Peak spreading in linear gradient elution chromatography with a thin monolithic disk.

The peak spreading of DNAs of various sizes [12-mer, 20-mer, 50-mer and 95-mer poly(T)] in linear gradient elution (LGE) chromatography with a thin monolithic disk was investigated by using our method developed for determining HETP in LGE. Electrostatic interaction-based chromatography mode (ion-exchange chromatography, IEC) was used. Polymer-based monolithic disks of two different sizes (12 mm diameter, 3mm thickness and 0.34 mL; 5.2 mm diameter, 4.95 mm thickness and 0.105 mL) having anion-exchange groups were employed. For comparison, a 15-µm porous bead IEC column (Resource Q, 6.4mm diameter, 30 mm height and 0.97 mL) was also used. The peak width did not change with the flow velocity for the monolithic disks where as it became wider with increasing velocity. For the monolithic disks the peak width normalized with the column bed volume was well-correlated with the distribution coefficient at the peak position K(R). HETP values were constant (ca. 0.003-0.005 cm) when K(R)>5. Much higher HETP values which are flow-rate dependent were obtained for the porous bead chromatography. It is possible to obtain 50-100 plates for the 3mm monolithic disk. This results in very sharp elution peaks (standard deviation/bed volume=0.15) even for stepwise elution chromatography, where the peak width is similar to that for LGE of a very steep gradient slope.

Interaction mechanism of mono-PEGylated proteins in electrostatic interaction chromatography.

The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEGylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.

[Normative data on tests for frontal lobe functions: Trail Making Test, Verbal fluency, Wisconsin Card Sorting Test (Keio version)].

Trail Making Test (TMT Part A, B), Verbal fluency test (phonemic, semantic) and Wisconsin Card Sorting Test (Keio version : KWCST) are useful to evaluate frontal lobe functions and are commonly used in clinical settings. However, few normative data for aged Japanese have been reported. We collected normative data on these tests in elderly population, and examined the effects of age and education on the performance of these tests. Seventy-six healthy adults, aged 45 to 74 years, participated in this study. Subjects were classified into three groups by age (45-54, 55-64, 65-74). Fifty-five subjects repeated the same tests after 6 months to examine the test-retest reliability. Performance of the TMT Part A was correlated with age (r= 0.530) and that of Part B was correlated with age (r=0.500) and education (r=-0.340). Performance on both phonemic and semantic fluency was correlated with education (phonemic: r=0.357, semantic: r=0.279). Number of Categories Achieved (CA) of KWCST was correlated with education (r=0.376). The test-retest reliability of all these tests except for semantic verbal fluency and difficulty maintaining set (DMS) of KWCST was good enough for longitudinal studies.