Method for baseline correction in a chromatogram

Patent metadata

Field	Value
Publication number	US-10598640-B2
Application number	US-201615066429-A
Country	US
Kind code	B2
Filing date	Mar 10, 2016
Priority date	Mar 27, 2015
Publication date	Mar 24, 2020
Grant date	Mar 24, 2020

Abstract

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.

First claim

The invention claimed is:

1. A method for correcting a baseline in a chromatogram using a buffer with at least one UV-absorbing component which is a weak acid or a weak base, the method comprising the steps of:
a) providing a chromatography system comprising:
(i) a chromatography column;
(ii) a pH detector located downstream of an outlet of the chromatography column and configured for measuring the pH values;
(iii) a UV detector located between the chromatography column outlet and the pH detector;
(iv) a conductivity connector located downstream of the pH detector and configured for measuring an ionic strength the buffer; and
(v) a control unit having one end connected to a first pump and a second pump of the chromatography column and a second end connected to the UV detector, the pH detector, and the conductivity detector, the control unit being configured to control the pumps and to receive and measure signals from the detectors;

(b) during operation, controlling, at the control unit, a mobile phase buffer composition, by:

(i) switching off the first pump and switching on the second pump to obtain a step gradient; (ii) linearly decreasing the flow rate of the first pump and linearly increasing the flow rate of the second pump to obtain a linear buffer gradient; or (iii) changing the relative flow rates of the first pump and the second pump according to predetermined non-linear function to obtain a non-linear gradient;
(c) mixing a first buffer and a second buffer delivered at determined flow rates by the first and second pumps, respectively, to form the mobile phase buffer composition to be conveyed to the chromatography column through the UV detector, the pH detector and the conductivity detector, by calculating a mixing ratio of the first buffer and the second buffer needed to obtain predetermined values of pH and conductivity;
(d) injecting a sample having components, via an injection valve, through the chromatography column, each component exiting the chromatography column at a different point of time and being detected by the UV detector;
(e) providing, at the control unit, UV extinction coefficients for a weak acid form and a weak base form of the at least one UV-absorbing component and a dissociation constant for the UV-absorbing component;
(f) obtaining a chromatogram having UV absorption data from the UV detector under a set of conditions where a range of pH and/or a conductivity values are measured;
(g) calculating, at the control unit, concentrations of the weak acid and weak base forms of the at least one UV absorbing buffer component under the set of conditions used in the chromatogram; and
(h) calculating, via the pH and conductivity signals measured by the control unit, UV absorption values of the acid and base forms under the same set of conditions and subtracting the calculated UV absorption values from the UV absorption values measured by the control unit.

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

3. The method of claim 2 , wherein an ion size parameter a in the Debye-Huckel 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 the species is used as weighting parameter.

4. The method of claim 3 , wherein the ion size parameter a of the Debye-Huckel equation is determined as

a
=

∑

I
i

⁢

a
i

I

,

wherein I i , is the ionic strength and a i is the ion size parameter of species i and I the total ionic strength.

5. The method of claim 2 , wherein the ion size parameter a of the Debye-Huckel equation is approximated as a=0.5*(mass) 1/3 +shell,
wherein “shell” is fixed at one value for a positively charged species and fixed at a different value for a negatively charged species, and fixed at a value in the range of 3.8-4.2 for positively charged ionic species; and “shell” is fixed at a value in the range of 0-0.2 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 the chromatogram is obtained through elution with a gradient in pH and/or conductivity.

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

10. The method of claim 1 , wherein the concentrations of the acid and base forms are calculated from pH values measured by the pH detector.

11. The method of claim 1 , wherein the wavelength X is within the range of 190-260 nm.

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

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

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

Assignees

• Ge Healthcare Bio Sciences Ab

Inventors

• Malmquist Gunnar
• Hall Martin
• Stafstrom Nils

Classifications

• G01N30/74

  Optical detectors {(measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J)} · CPC title

• G01N30/8624 Primary

  Detection of slopes or peaks; baseline correction · CPC title

• G01N30/8641 Primary

  Baseline · CPC title

• G01N30/34

  of fluid composition, e.g. gradient (G01N30/36 takes precedence) · CPC title