CN105561631A - Regenerating method of normal phase padding column in high performance liquid chromatograph - Google Patents

Regenerating method of normal phase padding column in high performance liquid chromatograph Download PDF

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CN105561631A
CN105561631A CN 201510929732 CN201510929732A CN105561631A CN 105561631 A CN105561631 A CN 105561631A CN 201510929732 CN201510929732 CN 201510929732 CN 201510929732 A CN201510929732 A CN 201510929732A CN 105561631 A CN105561631 A CN 105561631A
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normal phase
min
flow rate
column
tetrahydrofuran
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CN 201510929732
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Chinese (zh)
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袁泳
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袁泳
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/42Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424Elution mode
    • B01D15/426Specific type of solvent

Abstract

The invention discloses a regenerating method of a normal phase padding column in a high performance liquid chromatograph. The method comprises following steps: (1) reversing the normal phase padding column, back flushing the normal phase padding column with 40-44 times volume of tetrahydrofuran, and controlling a flow rate of the tetrahydrofuran to be 2-4L/min; (2) back flushing the normal phase padding column with 50-56 times volume of methyl alcohol, and controlling a flow rate of the methyl alcohol to be 2-4L/min; (3) back flushing the normal phase padding column with 48-50 times volume of tetrahydrofuran, and controlling a flow rate of the tetrahydrofuran to be 4-6L/min; (4) back flushing the normal phase padding column with 20-30 times volume of dichloromethane, and controlling a flow rate of the dichloromethane to be 6-10L/min; (5) back flushing the normal phase padding column with 10-20 times volume of anhydrous normal hexane, and controlling a flow rate of the anhydrous normal hexane to be 5-8L/min. The method has beneficial effects of good regenerating effect and small water content in a normal-phase chromatography column.

Description

高效液相色谱仪中正相填料柱的再生方法 HPLC method for regenerating column packing phase CKS

技术领域 FIELD

[0001]本发明涉及有机物分析领域,尤其涉及高效液相色谱仪中正相填料柱的再生方法。 [0001] The present invention relates to the analysis of organic compounds, and in particular relates to a method of reproducing CKS phase high performance liquid chromatography column packing.

背景技术 Background technique

[0002]高效液相色谱仪的系统由储液器、栗、进样器、色谱柱、检测器、记录仪等几部分组成。 [0002] The HPLC system consists of several parts of the reservoir, Li, injector, column, detector, and the like recorder. 储液器中的流动相被高压栗打入系统,样品溶液经进样器进入流动相,被流动相载入色谱柱(固定相)内,由于样品溶液中的各组分在两相中具有不同的分配系数,在两相中做相对运动时,经过反复多次的吸附-解吸的分配过程,各组分在移动速度上产生较大的差别,被分离成单个组分依次从柱内流出,通过检测器时,样品浓度被转换成电信号传送到记录仪,数据以图谱形式打印出来。 Reservoir of the mobile phase is a high-pressure system into the Li, the sample solution flows through the injector into the phase, the mobile phase is loaded the column (stationary phase), the sample solution since each component has two phases different distribution coefficients, when the relative movement in both phases, after the repeated adsorption - desorption allocation process, the components generating a large difference in the moving speed, was separated into the individual constituents of the effluent from the column , through the detector, the sample concentration is converted into an electrical signal to a recorder, data is printed out in the form of maps.

[0003]通常,样品中含有一些对分析者来说不感兴趣的东西。 [0003] Typically, the sample contains something for analysts who are not interested. 盐、脂质、含脂物质、腐质酸、疏水蛋白质和其它一些生物物质是一些可能在使用时与HPLC柱发生相互作用的物质。 Salts, lipids, lipid-containing substances, humic acid, hydrophobic proteins and other biological substances are some of the material may interact with the HPLC column during use. 这些物质有比分析者的目标物或少或大的保留值。 These substances have retention value less or greater than the analyst's target. 那些保留值较小的物质如盐类一般来说在空体积时就被冲出色谱柱。 Retention of the smaller species such as salts generally at the void volume was out of the column. 这些非目标产物的干扰能被检测器检测到而且能形成色谱峰,气泡,基线上移或者是负峰。 The non-interference of the target product can be detected by the detector and capable of forming a peak, bubbles, or negative peak shifting baseline. 如果样品成分在柱子中有很强的保留而且流动相溶液成分不足以把这些物质洗脱下来,多次上样后,这些吸附在柱子表面的物质通常就会积累在柱头。 If the sample has a strong component retained in the column and the flow-phase solution composition of these materials is insufficient to elute off, after repeated loading, the column adsorbed substances will accumulate in the surface generally stigma. 这些行为通常只有通过平行实验才能发现。 These behaviors are usually only found in parallel experiments. 有着中等保留值的样品能被缓慢洗出而且表现为宽峰,基线扰动,或者基线漂移。 Samples with moderate retention values ​​can be slow to wash out and showed a broad peak baseline disturbance, or baseline drift. 有时候这些被吸附的样品成分累积到一定程度足以使他们开始形成新的固定相。 Sometimes these adsorbed sample constituents build up to levels sufficient to cause them to start to form a new stationary phase. 分析物能与这些杂质作用形成一定的分离机理。 Certain analyte separation mechanism capable of forming these as impurities. 保留时间会波动,拖尾会出现。 Retention time fluctuate, tailing will appear. 如果足够的污染产生,柱子的反压能超过栗所能承受的最大压力,使柱子无法工作以及在堵塞处产生空体积。 If sufficient pollution, the column backpressure exceed Li can withstand the maximum pressure, and the column was unable to work and generate clogging the void volume. 这时,我们就要对色谱柱进行再生。 At this time, we will carry out the regeneration of the column. 而现有的反相填料柱再生方法一般是通过水一甲醇一异丙醇一二氯甲烷一异丙醇一甲醇一水进行清洗,没有专门的正相填料柱再生方法。 The conventional regeneration process is typically reversed phase column packing cleaned by a water-methanol-isopropanol-methanol in dichloromethane an isopropanol-water, no special reproducing method normal phase column packing.

发明内容 SUMMARY

[0004]本发明要解决的技术问题是现有的高效液相色谱仪中正相填料柱再生方法效果差,无专门的再生方法,为此提供一种高效液相色谱仪中正相填料柱的再生方法。 [0004] The present invention is to solve the technical problems of the prior CKS phase high performance liquid chromatography column packed with poor regeneration process, no special reproducing method therefore provides a high performance liquid chromatography with a packed column regeneration CKS method.

[0005]本发明的技术方案是:高效液相色谱仪中正相填料柱的再生方法,它包括以下步骤:(I)、倒转正相填料柱,用40-44倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在2-4L/min; (2)、用50-56倍体积的甲醇反冲正相冲填料柱,甲醇流速控制在2_4L/min;(3)、用48-50倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在4-6 L/min; (4)、用20-30倍体积的二氯甲烷反冲正相填料柱,二氯甲烷流速控制在6-10 L/min; (5)、用10-20倍体积的无水正己烷反冲正相填料柱,无水正己烷流速控制在5-8 L/min。 [0005] aspect of the present invention are: high performance liquid chromatography with a packed column CKS reproducing method, comprising the steps of: (the I), reversing the positive phase column packed with 40-44 volumes of tetrahydrofuran recoil normal phase packed column, flow rate control in tetrahydrofuran 2-4L / min; (2), with 50-56 volumes of methanol recoil impulse normal phase column packing, methanol flow rate control in 2_4L / min; (3), with 48-50 fold tetrahydrofuran recoil volume normal phase column packing, tetrahydrofuran flow rate control in 4-6 L / min; (4), with 20-30 volumes of dichloromethane recoil normal phase column packing, flow rate control in dichloromethane 6- 10 L / min; (5), dried over anhydrous hexane recoil 10-20 volumes of normal phase column packing, the velocity of flow of anhydrous hexane 5-8 L / min.

[0006]本发明的有益效果是采用四氢呋喃-甲醇-四氢呋喃-二氯甲烷-无水正己烷流速逐渐提高、与正相填料柱相比的体积倍数逐渐降低的方法来再生正相填料柱,再生效果好,正相色谱柱中水分含量少。 [0006] Advantageous effects of the present invention is the use of tetrahydrofuran - methanol - tetrahydrofuran - methylene chloride - anhydrous hexane and gradually increase the flow rate, as compared with the method of packing a normal phase column volume decreasing multiples to regenerate normal phase column packing, regeneration effective, less normal phase chromatography column moisture content.

具体实施方式 detailed description

[0007]下面结合实施例对本发明做进一步说明。 [0007] The following embodiments in conjunction with embodiments of the present invention will be further described.

[0008]实施例1:高效液相色谱仪中正相填料柱的再生方法,它包括以下步骤:(1)、倒转正相填料柱,用40倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在2L/min; (2)、用50倍体积的甲醇反冲正相冲填料柱,甲醇流速控制在2L/min; (3)、用48倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在4L/min; (4)、用20倍体积的二氯甲烷反冲正相填料柱,二氯甲烷流速控制在6L/min; (5)、用1倍体积的无水正己烷反冲正相填料柱,无水正己烷流速控制在5L/min。 [0008] Example 1: high performance liquid chromatography column packing phase CKS reproducing method, comprising the steps of: (1), reversing the positive phase packing column with 40 volumes of tetrahydrofuran recoil normal phase column packing, tetrahydrofuran Flow rate control 2L / min; (2), 50 volumes of methanol recoil normal phase column packed with red, methanol flow rate control in 2L / min; (3), with 48 times the volume of tetrahydrofuran recoil normal phase column packing, tetrahydrofuran in the flow rate control 4L / min; (4), with a 20-fold volume of dichloromethane recoil normal phase column packing, flow rate control in dichloromethane 6L / min; (5), with 1 volume of anti anhydrous n-hexane Reversal phase packing column, flow rate control of anhydrous hexane at 5L / min.

[0009]实施例2:高效液相色谱仪中正相填料柱的再生方法,它包括以下步骤:(I)、倒转正相填料柱,用42倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在3L/min; (2)、用52倍体积的甲醇反冲正相冲填料柱,甲醇流速控制在3L/min; (3)、用49倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在5 L/min; (4)、用25倍体积的二氯甲烷反冲正相填料柱,二氯甲烷流速控制在8 L/min; (5)、用15倍体积的无水正己烷反冲正相填料柱,无水正己烧流速控制在7 L/min。 [0009] Example 2: High performance liquid chromatography column packing phase CKS reproducing method, comprising the steps of: (the I), reversing the positive phase column packed with 42 volumes of tetrahydrofuran recoil normal phase column packing, tetrahydrofuran Flow rate control 3L / min; (2), using 52 volumes of methanol recoil impulse normal phase column packing, methanol flow rate control in 3L / min; (3), with 49 times the volume of tetrahydrofuran recoil normal phase column packing, tetrahydrofuran controlling the flow rate 5 L / min; (4), using 25 volumes of dichloromethane recoil normal phase column packing, flow rate control in methylene chloride 8 L / min; (5), dried over anhydrous 15 volumes of n-hexyl alkoxy recoil normal phase column packed with anhydrous n-hexyl burn flow control in 7 L / min.

[0010]实施例3:高效液相色谱仪中正相填料柱的再生方法,它包括以下步骤:(I)、倒转正相填料柱,用44倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在4L/min; (2)、用56倍体积的甲醇反冲正相冲填料柱,甲醇流速控制在4L/min; (3)、用50倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在6 L/min; (4)、用30倍体积的二氯甲烷反冲正相填料柱,二氯甲烷流速控制在1 L/min; (5)、用20倍体积的无水正己烷反冲正相填料柱,无水正己烧流速控制在8 L/min。 [0010] Example 3: High performance liquid chromatography with a packed column CKS reproducing method, comprising the steps of: (the I), reversing the positive phase column packed with 44 volumes of tetrahydrofuran recoil normal phase column packing, tetrahydrofuran Flow rate control 4L / min; (2), 56 volumes of methanol recoil normal phase column packed with red, methanol flow rate control in 4L / min; (3), with 50 times the volume of tetrahydrofuran recoil normal phase column packing, tetrahydrofuran controlling the flow rate 6 L / min; (4), using 30 volumes of dichloromethane recoil normal phase column packing, flow rate control in dichloromethane 1 L / min; (5), dried over anhydrous 20 volumes of n-hexyl alkoxy recoil normal phase column packed with anhydrous n-hexyl burning velocity of flow 8 L / min.

[0011]本发明可以有效去除正相填料柱中的水分,保证固定相的水分含量变化较小,从而保持水分条件的一致性。 [0011] The present invention can effectively remove moisture normal phase column packing to ensure that small changes in the moisture content of the stationary phase, thereby maintaining the consistency of water conditions.

Claims (1)

  1. 1.高效液相色谱仪中正相填料柱的再生方法,其特征是它包括以下步骤:(I)、倒转正相填料柱,用40-44倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在2-4L/min;(2)、用50-56倍体积的甲醇反冲正相冲填料柱,甲醇流速控制在2-4L/min; (3)、用48-50倍体积的四氢呋喃反冲正相填料柱,四氢呋喃流速控制在4-6 L/min; (4)、用20-30倍体积的二氯甲烷反冲正相填料柱,二氯甲烷流速控制在6-10 L/min;(5)、用10-20倍体积的无水正己烷反冲正相填料柱,无水正己烷流速控制在5-8 L/min。 1. HPLC reproducing method CKS phase column packing, characterized in that it comprises the steps of: (I), n reverse phase column packed with 40-44 volumes of tetrahydrofuran recoil normal phase column packing, tetrahydrofuran Flow rate control 2-4L / min; (2), with 50-56 volumes of methanol recoil impulse normal phase column packing, methanol flow rate control in 2-4L / min; (3), with 48-50 volumes of tetrahydrofuran recoil normal phase column packing, tetrahydrofuran flow rate control in 4-6 L / min; (4), with 20-30 volumes of dichloromethane recoil normal phase column packing, flow rate control in dichloromethane 6-10 L / min; (5), dried over anhydrous hexane recoil 10-20 volumes of normal phase column packing, the velocity of flow of anhydrous hexane 5-8 L / min.
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