Perfusion column

Koal et al. (2004) measured four immunosuppressants (cyclosporine A, tacrolimus, sirolimus, and everolimus) in whole blood samples from transplant recipients. The samples were treated first with a protein precipitation step. The supernatant was extracted with a Poros Rl/20 perfusion column (30 x 2.1 mm, 20 tm, Applied Biosystems, Darmstadt, Germany) online. A Luna phenyl hexyl column (2 x 50 mm, Phenomenex, Schaffenburg, Germany) was used for separation. The total run time was 2.5 min. The lower limit of quantitation was 10 ng/mL for cyclosporine A and 1 ng/mL for the other three analytes. 

Anion exchange perfusion column POROS HQ/10 packed with cross-linked polystyrene-divinylbenzene beads. 

Kassel, D. B. Shushan, B. Sakuma, T. Salzmann, J. P. 1994. Evaluation of packed capillary perfusion column HPLC/MS/MS for the rapid mapping and sequencing of enzymatic digests. Anal. Chem., 66,236-243. 

One soil bacterium isolated from the soil perfusion columns and later identified as P. striata was capable of evolving 14C02 from the ring labeled portion of CIPC (28). The isopropyl moiety was lost as some volatile component that could not be recovered in the C02 traps. Products resulting from metabolism of carbon labeled CIPC have not yet been identified. 

Figure 4. CO2 evolution from ring and chain labeled C7PC-14C and carbonyl labeled banol from soil perfusion columns...

Frey et al. [61] compared the plate height equations under linear conditions for chromatography with traditional and with perfusion columns. In the latter case, convection takes place in large macropores inside the packing particles. For a column packed with a material having a uniform porosity, in the pores of which there is no convection, the reduced plate height is expressed by the following relationship [29-31]... 

The concentration of creatinine, medinal, and MMS was determined at the inlet and the exit of the perfusion column after 15, 30, 45, and 60 min, using standard methods and techniques. Simultaneously, the loss of platelets and leucocytes, as well as of free hemoglobin, from blood plasma was carried out. 

Concentration and MWD of F-PHEA After Absorption. F-PHEA was determined in perfusate samples by quantitative GPC relative to a freshly prepared F-PHEA standard run on the same day. Either a mixed-bed column (12 x 300 mm Sephacryl S-200 Sephadex G-25 SF 3 1, Pharmacia LKB) or a Separon HEMA-Bio 40 column (8 x 250 mm 10 pm particle size, Tessek A/S, Aarhus, Denmark) was used with a 20 pL injection volume. A mobile phase of pH 7.4 phosphate buffered saline (0.05 M phosphate, 0.15 M NaCl) was supplied (Model LC-7A Bio Liquid Chromatograph, Shimadzu Corporation, Kyoto, Japan) at 0.5 or 1 mL/min. Fluorescent detection was employed (Model RF-535 Fluorescence HPLC Monitor,... 

Shimadzu Xex = 486nm, A,em = 516 nm) with a chart recorder (Fisher Recordall, Series 5000, Springfield, NJ). F-PHEA concentrations in perfusate were determined by area comparison of the eluted curves to that of the standard. Curves of logMW vs. elution volume, Ve, were constructed for each column using the integral-MWD method as described previously (9). Molecular weight distributions of absorbed material were determined from the calibration curves and the sample s chromatograph by curve summation (9). 

The effect of pore size on CEC separation was also studied in detail [70-75]. Figure 9 shows the van Deemter plots for a series of 7-pm ODS particles with pore size ranging from 10 to 400 nm. The best efficiency achieved with the large pore packing led to a conclusion that intraparticle flow contributes to the mass transfer in a way similar to that of perfusion chromatography and considerably improves column efficiency. The effect of pore size is also involved in the CEC separations of synthetic polymers in size-exclusion mode [76]. 

Most of the applications of HPLC for protein analysis deal with the storage proteins in cereals (wheat, corn, rice, oat, barley) and beans (pea, soybeans). HPLC has proved useful for cultivar identihcation, protein separation, and characterization to detect adulterations (illegal addition of common wheat flour to durum wheat flour) [107]. Recently Losso et al. [146] have reported a rapid method for rice prolamin separation by perfusion chromatography on a RP POROS RH/2 column (UV detection at 230nm), sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and molecular size determination by MALDl-MS. DuPont et al. [147] used a combination of RP-HPLC and SDS-PAGE to determine the composition of wheat flour proteins previously fractionated by sequential extraction. 

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