High performance liquid chromatography column efficiency enhancement by zero dead volume recycling and practical approach using park and recycle arrangement.

A new instrumental approach to recycling HPLC is described. The concept is based on fast reintroduction of incremental peak sections back onto the separation column. The re-circulation is performed within a closed loop containing only the column and two synchronized switching valves. By having HPLC pump out of the cycle, the method minimizes peak broadening due to dead volume. As a result the efficiency is dramatically increased allowing for the most demanding analytical applications. In addition, a parking loop is employed for temporary storage of analytes from the middle section of the separated mixture prior to their recycling.

Comparison of different types of outlet frits in slurry-packed capillary columns.

Fused-silica capillary columns for high-performance liquid chromatography with 320 and 250 µm inner diameter were prepared by slurry packing with 5 and 3 µm Nucleosil C18 stationary phase. Different types of mechanical and monolithic outlet frits were used and their influence on the resulting column performance was evaluated. Columns with quartz wool exhibited symmetrical peaks and low theoretical plate height, and the preparation time was short. The performance of monolithic frits varied based on type of monolith, length of the frit, and silanization procedure. The best frit performed similarly to the quartz wool ones, but the preparation took several hours. Their main advantage lies in the possibility of on-column detection, because the detection window can be burnt immediately behind the frit.

Differentiation among various kinds of cheese by identification of casein using HPLC-chip/MS/MS.

In proteomics, proteins can be identified by enzymatic cleavage of the target protein using an enzyme of the known specificity (primarily trypsin), sequencing the obtained specific peptides by MS and comparing the amino acid sequence of the peptides with a protein database. The sophisticated approach described above was used in this study to determine and verify the original species of cheeses. Proteins were extracted from three different cheese samples which were produced from cow, sheep and goat milks. The isolated proteins were cleaved with trypsin and the peptides obtained were sequenced and identified by a HPLC-chip/MS/MS microfluidic system. Two different extraction methods and two various chromatographic sorbents packed in plastic chips were studied. Beta-lactoglobulin and four kinds of casein were found in the cheese samples. The species of kappa-casein were identified unambiguously in all the three cheese samples and, thus, kappa-casein can be used to determine the origin of milk of the cheese. The other proteins found in the samples show very similar primary structures and cannot be recommended for identification of the cheese milk origin.