuclease A, cytochrome c, and transferrin under reversed-phase gradient conditions. The low backpressure characteristics of C-CP fiber columns enable operation at high linear velocities (up to 75 mm s−1 at 1.5 mL min−1). In contrast with the performance of other phases, such velocities enable enhanced resolution of the three-protein mixture, because peak widths decrease with velocity. Increased column length resulted in increased resolution, because the peak widths remained essentially constant, although retention times increased. In addition, it was found that the peak capacity increased with column length and linear velocity. Radial compression of the microbore tubing enhanced the homogeneity of the packing and, thereby, separation efficiency and resolution. Radial compression of columns resulted in a decrease in the interstitial fraction (~5 %), but increased resolution of ~14 % between ribonuclease A and cytochrome c. Even so, a linear velocity of 75 mm s−1 required a backpressure of 9.5 MPa only. It is clear that the fluid and solute-transport properties of the C-CP fiber microbore columns afford far better performance than is obtainable by use of standard format columns. The ability to achieve high separation efficiencies, rapidly and with low volume flow rates, holds promise for high-capacity protein separations in proteomics applications.

Microbore polypropylene capillary channeled polymer (C-CP) fiber columns for rapid reversed-phase HPLC of proteins

We describe a simple method for the direct inline separation of two immiscible liquids based on the selective wetting and permeation of a porous polytetrafluoroethylene capillary by one of the liquids. Using water dispersed in fluorous carrier fluid as a test system, quantitative recovery of the water from the carrier fluid is achieved over a wide range of flow conditions, with no contamination by the fluorous component even when present in large (ten-fold) excess. The exiting water stream may be readily redispersed by injecting additional carrier fluid downstream, allowing for repeated switching between the segmented and continuous flow regimes – a critical requirement for multistep chemical processing. The separator is shown to simplify in-line sample analysis by allowing measurements to be carried out quasi-statically without the need for fast instrumentation synchronised to the segmented water flow.

/pdf/microscale-separation-of-immiscible-liquids-using-a-porous-1sscibvtd2.pdf

Microscale separation of immiscible liquids using a porous capillary

A proteomic study of rat urine was undertaken using two-dimensional gel electrophoresis, microbore high performance liquid chromatography, mass spectrometry and N-terminal sequencing. Five known urinary proteins were identified but two novel peptide fragments matched a large number of rat expressed sequence tags (ESTs) from a liver library. By combining protein chemical and nucleotide data, two 101-residue open reading frames with 90% amino acid identity were determined, rat urinary protein 1 (RUP-1) and RUP-2. The data established signal peptide removal and provided evidence for N-glycosylation. A third related sequence, rat spleen protein (RSP-1) was confirmed from EST searches. These three proteins have been submitted to SWISS-PROT as P81827, P81828 and Q9QXN2, respectively. A fourth novel homologue was found in porcine and bovine ESTs from embryo libraries. Alignment with known homologues showed conserved cysteine positions characteristic of a secreted subfamily of Ly-6 proteins. In two cases, antineoplastic urinary protein and caltrin, these homologues have unverified functional annotations. The RUP sequences showed high scoring matches to three unrelated rat mRNAs subsequently established to be chimeric. Two of these share extended sectional identity to RUP-1 but the third may represent another novel Ly-6 homologue. These chimeras have caused serious annotation errors in secondary databases.

The characterisation of novel secreted Ly-6 proteins from rat urine by the combined use of two-dimensional gel electrophoresis, microbore high performance liquid chromatography and expressed sequence tag data.

Perfusion reversed-phase high-performance liquid chromatography for protein separation from detergent-containing solutions: an alternative to gel-based approaches.

Disposable microbore high-pressure liquid chromatography columns for protein and peptide separations.

Synergistic scale-down of three protein micropreparation techniques

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Disposable microbore high-pressure liquid chromatography columns for protein and peptide separations.

Synergistic scale-down of three protein micropreparation techniques