Chromatographic activity of residual silanols of alkylsilane derivatized silica surfaces

Scholten, Alex; Claessens, H.A.; Haan, J.W. de; Cramers, C.A.M.G.

doi:10.1016/s0021-9673(96)00765-0

Cited by 38 publications

(21 citation statements)

References 31 publications

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“…While the chromatographic surfaces were "end-capped," unreacted silanols undoubtedly remain, given the carbon loadings and surface areas of the matrices. This is consistent with published observations on other commercial matrices (Scholten et al, 1997). Estimates of the pK a of the unreacted silanol sites range from 3.5-4.0 for general RPC surfaces (Mant and Hodges, 1991) to near 7 for the specific surface used here (Ole Schou, pers.…”

Section: Discussionsupporting

confidence: 91%

Protein unfolding during reversed‐phase chromatography: II. Role of salt type and ionic strength

McNay

O’Connell

Fernandez

2001

Biotech & Bioengineering

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While reversed-phase chromatography (RPC) may be a powerful method for purification of proteins at the analytical scale, both preparative and analytical applications have been hindered by the complex chromatographic behavior of proteins compared to small molecules. Further, preparative applications have been limited because of poor yields caused by the denaturing conditions involved. One means for modulating both the stability and chromatographic behavior of proteins is through the use of added salt. In this investigation, we show how salt type and ionic strength affect protein conformation on RPC surfaces. Exposure of amide groups of adsorbed BPTI was monitored using nuclear magnetic resonance (NMR) spectroscopy and hydrogen-deuterium isotope exchange. Sodium chloride, sodium acetate, and ammonium sulfate were studied at ionic strengths up to I = 0.375, with adsorption hold times being 5 min and 2 h. We found that increasing ionic strength decreased exposure of the exchange reporter groups in essentially all cases. However, even at the same ionic strength the level and distribution of residue protection varied with salt type and hold time. NaCl does not protect certain reporter groups at all, while those that it does protect to some degree at short hold times can exchange slightly more at longer times. The pattern and level of protection for NaAc at short times is similar to that for NaCl, but at longer times more uniform protection is seen as the reporter groups completely exposed at short times become more protected. For (NH(4))(2)SO(4) the pattern of protection at short hold time is similar to those of the other salts, although it protects all groups much more. This would be expected from the Hofmeister series. However, at longer times the level of protection with (NH(4))(2)SO(4) decreases below that of the other salts, while it uniquely protects all groups to nearly the same level. Such subtle variations in the protein structure would not have been detected without the measurements and analysis used here. Chromatographic retention times and peak shapes were obtained for the above systems. Variations of behavior were seen that could not be correlated with any of the above protection patterns and levels or even with heuristics such as the Hofmeister series. This suggests further conformational changes upon elution may be critical to the retention process. However, an excellent correlation was found between peak width at half-height and the average degree of unfolding, as indicated by the average level of isotopic exchange. Thus, while further studies are needed to definitively determine the connection between protein unfolding and retention, use of this correlation may improve designing and screening for chromatographic conditions that minimize protein unfolding.

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Section: Discussionsupporting

confidence: 91%

Protein unfolding during reversed‐phase chromatography: II. Role of salt type and ionic strength

McNay

O’Connell

Fernandez

2001

Biotech & Bioengineering

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“…Thus, in examining the spectra shown in Figure 3, the peak that could be derived from the remaining methoxy groups of trifunctional AEAPTS was not detected in the +60 ppm region [13]. This fact clearly indicates that methoxy groups were hydrolyzed during the washing procedure of the preparation step.…”

Section: Nmr Spectramentioning

confidence: 95%

“…FTIR spectra were obtained in the 4 000 to 450 cm -1 region by means of the reflectance technique in a Bomen MB series spectrometer. Solid state NMR measurements of 13 …”

Section: Characterizationmentioning

confidence: 99%

“…The presence of the peak at -99 ppm is a clear indication that silanol groups are still present in the silica matrix, after the modification process. From the chromatographic point of view, this is not a desirable situation and an attempt to improve the surface is to force an endcapping reaction using a more reactive silylating agent such as chlorotrimethylsilane or hexamethyldisilazane as suggested [13].…”

Section: Nmr Spectramentioning

confidence: 99%

“…Another feature to be considered in the characterization procedure is the solid state 13 C NMR spectroscopy which was applied to characterize the structure of AEAPTS ligand attachment on the silica surface. Thus, in examining the spectra shown in Figure 3, the peak that could be derived from the remaining methoxy groups of trifunctional AEAPTS was not detected in the +60 ppm region [13].…”

Section: Nmr Spectramentioning

confidence: 99%

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New Stationary Phase Prepared by Immobilization of a Copper-Amine Complex on Silica and Its Use for High Performance Liquid Chromatography

Silva¹,

Jardim²,

Airoldi³

1999

J. High Resol. Chromatogr.

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A new solventless process to hydrophobize silica powders in fluidized beds

2008

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A new hydrophiztaion technique was developed in a fluidized bed reactor to modify the surface of silica‐based powders with n‐octadecyltrichlorosilane (ODTCS). The reaction was performed at dry phase by mixing two classes of particles during the fluidization: “target‐particles” to be treated (a fine microporous silica powder) and “ODTCS‐carrier” particles consisting of coarser porous alumina beads containing an adequate amount of reagent. The results showed that ODTCS was successfully anchored on silica surface and achieved high surface coverage (1.4 groups/nm2) in only few minutes with an interesting hydrophobic behavior. The energy consumption is reduced if the reaction is carried out at low temperatures, but using humidified air (7% RH). These conditions promote, however, a vertical polymerization of the reagent on solids surface. © 2008 American Institute of Chemical Engineers AIChE J, 2008

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Chromatographic activity of residual silanols of alkylsilane derivatized silica surfaces

Cited by 38 publications

References 31 publications

Protein unfolding during reversed‐phase chromatography: II. Role of salt type and ionic strength

Protein unfolding during reversed‐phase chromatography: II. Role of salt type and ionic strength

New Stationary Phase Prepared by Immobilization of a Copper-Amine Complex on Silica and Its Use for High Performance Liquid Chromatography

A new solventless process to hydrophobize silica powders in fluidized beds

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