Analysis Method

Abstract

A first chromatogram is obtained by analyzing a second standard sample by size exclusion chromatography analysis using a first detector. Also, a second chromatogram is obtained by analyzing a solvent for the second standard sample by size exclusion chromatography analysis using the first detector. Then, from the difference between the first chromatogram and the second chromatogram, a third elution time in size exclusion chromatography analysis of the second standard sample using the first detector is determined.

Claims

1-4. (Cancelled)

5. An analytical method comprising: analyzing a plurality of first standard samples each having a different molecular weight by size exclusion chromatography analysis using a first detector, thereby obtaining a plurality of first elution times; analyzing a second standard sample by size exclusion chromatography analysis using the first detector, thereby obtaining a first chromatogram; analyzing a solvent for the second standard sample by size exclusion chromatography analysis using the first detector, thereby obtaining a second chromatogram; analyzing the second standard sample by size exclusion chromatography analysis using a second detector, thereby obtaining a second elution time; determining a third elution time in size exclusion chromatography analysis of the second standard sample using the first detector, from a difference between the first chromatogram and the second chromatogram; reflecting a difference between the second elution time and the third elution time in each of the plurality of first elution times, thereby determining a plurality of fourth elution times; creating a calibration curve showing a relationship between the plurality of fourth elution times and corresponding molecular weights; analyzing a substance to be measured by size exclusion chromatography analysis using the second detector, thereby obtaining a third chromatogram; and converting the third chromatogram using the calibration curve, thereby obtaining a molecular weight distribution of the substance.

6. The analytical method according to claim 5, wherein the first detector is a differential refractometer.

7. The analytical method according to claim 5, wherein the second standard sample has a ratio of a number average molecular weight to a weight average molecular weight of 1.30 or less.

8. The analytical method according to claim 5, wherein the second detector is an ultraviolet absorption detector.

9. The analytical method according to claim 5, wherein the first detector is a differential refractometer and the second detector is an ultraviolet absorption detector.

10. A method of creating a calibration curve, the method comprising: obtaining a plurality of first elution times by analyzing a plurality of first standard samples each having a different molecular weight by size exclusion chromatography analysis using a first detector; obtaining a first chromatogram by analyzing a second standard sample by size exclusion chromatography analysis using the first detector; obtaining a second chromatogram by analyzing a solvent for the second standard sample by size exclusion chromatography analysis using the first detector; obtaining a second elution time by analyzing the second standard sample by size exclusion chromatography analysis using a second detector; determining a third elution time in size exclusion chromatography analysis of the second standard sample using the first detector, from a difference between the first chromatogram and the second chromatogram; determining a plurality of fourth elution times by reflecting a difference between the second elution time and the third elution time in each of the plurality of first elution times; and creating the calibration curve showing a relationship between the plurality of fourth elution times and corresponding molecular weights.

11. The method according to claim 10, wherein the first detector is a differential refractometer.

12. The method according to claim 10, wherein the second standard sample has a ratio of a number average molecular weight to a weight average molecular weight of 1.30 or less.

13. The method according to claim 10, wherein the second detector is an ultraviolet absorption detector.

14. The method according to claim 10, wherein the first detector is a differential refractometer and the second detector is an ultraviolet absorption detector.

15. The method according to claim 14, further comprising determining a molecular weight distribution of a substance to be measured using the calibration curve.

16. The method according to claim 15, wherein determining the molecular weight distribution of the substance to be measured comprises: obtaining a third chromatogram by analyzing the substance to be measured by size exclusion chromatography analysis using the second detector; and obtaining the molecular weight distribution of the substance by converting the third chromatogram using the calibration curve.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a flowchart for describing an analytical method according to an embodiment of the present invention.

[0021] FIG. 2 is a characteristic diagram showing a molecular weight distribution obtained in an experiment using an analytical method according to an embodiment of the present invention.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0022] Hereinafter, an analytical method according to an embodiment of the present invention will be described with reference to FIG. 1. Note that, in the analysis, the first detector and the second detector are connected in series for use on the outlet side of the column for size exclusion chromatography. This is a general device configuration for this type of analysis.

[0023] At first, in a first step Sioi, a plurality of first elution times [T.sub.A (ST.sub.k)] are obtained by analyzing a plurality of first standard samples (ST.sub.k) each having a different molecular weight by size exclusion chromatography analysis using the first detector. The first standard samples are, for example, polymethyl methacrylate (PMMA) standard samples. The first detector is, for example, a differential refractometer (refractive index (RI) detector).

[0024] Next, in a second step S102, a first chromatogram [F.sub.Sk (T.sub.A)] is obtained by analyzing a second standard sample (S.sub.k) by size exclusion chromatography analysis using the first detector. The second standard sample is, for example, polyethylene terephthalate.

[0025] Next, in a third step S103, a second chromatogram [F.sub.blank (T.sub.A)] is obtained by analyzing a solvent for the second standard sample by size exclusion chromatography analysis using the first detector. The solvent for the second standard sample is, for example, 1,14,3,3,3-hexafluoro-2-propanol containing sodium trifluoroacetate.

[0026] Next, in a fourth step S104, a second elution time [T.sub.B (S.sub.k)] is obtained by analyzing the second standard sample by size exclusion chromatography analysis using a second detector. The second detector is, for example, an ultraviolet absorption detector (ultraviolet (UV) detector).

[0027] Next, in a fifth step S105, from the difference [F.sub.Sk (T.sub.A)-F.sub.blank (T.sub.A)] between the first chromatogram [F.sub.Sk (T.sub.A)] and the second chromatogram [F.sub.blank (T.sub.A)], a third elution time [T.sub.A (S.sub.k)] in size exclusion chromatography analysis of the second standard sample using the first detector is determined.

[0028] Next, in a sixth step S106, a plurality of fourth elution times [T.sub.B (ST.sub.k)] is determined by reflecting the difference [ΔT (S.sub.k)=T.sub.B(S.sub.k)-T.sub.A (S.sub.k)] between the second elution time [T.sub.B (S.sub.k)] and the third elution time [T.sub.A (S.sub.k)] in each of the plurality of first elution times [T.sub.A (ST.sub.k)].

[0029] Next, in a seventh step S107, a calibration curve showing the relationship between the plurality of fourth elution times [T.sub.B (ST.sub.k)] and corresponding molecular weights (M) is created (a calibration curve expression is calculated).

[0030] Next, in an eighth step S108, a third chromatogram is obtained by analyzing a substance to be measured by size exclusion chromatography analysis using the second detector.

[0031] Next, in a ninth step S109, the third chromatogram is converted using the calibration curve, thereby obtaining the molecular weight distribution of the substance.

[0032] According to the embodiment mentioned above, since the third elution time in size exclusion chromatography analysis of the second standard sample using the first detector is determined from the difference between the first chromatogram and the second chromatogram, fluctuations or the like in the detection results of the first detector due to the solvent for the second standard sample are removed. As a result, according to the above embodiment, an accurate calibration curve can be created using multiple detectors in size exclusion chromatography, and accurate analysis can be performed.

[0033] Hereinafter, how the present inventors have reached embodiments of the present invention will be described. As mentioned above, when using a standard substance that can be detected by both RI detector and UV detector to determine the difference ΔT between the elution time in the case of using the RI detector and the elution time in the case of using the UV detector, the ΔT was sometimes found to differ when the standard samples had different average molecular weights. As a result of diligent research on this phenomenon, the present inventors have found out that fluctuations (variations) occur in the measurement results of the RI detector and that this is caused by the solvent used for the measurement.

[0034] The RI detector measures the difference in refractive index (differential refractive index) between the solvent sealed in the reference cell and the solution in which the substance to be measured (polymeric material) is dissolved, flowing through the flow cell and eluted from the column. Here, when only the solvent was measured in the same way, a minor amount of gas, water, or the like included in the solvent was separated in the process of passing through the column, causing a slight change in the refractive index of the eluate, and the differential refractive index measured with the RI detector after passing through the column changed slightly depending on the samples. These fluctuations caused by the solvent appear as fluctuation in the chromatogram obtained from the measurement of the substance to be measured by the RI detector.

[0035] Since the fluctuations mentioned above are small compared to the change in refractive index due to the elution of the samples that have passed through the column, when the analysis of molecular weight distribution is performed only with the RI detector, such fluctuations have small influence on the approximate shape of the molecular weight distribution and the values of the number average molecular weight and weight average molecular weight, and thus hardly affect the analytical results.

[0036] However, as a result of diligent research by the present inventors, it has been found that, in order to determine the difference ΔT between the elution time in the case of using the RI detector and the elution time in the case of using the UV detector, it is necessary to accurately determine a single point, the peak value of each, and for this purpose, the fluctuations mentioned above cannot be ignored. In embodiments of the present invention, the influence of the fluctuations mentioned above is eliminated by subtracting the chromatogram of the solvent from the chromatogram of the substance to be measured dissolved in the solvent.

[0037] Next, more detailed description will be given using the results of experiments.

[0038] Measuring device [0039] Device: An SEC device from Waters Corporation, AQUICTY APC. [0040] Columns: APC-XT, 186006995, 186006998, 186007003, and 186007254.

[0041] First standard samples [0042] Commercially available PMMA standard samples (six types with peak top molecular weights of 102500, 56900, 24400, 10900, 8350, and 4250) were used.

[0043] Second standard sample [0044] Polyethylene terephthalate.

[0045] In the experiments, nine types of polyethylene terephthalate with different peak top molecular weights Mp and weight average molecular weight/weight average molecular weight (Mw/Mn) were provided in order to check for fluctuations caused by the solvent (see Table 1). Note that it is desirable that the second standard sample should be a polymeric material with a molecular structure similar to that of the substance to be measured, but it is not necessary for their compositions to be completely identical.

TABLE-US-00001 TABLE 1 S.sub.1 S.sub.2 S.sub.3 S.sub.4 S.sub.5 S.sub.6 S.sub.7 S.sub.8 S.sub.9 Mp 15000 15000 15000 15000 15000 30000 60000 8000 4000 Mw/Mn 1.5 1.30 1.20 1.10 1.05 1.05 1.05 1.05 1.05

[0046] Sample to be measured [0047] Polyethylene terephthalate (molecular weight distribution unknown).

[0048] Preparation of samples

[0049] Each of the first standard samples, the second standard sample, and the sample to be measured was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) containing .sub.5 mmol/L of sodium trifluoroacetate at 1 mg/1 mL. Each solution was placed in a sample bottle, each sample bottle was covered with a lid and left to stand overnight, and the solution was filtered through a PTFE syringe filter with a pore diameter of 0.2 μm into a vial for measurement.

[0050] Measurement conditions [0051] Solvent: HFIP containing 5 mmol/L of sodium trifluoroacetate. [0052] Column temperature: 40° C. [0053] Flow rate: 0.25 mL/min. [0054] Sample concentration: 1 mg/mL. [0055] Injection volume: 0.2μL/time [0056] Detectors: RI detector (40° C.) and UV detector (254 nm).

[0057] Size exclusion chromatography analysis [0058] Analysis of the solvent, the first standard samples (ST.sub.k), the second standard sample (S.sub.k), and the sample to be measured was performed.

[0059] Calibration curve

[0060] From the analytical results of the first standard samples by the RI detector, a cubic calibration curve was created.

[0061] Experiment 1 [ooso] The analysis of the solvent and S.sub.5, S.sub.6, S.sub.7, S.sub.8, and S.sub.9 (second standard samples) shown in Table 1 was performed, and ΔT (S.sub.k) was determined for each of them. The third elution time [T.sub.A (S.sub.k)], the second elution time [T.sub.B (S.sub.k)], the determined ΔT (S.sub.k), and the RI detection output value F.sub.blank (T.sub.A (S.sub.k)) at the third elution time [T.sub.A (S.sub.k)] for the solvent analysis are shown in Table 2 below. Note that the Mp and Mw/Mn in the table are known values from separate measurements.

TABLE-US-00002 TABLE 2 S.sub.5 S.sub.6 S.sub.7 S.sub.8 S.sub.9 Mp 15000 30000 60000 8000 4000 Mw/Mn   1.05   1.05   1.05   1.05   1.05 T.sub.A(S.sub.k)   11.05   10.70   10.30  11.80  12.90 T.sub.B(S.sub.k)   11.23   11.15   10.50  11.88  13.12 ΔT(S.sub.k) =   0.18   0.45   0.20   0.08   0.22 T.sub.B(S.sub.k) − T.sub.A(S.sub.k) F.sub.blank(T.sub.A(S.sub.k))   0.02   0.22   0.01  −0.13   0.03

[0062] For S.sub.5, S.sub.7, and S.sub.9, ΔT (S.sub.k) was approximately 0.20 and almost equal with each other, but for S.sub.6 and S.sub.8, ΔT (S.sub.k) was far off from 0.20. Comparing F.sub.blank (T.sub.A (S.sub.k)), it was found that S.sub.5, S.sub.7, and S.sub.9, which had ΔT (S.sub.k) of around 0.20, had F.sub.blank (T.sub.A (S.sub.k)) of in the vicinity of zero, which was almost equal with each other, while S.sub.6 and S.sub.8, which had ΔT (S.sub.k) of far off from 0.20, also had a value of F.sub.blank (T.sub.A (S.sub.k)) off from o (the underlined numbers in the table). This is thought to be because, when the elution of S.sub.k reaches its maximum near the time zone where the baseline of the chromatogram by the RI detector was fluctuating in the solvent analysis, there was a gap between the time when the elution of S.sub.k actually reached its maximum and the time when the output of the RI detector reached its maximum. From the above, it was suggested that ΔT (S.sub.k)=T.sub.B (S.sub.k)-T.sub.A (S.sub.k) may not indicate the true difference in elution time between the two detectors.

[0063] Experiment 2

[0064] First, the analysis of the solvent and the second standard sample shown in Table 1 were performed, and for each of them, the third elution time [T.sub.A (S.sub.k)] in size exclusion chromatography analysis of the second standard sample using the RI detector was determined from F.sub.Sk (T.sub.A)-F.sub.blank (T.sub.A). T.sub.A (S.sub.k) is the time when F.sub.Sk (T.sub.A)-F.sub.blank (T.sub.A) reaches its maximum. In addition, from the determined T.sub.A (S.sub.k), ΔT (S.sub.k)=T.sub.B (S.sub.k)-T.sub.A (S.sub.k) was determined. Each value determined is shown in Table .sub.3 and Table .sub.4 below. Note that the Mp and Mw/Mn in the table are known values from separate measurements. In addition, S.sub.5 is repeatedly shown in Table .sub.3 and Table .sub.4.

TABLE-US-00003 TABLE 3 S.sub.1 S.sub.2 S.sub.3 S.sub.4 S.sub.5 Mp 15000 15000 15000 15000 15000 Mw/Mn   1.5   1.30   1.20   1.10   1.05 T.sub.a(S.sub.k)   10.97   11.00   11.05   11.04   11.03 T.sub.B(S.sub.k)   11.23   11.23   11.23   11.23   11.23 ΔT(S.sub.k) =   0.26   0.23   0.18   0.19   0.20 T.sub.B(S.sub.k) − T.sub.a(S.sub.k)

TABLE-US-00004 TABLE 4 S.sub.5 S.sub.6 S.sub.7 S.sub.8 S.sub.9 Mp 15000 30000 60000 8000 4000 Mw/Mn   1.05   1.05   1.05   1.05   1.05 T.sub.a(S.sub.k)   11.03   10.93   10.31  11.70  12.91 T.sub.B(S.sub.k)   11.23   11.15   10.50  11.88  13.12 ΔT(S.sub.k) =   0.20   0.22   0.19   0.18   0.21 T.sub.B(S.sub.k) − T.sub.a(S.sub.k)

[0065] For all of S.sub.5, S.sub.6, S.sub.7, S.sub.8, and S.sub.9, ΔT (S.sub.k) is approximately 0.20 and almost equal with each other, and the correction by F.sub.Sk (T.sub.A)-F.sub.blank (T.sub.A) is effective. Comparing the results of S.sub.1, S.sub.2, S.sub.3, S.sub.4, and S.sub.5, as the Mw/Mn becomes larger, ΔT (S.sub.k) tends to be further away from 0.20. Then, it was found that, for the sample: S.sub.k for calculating the elution time difference between the RI detector and the UV detector, (Mw/Mn)≤1.30 is preferable, and (Mw/Mn)≤1.10 is more preferable.

[0066] Experiment 3

[0067] Next, Experiment 3 will be described. Note that the RI detector is defined as a detector A, and the UV detector is defined as a detector B.

[0068] Procedures

[0069] Step 1: A sample obtained by filtering the solvent used for the measurement (blank sample) was analyzed with the detector A, thereby obtaining the second chromatogram F.sub.blank (T.sub.A).

[0070] Step 2: The first standard samples ST.sub.k (k=1, 2, 3, 4, 5, and 6) were analyzed with the detector A, and the first elution time T.sub.A (ST.sub.k) for each of them was determined.

[0071] Step 3: The second standard sample S.sub.5, which can be detected by both detector A and detector B, was analyzed, thereby obtaining the first chromatogram F.sub.S.sub.5 (T.sub.A).

[0072] Step 4: The sample X, which was the subject to be measured, was measured with the detector B.

[0073] Step 5: The time T.sub.A (S.sub.5) at which the difference of F.sub.S.sub.5 (T.sub.A)-F.sub.blank (T.sub.A) reached its maximum was determined, and it was found to be 11.03 min.

[0074] Step 6: The difference between T.sub.A (S.sub.5)=11.03 min and the second elution time T.sub.B(S.sub.k)=11.23 min determined by size exclusion chromatography analysis of the second standard sample S.sub.5 using the detector B was determined, and it was found that ΔT (S.sub.5)=T.sub.B (S.sub.5)-T.sub.A (S.sub.5)=0.20 min.

[0075] Step 7: The first elution time T.sub.A (ST.sub.k), which was the time of peak top obtained from the analysis of each of the first standard samples ST.sub.k (k=1, 2, 3, 4, 5, and 6) by the detector A, was converted into T.sub.B (ST.sub.k) by using ΔT (S.sub.5) of 0.20 min, and a calibration curve expression showing the correspondence relationship between TB and the molecular weight M was calculated.

[0076] Step 8: The analytical results of the sample X, which was the subject to be measured by the detector B, were converted with the calculated calibration curve, thereby obtaining the molecular weight distribution of the sample X (see FIG. 2).

[0077] As described above, according to embodiments of the present invention, since the first chromatogram of the second standard sample is obtained using the first detector, the second chromatogram of the solvent for the second standard sample is obtained using the first detector, and the third elution time of the second standard sample using the first detector is determined from the difference between the first chromatogram and the second chromatogram, an accurate calibration curve can be created using multiple detectors in size exclusion chromatography.

[0078] Note that the present invention is not limited to the embodiments described above, and it is apparent that those having ordinary skill in the art can make many modifications and combinations without departing from the technical spirit of the present invention.