High performance capillary electrophoresis - T.E.A.M.
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High performance capillary electrophoresis - T.E.A.M.

High performance capillary electrophoresis - T.E.A.M. High performance capillary electrophoresis - T.E.A.M.

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03.01.2015 Views

Modes Type Comment Silylation coupling ● Numerous usable functional groups (Si-O-Si-R) ● Generally simple to prepare R = Polyacrylamide ● Slioxane bond stable between pH 4 and 7 Aryl penta fluoro ● Limited long term stability Protein or amino acid Sulfonic acids Maltose PEG Polyvinyl pyrrolidinone Direct Si-C coupling Polyacrylamide via Grignard ● Si-C binding eliminates need for silylation ● pH stable between 2 and 10 ● Difficult to prepare Adsorbed polymers ● Poor long-term stability Cellulose, PEG, PVA ● Low pH range (2 to 4) ● Relatively hydrophobic Table 12 Bonded or adhered phases Adsorbed, crosslinked polymers Polyethyleneimine GC phases PEG Phenylmethyl silicone LC phases C 2 , C 8 , C 18 ● Reverses EOF ● Useful for basic proteins ● Stable at physiological pH ● Hydrolytically unstable ● Can increase protein adsorption Depending on the deactivation, the EOF can be eliminated or reversed. Neutral deactivation with polyacrylamide or polyethylene glycol, for example, eliminates EOF. This results from both decreased effective wall charge and increased viscosity at the wall. Deactivation with cationic groups reverses the EOF. Deactivation with amphoteric species, such as proteins or amino acid, yield reversible EOF depending on the pI of the coating and pH of the buffer (figure 26). 56

Modes EOF [mm/min] 50 40 30 20 10 These covalent modifications are intended to be permanent and to require little or no maintenance. Since the capillaries are usually washed after use (adsorption may occur even with the coating), they must be stable to washing solutions and to hydrodynamic flow. Unfortunately, the stability of most coatings is limited. It is anticipated that numerous types of stable coatings will soon be purchasable, similar to LC and GC columns. 0 -10 -20 0 2 4 6 8 10 pH Figure 26 Reversible electroosmotic flow in a a-lactalbumin-coated capillary 12 3.1.2.2 Dynamic deactivation Addition of modifiers to the running buffer is an alternative to the bonded or adhered phases. An advantage of dynamic coatings is stability. Since the modifier is in the buffer, the coating is continuously regenerated and permanent stability is not required. As with covalent coatings, additives can interact with the wall and alter charge and hydrophobicity. These modifiers are both easier to implement and optimize since they are prepared by simple dissolution of the modifier in the buffer. Several dynamic deactivation methods are listed in table 13 and an illustration of the use of cationic surfactants to reverse the EOF was shown in figure 22. A potential disadvantage of the dynamic modification approach is that solutes as well as the capillary surface are affected. Biological-type conditions will be sacrificed by the use of pH extremes and addition of surfactants. Another limitation can be the equilibration time needed to obtain a reproducible surface and constant EOF. Furthermore, postcolumn analyses such as mass spectrometry and enzymatic assays are sensitive to additives, especially those in high concentrations. 57

Modes<br />

Type<br />

Comment<br />

Silylation coupling<br />

● Numerous usable functional groups<br />

(Si-O-Si-R)<br />

● Generally simple to prepare<br />

R = Polyacrylamide ● Slioxane bond stable between pH 4 and 7<br />

Aryl penta fluoro<br />

● Limited long term stability<br />

Protein or amino acid<br />

Sulfonic acids<br />

Maltose<br />

PEG<br />

Polyvinyl pyrrolidinone<br />

Direct Si-C coupling<br />

Polyacrylamide via Grignard<br />

● Si-C binding eliminates need for<br />

silylation<br />

● pH stable between 2 and 10<br />

● Difficult to prepare<br />

Adsorbed polymers<br />

● Poor long-term stability<br />

Cellulose, PEG, PVA ● Low pH range (2 to 4)<br />

● Relatively hydrophobic<br />

Table 12<br />

Bonded or adhered phases<br />

Adsorbed, crosslinked polymers<br />

Polyethyleneimine<br />

GC phases<br />

PEG<br />

Phenylmethyl silicone<br />

LC phases<br />

C 2<br />

, C 8<br />

, C 18<br />

● Reverses EOF<br />

● Useful for basic proteins<br />

● Stable at physiological pH<br />

● Hydrolytically unstable<br />

● Can increase protein adsorption<br />

Depending on the deactivation, the EOF can be eliminated<br />

or reversed. Neutral deactivation with polyacrylamide or<br />

polyethylene glycol, for example, eliminates EOF. This<br />

results from both decreased effective wall charge and<br />

increased viscosity at the wall. Deactivation with cationic<br />

groups reverses the EOF. Deactivation with amphoteric<br />

species, such as proteins or amino acid, yield reversible<br />

EOF depending on the pI of the coating and pH of the buffer<br />

(figure 26).<br />

56

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