JPS6022660A - Calculator for retention time of chromatogram - Google Patents

Abstract

PURPOSE:To calculate exactly retention time by storing preliminarily the peak curve of each component of a chromatogram in a storage means and detecting the part of the shape approximate to a part of the chromatogram from at least a part of the curve stored in the above-mentioned way. CONSTITUTION:An analog or digital chromatogram signal is inputted from an input terminal 11 and said signal and the shape of the known function curve signal corresponding to the peak curve supplied from a storage means 12 are compared in a comparing means 13 and are approximated. The retention time in said peak part is calculated in this stage by detecting the most approximate part of the curve. The retention time refers to the time when the max. value of the known function curve value corresponding to the peak curve inherent to each component supplied from the storage means is taken. The peak value of the peak curve with which the retention time is determined in the above-mentioned manner is calculated in a calculator 14 for the peak value according to need.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus for calculating the retention time of each component from a chromatogram obtained by analyzing a sample containing at least two components by chromatography.

<Background> The present inventor previously disclosed in Japanese Patent Application No. 58-9462'0 "Chromatogram Peak Value Calculation Apparatus" a known function curve corresponding to the maximum point of a chromatogram and a peak curve unique to each component. We proposed a device that approximates by matching the thick points of However, in this device, when the hem of a neighboring piece is located at the maximum point of the peak of interest, the apparent maximum point of the peak of interest may shift, resulting in a shift in the pick holding time.

Therefore, when the maximum point of the apparent chromatogram is made to correspond to the known function curve corresponding to the peak curve specific to each component, the approximation may not be performed correctly.

<Objective of the Invention> An object of the present invention is to provide an apparatus that can correctly calculate the suspension time even when the base of a nearby peak overlaps the thick point of the peak of interest.

<Summary of the Invention> The present invention is an apparatus for calculating retention time from a chromatogram with nine different peaks of multiple components, which stores the peak curves of each component of the talomadogram in advance in a storage means, and calculates the retention time automatically. Iiii the part of the shape that is close to a part of the madogram
detection from at least a portion of the stored curve, thereby estimating the retention time.

It is preferable that the known function curve is a function curve obtained by analyzing a certain erectile single component, and this curve may be expressed by a mathematical formula or cannot be expressed by a mathematical formula. Also good. Function curves that can be expressed by the above formula include, for example, Gaussian (normal) distribution curves, binomial distribution curves, chi-square distribution curves, statistical distribution function curves such as F distribution curves, trigonometric function curves, logarithmic function curves, There are function curves such as exponential function curves, and function curves created by combinations of these function curves. Talomadogram detection includes ultraviolet absorption, photorefraction, fluorescence, color,
This can be done using electrical conductivity, etc., but it can be done inexpensively using ultraviolet 2 absorption. The chromatograms that are the object of this invention are those obtained by dull permeation chromatography, ion exchange chromatography, partition chromatography, adsorption chromatography, Seize exclusion chromatography, thin layer chromatography, gas chromatography, and the like.

You can also set the order of each component in advance when approximating a known function curve to a one-frame matogel.
In this case, the approximation can often be made easier.

<Embodiment> FIG. 1 shows an example of a device according to the present invention. Input Mizuko 1
An analog or digital chromatogram signal is input from 1, and the shape of the known function curve signal corresponding to the peak curve supplied from the small installation 2 is compared with this by the comparing means 13 and calculated.

At this time, by detecting the closest curve portion, the retention time of the peak portion is calculated. Here, the retention time is the time required to take the thickest value of the known function curve signal corresponding to the peak curve specific to each component supplied from the unique means. If necessary, the peak value of the peak curve whose retention time has been determined in this way is calculated by the peak value calculating device 14.

The main components contained in the target sample are known in advance, and the peak retention time and standard difference for each component are also known in advance, and by comparing and approximating such a curve to a chromatogram. A case in which a suitable retention time is determined for each component in the input chromatogram signal and the peak value of each component is determined using the retention time will be explained in detail below.

The storage means 12 stores only the basic waveform, in this example a normal distribution curve g([) assuming that the peak curve of each component can be approximated by a normal distribution curve.

Here, tH time, 111 is the height of one mountain (peak part) in the chromatogram, and δ! represents the spread of the mountain (Yanagi standard deviation), and R11il-j represents the retention time, respectively. The input chromatogram signal r (for example, a chromatogram obtained by introducing serum into a GPC ('17' permeation chromatography) filled with polyvinyl alcohol gel, for example, the curve in Figure 2) Components in this chromatogram, 1 gM (immunoglobulin M)
, IgA (immunoglobulin A), IgG (immunoglobulin G), Tf (+-transferrin) and 'Aib (
(albumin) are approximated by normal distribution curves 15, 16, 17, 18 and 19 as shown in FIG. In other words, each of these components IgM.

Spread of each component in the chromatograms of IgA, IgG, Tf and Aib I) 'IgM, 'Ig
A, 'IgG, 'T (and aAJ2b and H mean time (retention time) R11gM, tlgA.

Approximate values of RtIgG, RlTf, and RtA knob are known in advance. Even if the same sample is analyzed using the same chromatographic system, the retention time and standard ulli difference will vary slightly due to slight changes in the system's temperature and pH, or flow due to air bubbles. . When a chromatograph is approximated by a function curve specific to each component, a slight change in standard/$ deviation can be ignored as an error range, but even a slight change in retention time may cause a serious problem. There are many. For example, if there are two peaks forming a normal distribution with a retention time of 700 seconds and a standard deviation of 20 seconds, if flow I decreases by 1%, the retention time will be 707 seconds and the standard deviation will be 20.2 seconds. Become. The time difference (W committee =, 2.37σ) changes from 47.4 seconds to 479 seconds. In other words, an error of 1 unit in the standard deviation will only increase the half-width of the peak curve approximated by a normal distribution by 0.5 seconds, whereas an error in the retention time of 1 unit will amount to 7 seconds, which is a serious problem. Become. That is, σi (! = IgM, IgA, IgG,
Tf.

Aib) is known, and if the retention time Rti is accurately calculated, the height J of each component can be determined according to the input chromatogram, and the capacity of each of these components can be accurately known.

The determination of each peak value in this example is performed, for example, as follows. That is, from the input chromatogram f (t), first i
gM, IgG, and Aib components are detected, and these components are subtracted from the input chromatogram f(t),
Calculate the retention time and peak value of each component of IgA and Tf from the remaining components. In this way, the influence of overlapping adjacent components is avoided.

To calculate the retention time, for example, in each peak part of the chromatogram, find the part whose curved shape is most similar to the curved shape of a normal distribution, and calculate the retention time by extending it. . This is because at the peak top of a peak curve that has a normal distribution shape, even if small peaks overlap, they are affected by the small peak, but by using a relatively steep part above the half width, small peaks can overlap. This method takes advantage of the fact that the shadow of the peak can be avoided.

Next, a specific example of processing for calculating a peak value using the present invention will be described with reference to FIG. First, in step S1, the peak curve g(1) is read out from the storage means and input to the comparison means. The signal input at this time is a normal distribution curve g (
It has the shape of J.

Rti+01 can be changed. Furthermore, σ1(l
=■gM, ■gA, ■gG, Tf, Aib).

In this example, the values are 12.0 and 17.0, respectively. s, 19.
4, 14.3°228 seconds.

Next, the chromatogram f input in step S2
(t) Retention time R11gM of IgM, IgG, and Aib.

Determine RllgG and RtJb, respectively. That is, first, it is known that the default values of RlIgMIRtIgG and RLAib are 475 (seconds), 712 (seconds), and 865 (seconds), respectively.
, (475-495 seconds, 705-725 seconds, 860-8
Change at 90 seconds. Assuming that this time t is one holding time R1i, the chromatogram value at this time is f: f (t), and 1 near the other time t.
Using the chromatogram value f (it) at time 1, the peak height J (t, tt) can be expressed as follows. Here, time 11 is t-13≦11≦t-B
Then, take the average value of each J (t, it) obtained at that time and calculate h i (t). In other words, the peak height at time 11 is t-13≦11≦t-si. The relative square error △hr (') of sJ (t, tt) is.This squared error △J
When that part of the chromatogram f is most approximated to a normal distribution, that is, the part whose shape is closest to the memorized peak curve is detected, and the time t at this time is the retention time Thus, in this example, the retention time R1IgM - 488 seconds, RHgc = 7
16 seconds, RLAib = 885 seconds was calculated.

Next, in this example, in step s3, the peak heights hIgM, 111gG, and hAAib were determined by l1i(Ri) using the calculated retention times. In this example, 34.2, 374.5, 521 (X
10-' AB U ) Te was there. Next Nisten 7'
In step S4, σ1 of step Sl, each Rti obtained in step s2, and hl obtained in step S3 are expressed as g(
By substituting the following, the composite waveforms of IgM', IgG, and iB are obtained as grgM(,t'') and glgc(t').

Set gApb(,i). In step S5, these combined curves g;(Q are subtracted from the chromatogram f(t) to obtain a waveform f'().

In step S6, the same operation as step S2 is performed with f'(t
) IgA, 'rl. However, for IgA, t is set to 660≦[≦680, and for T(, t is set to 735≦t≦750, and △h1(t)
IgA, Tf retention time Rt from time t that minimizes
Add an i. In this example, average gA = 673 seconds, RlT
f-747 seconds was obtained. Furthermore, in step S7, the peak height hTgA is determined in the same manner as in step S3.

Ibrl is determined, in this example each 90. L
.

317,j (XIO'A, BU) was obtained. In step S8, IgA is added in the same manner as in step S4.

A waveform of T is created. In step S9, the peak value (peak height, waveform area, half width, etc.) of each component and the a7 totogram are printed out.

<Effects> As described above, the apparatus of the present invention compares and approximates the curve shape of a chromatogram with the curve shape of a known function curve corresponding to the peak curve unique to each component, so that the closest approximation can be obtained. By doing this, the retention time can be calculated accurately. Therefore, it becomes possible to accurately calculate peak values such as peak height or peak area of each component.

Therefore, chromatography is the first step on the chromatogram.
The present invention is even more effective for convex mudgraphy using, for example, a GPC column filled with polyvinyl alcohol gel, which tends to form peaks.

[Brief explanation of the drawing]

FIG. 1 shows an example of a chroma 1-gram peak value calculating device according to the present invention. Block 1 is block 1, and FIG.
A diagram showing a chromatogram obtained by analysis with a C column,
FIG. 3 is a diagram showing a curve approximated to each (11, - component) of an analytical waveform obtained by an apparatus adapted to the present invention.
FIG. 4 is a flowchart showing an example of the operation of the apparatus of the present invention. Patent Applicant Representative Asahi Kasei Koso Co., Ltd. Representative Takuo Kusano 1 Figure 72 Figure 73 Figure Procedures Sleeve Main Gate (Voluntary) Commissioner of the Japan Patent Office 1, Indication of Case Patent Application 1982-1? , 13472, Title of the invention Chromatogram retention time calculation device 8, Relationship with the case of the person making the amendment Patent applicant Asahi Kasei Kogyo Co., Ltd. 4, Representative Uto 66J5 Aigoku Building, 4-2-21 Shinjuku, Urashuku-ku, Tokyo Patent attorney Kusano Nihe11T2τroyu (
'T"'°' 5. Subject of amendment Specification rising invention σkawaki': Jii of 11 na 1 shi"; 10. Contents of amendment 337-

Claims (1)

[Claims] (1) A device that calculates the retention time of each component from a chromatogram in which the peaks of multiple components are aligned, comprising means for storing the peak curve of each component, and a curved shape of at least a portion of the chromatogram. A taromadogram retention time calculation device comprising means for calculating a retention time by detecting a shape portion closest to at least a portion of the stored peak curve.