Large scale industrial equipment for liquid chromatography and its application

Abstract: Based on the new product dac800 industrial high-pressure preparation liquid chromatography separation and purification equipment developed by our company, the basic composition of large-scale industrial preparation liquid chromatography separation and purification equipment is summarized, and some basic factors affecting linear amplification to industrial production are discussed It is hoped to provide guidance and methods for pharmaceutical enterprises to select and use the equipment in the future, so as to meet the requirements of pharmaceutical industry for large-scale separation and purification of product rate, low cost and high purity, so as to enhance the international competitiveness of Chinese pharmaceutical enterprises Key words: dac800; industrial preparative liquid chromatography; chromatographic separation equipment; large-scale separation and purification preparative chromatography is an effective preparative separation technology of Zui in separation science, which is an indispensable separation method in many research fields and modern synthetic medicine, biopharmaceutical and plant extraction However, due to the influence of traditional pharmaceutical technology, up to now, the industrial high-pressure preparation liquid chromatography purification equipment is still in the stage of waiting for recognition and acceptance, and there are few pharmaceutical manufacturers applying this technology Especially in the process of biological and chemical production, in order to meet the challenges of commercial competition in product cost and quality standards and environmental protection pressure, it is necessary to explore the enlargement of chromatographic production process and the optimization of Zui operation People have a deeper understanding that it can save development time, select solvents and stationary phases, make the process more stable and durable, and finally reduce the cost and accelerate the market-oriented process of Zui In this paper, we will introduce some main technical points in the process of using dac800 large-scale industrial liquid chromatography device, so as to provide a reference for the next step of equipment promotion and application A brief introduction of dac800 type industrial preparation liquid chromatography separation equipment dac800 large-scale industrial preparation liquid chromatography separation equipment [front view] dac800 large-scale industrial preparation liquid chromatography separation equipment [aerial view] The equipment consists of 800mm diameter dynamic axial pressurized chromatographic column, high-pressure infusion pump with large flow, sample pump, online detector and data processing software, automatic fraction collector, explosion-proof control system and auxiliary devices The function of the chromatographic column is to separate the components in the mixture The general requirements for chromatographic column are high column efficiency, good selectivity and high separation speed The function of the infusion pump is to continuously send the eluent into the column system under high pressure, so that the liquid mixture containing the target substance can complete the separation process in the column The function of the injection pump is to introduce the sample to be separated into the chromatographic column On line detector, high pressure infusion pump and chromatographic column are three key components of HPLC The components separated by the chromatographic column enter the detector together with the mobile phase According to the physical or chemical characteristics of the sample, the information is converted into the electrical signal which is easy to measure, and recorded to obtain the chromatogram of the sample component separation According to the position, shape and size of the peaks, the separation was judged, and qualitative and quantitative analysis was carried out With the development of computer science and technology, remote control has become a major feature of industrial liquid chromatography system The operator can control and monitor the equipment through PC in the final control room The distillate collection system is to make the solvent after elution by dynamic axial compression column flow to different storage tanks orderly through process control A sampling port shall be set for each fraction collection pipeline, so as to extract samples for analysis and observation at any time The equipment control system consists of forward explosion-proof electrical control cabinet, two-way and three-way electric control pneumatic ball valve, pump rear flowmeter, pressure transmitter, gas and liquid pipes and control elements The forward explosion-proof electrical control cabinet is equipped with a drive board, a motor frequency converter, a system core control unit (MCU) and a liquid crystal display unit to drive the solvent pump and the sample pump The auxiliary device includes a homogenate loading machine and a turning and lifting device: the function of the homogenate loading machine is to mechanically mix the packing applied to the dynamic axial column to make it evenly mixed with the loading liquid; and to inject the evenly stirred packing into the column tube The turnover and lifting device is used for the initial assembly, regular maintenance and replacement of the packing in the column Two key points in the application of large-scale industrial preparative liquid chromatography separation equipment 1 Packing column (1) the selection of suitable packing particle size is the key problem in preparative chromatography separation The particle size of packing mainly affects the application of preparative chromatography from column efficiency and pressure The pressure affects the operation cost, the column effect affects the separation effect, but the two restrict each other The effective way to improve the column efficiency is to use relatively small particles to reduce purification time, solvent consumption, product dilution and purification cost Compared with the traditional preparative chromatographic packing (particle size at least 100 μ m), the typical preparative packing is 10-20 μ m, and the column bed length is also shorter, generally 250-450 mm In addition, it is also very important to ensure the narrow particle size distribution of the filler, so as to avoid too small particles producing post column pressure and too large particles leading to low column efficiency Generally speaking, the use of small particle packing in the preparation of chromatography can obtain high column efficiency, especially for the difficult separation system, can obtain high resolution On the premise of meeting the separation requirements, the use of larger particle fillers can obtain larger production capacity, and the price of fillers is relatively cheap, so it can effectively reduce production costs Spherical silica gel (normal phase) and C18 (reversed phase) are commonly used as stationary phase fillers for the preparation of liquid chromatography Zui (2) The traditional packing method is divided into dry method and homogenate method The dry method is clumsy, the column performance is not good, and the filler particles are easy to disperse in the column wall area during the filling process The density distribution of homogenate filling column is not uniform, the density near the wall is higher, and the reproducibility of column performance is poor When the column with large diameter is filled by traditional method, the bed is unstable and the separation performance will be reduced Dynamic axial compression (DAC) technology is recognized as the best Zui technology for filling and preparing chromatographic column (the inner diameter of the column is greater than 50 mm) Its principle is to adopt piston packing (homogenate filling) and maintain the compression state of the column bed during the operation to ensure its stability [4] The column packed by DAC method has uniform bed, stable performance, high density, high column efficiency and good reproducibility The column packed by DAC process is gradually dominating the whole preparative column market The performance of chromatographic column depends on the strength of filling pressure and the length of compression time The behavior of dynamic axial compression bed is very complex In the compression process of chromatographic column, although the pressure applied to the piston is equal, the pressure transfer in the bed layer is not as uniform as the pressure transfer in the liquid, and the particles contacting the piston are subject to direct pressure, while the other end of the bed layer is only subject to the compression of filter plate and flange, and the pressure on the piston end of the bed layer is greater than that on the fixed end, because of the column There is friction between the wall and the particles, so even in the same section along the column axis, the pressure distribution is different, that is, there are pressure gradients in the axial and radial directions Generally speaking, for regular packing of 10 μ m, the recommended packing pressure is 7MPa Under the same conditions (packing and packing pressure of the same particle size), with the increase of the column, the axial pressure distribution in the column is larger The longer the column is, the greater the pressure difference between the piston and the other end, the more unstable and uneven the column bed is, and the smaller the packing density will be, which will result in the axial pressure gradient and density gradient of the chromatographic column Therefore, the general packing length is not more than 450mm In addition, due to the smaller packing particles and higher column bed pressure, the mobile phase in the column is subject to greater flow resistance, so high-pressure infusion pump is required to provide stable flow and good reproducibility mobile phase to the column during separation When selecting high flow and high pressure infusion pump, it should have the following properties: high output pressure, good sealing performance; wide flow range, stable flow, basically no pulse of the output mobile phase; corrosion resistance, and self-cleaning device; small dead volume of pump cavity, which is conducive to change eluent 2 Explore the process parameters to achieve linear amplification Considering the possible problems of direct amplification production and large investment, it is generally necessary to conduct pilot test (use the preparation column with a diameter of 50-150 mm generally) for process exploration first, and then directly enlarge to large-scale industrial specifications The specific process is different with different product types and chromatographic methods, which need to be treated differently, but there are some general principles for reference: (1) to determine the best process parameters of Zui Each chromatography has the following related factors: stationary phase (including type and particle size), mobile phase (eluent), column size (column diameter and length), flow rate, elution method (equivalency or gradient), etc Under the condition of a certain degree of separation, the larger the flow rate is, the better The determination of the flow rate is restricted by three factors: the first is the degree of separation; the second is the performance of the packing itself (for example, many packing materials used for protein separation are relatively soft and cannot bear too high flow rate); the third is the hardware equipment, namely the working capacity of the pump (2) consider the limiting factors comprehensively Some parameters, such as annual output requirement, purity requirement, raw material price, operation index of equipment, etc., need to be obtained before scale-up production For example, the flow rate and pressure of the pump may be limiting factors It is also possible that the flow rate of the pump is allowed to be very high, but at this flow rate, detection and collection of components become a problem If the raw material is very expensive, the resolution should be large to ensure the yield If possible, the larger the sample loading, the better Increase the sample loading in two ways Simply increasing the sample concentration (small volume) is called concentration overload; keeping the appropriate concentration, only increasing the sample volume is called volume overload However, the upper limit of sample concentration needs to be determined by experiments In general, the volume overload at a certain concentration is used in practical operation, and the overload is limited by the separation degree (3) clear key considerations There are two factors to increase the yield: one is to increase the sample loading; the other is to increase the flow rate on the basis of ensuring a certain degree of separation, that is, the product reaches a certain purity However, there are different optimization parameters for different chromatograms, and the change of their values has a great influence on the yield The optimization should focus on these parameters 3 integrated control and remote operation: the solution after elution by dynamic axial compression column is analyzed and detected by the detector and then distributed to different storage tanks for collection The user can automatically complete the collection work according to the sequence of component outflow after sample separation, or according to the time, or according to the start and end signals of the chromatographic peak, and according to the preset program of the control software Due to the large flow rate, the flow cell of the general detector can not complete the instantaneous large flow post column liquid detection Therefore, the special flow cell with high flow rate must be used to ensure that the post column solution with large flow rate can be detected and separated quickly and the purity of the recovered fraction can be ensured Conclusion industrial preparative chromatography will become one of the main methods in the field of separation and purification because of its incomparable characteristics and strong separation ability The packing with high quality and low price and the column with reasonable design are used,