JP4110142B2 - Liquid chromatograph and preheat condition setting method - Google Patents

Description

  The present invention relates to a liquid chromatograph, and more particularly to a column oven.

In general, a column oven of a liquid chromatograph is provided with a portion for heating an eluent sent from a pump before introducing it into a column, so-called preheating means. For example, Japanese Patent Application Laid-Open Nos. 11-258222 and 2000-111536 show such devices.
The purpose of preheating is to make the eluent before introduction into the column as close as possible to the temperature of the column.

Matching the eluent and column temperature gives good results for the reproducibility of the separation peak retention time. However, depending on the type of separation column, this coincidence of temperature may adversely deteriorate the shape of the separation peak and impair the degree of separation of the peak.
In general, if the peak shape of the chromatogram obtained by the analysis without preheating is reading (with a tail before the peak), preheating and raising the temperature will result in a chromatogram of the resulting chromatogram. It can be observed that the peak shape changes in the direction of tailing (a state in which the tail is pulled after the peak). This phenomenon is due to a change in linear flow velocity due to a temperature difference between the center portion of the column and the wall surface portion and a column wall surface effect on the fluid. That is, when the “change in linear flow velocity” and the “column wall surface effect” cancel each other, the best peak shape is obtained.
In addition to the temperature of the eluent before column introduction as described above, the influence on the detection result also affects the temperature of the solution after passing through the column. That is, if the solution exited from the column oven enters the detector without returning to room temperature, the noise and drift of the detector increase, resulting in a problem that the detection sensitivity is lowered.
Therefore, before the solution exiting the oven reaches the detector, the temperature of the solution needs to be returned to room temperature (post-cool). Generally, this is dealt with by lengthening the flow path (tube) connecting the column and the detector. However, with this method, the separation of the column is impaired due to band spreading due to laminar flow in the tube.
In a liquid chromatograph that performs multi-component separation analysis, chromatograms required by a measurer are roughly classified as follows.
(1) The case where importance is placed on the reproducibility of the retention time of each separation peak even if the shape of the separation peak is somewhat bad.
This is mainly required when analyzing samples with few impurities or when emphasizing qualitative and quantitative analysis.
(2) When emphasizing the shape of the separation peak.
This is required when analyzing a sample with many impurities and high chromatogram peak density.
An object of the present invention is to provide a liquid chromatograph in which an optimum chromatogram required by a measurer can be easily obtained by controlling the temperature of a solution before and after a column.
For the above purpose, the present invention sets the conditions for setting the desired chromatogram conditions before the start of analysis, and conforms to the set conditions for the chromatogram after analyzing the standard sample multiple times. If the condition is not met, control is performed so as to change the set temperature of the preheating section.
Further, the preheat section includes a plurality of preheat pipes having different capacities. Furthermore, a separation column is provided for each preheat pipe.
In addition, a post-cool unit that controls the temperature of the eluent to match the ambient temperature is provided between the column oven and the detector.
According to the configuration of the present invention, it is possible to easily set optimum conditions for outputting a chromatogram desired by a measurer, such as reproducibility and symmetry.
Further, noise and drift appearing in the chromatogram can be suppressed.

FIG. 1 is a schematic configuration diagram of the present invention.
FIG. 2 shows a chromatogram after analyzing a standard sample a plurality of times.
FIG. 3 is a diagram showing an embodiment of the preheating unit 5.
FIG. 4 is a diagram showing an embodiment of the preheating unit 5.
FIG. 5 is a flowchart for setting preheat conditions.
FIG. 6 is a condition setting screen displayed when preheating condition setting is performed.
FIG. 7 is a chromatogram used for explanation for obtaining the degree of symmetry and the like.
FIG. 8 is a diagram showing an embodiment of the preheating unit 5.
FIG. 9 is a diagram showing an example of the preheating unit 5.
FIG. 10 is another schematic configuration diagram of the present invention.

上式において、ｘはピークトップの保持時間、ｎはデータ取得回数を示す。

この「再現性」が選択されている場合は、環境温度（室温）の変化に耐えるように、最初から十分な加熱が行われる。
「対称度」（非対称係数とも言う）は、文字通りピークの対称性を示す値であり、ピークトップの保持時間を中心として、その前後のピーク幅の比率を求めたものである。対称性を表す方法として、複数の計算式がある。第７図にピークの例を示すが、対称度をＳ、ピーク幅をＡ、前半のピーク幅をＢとした場合、計算式の一例として、Ｓ＝Ａ／２Ｂで表すことが出来る。この計算式の場合、対称度が１に近いほど対称性が良いといえ、１以上の数値はテーリング、１未満の数値はリーディングを示すことになる。本発明の場合は、複数回のデータ取得を行うため、ピーク幅Ａ，Ｂは、得られたデータの平均値を用いる。また、ピーク幅Ａ，Ｂを求める高さ位置ｈ′は、ピークトップの高さｈの５〜１０％付近の値を用いる。第６図の設定画面では、この対称度の数値は上限値と下限値を入力することになる。
「理論段数」は、ピークトップの保持時間ｔとピークのベースライン幅ｗとの関係によって求められるものであり、理論段数を求める式としても複数の計算式があるが、一例としてｎ＝（４ｔ／ｗ）^２で求めることができる。理論段数は、ピークの先鋭度を判断する指標として利用可能であるが、本発明においては使用しなくても良い。
ステップ５０５では、上記に示した判断基準に基づき、得られたデータの判断を行う。もし、判断基準に合わないデータであれば、ステップ５０６においてプレヒート部５の温度設定条件の変更を行い、再度ステップ５０２に戻る。判断基準に合うデータが得られた場合には、ステップ５０７へ行きプレヒート条件の設定が終了となる。
上記設定が終了すると、未知試料の分析を開始する。上記のようにプレヒート条件が設定されているため、未知試料の分析においても目的に応じたピークを得ることが可能になる。また、未知試料を繰り返し分析する場合、上記のプレヒート条件設定の処理を定期的に行えば、より確実に目的とするピーク形状を得ることが出来る。
第８図に、プレヒート部５の他の実施例を示す。
この実施例の特徴は、長さの異なるプレヒート配管８２，８３を備え、流路切替バルブ８４によって、いずれか一方の配管へ溶液を導くことが出来ることに有る。
プレヒート配管８２，８３は、ヒートブロック８１内を通過するように配置される。ヒートブロック８１には、第３図と同様の温度制御ユニットが設けられる（図示せず）。ヒートブロック８１の制御条件設定は、第５図に示したフローチャートと同様の手順で行う。本実施例では、２種類のプレヒート配管を備えていることから、より広い範囲の条件設定を行うことが出来る。
また、それぞれ容量の異なるプレヒート配管８２，８３は、例えば、標準用，セミミクロ用として使い分けることが出来、分析条件により切り替えて用いることが出来る。これにより、高流量域で使用するためにプレヒート容量を大きくする必要のある通常の分析の場合と、低流量域で使用し、デッドボリュームを最小限にしたい（プレヒート容量を最小限にしたい）セミミクロ用分析を流路切換バルブ４の切り換えにより容易に最適なプレヒート流路に切り替えることが可能となる。更に、第５図のフローチャートの条件設定を分析に先立って各配管ごとにプレヒート条件を設定することで、より最適な所望の分析結果を得ることが可能となる。
第９図に、第８図の構成を応用した他の実施例を示す。
本実施例は、第８図で示したプレヒート部５に分離カラムを組み合わせた例であり、容量の異なるプレヒート配管８２，８３のそれぞれに大きさの異なるカラム９１，９２を接続したものである。ヒートブロック８２には、第３図と同様の温度制御ユニットが設けられる（図示せず）。
本実施例では、２種類の分離カラムとその分析に適したプレヒート配管を備え、切り換えながら分析を行うことが出来る。プレヒートの条件設定は、各プレヒート配管ごとに行う。
第１０図に、カラムオーブン７の後段にポストクール部１３を備えた他の実施例を示す。
本実施例では、分離カラム６の後段にポストクール部１３を備えたことに特徴がある。ポストクール部１３は、カラムオーブン７とは熱的に分離されて配置され、その構成は、第３図や第４図で示したプレヒート部５と同様のもので良い。
ポストクール部１３は、カラムオーブン７によって設定温度に恒温化された液温を環境温度（室温）付近に戻すように温度を制御する。
本実施例では、ポストクール部１３により、検出器８におけるノイズ，ドリフトを抑えることが出来る。
以上説明したように、本発明の構成によれば、再現性や対称度など、測定者の望むクロマトグラムを出力するための最適な条件設定を容易に行うことが可能となる。
また、クロマトグラムに表れるノイズやドリフトを抑制することができる。FIG. 1 is a schematic view of a liquid chromatograph apparatus.
The solutions A and B (1, 2) are fed by the pump 3 while changing the mixed composition with time. Thereafter, the sample to be analyzed is added to the solution by an injector in the autosampler 4. The solution to which the sample is added passes through the preheating unit 5 and is then introduced into the separation column 6. In order to perform multi-component separation with good reproducibility, the separation column 6 is installed in a column oven 7 maintained at a constant temperature. Each component separated by the separation column 6 is detected by the detector 8, and the data is processed and stored by the data processing device 9. In addition, the data processing device 9 normally controls each unit.
In the separation column 6, the linear flow velocity of the fluid is faster at the center. Therefore, the fluid flowing through the center part elutes earlier from the separation column 6. That is, each component of the sample injected into the separation column 6 elutes from the column later than that flowing through the center of the column. In the chromatogram detected in this state, the peak shape of the component has a portion that elutes later, so tailing occurs.
Next, consider a case where a sample is introduced into the separation column 6 in a state where preheating is not performed. The solution introduced into the separation column 6 at a temperature lower than the column ambient temperature is warmed more rapidly as the column wall portion. Therefore, a phenomenon occurs in which the linear flow velocity of the fluid introduced into the separation column 6 is faster on the column wall surface. In this case, each component of the sample injected into the separation column 6 is eluted early at the portion that has flowed through the column wall surface portion, and is slowly eluted at the portion that has flowed through the center portion of the column. Therefore, the peak shape of the detected peak causes reading.
By appropriately controlling the temperature of the solution introduced into the column so that the above two phenomena can be offset, the linear flow rate of the solution in the separation column 6 can flow at a constant speed regardless of the column diameter. Become. At this time, the peak of the obtained component is a peak with good symmetry.
The effect of preheating on the peak shape is shown in FIG. FIG. 2 shows the chromatograms obtained by performing the analysis a plurality of times, with the same holding time being superimposed.
FIG. 2 (a) shows the state of the peak when the preheating is sufficiently performed. Since the portion that has flowed through the column wall part elutes with a delay, the peak shape shows tailing. However, since the temperature of the solution introduced into the separation column 6 is the same as the temperature of the separation column 6, there is little shift of each chromatogram, and it can be said that the reproducibility of the retention time shows a good result.
FIG. 2 (b) shows the state when preheating is not performed. Since the portion that has flowed through the column wall portion elutes quickly, the peak shape shows a reading. Further, since the temperature of the solution introduced into the separation column 6 has a large temperature difference from the temperature of the separation column 6, the shift of each chromatogram is large and the reproducibility of the retention time is inferior.
(C) shows the state of the peak when preheating is controlled so that the flow rate of the solution in the column is constant in the radial direction of the column. In this case, a symmetrical peak is obtained as the peak shape. In the reproducibility of the holding time, a result between when the preheating is sufficiently performed and when the preheating is not performed is obtained.
An example of the preheating part 5 in this invention is shown in FIG.
The pipe 10 through which the solution passed through the autosampler 4 is fed is fitted into the heat block 11. A temperature control unit 15 for heating or cooling the heat block 11 is connected to the heat block 11, and the temperature of the heat block 11 is controlled separately from the temperature of the column oven by controlling the temperature control unit 15. As the temperature control unit 15, for example, a unit using a Peltier element is used.
Moreover, an example of another preheating part is shown in FIG.
In this example, the preheating part 5 has a hollow structure, and the case 14 has a structure having a preheating space 12. The pipe 10 through which the solution that has passed through the autosampler 4 is fed is coiled and is disposed in the preheat space 12. A temperature control unit 16 for heating or cooling the air in the preheating space 12 is connected to the preheating unit 5, and the temperature in the preheating space 12 is controlled separately from the column oven. The temperature control unit 16 is, for example, a type that performs heat exchange using a Peltier element when the air in the preheat space 12 and the outside air are switched.
The piping in the preheating part 5 shown above uses a metal that can easily exchange heat, for example, a stainless steel piping.
FIG. 5 shows a flowchart for setting preheating conditions. The preheating condition setting process shown in this flowchart is processed in the data processing device 9 having a display such as a CRT.
When the condition setting process is started (501), first, a certain amount of sample (standard sample) is injected into the eluent by the autosampler 4 (502), and data is acquired by the detector 8 through the separation column 6 ( 503), the data processor 9 creates a chromatogram. The data processing device 9 counts the number of times of acquired data and determines whether or not it has been acquired a preset number of times (504). As the number of times set is larger, the reliability is improved, but usually about six times is set. If the number of data acquisitions is less than the set number, the process returns to step 502 above. When the number of data acquisition times reaches a predetermined number, it is determined whether a peak corresponding to the purpose is obtained from the obtained peak (505).
The criterion in step 505 is defined in advance in the data processing device 9 by the measurer. Specifically, it is selected whether the criterion to be emphasized in the determination is “reproducibility of holding time” or “peak shape”.
FIG. 6 shows an example of a setting screen displayed on the display means of the data processing device 6. Numeric values that serve as thresholds for “reproducibility”, “symmetry”, and “theoretical plate number” can be set by the measurer. Each item has a check box, and a priority criterion item can be designated. In the example of FIG. 6, “reproducibility” is selected. “Reproducibility” corresponds to the above “reproducibility of holding time”, and “symmetry” corresponds to the above “peak shape”.
“Reproducibility” is for judging the degree of variation in the retention time of the peak top of the chromatogram, and a numerical value represented by RSD (relative standard deviation) is used. RSD is represented by the following equation.

In the above equation, x represents the peak top retention time, and n represents the number of data acquisition times.

When this “reproducibility” is selected, sufficient heating is performed from the beginning to withstand changes in ambient temperature (room temperature).
“Symmetry” (also referred to as an asymmetric coefficient) is a value literally indicating the symmetry of the peak, and the ratio of the peak width before and after the peak top retention time is obtained. As a method of expressing symmetry, there are a plurality of calculation formulas. FIG. 7 shows an example of a peak. When the symmetry is S, the peak width is A, and the peak width of the first half is B, it can be expressed as S = A / 2B as an example of a calculation formula. In this calculation formula, the closer the degree of symmetry is to 1, the better the symmetry. A numerical value of 1 or more indicates tailing, and a numerical value of less than 1 indicates reading. In the case of the present invention, since data acquisition is performed a plurality of times, the average values of the obtained data are used as the peak widths A and B. The height position h ′ for obtaining the peak widths A and B uses a value in the vicinity of 5 to 10% of the height h of the peak top. In the setting screen of FIG. 6, the upper limit value and the lower limit value are input as the numerical value of the symmetry.
The “theoretical plate number” is obtained from the relationship between the peak top retention time t and the peak baseline width w, and there are a plurality of calculation formulas for calculating the theoretical plate number. As an example, n = (4t / W) ² can be obtained. The number of theoretical plates can be used as an index for judging the sharpness of the peak, but may not be used in the present invention.
In step 505, the obtained data is judged based on the judgment criteria shown above. If the data does not meet the criteria, the temperature setting condition of the preheating unit 5 is changed in step 506 and the process returns to step 502 again. If data that meets the criteria is obtained, the process goes to step 507 to finish setting the preheating conditions.
When the above setting is completed, the analysis of the unknown sample is started. Since the preheating conditions are set as described above, it is possible to obtain a peak according to the purpose even in the analysis of an unknown sample. Further, when an unknown sample is repeatedly analyzed, the target peak shape can be obtained more reliably by performing the above preheating condition setting processing periodically.
FIG. 8 shows another embodiment of the preheating unit 5.
The feature of this embodiment is that preheat pipes 82 and 83 having different lengths are provided, and the solution can be guided to one of the pipes by the flow path switching valve 84.
The preheat pipes 82 and 83 are arranged so as to pass through the heat block 81. The heat block 81 is provided with a temperature control unit similar to that shown in FIG. 3 (not shown). The control condition setting of the heat block 81 is performed in the same procedure as the flowchart shown in FIG. In this embodiment, since two types of preheat pipes are provided, a wider range of conditions can be set.
Further, the preheat pipes 82 and 83 having different capacities can be selectively used for, for example, standard use and semi-micro use, and can be switched depending on analysis conditions. As a result, in the case of normal analysis where the preheat capacity needs to be increased in order to use it in the high flow rate range, and in the case of using the low flow rate range, the dead volume is minimized (the preheat capacity is minimized). The analysis can be easily switched to the optimum preheat channel by switching the channel switching valve 4. Furthermore, it is possible to obtain a more optimal desired analysis result by setting preheating conditions for each pipe prior to analysis of the condition setting of the flowchart of FIG.
FIG. 9 shows another embodiment in which the configuration of FIG. 8 is applied.
This embodiment is an example in which a separation column is combined with the preheating section 5 shown in FIG. 8, and columns 91 and 92 having different sizes are connected to preheating pipes 82 and 83 having different capacities. The heat block 82 is provided with a temperature control unit similar to that shown in FIG. 3 (not shown).
In this embodiment, two types of separation columns and preheat piping suitable for the analysis are provided, and analysis can be performed while switching. Preheat condition setting is performed for each preheat pipe.
FIG. 10 shows another embodiment in which a post-cool portion 13 is provided at the rear stage of the column oven 7.
The present embodiment is characterized in that a post-cool portion 13 is provided in the subsequent stage of the separation column 6. The post-cooling unit 13 is arranged to be thermally separated from the column oven 7, and the configuration thereof may be the same as that of the preheating unit 5 shown in FIG. 3 and FIG.
The post-cooling unit 13 controls the temperature so that the temperature of the liquid set to the set temperature by the column oven 7 is returned to the vicinity of the environmental temperature (room temperature).
In this embodiment, the post-cool unit 13 can suppress noise and drift in the detector 8.
As described above, according to the configuration of the present invention, it is possible to easily set optimum conditions for outputting a chromatogram desired by the measurer, such as reproducibility and symmetry.
Further, noise and drift appearing in the chromatogram can be suppressed.

Claims (8)

An injector for injecting the sample into the eluent, a preheating unit for controlling the temperature of the sample solution before flowing into the column oven, a separation column for separating the sample, a column oven for controlling the temperature of the separation column, and the sample after separation In a liquid chromatograph equipped with a detector for detecting, and a data processing device for creating a chromatogram based on data obtained from the detector and controlling each part,
The data processing device includes:
Before starting the analysis, determine whether the condition setting means for setting the desired chromatogram conditions and the chromatogram after analyzing the standard sample multiple times meet the conditions set by the condition setting means. And a preheat part control means for controlling the preheat part to change the set temperature when the conditions are not met. In claim 1,
The condition setting means inputs at least a reproducibility of the chromatogram peak or a tolerance value of the symmetry degree of the chromatogram peak, and prioritizes either the reproducibility or the symmetry degree when determining the condition setting. A liquid chromatograph characterized by having means for designating cracks. In claim 2,
The liquid chromatograph characterized in that the condition setting means has means for inputting a value within an allowable range of the number of theoretical plates. In claim 1,
The preheat section includes a heat block having a path space in which piping through which an eluent flows is arranged, and a temperature control unit for controlling the temperature of the heat block. In claim 1,
The preheat section includes a case having a space in which a pipe through which an eluent flows is arranged, and a temperature control unit for controlling the temperature of air in the space. In claim 1,
The preheating unit includes a first preheat pipe, a second preheat pipe, and the first and second preheat pipes connected to each other, and the introduced sample solution is any of the first and second preheat pipes. A flow path switching valve for feeding liquid and a heat block having a path space in which the first and second preheat pipes are arranged, and the first and second preheat pipes have different capacities. A liquid chromatograph characterized by An injector for injecting the sample into the eluent, a preheating unit for controlling the temperature of the sample solution before flowing into the column oven, a separation column for separating the sample, a column oven for controlling the temperature of the separation column, and the sample after separation In the preheating condition setting method for the preheating part in the liquid chromatograph, comprising a detector for detecting the data, a chromatogram based on data obtained from the detector, and a data processing device for controlling each part,
Before starting the analysis, setting the desired chromatogram conditions;
Analyzing the standard sample multiple times;
Determining whether the chromatogram after analyzing the standard sample a plurality of times meets the set conditions, and if not, to control to change the set temperature of the preheating unit; A preheating condition setting method characterized by comprising: In claim 7 ,
The conditions set before analysis are at least the reproducibility of chromatogram peaks or the acceptable range of symmetry of chromatogram peaks, and priority is given to either reproducibility or symmetry when judging after analysis. A preheating condition setting method characterized by specifying whether or not to be performed.

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