Evaluation of stationary phases for 2-dimensional HPLC of proteins Part 1. Validation of commercial RP-columns.

RP-separation with TFA-based water/acetonitrile eluents is widely used for peptides and small proteins but is well known difficult for large or membrane proteins. Especially in proteomics or other complex biological matrices reliable elution patterns are difficult to achieve. New commercial stationary phases are validated regarding long term stability, protein recovery, carry over, symmetry and selectivity using 10 different proteins with different molar weights, isoelectric points and glycosylation. It could be demonstrated that some stationary phases had poor protein elution performances. They did not elute a protein at all or with minor recovery, peak symmetries. Sometimes bad and formidable carry over effects for peak areas in the following run were observed. Selectivity in separation of different isomers or glycosylated proteins is also different. The results suggest that neither surface area nor pore diameter play an important role in the application of reversed phases for HPLC of proteins. The investigations leads one to suppose that the bonding chemistry seems to be an important aspect. Most critical fact is that some RP-phases did not elute a protein at all others only 20% of the injected protein mass, which makes the objective of an RP-chromatogram highly questionable.

Quantitative determination of albumin in urine by on-line immunoadsorptive cleanup and reversed-phase chromatography.

Albumin in urine is selectively adsorbed on an immunoadsorber (human serum albumin-specific antibodies coupled covalently with a silica stationary phase) and after elution with 0.1% HCl is quantitatively determined by reversed-phase chromatography with detection of native fluorescence. The optimization of sample preparation and characteristics of the method such as recovery, linearity, reproducibility, detection limit, and selectivity are discussed.

Determination of digoxin in serum by on-line immunoadsorptive clean-up high-performance liquid chromatographic separation and fluorescence-reaction detection.

The use of valve-switching with an anti-digoxin immunoadsorptive clean-up cartridge prior to reversed-phase elution of serum is described. Eluted digoxin is detected by reaction of the unsplit eluent stream with hydrochloric acid in a PTFE coil, followed by fluorescence detection. Routes for optimization and evaluation of the immunoadsorptive clean-up are reported, and the effects on the recovery rate examined. The detection limit of 300 pg/ml and reproducibility of less than 7% for the application of the analytical procedure to serum are reasonably good.