Reagents for preparing a sample containing biomolecular analytes for capillary electrophoretic separation and methods for making the reagents

• US 6,465,692 B1
• Filed: 12/22/1999
• Issued: 10/15/2002
• Est. Priority Date: 12/22/1999
• Status: Expired due to Term

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Inventors: Liu, Yu, Ratnayake, Chitra K., Gu, Jirong, Reddy, M. Parameswara
• Primary Examiner(s): Kumar, Shailendra
• Application Number:
  US09/469,675
• Time in Patent Office: 1,028 Days
• Field of Search: 564/215, 564/216, 514/629, 204/451
• US Class Current: 564/215
• CPC Class Codes: C07C 231/24 Separation; Purification
• CPC Class Codes: C07C 233/03 with carbon atoms of carboxamide groups bound to hydrogen atoms

Abstract

Formamide with low conductivity and a neutral or slightly basic pH is provided. Such formamide may be used in a sample loading solution for capillary electrophoretic separation of biomolecular analytes to enhance the efficiency of sample injection into the capillary and the intensity of the signal. Formamide of the present invention may be obtained by first purifying the formamide to a conductivity of below 7 micro mho, and then adjusting the pH of the purified formamide with a base to a range of about pH 6.5 to 7.5. The purification may be carried out by first removing the water from the formamide, and then distilling the dried formamide until the conductivity of formamide is below about 7 micro mho.

Claims

1. Formamide with a conductivity below 10 micro mho and a pH in the range of about 6 to 8.5.

2. The formamide of claim 1 being substantially moisture free.

3. The formamide of claim 1 being distilled.

4. A method of using the formamide in claim 1 for the analysis of biomolecular analytes.

5. The method of claim 4, wherein the biomolecular analyte is DNA.

6. A method of preparing formamide for use in electrophoretic separation of biomolecular analytes, comprising:

(a) purifying the formamide until the conductivity of the formamide is below about 10 micro mho; and

(b) adjusting the pH of the purified formamide from step (a) to a range of about 6 to 8.5.

7. The method of claim 6, wherein purifying step (a) further comprises the steps of:

(a) removing the water contained in the formamide to produce a formamide that is substantially moisture free; and

(b) distilling the substantially moisture-free formamide to generate a purified formamide with a conductivity that is less than 10 micro mho.

8. The method of claim 7, wherein in step (a) the formamide is treated with moisture-free molecular sieves for removing the water of the formamide.

9. The method of claim 7, wherein in step (b) the distilling step is repeated.

10. The method of claim 9, wherein the purified formamide has a conductivity that is about 4 micro mho.

11. The method of claim 6, wherein in step (b) the pH of the purified formamide is adjusted by a base.

12. The method of claim 11, wherein the base is a non-nucleophilic base.

13. The method of claim 11, wherein the base is an organic tertiary amine.

14. The method of claim 13, wherein the base is selected from the group consisting of triethyl amine (TEA), Diisopropylethyl amine (DIPEA), and tributyl amine (TBA).

15. The method of claim 14, wherein the base is TBA.

16. A reagent for preparing a sample containing biomolecular analytes for capillary electrophoretic separation, comprising formamide having a conductivity below 10 micro mho and a pH in the range of about 6 to 8.5.

17. The reagent of claim 16, wherein the formamide is substantially moisture free.

18. The reagent of claim 16, wherein the reagent further comprises a branched polymer.

19. A method of using the reagent in claim 18 for the analysis of biomolecular analytes.

20. The method of claim 19, wherein the biomolecular analyte is DNA.

21. The method of claim 18, wherein the branched polymer is selected from the group consisting of branched polyvinylpyrrolidone, and the like.

22. The method of claim 21, wherein the branched polymer is branched polyvinyl pyrrolidone.

23. The method of claim 22, wherein the molecular weight of the branched polyvinylpyrrolidone is in a range of about 1,200,000 to 2,000,000 daltons.

24. A method for preparing a sample containing biomolecule analytes for capillary electrophoretic separation comprising the steps of:

(a) providing a reagent comprising formamide having a conductivity below 10 micro mho and a pH in the range of about 6 to 8.5; and

(b) mixing the sample with the reagent prior to the capillary electrophoretic separation of the analytes.

25. The method of claim 24, wherein the formamide is moisture free.

26. The method of claim 24, wherein the formamide is double distilled.

27. The method of claim 24, wherein the formamide has a conductivity that is about 4 micro mho.

28. The method of claim 24, wherein the pH of the formamide is adjusted by a base.

29. The method of claim 28, wherein the base is a non-nucleophillic base.

30. The method of claim 28, wherein the base is an organic tertiary amine.

31. The method of claim 30, wherein the base is selected from the group consisting of tri-ethyl amine (TEA), Di-isoporpylethyl amine (DIPEA), and tributyl amine (TBA).

32. The method of claim 31, wherein the base is TBA.

33. The method of claim 24, wherein the reagent further comprises a branched polymer.

34. The method of claim 33, wherein the branched polymer is selected from the group consisting of branched polyvinylpyrrolidone, and the like.

35. The method of claim 34, wherein the molecular weight of the branched polyvinylpyrrolidone is in a range of about 1,2000,000 to 2,000,000 daltons.

36. The method of claim 24, wherein the biomolecule analyte is DNA or RNA.