KR101017572B1 - Packing method of prep hplc column - Google Patents

Packing method of prep hplc column Download PDF

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KR101017572B1
KR101017572B1 KR1020100002079A KR20100002079A KR101017572B1 KR 101017572 B1 KR101017572 B1 KR 101017572B1 KR 1020100002079 A KR1020100002079 A KR 1020100002079A KR 20100002079 A KR20100002079 A KR 20100002079A KR 101017572 B1 KR101017572 B1 KR 101017572B1
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column
slurry
filling
outlet
liquid chromatography
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KR1020100002079A
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Korean (ko)
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수 창 이
진 한 임
윤 호 최
성 애 홍
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(주) 영진바이오크롬
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B01D15/206—Packing or coating
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/56—Packing methods or coating methods
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/56—Packing methods or coating methods
    • G01N2030/562—Packing methods or coating methods packing
    • G01N2030/565—Packing methods or coating methods packing slurry packing

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  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

PURPOSE: A filling method of a high performance liquid chromatography column is provided to apply the uniform pressure to the top, the middle, and the bottom of the column for maintaining the filling density inside the entire column. CONSTITUTION: A filling method of a high performance liquid chromatography column comprises the following steps: storing a slurry tank(1) with slurry containing filling media and silica gel; injecting the slurry from the slurry tank into the column(30) using a slurry supplying pump(2); and pressurizing the slurry inside the column with a piston(6) of a hydraulic cylinder, for discharging the filling media inside the slurry to the outside of the column through a discharging unit(32).

Description

분취용 고성능 액체크로마토그래피 컬럼의 충진 방법 {Packing method of Prep HPLC Column}Packing method of prep HPLC column for preparative high performance liquid chromatography column

본 발명은 분취용 고성능 액체크로마토그래피 컬럼의 충진 방법에 관한 것으로서, 구체적으로는 컬럼 내에 충진되는 충진물의 충진 밀도를 컬럼 내 충진층 전체에 걸쳐 균일하게 유지시켜면서 고밀도로 충진시켜, 컬럼 충진층의 길이가 70cm 이상인 컬럼도 충진할 수 있는 분취용 고성능 액체크로마토그래피 컬럼 의 충진 방법에 관한 것이다.The present invention relates to a method of filling a preparative high performance liquid chromatography column, and in particular, to maintain the filling density of the filler packed in the column at a high density while maintaining a uniform packing throughout the packed layer in the column, The present invention relates to a method for filling a preparative high performance liquid chromatography column capable of filling a column of 70 cm or more in length.

보다 구체적으로는 얻어진 크로마토그램 피크 모양의 대칭성이 좋고 샤프한, 즉, 컬럼의 성능을 나타내는 이론단수가 향상되어, 크로마토그래피의 분리 효율을 크게 향상시킨 분취용 컬럼 의 충진 방법에 관한 것이다.
More specifically, the present invention relates to a method of filling a preparative column in which the symmetry of the obtained chromatogram peak shape is good and sharp, that is, the number of theoretical plates representing the performance of the column is improved, and the separation efficiency of chromatography is greatly improved.

분취용 고성능 액체크로마토그래피 컬럼을 충진하는 종래 방법으로는 도 1 내지 도 3에 도시된 바와 같이 충진용매와 충진제인 실리카 겔이 혼합된 슬러리가 담겨진 슬러리 탱크(1)로부터 슬러리를 컬럼(3)에 주입한 다음, 상기의 컬럼에 주입된 슬러리를 유압실린더의 피스톤(6)으로 가압하여 슬러리내 충진용매를 상기 컬럼의 유출부(8)를 통해 컬럼(3) 밖으로 배출한 다음, 상기 컬럼(3)에 이동상 용액을 주입하여 컬럼내 잔존하는 충진용매를 이동상 용액으로 치환하여 컬럼을 충진하는 방법이 사용되어 왔다.Conventional methods for filling preparative high performance liquid chromatography columns include slurry from column (3) from slurry tank (1) containing slurry mixed with filler solvent and silica gel as shown in FIGS. After the injection, the slurry injected into the column is pressurized by the piston 6 of the hydraulic cylinder to discharge the filling solvent in the slurry out of the column 3 through the outlet 8 of the column, and then the column 3 A method of filling a column by injecting a mobile phase solution into the c) and replacing the remaining filling solvent in the column with a mobile phase solution has been used.

도 1 내지 도 3은 색층분석용 컬럼을 충진하는 종래 방법의 각 공정들을 도시한 공정 개략도들이다.1 to 3 are process schematics showing respective processes of the conventional method of filling a chromatographic column.

구체적으로, 도 1은 충진용매와 실리카 겔이 혼합된 슬러리가 담겨진 슬러리 탱크로부터 슬러리를 컬럼(3)에 주입하는 공정의 개략도이고, 도 2는 컬럼에 주입된 슬러리를 유압실린더의 피스톤(6)으로 가압하여 슬러리내 충진용매를 컬럼의 유출부(8)를 통해 컬럼(3) 밖으로 배출하는 공정의 개략도이고, 도 3은 컬럼(3)내에 이동상 용액을 주입하여 컬럼(3)내 잔존하는 충진 용매를 이동상 용액으로 치환하는 공정의 개략도이다.Specifically, FIG. 1 is a schematic diagram of a process of injecting a slurry into a column 3 from a slurry tank containing a slurry in which a filling solvent and silica gel are mixed, and FIG. 2 is a piston 6 of a hydraulic cylinder. Is a schematic diagram of a process of discharging the filling solvent in the slurry to the outside of the column 3 through the outlet 8 of the column, and FIG. 3 shows the filling remaining in the column 3 by injecting a mobile phase solution into the column 3. It is a schematic of the process of substituting a solvent for a mobile phase solution.

그러나, 상기의 종래방법은 유압 실린더의 피스톤(6)으로 가압시 컬럼 내부의 압력을 균일하게 유지시킬 수 없어서 도 2에 잘 도시된 바와 같이 컬럼 중간부(a)의 충진물 충진 밀도가 컬럼 상부(b) 및 컬럼 하부(c)의 충진물 충진 밀도보다 낮게 되어서 컬럼 전체적으로 충진층의 균질성을 확보할 수 없었고, 그로인해 크로마토그래피의 분리 효율이 떨어지는 문제가 있었다.However, the conventional method cannot maintain the pressure inside the column uniformly when pressurized by the piston 6 of the hydraulic cylinder, so that the packing density of the filler in the middle part of the column (a) as shown in FIG. b) and the packing density lower than the packing density of the column bottom (c), it was not possible to ensure the homogeneity of the packed layer throughout the column, thereby reducing the separation efficiency of chromatography.

일반적으로 분취용 컬럼의 충진 작업시 컬럼내 충진층의 길이가 길어지면 이론단수가 높아져 고순도로 물질을 분취할 수 있는 장점이 있다.In general, when the length of the packed bed in the column is increased during the filling operation of the preparative column has the advantage that the theoretical fraction can be increased to fractionate the material with high purity.

그러나, 상기의 종래 충진 방법에서는 충진 작업시 컬럼 내부의 압력을 균일하게 유지할 수 없기 때문에 컬럼내 충진층 길이를 30~50㎝ 정도로 충진하고 있었다.However, in the above-mentioned conventional filling method, since the pressure inside the column cannot be maintained uniformly during the filling operation, the packed bed length in the column is about 30 to 50 cm.

만약 상기의 종래 충진방법으로 컬럼내 충진층 길이를 50㎝ 이상으로 하는 경우 컬럼내 충진물의 충진 밀도가 불균일하게 되어, 다시말해 컬럼의 상부 및 하부에는 충진물의 충진 밀도가 높아지고 컬럼의 중간부는 충진물의 충진밀도가 낮아져 분리성능이 떨어지고, 이론단수가 낮아지는 문제가 발생 되었다.If the length of the packed bed in the column is 50 cm or more by the conventional filling method, the packing density of the packing material in the column becomes uneven, that is, the packing density of the packing material is increased in the upper and lower parts of the column, and the middle part of the column is filled. As the packing density is lowered, the separation performance is lowered and the number of theoretical plates is lowered.

상기 컬럼의 이론단수는 컬럼 충전 작업 완료 후 표준샘플을 컬럼내에 주입하여 자외선(UV) 디텍터에 의해 나타나는 크로마토그램 그래프 상의 피크 높이와 좌우대칭성 등에 관한 수식으로 계산하며, 이론단수가 높을수록 컬럼 성능이 우수한 것을 나타낸다.
The theoretical number of columns of the column is calculated by formulating the peak height and left-right symmetry on the chromatogram indicated by the ultraviolet (UV) detector by injecting a standard sample into the column after the column filling operation is completed. It shows excellent.

본 발명의 과제는 분취용 고성능 액체크로마토그래피 컬럼의 충진시에 컬럼의 상부, 중간부 및 하부 모두에 균일한 압력을 부여함으로써, 컬럼내 충진물의 충진밀도를 컬럼 충진층 전체에 걸쳐서 균일하게 유지시킬 수 있어서, 분취용 고성능 액체크로마토그래피 컬럼의 성능을 크게 향상시킴과 동시에 컬럼내 충진용매를 완전하게 배출시켜 충진효율을 높혀 컬럼 충진층의 높이를 70㎝ 이상으로 할 수 있고, 컬럼의 성능을 나타내는 이론단수를 향상시킬 수 있는 분취용 고성능 액체크로마토그래피 컬럼의 충진 방법을 제공하는 것이다.An object of the present invention is to provide a uniform pressure to all of the top, middle and bottom of the column during the filling of the preparative high performance liquid chromatography column, thereby maintaining a uniform packing density throughout the column packed bed. It can greatly improve the performance of the preparative high performance liquid chromatography column and at the same time completely discharge the filling solvent in the column to increase the filling efficiency so that the height of the column packing layer can be 70 cm or more, It is to provide a method of filling a high performance liquid chromatography column for fractionation that can improve the theoretical number.

이와 같은 과제를 달성하기 위해서 본 발명에서는 앞에서 설명한 종래방법으로 분취용 고성능 액체크로마토그래피 컬럼을 충진할 때, 컬럼의 유출부에 압력 조절 밸브를 설치하여, 컬럼에 주입된 슬러리를 유압실린더 피스톤으로 가압하여 슬러리내 충진용매를 컬럼의 유출부를 통해 컬럼 밖으로 배출시킬 때 컬럼의 유출부에 배압(Back pressure)를 걸어 컬럼내 슬러리 충진층의 상부, 하부 및 중간부의 압력을 균일하게 유지시켜 주는 것을 특징으로 한다.
In order to achieve the above object, in the present invention, when filling the preparative high performance liquid chromatography column by the conventional method described above, a pressure regulating valve is installed at the outlet of the column to pressurize the slurry injected into the column with a hydraulic cylinder piston. When the filling solvent in the slurry is discharged out of the column through the outlet of the column, the back pressure is applied to the outlet of the column to maintain the pressure of the upper, lower and middle portions of the slurry filling layer in the column uniformly. do.

본 발명은 분취용 고성능 액체크로마토그래피 컬럼내 충진물의 충진밀도를 컬럼 충진층 전체에 걸쳐서 균일하게 유지시킬 수 있어서, 분취용 고성능 액체크로마토그래피 컬럼의 성능을 크게 향상시킴과 동시에 컬럼내 충진용매를 완전하게 배출시켜 충진효율을 높혀 컬럼 충진층의 높이를 70㎝ 이상으로 할 수 있고, 컬럼의 성능을 나타내는 이론단수를 향상시킬 수 있다.
The present invention can maintain the packing density of the filling in the preparative high performance liquid chromatography column uniformly throughout the column packing layer, greatly improving the performance of the preparative high performance liquid chromatography column and at the same time completely filling the filling solvent in the column. It can be discharged so as to increase the filling efficiency to increase the height of the column packed bed to 70 cm or more, it is possible to improve the theoretical number indicating the performance of the column.

도 1 내지 도 3은 분취용 컬럼을 충진하는 종래방법의 각 공정들을 도시한 공정 개략도.
도 4는 본 발명에 따라 분취용 컬럼을 충진하는 공정 흐름도(플로우차트)
도 5는 본 발명에 따라 분취용 컬럼을 충진하는 공정 개략도.
도 6은 실시예 1로 얻어진 크로마토그램 그래프.
도 7은 실시예 2로 얻어진 크로마토그램 그래프.
도 8은 비교실시예 1로 얻어진 크로마토그램 그래프.
도 9는 비교실시예 2로 얻어진 크로마토그램 그래프.1 to 3 is a process schematic showing each process of the conventional method for filling a preparative column.
4 is a process flow chart (flow chart) for filling a preparative column in accordance with the present invention.
5 is a process schematic diagram of filling a preparative column in accordance with the present invention.
6 is a chromatogram graph obtained in Example 1.
7 is a chromatogram graph obtained in Example 2.
8 is a chromatogram graph obtained in Comparative Example 1.
9 is a chromatogram graph obtained in Comparative Example 2.

이하, 첨부한 도면을 통하여 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

먼저, 본 발명에 따른 분취용 고성능 액체크로마토그래피 컬럼의 충진방법은 도 4 내지 도 5에 도시된 바와 같이 충진용매와 충진제인 실리카 겔이 혼합된 슬러리가 담겨진 슬러리 탱크(1)로부터 슬러리 공급펌프(2)로 슬러리를 컬럼(30)에 주입한 다음, 상기 컬럼(30)에 주입된 슬러리를 유압실린더의 피스톤(6)으로 가압하여 슬러리내 충진용매를 상기 컬럼의 유출부(32)를 통해 컬럼 밖으로 배출한 다음, 상기 컬럼(30)내에 이동상 용액을 주입하여 컬럼내 잔존하는 충진용매를 이동상 용액으로 치환하여 분취용 고성능 액체크로마토그래피 컬럼을 충진할 때, 상기 컬럼(30)의 유출부(32)에 컬럼 압력 조절 밸브(column pressure control valve, 33)를 설치하여 컬럼에 주입된 슬러리를 유압실린더 피스톤(6)으로 가압하여 슬러리내 충진용매를 컬럼의 유출부(32)를 통해 컬럼(30) 밖으로 배출시킬 때 컬럼의 유출부(32)에 배압(Back pressure)를 걸어 컬럼(30)내 슬러리 충진층의 상부(b), 하부(c) 및 중간부(a)의 압력을 균일하게 유지시켜 주는 것을 특징으로 한다.First, the filling method of the preparative high performance liquid chromatography column according to the present invention is a slurry feed pump from a slurry tank (1) containing a slurry in which a silica gel as a filler and a filler is mixed as shown in FIGS. 2) injecting the slurry into the column 30, and then pressurized the slurry injected into the column 30 to the piston (6) of the hydraulic cylinder to pass the filling solvent in the slurry through the outlet 32 of the column After discharging, the mobile phase solution is injected into the column 30 to replace the remaining filling solvent in the column with the mobile phase solution to fill a preparative high performance liquid chromatography column. Column pressure control valve (33) is installed in the column to pressurize the slurry injected into the column with the hydraulic cylinder piston (6) and the filling solvent in the slurry is curled through the outlet 32 of the column. Back pressure is applied to the outlet 32 of the column when discharging it out of the rum 30 to reduce the pressure of the top (b), bottom (c) and middle (a) of the slurry packed bed in the column (30). It is characterized by maintaining uniform.

도 4는 본 발명에 따라 분취용 컬럼을 충진하는 공정 흐름도(플로우 차트)이고, 도 5는 본 발명에 따라 분취용 컬럼을 충진하는 장치 일례의 공정 개략도이다.4 is a process flow chart (flow chart) for filling a preparative column in accordance with the present invention, and FIG. 5 is a process schematic diagram of an example of an apparatus for filling a preparative column in accordance with the present invention.

본 발명에서는 상기 충진제로는 실리카겔(Silicagel), 옥타데실실리카겔(C18 혹은 ODS), 옥틸실리카겔(C8), 테트라실리카겔(C4), 메틸실리카겔(C1)등과 같이 분취용 컬럼에 사용되는 모든 충진제 들이 사용될 수 있고, 충진용매로는 메탄올, 디클로로메탄 또는 아세톤 등이 사용될 수 있고, 이동상 용액으로는 디클로로메탄과 메틸알코올의 혼합액 등이 사용될 수 있다.In the present invention, as the filler, all fillers used in preparative columns such as silica gel, octadecyl silica gel (C18 or ODS), octyl silica gel (C8), tetrasilica gel (C4), methyl silica gel (C1), etc. may be used. As a filling solvent, methanol, dichloromethane or acetone may be used, and as a mobile phase solution, a mixed solution of dichloromethane and methyl alcohol may be used.

본 발명에서는 충진제의 종류, 충진용매의 종류 및 이동상 용액의 종류를 특별하게 한정하는 것은 아니다.In the present invention, the type of the filler, the type of the filler solvent and the type of the mobile phase solution are not particularly limited.

상기 컬럼(30)의 유출부(32)에 설치된 컬럼 압력 조절 밸브(33)로 컬럼(30)의 상단(b)에 걸어주는 배압은 유압실린더의 피스톤(6)에 의해 가해지는 압력 대비 85~98%의 압력 수준인 것이 바람직하다.The back pressure applied to the upper end (b) of the column 30 by the column pressure regulating valve 33 installed at the outlet 32 of the column 30 is 85 to the pressure applied by the piston 6 of the hydraulic cylinder. Preferably, the pressure level is 98%.

컬럼 압력 조절 밸브(33)로 걸어주는 배압이 상기 범위보다 낮으면 컬럼(30)내 충진물의 충진밀도가 불균일하게 되고, 상기 범위보다 높으면 충진에 소요되는 시간이 길어지게 된다.If the back pressure applied to the column pressure regulating valve 33 is lower than the above range, the filling density of the filling in the column 30 becomes non-uniform, and if it is higher than the above range, the time required for filling becomes longer.

본 발명에 따른 분취용 고성능 액체크로마토그래피 컬럼의 충진방법은 도 4에 도시된 바와 같이 충진용매와 실리카겔이 혼합된 슬러리를 컬럼내 주입하는 단계(S100)와, 컬럼 유출부에 설치한 컬럼 압력 조절 밸브로 컬럼 유출부에 배압을 가하여 주는 단계(S102)와, 배압이 가하여진 상태에서 컬럼에 주입된 슬러리를 유압실린더 피스톤으로 가압하여 슬러리내 충진용매를 컬럼의 유출부를 통해 컬럼 밖으로 배출해 주는 단계(S106)와, 컬럼내 잔존하는 충진용매를 이동상 용액으로 치환하는 단계(S104)로 구성된다.Filling method of preparative high performance liquid chromatography column according to the present invention is a step of injecting a slurry mixed with a filling solvent and silica gel in the column as shown in Figure 4 (S100), and the column pressure control installed in the column outlet The step (S102) of applying a back pressure to the column outlet with a valve, and pressurizing the slurry injected into the column in the state that the back pressure is applied by a hydraulic cylinder piston to discharge the filling solvent in the slurry out of the column through the outlet of the column ( S106) and the step (S104) of replacing the remaining filling solvent in the column with the mobile phase solution.

본 발명을 앞에서 설명한 바와 같이 컬럼(30)의 유출부(32)에 컬럼 압력 조절 밸브(33)를 설치하여 컬럼에 주입된 슬러리를 유압실린더 피스톤(6)으로 가압하여 슬러리내 충진용매를 컬럼의 유출부(32)를 통해 컬럼(30) 밖으로 배출시킬 때 컬럼의 유출부(32)에 배압(Back pressure)를 걸어 컬럼(30)내 슬러리 충진층의 상부(b), 하부(c) 및 중간부(a)의 압력을 균일하게 유지시켜주기 때문에 컬럼내 충진물의 충진밀도를 균일하게 할 수 있어 컬럼의 성능을 향상시킬 수 있다.As described above, the column pressure regulating valve 33 is installed at the outlet 32 of the column 30 to pressurize the slurry injected into the column with the hydraulic cylinder piston 6 so that the filling solvent in the slurry Back pressure is applied to the outlet 32 of the column when it is discharged out of the column 30 through the outlet 32. The top (b), the bottom (c) and the middle of the slurry packed bed in the column 30 Since the pressure in the part (a) is maintained uniformly, the packing density of the packing material in the column can be made uniform, thereby improving the performance of the column.

또한, 본 발명은 컬럼내 충진 용매를 완전하게 배출시켜 충진 효율을 높임으로서 높은 이론단수의 컬럼, 즉, 충진층의 길이가 70㎝ 이상인 컬럼을 충진할 수 있다.In addition, the present invention can be filled with a high theoretical number of columns, that is, a column having a length of 70 cm or more by completely discharging the filling solvent in the column to increase the filling efficiency.

실시예Example 1 One

평균 입자경 10㎛(Pore size 120Å)인 실리카겔을 3.5㎏을 칭량하여 아세톤 12ℓ에 슬러리를 제조하였다. 다음으로, 직경x길이가 100㎜x700㎜인 팩커(Packer)를 연결한 DAC (Dynamic Axial Compression) 방식의 컬럼(직경 : 100㎜, 길이 : 1,100㎜)내에 슬러리 펌프를 사용하여 상기와 같이 제조된 슬러리를 주입하고, 24시간 이상 방치한 후, 상등액과 Packer를 제거하고, 상기 컬럼 상부에는 상부 앤드 캡(End Cap)을 장착하고, 상기 컬럼의 유출부에는 컬럼 압력 조절 밸브를 설치하였다.A slurry was prepared in 12 L of acetone by weighing 3.5 kg of silica gel having an average particle diameter of 10 µm (Pore size 120 mm 3). Next, using a slurry pump in a DAC (Dynamic Axial Compression) column (diameter: 100 mm, length: 1,100 mm) connected to a packer (diameter x length 100 mm x 700 mm) was prepared as described above. After the slurry was injected and left for 24 hours or more, the supernatant and the packer were removed, an upper end cap was mounted on the column top, and a column pressure regulating valve was installed at the outlet of the column.

계속해서, 상기 컬럼 압력 조절 밸브로 컬럼의 유출부에 90kg/㎠의 배압을 걸어주면서, 상기 컬럼에 주입된 슬러리를 유압실린더의 피스톤으로 100kg/㎠의 압력으로 가압해, 즉, 컬럼의 하단에 걸리는 압력과 컬럼의 상단에 걸리는 압력 차이(△P)를 10kg/㎠로 유지하면서 상기 컬럼내 충진용매(아세톤)을 컬럼 밖으로 배출시켜 충진층 길이(Bad length)가 77㎝인 컬럼을 충진하였다.Subsequently, while applying a back pressure of 90 kg / cm 2 to the outlet of the column with the column pressure regulating valve, the slurry injected into the column is pressurized at a pressure of 100 kg / cm 2 with the piston of the hydraulic cylinder, that is, at the bottom of the column. The filling solvent (acetone) in the column was discharged out of the column while maintaining the pressure difference (ΔP) applied to the top of the column at 10 kg / cm 2 to fill a column having a bed length of 77 cm.

계속해서, 이동상 펌프를 사용하여 디클로로메탄과 메틸알코올을 98:2의 부피비로 혼합한 이동상 용액을 150㎖/분의 유량으로 상기 컬럼에 주입하여 컬럼내에 잔존하는 충진 용매인 아세톤을 상기 이동상 용액으로 치환하여 분취용 고성능 액체크로마토그래피 컬럼의 충진 작업을 완료하였다.Subsequently, a mobile phase solution in which dichloromethane and methyl alcohol were mixed at a volume ratio of 98: 2 was injected into the column using a mobile phase pump at a flow rate of 150 ml / min, and acetone, a filling solvent remaining in the column, was transferred to the mobile phase solution. Substitution was completed to fill the preparative high performance liquid chromatography column.

상기와 같이 충진한 분취용 컬럼에 1% 니트로벤젠 1㎖를 샘플 인젝터(Sample Injector)로 주입하여 자외선(UV) 파장 254㎚에서 얻어진 크로마토그램 그래프는 도 6과 같았다.6 ml of 1% nitrobenzene was injected into a sample injector into the preparative column packed as described above, and the chromatogram graph obtained at an ultraviolet (UV) wavelength of 254 nm was as shown in FIG. 6.

도 6은 좌우 대칭성이 좋은 피크(Peak)를 나타내고 이론단수가 20,000/m로 우수하였다.
FIG. 6 shows a peak having good symmetry and excellent theoretical number of 20,000 / m.

실시예Example 2 2

슬러리를 컬럼에 주입하기 이전에 미리 컬럼의 상부와 컬럼의 유출부 각각에 상부 앤드 캡과 컬럼 압력 조절 밸브를 각각 설치한 다음, 그 후에 슬러리를 컬럼내로 주입하고 나서 방치하지 않고 바로 실시예 1과 동일한 방법으로 컬럼을 충진해, 충진층 길이가 85㎝인 분취용 고성능 액체크로마토그래피 컬럼의 충진작업을 완료하고, 실시예 1과 동일한 조건으로 측정한 크로마토그램 그래프는 도 7과 같았다.Prior to injecting the slurry into the column, an upper end cap and a column pressure control valve were respectively installed at the top of the column and the outlet of the column, and thereafter, the slurry was injected into the column and immediately left without leaving. The column was filled in the same manner to complete the filling of the preparative high performance liquid chromatography column having a packed bed length of 85 cm, and the chromatogram graph measured under the same conditions as in Example 1 was as shown in FIG. 7.

도 7은 좌우 대칭성이 좋은 피크(Peak)를 나타내고, 이론단수가 12,000/m로 우수하였다.
Fig. 7 shows peaks having good left and right symmetry, and the theoretical number of stages was excellent at 12,000 / m.

비교실시예Comparative Example 1 One

컬럼의 유출부에 컬럼 압력 조절 밸브를 설치하지 않은 것을 제외하고는 실시예 1과 동일한 방법으로 충진층 길이가 81㎝인 분취용 고성능 액체크로마토그래피 컬럼의 충진작업을 완료하고, 실시예 1과 동일한 조건으로 측정한 크로마토그램 그래프는 도 8과 같았다.Filling of a preparative high performance liquid chromatography column with a packed bed length of 81 cm was completed in the same manner as in Example 1, except that no column pressure regulating valve was installed at the outlet of the column. The chromatogram graph measured under the conditions was as shown in FIG.

도 8은 좌우 비대칭성인 피크(Peak)를 나타내고, 이론단수의 측정도 불가능하였다.8 shows peaks that are asymmetrical left and right, and the theoretical singularity could not be measured.

비교실시예Comparative Example 2 2

컬럼의 유출부에 컬럼 압력 조절 밸브를 설치하지 않은 것을 제외하고는 실시예 2과 동일한 방법으로 충진층 길이가 95㎝인 분취용 고성능 액체크로마토그래피 컬럼의 충진작업을 완료하고, 실시예 1과 동일한 조건으로 측정한 크로마토그램 그래프는 도 8과 같았다.The filling operation of the preparative high performance liquid chromatography column having a packed bed length of 95 cm was completed in the same manner as in Example 2, except that no column pressure regulating valve was installed at the outlet of the column. The chromatogram graph measured under the conditions was as shown in FIG.

도 8은 좌우 비대칭성인 피크(Peak)를 나타내고, 이론단수의 측정도 불가능하였다.8 shows peaks that are asymmetrical left and right, and the theoretical singularity could not be measured.

1 : 슬러리 탱크 2 : 슬러리 공급펌프
3, 30 : 컬럼 4 : 슬러리 공급관
5 : 충진제 6 : 유압실린더의 피스톤
7 : 컬럼내 슬러리 충진층 8,32 : 컬럼의 유출부
a : 컬럼 중간부 b : 컬럼 상부
c : 컬럼 하부 9,31 : 배출되는 충진용매
33 : 컬럼 압력 조절 밸브1: slurry tank 2: slurry supply pump
3, 30: column 4: slurry feed pipe
5: Filler 6: Piston of Hydraulic Cylinder
7: slurry packed bed in column 8,32: outlet of column
a: column middle b: column top
c: column bottom 9,31: discharged solvent
33: column pressure regulating valve

Claims (2)

충진 용매와 충진제인 실리카겔이 혼합된 슬러리가 담겨진 슬러리 탱크로부터 슬러리를 분취용 컬럼에 주입한 다음, 상기 컬럼에 주입된 슬러리를 유압실린더의 피스톤으로 가압하여 슬러리 내 충진용매를 상기 컬럼의 유출부를 통해 컬럼 밖으로 배출한 다음, 상기 컬럼 내에 이동상 용액을 송액해 컬럼내 잔존하는 충진 용매를 이동상 용액으로 치환하여 분취용 고성능 액체크로마토그래피 컬럼을 충진함에 있어서,
상기 컬럼의 유출부에 컬럼 압력 조절 밸브(column pressure control valve)를 설치하여 컬럼에 주입된 슬러리를 유압실린더 피스톤으로 가압하여 슬러리내 충진 용매를 컬럼의 유출부를 통해 컬럼 밖으로 배출시킬 때 컬럼의 유출부에 배압(Back pressure)를 걸어 컬럼내 슬러리 충진층의 상부, 하부 및 중간부의 압력을 균일하게 유지시켜 주는 것을 특징으로 하는 분취용 고성능 액체크로마토그래피 컬럼의 충진 방법.The slurry is injected into the preparative column from the slurry tank containing the slurry mixed with the filling solvent and the silica gel filler, and then the slurry injected into the column is pressed by the piston of the hydraulic cylinder, and the filling solvent in the slurry is passed through the outlet of the column. After discharging out of the column, the mobile phase solution is sent in the column to replace the remaining filling solvent in the column with the mobile phase solution to fill a preparative high performance liquid chromatography column.
A column pressure control valve is installed at the outlet of the column to pressurize the slurry injected into the column with a hydraulic cylinder piston to discharge the filling solvent in the slurry out of the column through the outlet of the column. A method of filling a high performance liquid chromatography column for preparative liquid chromatography, wherein the back pressure is applied to maintain the pressures of the upper, lower, and middle portions of the slurry filling layer in the column uniformly. 제1항에 있어서, 상기 컬럼의 유출부에 설치된 컬럼 압력 조절 밸브로 유압실린더의 피스톤에 의해 가해지는 압력 대비 85~98%의 배압을 상기 컬럼의 유출부에 걸어주는 것을 특징으로 하는 분취용 고성능 액체크로마토그래피 컬럼의 충진 방법.The preparative high performance according to claim 1, wherein a back pressure of 85 to 98% of the pressure applied by the piston of the hydraulic cylinder is applied to the outlet of the column by a column pressure control valve installed at the outlet of the column. Filling method of liquid chromatography column.
KR1020100002079A 2010-01-11 2010-01-11 Packing method of prep hplc column KR101017572B1 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101740596B1 (en) 2016-05-30 2017-05-26 (주) 영진바이오크롬 System for filling column of high performance liquid chromatography
CN109358146A (en) * 2018-12-25 2019-02-19 成都格莱精密仪器有限公司 A kind of column filler that can quickly load chromatographic column
KR20190084524A (en) * 2018-01-08 2019-07-17 (주) 영진바이오크롬 Packing system and method of preparative hplc column
KR102532719B1 (en) * 2021-12-16 2023-05-16 한국표준과학연구원 Liquid chromatography column filler

Citations (4)

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Publication number Priority date Publication date Assignee Title
JPH0666779A (en) * 1992-08-20 1994-03-11 Kurita Water Ind Ltd Method for filling up liquid chromatographic column with slurry with pressurized gas
JPH1148348A (en) 1997-08-05 1999-02-23 Daisen Kogyo:Kk Vibratory compression filling method and evacuation valve device used therefor
JP2000210965A (en) 1999-01-25 2000-08-02 Daisen Kogyo:Kk Method for filling raw material bead
JP2004045309A (en) * 2002-07-15 2004-02-12 Mitsubishi Kakoki Kaisha Ltd Column for liquid chromatography, and separating agent filling method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0666779A (en) * 1992-08-20 1994-03-11 Kurita Water Ind Ltd Method for filling up liquid chromatographic column with slurry with pressurized gas
JPH1148348A (en) 1997-08-05 1999-02-23 Daisen Kogyo:Kk Vibratory compression filling method and evacuation valve device used therefor
JP2000210965A (en) 1999-01-25 2000-08-02 Daisen Kogyo:Kk Method for filling raw material bead
JP2004045309A (en) * 2002-07-15 2004-02-12 Mitsubishi Kakoki Kaisha Ltd Column for liquid chromatography, and separating agent filling method

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101740596B1 (en) 2016-05-30 2017-05-26 (주) 영진바이오크롬 System for filling column of high performance liquid chromatography
KR20190084524A (en) * 2018-01-08 2019-07-17 (주) 영진바이오크롬 Packing system and method of preparative hplc column
KR102118982B1 (en) 2018-01-08 2020-06-16 (주)영진바이오크롬 Packing system and method of preparative hplc column
CN109358146A (en) * 2018-12-25 2019-02-19 成都格莱精密仪器有限公司 A kind of column filler that can quickly load chromatographic column
KR102532719B1 (en) * 2021-12-16 2023-05-16 한국표준과학연구원 Liquid chromatography column filler

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