Applications Of Coated Capillaries

Free solution separations of proteins remain the most widespread application of coated capillaries. A few examples are discussed later and also shown in Table 1. Basic protein separations (pI > 7) are typically done under acidic conditions because they carry a net positive charge. Under both high and minimal EOF conditions the applied polarity is positive. Figure 1 demonstrates a high-resolution, high-efficiency separation (500,000 plates/50 cm) of a mixture of five basic proteins using a preformed polymer-coated capillary. Acidic proteins (pI < 7) are typically run under basic conditions and carry a net negative charge. In neutral coated capillaries, where the EOF is minimized, these separations are normally done under negative or reversed polarity conditions. Mixtures of acidic and basic proteins are typically run under pH conditions that facilitate ionization of all the proteins while avoid-

Table 1

Protein Applications Using Various Coated Capillaries

Capillary coating

Proteins tested

Polyvinylpyrollidone coated capillary

Epoxy-diol coating Maltose coating

Standard proteins varying in pi from 4.5 to 11 and in size from 12-77 kDa Standard basic proteins at pH 4.0 Standard basic proteins at pH 4.0

Polyethyleneimine coating-

(cationic coating) Polyacrylamide-coated capillary through Si-C bond

Standard basic proteins at pH 7.0

Model protein separations (mainly acidic) at pH 9.5 ranging in pi of 4.3-7.6; model protein separations (mainly basic) at pH 2.7 ranging in pi of 4.5-10.7.

Alpha lactalbumin coated capillary Allyl-methyl cellulose coated capillary Allyl-dextran coated capillary

Model protein separations

(mainly basic and myoglobin) Model protein separations (acidic) at pH 9.8; Model protein separations (basic) at pH 7.0 stable coating

Reference

Comments

Electrolyte: 38.5 mM H3P04, 20 mM NaH2P04, pH 2.0

Electrolyte: phosphate buffer at pH 4.0; 35.000-50.000 plates achieved in a 52 cm capillary at an applied voltage of 20 kV Electrolyte: phosphate buffer at pH 4.0.; 25.000 plates in a 39.5 cm capillary at an applied voltage of 10 kV Electrolyte: 20 mM hydroxylamine-

HC1 at pH 7.0 Electrolyte: 50 mM glutamine; triethylamine at pH 9.5; 30 mM citric acid (pH 2.7 adjusted with 1 MNaOH). (Note: Electrolyte at pH 9.5 has an amine additive that can potentially interact with residual unmasked silanol groups)

Electrolyte: 50 mM glycine-NaOH pH 9.8; 50 mM Tris-HCl pH 7.0. Cited to be pH and detergent

Crosslinked multilayer methylcellulose based coatings (neutral coating)

Model protein separations (basic proteins) at pH 3.0 and 4.5

Multilayer PEG-based coating with anodal flow

Model protein separations— acidic proteins at pH 6.5 (crude trypsin inhibitor) and basic proteins at pH 3.0

Hydrophilic-coated capillary (SGE. Milton Keynes. UK) or Celect PI (Supelco. Belmont. PA) Hydroxypropyl cellulose based coating

Milk proteins - caseins (a^CN, as2CN. bCN. kCN); Serum proteins (|3-lactoglobulin and a-lactalbumin)

1. Tryptic digests of cytochrome c

2. RNase A and RNase B glycoform separations

3. Horseradish peroxidase isoenzymes

4. Ovlabumin glycoforms

Smith and Rassi (1993) (22)

Electrolyte: 100 m M Phosphate adjusted to pH 3.0 and 4.5. 400.000-550.000 plates/80 cm at an applied voltage of 18 kV Acidic proteins Electrolyte: 100 mM phosphate adjusted to pH 6.5: 80.000 plates/80 cm at an applied voltage of 18 kV. Basic proteins. Electrolyte: 100 mM phosphate adjusted to pH 3: 80.000 plates/ 80 cm at an applied voltage of 18kV

Electrolyte: 10 mM sodium phosphate containing 6 M urea and 0.05% methylhyroxyethyl cellulose (pH 2.0-3.0).

1. Electrolyte: 25 mM Tris-HCl, 1-propanol.

2. Electrolyte: 25 mM Tris-HCl. pH 3.0. by addition of 2% (v/v) pH 3.0. by addition of 2% (v/v) 1-propanol

3. Electrolyte: 50 mM Tris-HCl. pH 2.8. by addition of 2% (v/v) 1-propanol 4. Electrolyte: 50 mM AMPD/H3PO4 pH 9.1.

(continued)

Table 1 (continued)

Protein Applications Using Various Coated Capillaries

Capillary coating

Proteins tested

Reference

Comments

Polyacrylamide coated capillary, Fluorocarbon capillary, J&W Scientific (Folsom, CA) C18-coated capillary, Supelco; ISCO (Lincoln, NE).

Polyacrylamide polymer coated capillary, Polyvinylpyrollidone polymer coated capillary Polyacrylamide polymer coated capillary. Poly(ethylene) oxide polymer coated capillary Copolymer of acrylamide and (methacrylamidopropyl) trimethylammonium chloride polymer coated capillary.

Hemoglobin variants; Antibodies-anti-aj-antitrypsin; Human IgG

Human growth hormone precursor Pre-bhGH of less than 2% was demonstrated for runs done with 5 coated capillaries. Model protein separations (basic proteins);

Hemoglobin variants Milk proteins (acidic proteins)

Model protein separations (basic proteins); Acidic proteins

Capillary isoelectric focusing separations

Jorgensen et al. (1998) (38) Electrolyte: 150 mM Tricine, 7.5%

Electrolyte: 50 mM sodium acetate, pH 4.5. 500,000 plates/ 50 cm at an applied voltage of 20 kV; 650,000 plates /65 cm at an applied voltage of 20 kV for hemoglobin variants separations Eluent: 100 mM Sodium phosphate, pH 8.4, with 6 M urea Electrolyte: 50 mM sodium acetate, pH 4.5; 300,000 plates/ 50 cm capillary for basic proteins at an applied voltage of 20 kV Electrolyte: 25 mM phosphate buffer at pH 7.0>200,000 plates/50 cm at an applied voltage of 20 kV

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