METHOD FOR BASELINE CORRECTION IN A CHROMATOGRAM

• US 20160282318A1
• Filed: 03/10/2016
• Published: 09/29/2016
• Est. Priority Date: 03/27/2015
• Status: Active Grant

• Current Assignee: Cytiva Sweden AB (Danaher Corporation)
• Original Assignee: GE Healthcare Bio-Sciences AB (GE Healthcare Technologies, Inc.)
• Inventors: Malmquist, Gunnar, Hall, Martin, Stafstrom, Nils
• Granted Patent: US 10,598,640 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G01N 30/34 of fluid composition, e.g. gradient G01N30/36 takes precedence
• CPC Class Codes: G01N 30/74 Optical detectors measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J
• CPC Class Codes: G01N 30/8624 Detection of slopes or peaks; baseline correction

Abstract

The invention discloses a method for correcting a baseline in a chromatogram obtained using a buffer with at least one UV-absorbing component which is a weak acid or a weak base. The method comprises the steps of:

a) providing a chromatography system with a chromatography column and a UV detector downstream of the chromatography column;
b) providing UV extinction coefficients at a wavelength λ for the acid form and the base form of the UV-absorbing component(s) and a dissociation constant for the component(s),
c) obtaining a chromatogram with the UV detector at wavelength λ using the buffer under a set of conditions where pH and/or conductivity is varied between these conditions,
d) for a plurality of said conditions calculating the concentrations of the acid and base forms using the dissociation constant(s) and values for pH and conductivity values, and
e) calculating UV absorption values of the acid and base forms from the concentrations and UV extinction coefficients under the conditions in question and subtracting the UV absorption values from the chromatogram.

Claims

1. A method for correcting a baseline in a chromatogram obtained using a buffer with at least one UV-absorbing component which is a weak acid or a weak base, said method comprising the steps of:

a) providing a chromatography system with a chromatography column and a UV detector downstream of said chromatography column;

b) providing UV extinction coefficients at a wavelength λ

for an acid form and a base form of said at least one UV-absorbing component and a dissociation constant for said component,

c) obtaining a chromatogram with said UV detector at wavelength λ

using said buffer under a set of conditions where pH and/or conductivity is varied between said conditions,

d) for a plurality of said conditions calculating the concentrations of said acid and base forms using said dissociation constant and pH and conductivity values, and

e) calculating UV absorption values of said acid and base forms from said concentrations and said UV extinction coefficients under said conditions and subtracting said UV absorption values from said chromatogram.

2. The method of claim 1, wherein the concentrations of said acid and base forms are calculated using the equation of Debye-Hü

ckel.

3. The method of claim 2, wherein the ion size parameter a in the Debye-Hü

ckel equation is determined as a weighted mean ion size of all species contributing significantly to the ionic strength of the buffer, and wherein the ionic strength of each of said species is used as weighting parameter.

4. The method of claim 2, wherein the ion size parameter a of the Debye-Hü

ckel equation is determined as

5. The method of claim 2, wherein the ion size parameter a of the Debye-Hü

ckel equation is approximated as

a=0.5*(mass)⅓

shell,

wherein “

shell”

optionally is fixed at one value for a positively charged species and fixed at a different value for a negatively charged species, and optionally fixed at a value in the range of 3.8-4.2, such as 4.0, for positively charged ionic species; and

“

shell”

is fixed at a value in the range of 0-0.2, such as 0, for negatively charged ionic species.

6. The method of claim 1, wherein the concentrations of said acid and base forms are calculated using an iterative procedure.

7. The method of claim 6, wherein the iterative procedure comprises:

i) determining the concentrations wherein a pre-defined ionic strength of the liquid mixture is addressed to the species according to a pre-defined distribution among the species;

ii) on the basis of the concentrations determined in the preceding step, calculating the ionic strength of each species in the buffer;

iii) determining a new set of concentrations;

taking account of the ionic strength calculated in ii), and

iv) repeating the steps ii) and iii) until a predetermined convergence criterion is met.

8. The method of claim 1, wherein said chromatogram is obtained through elution with a gradient in pH and/or conductivity.

9. The method of claim 1, wherein the concentrations of said acid and base forms are calculated from pH values determined from the composition of said buffer.

10. The method of claim 1, wherein said chromatography system further comprises a pH detector downstream of said chromatography column and wherein the concentrations of said acid and base forms are calculated from pH values measured by said pH detector.

11. The method of claim 1, wherein said wavelength λ

is within the range of 190-260 nm, such as 200-240 nm or 210-230 nm.

12. The method of claim 1, wherein said UV extinction coefficients and said dissociation constant are retrieved from a readable medium.

13. The method of claim 1, which is performed by a computer.

14. A computer program on a readable medium for correcting a baseline in a chromatogram according to the method of claim 1.

15. A chromatography system arranged to perform the method of claim 1.