Methods and apparatus for use in sequential chemical reactions

• US 5,605,839 A
• Filed: 05/29/1992
• Issued: 02/25/1997
• Est. Priority Date: 04/11/1990
• Status: Expired due to Fees

• Current Assignee: Ludwig Institute for Cancer Research, Ltd.
• Original Assignee: Ludwig Institute for Cancer Research, Ltd.
• Inventors: Moritz, Robert L., Burgess, Antony W., Simpson, Richard J.
• Primary Examiner(s): Snay, Jeffrey R.
• Application Number:
  US07/891,162
• Time in Patent Office: 1,733 Days
• Field of Search: 436/86-90, 436/149, 436/150, 436/151, 436/161, 436/173, 436/175, 436/177, 436/178, 436/180, 422/101, 422/82.01, 422/82.02, 422/186.04, 422/67, 422/50, 204/299 R, 204/302, 204/180.1, 204/186
• US Class Current: 436/89
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00698 Measurement and control of process parameters
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C07K 1/128 sequencing
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 27/44 using electrolysis to generate a reagent, e.g. for titration
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/4473 by electric means
• CPC Class Codes: G01N 30/06 Preparation
• CPC Class Codes: G01N 31/00 Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
• CPC Class Codes: G01N 33/6824 involving N-terminal degradation, e.g. Edman degradation
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

This invention relates to methods and apparatus for carrying out chemical reactions between a plurality of reactants and in particular it is amenable to micro or nano scale operation and to the sequential chemical reactions required during such processes as construction or sequencing of proteins, oligonucleotides and polysaccharides. The present invention further relates to a capillary liquid chromatography system for high-sensitivity component separation and microsequencing for use in association with the methods and apparatus herein described.

Claims

1. A method for conducting a chemical reaction, the method comprising:

immobilizing a first reactant at an electrode defining a reaction locus;

introducing an additional reactant to the reaction locus;

electrolytically activating a reaction between the first and additional reactants; and

removing remaining additional reactant or a reaction by-product from the reaction locus, wherein said method further comprises resolving, separating, or purifying at least one reactant by using a capillary liquid chromatographic system comprising a capillary column of ≦

0.50 mm I.D. packed with bonded-phase particles having from about 200 Å

to about 500 Å

pore size.

2. A method according to claim 1 wherein the capillary column is ≦

0.40 mm I.D.

3. A method according to claim 2 wherein the capillary column is ≦

0.32 I.D.

4. A method according to claim 1 wherein the capillary column is slurry-packed with from about 5 μ

m to about 20 μ

m diameter C₈ bonded-phase particles.

5. A method according to claim 4 wherein the C₈ bonded-phase particles are about 7 μ

m in diameter and have a pore size of approximately 300 Å

.

6. Apparatus for conducting a chemical reaction, the apparatus comprising an electrode disposed at a reaction locus:

means to immobilize a first reactant at the reaction locus;

means to introduce an additional reactant to the reaction locus;

means to energize the electrode to thereby electrolytically activate a reaction between the first and additional reactants or to electrophoretically attract the additional reactant to the reaction locus;

means to remove from the reaction locus, remaining additional reactant or a reaction by-product of the reaction, wherein said apparatus further comprises a capillary liquid chromatographic system to resolve, separate, or purify the reactant or reactant by-product comprising a capillary column of ≦

0.50 mm I.D. packed with bonded-phase particles having from about 200 Å

to about 500 Å

pore size.

7. Apparatus according to claim 6 wherein the capillary column is ≦

0.40 mm I.D.

8. Apparatus according to claim 7 wherein the capillary column is ≦

0.32 I.D.

9. Apparatus according to claim 6 wherein the capillary column is slurry-packed with from about 5 μ

m to about 20 μ

m diameter C₈ bonded-phase particles.

10. Apparatus according to claim 9 wherein the C₈ bonded-phase particles are about 7 μ

m in diameter and have a pore size of approximately 300 Å

.