34 Buyers’ Guide 2021
power of the new large pore monodense FPP 1.7um 400A material compared to that of a traditional 300Å material. Figure 9 shows a separation of Cytochrome C it is evident that a side peak is clearly present (as denoted by ‘B’ at 14.1 mins, which is not present for the competitor columns. These side peaks are possibly a higher order of aggregate impurities (or polymeric versions of Cytochrome C) present in the Cytochrome C. Interconversion of monomers and polymers in Cytochrome c has been known for nearly half a century, but the mechanism of its polymerisation is still unknown due to a lack of information on the structural and thermodynamic characteristics of the oligomerization process. The large pore FPP 1.7um 400A has a higher sensitivity to these impurities compared to the competitive column. Similar results are noted for monoclonal antibodies (mAbs) however the data is currently in publication elsewhere. The structures for A (monomeric) and B polymerics Cytochrome C are shown in Figure 8.
Conclusion:
New wide-pore and ultra-wide pore fully porous and superficially porous silica particles have been synthesised and surface treated with dimethyl butyl stationary phase for the separation of large protein molecules. The new materials proved to be stable with improved efficiency and peak shapes when employing low pH mobile phases containing additives like Trifluoroacetic acid (TFA) and Formic acid (FA) at high temperatures (40-60°C). The high column efficiency of the fully porous particle (FPP 1.7µm C4-400Å) compared to its commercially available competitive counterpart is primarily due to the narrow particle size distribution ratio and the
large surface area which contributed to effective sample loading. The ultra-wide pore superficially porous particle (SPP 2.6µm C4-1000Å) showed more sensitivity to aggregate impurities in cytochrome C compared to its competitive counterpart. These materials should find wide application in biopharmaceutical separations determining the purity of new drugs where high speed, high sensitivity and efficient separations without a drastic increase in column pressure are important.
References
1. B. M. Wagner, S. A. Schuster, B. E. Boyes, J. J. Kirkland, Superficially porous silica particles with wide pores for biomacromolecular separations, J. Chromtogr. A, 1264 (2012) 22-30
2. S. A. Schuster, B. M. Wagner, B. E. Boyes, J. J. Kirkland, Wider pore superficially porous particles for peptide separations by HPLC, J. Chromatogr. Sc. Vol. 48, Advanced Material Technology, Inc. Wilmington, DE 19810, August 2010.
3. LCGC North America, vol. 35, issue 4, April 2017, pages 22-23
4. A. Astefanei, I. Dapic, M. Camenzuli, Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations, Chromatographia 2017, 80(5), 665-687
5.
V.K.Langsi, B. A. Ashu-Arrah, J. D. Glennon, Sub-2-µm seeded growth mesoporous thin shell particles for high- performance liquid chromatography: Synthesis, functionalisation and characterisation, J. Chromatogr. A, 1402 (2015) 17-26.
6. S. A. Schuster, Improve your monoclonal antibody separations by leveraging
the advantages of Fused-core column, Advance Material Technology, Inc. Wilmington, DE 19810, July 15, 2018
7. J. P. Hanrahan, T. O’Mahony, R. Curley, J. J. Hogan, J. M. Tobin, An introduction to the concept of monodensity in silica particles and its effect on chromatographic performance, Chromatography Today, February/March 2017
8. M. Barrande, R. Bouchet, and R. Denoyel, Tortuosity of Porous Particles, Anal. Chem. 2007 (79), 9115-9121
9. R. Takahashi, S. Sato, T. Sodesawa and H. Nishida, Effect of pore size on the liquid- phase pore diffusion of nickel nitrate, PCCP 2002, issue 15.
10. European patent office number EP2365997B1
https://patents.google.com/patent/ EP2365997B1/en)
11. E. Olah, S. Fekete, J. Fekete, K. Ganzler, Comparative study of new shell-type sub- 2µm fully porous and monolith stationary phases focusing on mass transfer resistance, J. Chromatogr. A, 1217 (2010) 3642-3653
12. F. Gritti, I. Leonardis, D. Shock, P. Stevenson, A. Shalliker, G. Guiochon, Performance of columns packed with the new shell particles, Kinetex-C18J. Chromatogr. A, 1217 (2010) 1589-1603.
13. E.P. Barrett, L.G. Joyner, P.P. Halenda, The Determination of Pore Volume and Area Distributions in Porous Substances. I. Computations from Nitrogen Iso- therms, J. Am. Chem. Soc., 73 (1951) 373-380.
14. N. Marchetti, A. Cavazzini, F. Gritti, G. Guiochon, J. Chromatogr. A. 1163 (2007), 203-211
Glass Autosampler Vials Virtually Eliminate Surface Activity
Reduced Surface Activity (RSA™) glass autosampler vials are not coated vials and virtually eliminate the adsorption of basic compounds found with all other glass vials. The manufacturing process of RSA™ vials produces vials without surface activity such as basic compound adsorption. Unlike ordinary glass vials, these vials will not produce a pH change with aqueous diluent over time in the vial and with minimised surface metals, it is excellent for LCMS in that it does not contribute to sodium adducts.
Laboratories testing low abundance analytes such as low dosage form pharmaceuticals, unknown unknowns and natural products will see the greatest benefit even though all labs will find these vials to have value when repeating runs or investigations must be avoided.
The vials are available from Microsolv in 12x32mm clear and amber with volumes of 2ml, 1.5ml and 300ul. More information online:
ilmt.co/PL/ZJGm
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