JP2015021931A - Method to determine injection state of specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To precisely detect an abnormality of an injection state through a measurement by liquid chromatography.SOLUTION: According to the method of the present invention, a measurement by liquid chromatography detects a column eluate using: a first wavelength (from 405 nm to 420 nm) for detecting hemoglobin A1c using affinity liquid chromatography; and a second wavelength (from 490 nm to 520 nm) for monitoring a baseline fluctuation of an eluate, and an injection state of a specimen is determined based on an output change of both wavelengths.

Description

The present invention relates to a method for accurately determining a specimen injection state in liquid chromatography.

  Although measurement by liquid chromatography has a possibility of causing troubles due to various factors, various countermeasures are taken in advance to improve the accuracy and reliability of the measurement results. A “sample injection mechanism” for introducing a sample into an analysis column is well known as one of them.

  As a method for injecting a specimen into a column, for example, a two-position six-way valve that can switch between two flow path forms can be used (see FIG. 3). In the first position (load state), the loop for holding and weighing the sample is separated from the analysis flow path. In this state, the sample is introduced into the loop. In the second position (injection state), the loop is connected to the analysis flow path, and by switching from the first state to the second state, the sample held in the loop is introduced into the analysis flow path and the column Separated by

  In order to perform accurate analysis / quantification, the entire sample holding loop needs to be filled with the sample solution.

  At the time of sample injection, a problem as shown in FIG. 4 may occur.

  FIG. 4a shows a state in which the specimen is normally aspirated. The measuring loop is completely filled with the sample solution. FIG. 4B shows a case where the sample is not sent into the loop due to factors such as insufficient operation of the sample suction mechanism, and the eluent used in the previous measurement remains in the measurement loop. FIG. 4 c shows a case where the sample is not sent into the loop at all due to a shortage of the sample and the like, and the whole amount becomes air. FIG. 4d shows a case where the sample is sent into the loop, but is partially insufficient due to a shortage of the sample or the like and partially becomes air. FIG. 4e shows a case where bubbles are generated in the sample or in the sample aspirating process, and the bubbles are slightly mixed in the loop. When these events occur (FIGS. 4B to 4E), it is necessary to detect by some method and indicate to the user that the analysis result is not normal and the cause.

  In the case of general chromatography, a problem at the time of sample injection can be detected by monitoring the pressure applied to the entire system.

  FIG. 5 is a diagram schematically showing chromatograms and pressure fluctuations in an analysis system in which the system pressure is about 10 to 15 MPa, as in ion exchange chromatography using a gel of several microns (measurement target). : Hemoglobin A1c). When the specimen is normally injected, the pressure shows a substantially constant value as shown in FIG. 5a. When air is mixed into the specimen holding loop, the pressure is reduced at the same time as the injection as shown in FIGS. This is because air enters and compresses the system where a certain pressure is applied. The detector signal decreases in proportion to the decrease in absolute injection volume. Thus, it is possible to detect / determine an injection abnormality from pressure fluctuation. In addition, even when air is mixed, if the amount is small, it is crushed by the pressure and does not significantly affect the signal of the detector (FIG. 5b does not generate bubbles in the cell and does not affect the detector). In the case of FIG. 5c, bubbles are generated in the cell and affect the detector).

  However, in an analysis system in which the system pressure is only several MPa to several hundred KPa, the difference between the analysis pressure and the pressure when air is mixed becomes small, and it becomes difficult to detect / determine the injection abnormality from the pressure fluctuation. In addition, since the system pressure is low, even if a slight amount of air is mixed, bubbles are generated in the cell without being crushed by the pressure, and the detector signal is easily affected (see FIG. 6).

  As a result, when air is mixed at the time of sample injection and bubbles are generated in the detector cell, the amount of light in the cell increases despite the decrease in the absolute injection amount, affecting the detector signal. When a part of air is mixed in the sample measurement loop as shown in FIG. 4d, the signal tends to increase in spite of a decrease in the injection amount. FIG. 6b schematically shows changes in detector signal and pressure when bubbles are not generated in the detector cell, and FIG. 6c is a change in detector signal and pressure when bubbles are generated in the detector cell.

  The change in detector signal due to air often occurs in the vicinity where the void volume of the column is eluted.

  For example, the hemoglobin A1c (hereinafter referred to as A1c) measurement method by liquid chromatography is roughly classified into two methods, an ion exchange method and an affinity method. FIG. 1 is a flow chart and a representative chromatogram of A1c measurement by the ion exchange method. In the ion exchange chromatography method, a method is generally used in which a plurality of eluents having different salt concentrations are switched stepwise, fractionated into about 6 fractions, and the A1c content is calculated. FIG. 2 is a flow chart of A1c measurement by the affinity method and a representative chromatogram. In the affinity chromatography method, a method in which a component containing hemoglobin A0 (hereinafter referred to as A0) is eluted with a first eluent, and then a component containing A1c is eluted with a second eluent containing glucose or the like is generally used. Is.

  In the former ion exchange method, since both A1c and A0 are retained on the column, they are eluted after the void volume of the column. On the other hand, in the affinity method, A0 is hardly retained in the column and elutes near the void volume of the column. Therefore, the peak of A0 and the peak of the hemolyzed component of the specimen, the base fluctuation due to switching of the injection valve, and the like may overlap. However, when operating normally, the peak of the solution component obtained by diluting / hemolyzing the specimen and the base fluctuation due to the switching of the injection valve are extremely small compared to the peak of A0, so it does not greatly affect the quantitative result. Absent. In order to eliminate these influences, correction by two-wavelength measurement is also performed. Measure simultaneously at two wavelengths, the first wavelength (typically 415 nm) where the sample is absorbed and the second wavelength (typically 500 nm) where the sample is not absorbed, and subtract the latter from the former In many cases, a method of reducing the influence by subtracting the fluctuation of the baseline is used (see Patent Documents 1 to 3). All of these methods are intended to improve the measurement accuracy of the target component, and are not used for determining the injection state of the specimen into the apparatus.

  In the A1c separation by the ion exchange method, since both A1c and A0 are retained on the column, they are eluted after the void volume of the column. For this reason, although it is possible to detect and determine a signal abnormality due to air bubble mixing, it is difficult. On the other hand, in the affinity method, the component containing A0 is hardly held in the column and is eluted near the void volume of the column, so that it becomes difficult to detect and judge the abnormality due to the overlap of the base variation due to mixing of bubbles with the peak of A0.

International Publication No. 2007/111282 Pamphlet International Publication No. 2007/111283 Pamphlet JP 2007-315942 A

  The present invention was made for the purpose of accurately detecting abnormalities in the injection state in liquid chromatography, and is particularly suitable for separation by affinity in which A0 elutes in the vicinity of a column void in the measurement of A1c. .

As a result of intensive studies on the above problems, the present inventor has reached the present invention. That is, the present invention is as follows.
(1) In liquid chromatography, detection of column effluent using a first wavelength for detecting the detection target component and a second wavelength for monitoring the baseline fluctuation of the eluent without absorption in the detection target component A method for obtaining the output and determining the injection state of the specimen based on the output change due to both wavelengths.
(2) The method according to (1) above, wherein the detection target is hemoglobin A1c.
(3) The method according to (2) above, wherein the first wavelength is 405 nm to 420 nm, and the second wavelength is 490 nm to 520 nm.
(4) The method according to (2) or (3) above, which is an affinity liquid chromatography method.
(5) In the method according to any one of (2) to (4) above, at least a signal variation derived from hemoglobin A1c is measured at the first wavelength and a minute baseline variation is measured at the second wavelength , A method for determining that the sample has been successfully injected into the column.
(6) In the method described in any one of (2) to (4) above, at least at the first wavelength, a void peak is measured near the column void, and at the second wavelength, a void peak is measured near the column void. If it is determined that the eluent is injected instead of the sample being injected.
(7) In the method described in any of (2) to (4) above, at least at the first wavelength, no void peak is measured near the column void, and at the second wavelength, a void peak is present near the column void. A method of determining that nothing has been injected into the column if it is not measured.
(8) In the method according to any one of (2) to (4) above, when at least a signal variation derived from hemoglobin A1c is measured at the first wavelength and an excessive base variation is measured at the second wavelength, A method for determining that a sample and bubbles are injected into a column.
(9) In the method according to any one of (2) to (4) above, at least the signal fluctuation derived from hemoglobin A1c was not measured at the first wavelength, and excessive base fluctuation was measured at the second wavelength. In this case, it is determined that air has been injected without being able to inject the sample into the column.
The present invention is described in further detail below.

  In the present invention, the detection output of the column eluate is obtained by using the first wavelength for detecting the detection target component and the second wavelength for monitoring the baseline fluctuation of the eluent without absorption in the detection target component. Then, the injection state of the specimen is determined based on the output change due to both wavelengths. Here, as the first wavelength, a wavelength having absorption in the detection target is used. The second wavelength is a wavelength that is not absorbed by the detection target, but it is preferable to use a wavelength close to the first wavelength.

  In the present invention, the detection target is not particularly limited, and for example, A1c can be mentioned. In the case of A1c, it is preferable to use 405 to 420 nm as the first wavelength and 490 to 520 nm as the second wavelength.

  In the present invention, the type of liquid chromatography is not particularly limited, but affinity liquid chromatography is preferably used when A1c is to be detected.

  As a detector used in the present invention, using a single light source that can irradiate light of both the first wavelength and the second wavelength, irradiating the cell and dividing the transmitted light into the first wavelength and the second wavelength, A detector of each type is preferable. However, the same effect can be obtained by connecting a detector capable of detecting at the first wavelength and a detector capable of detecting at the second wavelength in series. In this case, it is preferable to minimize the influence of the peak spread due to the capacity of the detector and each connection pipe. It is desirable to arrange the first wavelength detector on the upstream side and the second wavelength detector on the downstream side (see FIG. 9).

  As a method of sample injection, for example, it is preferable to use a valve that can connect a sample holding loop to a two-position switching six-way valve that is generally used in liquid chromatography and introduce a certain amount of sample into the column. In the first position of the valve, the eluent from the pump is directly sent to the column, and during this time, the sample is introduced into the sample holding loop by using suction or discharge means. By switching to the second position, the eluent from the pump is sent to the column via the loop in which the sample is held. Thereby, the sample in the sample holding loop is introduced / separated into the column, and the intensity is measured by the detector.

  Hereinafter, the signal fluctuation of the detector depending on the sample injection state will be described with reference to FIG. 7 and Table 1, taking as an example the case where A1c is detected by affinity chromatography using the above-described sample injection method.

図中のａ〜ｄの検出器ベース変動（ピーク）は、以下の通りである。ａ：注入バルブ切り替えによる変動（ボイドピーク）、ｂ：溶離液切り替えによるによる変動（ステップグラジエント）、ｃ：気泡又は空気による変動、ｄ０：Ａ０由来の変動、ｄ１ｃ：Ａ１ｃ由来の変動。

The detector base fluctuations (peaks) a to d in the figure are as follows. a: Variation due to injection valve switching (void peak), b: Variation due to eluent switching (step gradient), c: Variation due to bubbles or air, d0: Variation derived from A0, d1c: Variation derived from A1c.

  The specimen injection state is roughly classified into the following six.

  E0: This is a case where the sample is reliably held in the sample holding loop and injected into the column (normal measurement). At the first wavelength, d0 is measured more than a certain amount, which overlaps with a and is seen near the void. D1c is also measured over a certain amount. Although b exists, it overlaps with other peaks and is not directly observed. At the second wavelength, the baseline fluctuation b due to the step gradient is measured, and a is measured in the vicinity of the void.

  E1: This is a case where the sample cannot be held in the sample holding loop due to some reason, and the eluent is injected instead of the sample being not injected. At the first wavelength, the baseline fluctuation b due to the step gradient is measured, and a is measured in the vicinity of the void. Even at the second wavelength, the baseline fluctuation b due to the step gradient is measured and a is measured in the vicinity of the void.

  E2: This is a case where nothing was injected into the column because the valve for injecting the sample did not switch from the load state to the injection state for some reason. At the first wavelength, only the baseline fluctuation due to the step gradient b is measured, and the fluctuation is minute. Even at the second wavelength, only the baseline fluctuation due to the step gradient b is measured, and the fluctuation is minute. In the vicinity of the void, no change is detected in either the first wavelength or the second wavelength.

  E3: This is a case where the sample is almost held in the sample holding loop, but a slight bubble is generated in the loop, and the sample and the bubble are injected into the column. At the first wavelength, the signal d0 derived from A0, the base fluctuation c due to bubbles, and a are added together, and an excessive peak is measured near the void. Apart from this, a peak derived from A1c also appears. In addition, although b exists, it overlaps with other peaks and is not observed directly. At the second wavelength, the bubble-derived base fluctuations c and a overlap and are measured as excessive fluctuations near the void. A minute b is also measured.

  E4: A case where a large amount of air is mixed in the sample holding loop, the sample cannot be retained at all, and air is injected without any sample or eluent being injected into the column. At the first wavelength, the base variation c derived from the air is measured in the vicinity of the void as an excessive variation, and a minute b is measured. However, no peak derived from A1c appears. At the second wavelength, the base variation c derived from air is measured near the void as an excessive variation. A minute b is also measured.

  E5: Other abnormalities.

In the present invention, among the signal fluctuations occurring at E0 to E5, characteristic signals can be selected in each of the states E0 to E5, and the injection state of the specimen can be determined using the selected one. Examples of characteristic signal fluctuation include the following among the above-mentioned ones.
E0: Signal fluctuation derived from hemoglobin A1c is measured at the first wavelength, and minute baseline fluctuation is measured at the second wavelength.
E1: At the first wavelength, a void peak is measured near the column void, and at the second wavelength, a void peak is measured near the column void.
E2: No void peak is measured near the column void at the first wavelength, and no void peak is measured near the column void even at the second wavelength.
E3: Signal fluctuation derived from hemoglobin A1c is measured at the first wavelength, and excessive base fluctuation is measured at the second wavelength.
E4: Signal fluctuation derived from hemoglobin A1c is not measured at the first wavelength, and excessive base fluctuation is measured at the second wavelength.

  In the present invention, the determination may be made based on at least such characteristic signal fluctuations, and may be further determined in consideration of other signal fluctuations.

  In the present invention, such determinations of E0 to E5 may be performed independently except for E5, or two or more of E0 to E5 may be combined.

  FIG. 8 is a flowchart showing an example of determining E0 to E5.

  At the time of detection using the first wavelength and the second wavelength, it is also possible to determine the injection state of the specimen from the output change and determine the cause of the abnormality at the injection unit.

It is the figure which showed the A1c measuring method by the general ion exchange chromatography method. a is a flow chart, and b is a typical chromatogram. It is the figure which showed the A1c measuring method by the general affinity chromatography method. a is a flow chart, and b is a typical chromatogram. It is the figure which showed typically the injection | pouring method of specimen injection | pouring. It is the figure which showed typically the malfunction in a sample injection mechanism. It is the figure which showed typically the chromatogram and pressure fluctuation at the time of the high pressure chromatography which a system pressure is about 10 Mpa-15 Mpa when air mixes at the time of sample injection. a is during normal measurement, and b is during aeration. It is the figure which showed typically the chromatogram and pressure fluctuation at the time of the sample injection | pouring in the low pressure chromatography whose system pressure is several MPa-about several hundred kPa when air mixes. a is a normal measurement, b is when air is mixed and no bubbles are generated in the cell, and c is when air is mixed and bubbles are generated in the cell. It is the figure which showed typically the change of the signal of a 1st wavelength and a 2nd wavelength by a test substance injection state. It is a flowchart which determines the content of abnormality based on the change of the signal of a 1st wavelength and a 2nd wavelength. It is the figure which showed the system of 2 wavelength measurement. a is a case of two-wavelength measurement by one detector, and b is a case of two-wavelength measurement by two detectors. 2 is a flow chart of the affinity chromatography method used in Example 1. FIG. FIG. 3 is a diagram showing a peak identification method in Example 1. In Example 1, it is the figure which showed the fluctuation | variation of a 1st wavelength and a pressure signal when a test substance is normally inject | poured, and when a bubble is mixed and injected. a is normal and b is a case where air bubbles are mixed. FIG. 13 is a diagram in which signal fluctuations at the first wavelength in FIGS. The shaded area indicates the identification range of the A0 peak. In Example 1, it is the figure which showed the signal fluctuation | variation of the 2nd wavelength when a test substance is normally inject | poured and when a bubble is mixed and injected. In Example 1, it is the figure which showed the fluctuation | variation of a 1st wavelength, a 2nd wavelength, and a pressure signal at the time of inject | pouring in the state with which the inside of the specimen holding loop was completely filled with air. In Example 1, it is the figure which showed the fluctuation | variation of the 1st wavelength and the 2nd wavelength when a sample is not introduce | transduced into a sample holding | maintenance loop at all and an eluent is injected instead. In Example 1, it is the figure which showed the fluctuation | variation of the 1st wavelength and the 2nd wavelength when the sample injection valve does not rotate.

  Hereinafter, the present invention will be described using examples. The present invention is not limited to these examples.

[Example 1]
A first visible detector capable of detection at the first wavelength and a second detector capable of detection at the second wavelength were connected in series downstream of the column for two-wavelength detection, and A1c measurement was performed by affinity chromatography. FIG. 10 shows a flow chart of the liquid chromatograph used.

  A two-position switching six-way valve (13) having a loop (14) capable of holding the solution is inserted between the eluent la pump (4) and the sample injection valve (6), and the eluent 1b is added to the above-mentioned 6 A step gradient was performed by holding the loop in the one-way valve and switching the valve.

  The pump (4) for feeding the eluent 1a used a metering pump FMM20 manufactured by KNF, and the pump (12) for filling the eluent 1b with a flow rate of 250 microliters per minute was also used.

  The detectors (10, 11) were connected in series with two UV-visible detectors UV-8020 manufactured by Tosoh, and used at a wavelength of 415 nm (upstream side) as the first wavelength and 500 nm (downstream side) as the second wavelength. . The column (8) was packed with TSKgel BORATE-5PW (manufactured by Tosoh Corporation) having an inner diameter of 1.5 mm and a length of 10 mm, and the temperature was adjusted to 60 ° C. and used. The specimen was used after diluting whole blood. The eluent 1a was glycine 100 mmol / L + Na sulfate 150 mmol / L (pH 9.2), and the eluent 1b was Tris 10 mmol / Na sulfate 50 mmol / L + D-sorbitol 100 mmol / L (pH 8.0). After equilibrating the column for a while with the eluent 1a, the sample was injected, switched to the eluent 1b after 0.55 minutes, and further separated by a step gradient returning to the eluent 1a after 0.75 minutes.

  The peaks separated and detected by the column are identified based on the identification table and quantitatively calculated. Table 2 shows a general identification table.

各ピークに対して基準となる溶出時間とその変動の許容幅を設定する。

The elution time used as a reference for each peak and the allowable range of the variation are set.

The peak of elution time (t _A0 -t _A0 × α) to (t _A0 + t _A0 × α) is A0, and the elution time is (t _A1c -t _A1c × β) to (t _A1c + t _A1c × β) After identifying the peak as A1c, quantitative calculation is performed (see FIG. 11).

  The identification table in Table 3 was used this time. Regarding the tolerance, both A0 and A1c were set to 10% of the standard elution time.

溶出時間が０．２５２〜０．３０８分までに検出されたピークをＡ０として、溶出時間が０．７８９〜０．９６５分までに検出されたピークをＡ１ｃとして同定し定量計算を実施した。

A peak detected at an elution time of 0.252 to 0.308 minutes was designated as A0, and a peak detected at an elution time of 0.789 to 0.965 minutes was identified as A1c, and quantitative calculation was performed.

FIG. 12a shows a case where the specimen is normally injected, and FIG. 12b shows a case where bubbles are mixed in the specimen holding loop. The upper graph shows the chromatogram measured at the first wavelength, and the lower graph shows the pressure fluctuation at that time. FIG. 13 is a diagram in which both chromatograms are overwritten.
Under this condition, the column pressure is about 100 kPa, which is considerably lower than that of general chromatography. Therefore, even if a small amount of air bubbles are mixed, there is little pressure fluctuation (decrease) at the time of sample injection, and there is a difference from the case of normal injection. It is difficult to determine the injection abnormality from the pressure fluctuation pattern.
With only the first wavelength (FIG. 12), both peaks eluted near the void are identified as A0 from the identification table.

  The difference between the two becomes clear when viewing the chromatogram of the second wavelength. When the sample is injected normally, only a slight baseline variation is observed at the second wavelength due to the solvent peak and the eluent change due to the step gradient. A similar bubble-derived peak is observed at the elution position of A0 (near the void volume) (see FIG. 14).

  As described above, when only the first wavelength having absorption in the detection target is used, it is determined as A0. However, by simultaneously monitoring the second wavelength having no absorption in the detection target, it is a base variation due to air mixing. It is possible to certify that.

  Next, FIG. 15 shows a case where the specimen holding loop is completely filled with air and injected, and the specimen is not introduced. FIG. 15 is a chromatogram measured at the first wavelength, b is the second wavelength, and c is a diagram showing pressure fluctuations at that time. When air is completely injected in this way, pressure fluctuation (decrease) occurs during sample injection, and abnormal injection can also be determined from the pressure fluctuation pattern.

  In such a case, at the first wavelength, a large peak elutes in the vicinity of the void and is identified as the peak of A0 from the identification table. However, since the peak of A1c does not appear at all, it is doubted that the specimen could not be injected. Since a large peak is eluted in the vicinity of the void even at the second wavelength, it can be determined that this peak is not A0.

  Next, FIG. 16 shows a case where the sample is not guided to the sample holding loop and the eluent is injected instead. FIG. 16a shows a chromatogram measured at the first wavelength, and FIG. 16b shows a chromatogram measured at the second wavelength. A large peak does not appear in the vicinity of the void at both the first wavelength and the second wavelength, and a slight baseline fluctuation b derived from the change in the eluent due to the step gradient is observed. In addition, a minute variation a due to the switching of the injection valve for both the first wavelength and the second wavelength appears near the void.

  Next, FIG. 17 shows a case where the specimen injection valve does not rotate. FIG. 17a is a diagram showing a chromatogram measured at the first wavelength, and FIG. 17b is a diagram showing a chromatogram measured at the second wavelength. For both the first wavelength and the second wavelength, no large peak appears in the vicinity of the void, and no fluctuation due to switching of the injection valve is observed. In addition, slight baseline fluctuations due to the eluent change due to the step gradient were observed for both the first wavelength and the second wavelength.

  In this way, even if injection abnormalities occur due to slight air mixing and it is difficult to determine due to pressure fluctuations, the injection state can be changed by combining the signal fluctuations of the first wavelength and the signal fluctuations of the second wavelength. It can be easily determined.

1a, 1b, 1c. Eluents 2a, 2b, 2c. 2. Switching valve Deaerator 4. Pump 5. Pressure sensor6. 6. Sample injection valve Sample loop 8. Analysis column9. Column thermostat 10. Detector 1
11. Detector 2
12 12. Eluent 1b filling pump Eluent switching valve 14. Eluent 1b retention loop 15. Detector light source 16. Cell 17. Half mirror 18. Wavelength selection filter 1
19. Wavelength selection filter 2
20. Photo sensor 1
21. Photo sensor 2
22. Branch and merge block

Claims (9)

In liquid chromatography, the detection output of the column effluent is obtained using the first wavelength for detecting the target component and the second wavelength for monitoring the baseline fluctuations of the eluent when there is no absorption in the target component. And determining the injection state of the specimen based on the output change due to both wavelengths. The method according to claim 1, wherein the detection target is hemoglobin A1c. The method according to claim 2, wherein the first wavelength is 405 nm to 420 nm, and the second wavelength is 490 nm to 520 nm. 4. The method according to claim 2 or 3, wherein the method is an affinity liquid chromatography method. The method according to any one of claims 2 to 4, wherein at least a signal variation derived from hemoglobin A1c is measured at the first wavelength, and a minute baseline variation is measured at the second wavelength, the specimen is normally applied to the column. How to determine that it has been injected. The method according to any one of claims 2 to 4, wherein a sample is injected when a void peak is measured in the vicinity of a void in the column at least at the first wavelength and a void peak is measured in the vicinity of the void in the column even at the second wavelength. Instead, it is determined that the eluent has been injected instead. The method according to any one of claims 2 to 4, wherein at least the void peak is not measured in the vicinity of the column void at the first wavelength, and the void peak is not measured near the column void even at the second wavelength. A method to determine that nothing has been injected. The method according to any one of claims 2 to 4, wherein at least a signal variation derived from hemoglobin A1c is measured at the first wavelength and an excessive base variation is measured at the second wavelength, the sample and the bubble are injected into the column. How to determine that has been done. The method according to any one of claims 2 to 4, wherein when at least the first wavelength does not measure a signal variation derived from hemoglobin A1c and an excessive base variation is measured at the second wavelength, the sample is injected into the column. A method of determining that air has been injected without being able to.

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