Stationary phases collapse

The physical structure of the stationary phase depends on the compatibility of the solvent with the bonded n-alkyl chains. Compatible nonpolar solvents tend to promote extension of the chains, allowing full penetration by the solvent. Conversely, fairly polar solvents tend to promote collapse of the chains upon each other, allowing negligible solvent penetration. The stationary phase therefore has the ability to adjust itself to maintain a relatively nonpolar character (113). Retention on monomeric bonded phases with octyl (C8) or longer chains are dominated by a partitioning-like mechanism (114). 

If polar sequences or short phosphopeptides fail to bind to the stationary phase upon loading, the acetonitrile content of the eluent could be reduced to 3%. A further decrease is not possible for standard Ci8-columns, as the stationary phase could collapse reducing the loading capacity and the reproducibility. Instead, TFA could be replaced by the more hydrophobic ion-pair reagent HFBA (heptafluorobutyric acid), or a stationary phase with a special coating compatible with pure aqueous eluents could be used (e.g., Aqua-column from Phenomenex). 

Void. An area in the LC column containing no packing. A void is the result of the stationary phase particles settling (collapsing). A void usually occurs at the top of a column and causes a large decrease in the efficiency. 

These have mainly been developed by McGuffin and Novotny [120] and coworkers and are characterised by low column diameter to particle size ratios of 2 to 5. This is much less than small bore packed columns (50-200) or conventional columns (500-2000). Below ratios of 2, it has been reported [101] that the packing structure collapses under the viscous flow and causes clogging of the column. The microcapillary columns are prepared by extruding a heavy walled glass tube, 0.5-2 mm i.d., packed with 10-50 pm particle size high temperatures resistant silica or alumina. For reverse phase work the stationary phases have then to be bonded in situ. 

If polar analytes are to be separated with reversed-phase HPLC, then very polar mobile phases must be used. An increase in the water content of the mobile phase leads to an increase in retention. With classical reversed phases, this is the case (at least for pressures under 200 bar) only when the proportion of organic solvent is above 5%. Below this, the retention suddenly collapses, so classical CIS columns cannot be used with 100% aqueous eluents. This is due to the high surface tension of water. More than 99% of the specific surface area ofthe stationary phase is located in the pores. If the proportion of organic solvent in the mobile phase falls below 5%, a drop forms at the entrance to the pores, which prevents the movement of analytes into and out of the pores. Only when the pressure rises above 200 bar is this again possible [7]. 

In highly aqueous mobile phases, which are often necessary for RPLC separations of polar compounds, non-polar bonded alkyls are poorly solvated and may collapse and stick together, changing significantly the properties of the bonded stationary phases. The so-called aqua bonded phases are designed to improve the solvation of the bonded material and to improve the retention of hydrophilic compounds when using highly aqueous mobile phases. 

Model chromatographic interfaces were examined using sum-frequency generation spectroscopy (SFG) in order to study the effect of various solvents on the structure and conformation of the stationary phase. Monolayers formed from mixed alkyltrichlorosilanes on fused silica were examined in contact with air, acetonitrile, isooctane, and water. Results show that monolayers of 100% Cig composition show little disorder in the alkyl chains for all the solvents examined, though a very distinct solvent shift is apparent in the methyl symmetric stretch. When mixed composition (Cis and Ci) monolayers were examined, significant disorder was induced in the alkyl chains for all the solvents. However, the largest change occurred when the monolayer was in contact with water. These results support a collapsed structure model for the mixed monolayer in contact with water. 

This lack of solvent-induced monolayer disorder was also observed by Pemberton and coworkers (7). Pemberton and coworkers used Raman spectroscopy to examine Cis alkylsiloxane monolayers on a SiQ/Ag substrate and found little disruption of the alkyl chains in contact with acetonitrile, as well as water. The results of Carr and Harris (19), however, showed that a Cis chromatographic stationary phase assumes a collapsed structure in water. The stationary phase can evolve from this collapsed state with the addition of an organic modifier, such as acetonitrile, to the aqueous solvent. The modifier intercalates into the monolayer, causing an increase in volume, polarity, and alkyl chain order. 

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