28.02.2013 Views

Handbook of Size Exclusion Chromatography and Related ...

Handbook of Size Exclusion Chromatography and Related ...

Handbook of Size Exclusion Chromatography and Related ...

SHOW MORE
SHOW LESS

Create successful ePaper yourself

Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.

industries (particularly the pharmaceutical industry), as well as in the chemical<br />

industry. Because the polarities <strong>of</strong> the mobile <strong>and</strong> stationary phases were the<br />

opposite <strong>of</strong> those in adsorption chromatography, this mode <strong>of</strong> liquid<br />

chromatography is generally referred to as reversed phase LC.<br />

Several polar bonded phases were developed based on the same bonding<br />

chemistry used to prepare C18, C8, <strong>and</strong> other alkyl bonded phases.<br />

Cyanopropyldimethylchlorosilane, 1,2-epoxy-3-propoxypropyltriethoxysilane,<br />

<strong>and</strong> aminopropyltriethoxysilane were reacted to obtain cyano, diol, <strong>and</strong> amino<br />

polar bonded phases, respectively.The cyanophase isaweaker adsorbing surface<br />

than plain silica, but it shares the benefit <strong>of</strong> bonded phases in that equilibrium is<br />

reached within minutes <strong>and</strong> retention is not strongly affected by traces <strong>of</strong> water in<br />

the mobile phase. Because <strong>of</strong> the presence <strong>of</strong> the propyl anchor group, the cyano<br />

phase has also been used as aweak alkyl bonded phase with aqueous/organic<br />

mobile phases. Under such conditions, the cyano group imparts special polar<br />

selectivity, such as seen in the analyses <strong>of</strong> tricyclic antidepressants <strong>and</strong> PTH<br />

(phenylthiohydantoin) amino acids. The amino phase has mainly been applied<br />

to the analysis <strong>of</strong> carbohydrates using water/acetonitrile mobile phases. The<br />

separation mode resembles adsorption (normal phase) chromatography in that an<br />

increase in the percentage <strong>of</strong> water decreases retention. Although the diol phase<br />

has been applied as asubstitute for silica in the analysis <strong>of</strong> steroids, for example,<br />

its main use has been as asupport in gel filtration chromatography,as discussed<br />

later in more detail. Columns packed with cyano, amino, or diol bonded<br />

phase silica are more popular in adsorption chromatography than plain silica<br />

columns (76).<br />

Figure10showsthetype<strong>of</strong>chemistryforthepreparation<strong>of</strong>thediolbonded<br />

phase, the usefulness <strong>of</strong> which was first demonstrated for SEC by Regnier <strong>and</strong><br />

Noel (5). The 1,2-epoxy-3-propoxypropyltriethoxysilane reagent is bonded to the<br />

silica following a reaction in toluene at 1208C for 12 h. After a washing step, the<br />

epoxide ring is opened by heating the bonded silica in strong acid for 1 h. In<br />

aqueous mobile phases, unreacted ethoxy groups are converted into silanol groups<br />

that can contribute to extra retention <strong>and</strong> adsorption effects. The bonding<br />

chemistry shown in Fig. 10 for preparing GFC phases is similar to the st<strong>and</strong>ard<br />

procedures for preparing deactivated phases for GPC. In this case, trimethylchlorosilane<br />

is bonded with silica in the presence <strong>of</strong> toluene as a solvent. Usually<br />

the reaction is repeated to maximize the coverage <strong>of</strong> trimethylsilyl groups. This<br />

“end-capping” step is also used as a second reaction in the preparation <strong>of</strong> reservedphase<br />

packing materials but is not common for most polar bonded phases.<br />

The diol functional group has been commercialized by several<br />

manufacturers (see Table 6), but other functional groups are worth mentioning.<br />

Engelhardt <strong>and</strong> co-workers have investigated the properties <strong>of</strong>, in particular, the<br />

amide bonded phase, which is prepared by reacting N-(3-triethoxysilylpropyl)<br />

acetamide with silica under similar conditions as used for the diol phase (51,77).<br />

© 2004 by Marcel Dekker, Inc.

Hooray! Your file is uploaded and ready to be published.

Saved successfully!

Ooh no, something went wrong!