Discussion on the reuse effect of sepaflash ® C18 reversed phase column and guide for column maintenance

Qian Meiyuan, Qiu Wenjun, Xubo Santai Technology Application Research Center The reversed-phase chromatographic column has been widely used for its binding of various functional groups on the silica gel stationary phase The reversed-phase column with octadecane groups bonded on the silica gel surface is called C18 reversed-phase column, which is suitable for the separation and purification of organic small molecules, natural product extracts, antibiotics, peptides and other samples In flash preparative chromatography, C18 reversed-phase column has also been widely used Because of its stability and durability, C18 reversed-phase column can bring better economic benefits to customers Therefore, this paper evaluates the reuse effect of C18 reversed-phase column The results show that under appropriate conditions, C18 reversed-phase column can be reused many times, but still ensure that the column efficiency does not drop The chromatographic column is sepaflash ® c1812g standard series chromatographic column (Order No.: sw-5201-012-ir) The experimental sample is the methanol solution of caffeine, acetophenone and toluene (colorless and transparent) The sample preparation method is as follows: weigh 0.1g of caffeine, 0.4g of Acetophenone and 7.5G of toluene, dissolve in 100ml of methanol solution, and make the sample completely dissolved by ultrasound for 1min Inject 0.5ml of syringe liquid, and then use water methanol system for gradient elution (flow rate is 30ml / min) The gradient was set from 0 to 10min, the methanol ratio increased from 10% to 90%, and then maintained at 90% for 2min After the operation, wash with high proportion of methanol, and then run with 10% methanol Repeat the injection several times, select the first, 11th and 21st injection, and get the separation result comparison chart as shown in Figure 1 Figure 1 Comparison of separation results of repeated injection of C18 reversed phase column It can be seen from Figure 1 that the retention time and peak height parameters of the sample have not changed significantly, and the separation effect is still very good It is proved that the separation column can be used repeatedly, and the column effect is basically unchanged 2 Before the maintenance and use of chromatographic column, please carefully read the instructions of chromatographic column, understand the types of chromatographic column, and select the appropriate chromatographic column In the selection of chromatographic column, the polarity size of the sample, the type and structural characteristics of the compound should be fully considered, and the suitable chromatographic column and separation and purification method should be selected according to the HPLC analysis conditions Different types of chromatographic columns use different mobile phases Using the wrong mobile phase will reduce the column efficiency and damage the column If the polar polysaccharides are separated and purified, hydrophilic reversed phase packing should be used For the first use of chromatographic column, the column should also be flushed and activated at low flow rate according to the manufacturer's factory instructions After activation, the covalent bond strength of chromatographic packing is enhanced, the column efficiency is improved, and the service life is prolonged Generally, the activation method is to wash 20-30 column volumes with methanol or acetonitrile, then wash 10 column volumes with methanol / acetonitrile: water (50:50), and then balance 10-30 column volumes with mobile phase 2.1 The cleaner the sample preparation before use, the longer the service life of the chromatographic column Many samples, especially biological samples, have complex components, which cause great damage to the chromatographic column The samples without pretreatment will shorten the service life of the chromatographic column Therefore, the sample needs to be pretreated during the preparation, including the selection of solvent, sample filtration, etc 2.1.1 The selection of sample preparation solvent usually needs to consider the solubility of the sample, the compatibility with the mobile phase and the applicability of the chromatographic packing This kind of solvent should have greater solubility to the sample, and be mutually soluble with the flow The elution intensity should be lower than that of the mobile phase or the initial mobile phase of gradient elution to avoid affecting the sample separation 2.1.2 Some properties of samples with other factors can also affect the service life of chromatographic column Strong acid, strong alkaline substances or biological macromolecules can interact with the stationary phase packing, or form an irreversible adsorption layer, change the surface characteristics of the packing, change the performance of the chromatographic column, resulting in greatly reduced separation effect In addition, too much sample will also affect the separation performance and service life of the column 2.2 The purity of mobile phase, the selection of solvent and the appropriate separation method are closely related to the performance and life of chromatographic column 2.2.1 The flow used for the selection of mobile phase shall be compatible with chromatographic column and sample, i.e the sample or sample solution and mobile phase are mutually soluble The mobile phase is required to have no chemical reaction with the sample and no dissolution or chemical reaction with the column packing Mobile phase is better to be used now, not to be placed for too long, or it will easily cause microbial growth and affect the service life of chromatographic column 2.2.2 Selection of mobile phase pH and buffer salt the mobile phase with extreme pH will destroy the covalent bond in the filler, "dissolve" silica gel, make the bonded phase lose, thus reducing column efficiency and shortening service life The pH value of silica gel based stationary phase should be in the range of 2.5 - 7.0 If it is necessary to use mobile phase with high pH or low pH, suitable chromatographic packing should be selected 2.2.3 In the process of controlling the flow rate, if the flow rate is too large and the pressure rises, it will cause the column packing to collapse and collapse Therefore, attention should be paid to controlling the flow rate in the process of separation 2.3 After using the cleaning and preservation column for a period of time, there will always be some impurities accumulated in the column, which will reduce the separation performance of the chromatographic column After cleaning these polluted chromatographic columns, part or even most of the separation capacity can be recovered Therefore, it is necessary to clean the chromatographic column carefully after use to prolong the service life of the chromatographic column 2.3.1 Cleaning of chromatographic column before and after use, the chromatographic column shall be rinsed with strong mobile phase After each experiment, the chromatographic column shall be rinsed If the mobile phase used in separation is free of acid, alkali or salt, it is recommended to wash 20 column volumes with 90% methanol aqueous solution; if the separated mobile phase contains buffer solution (usually salt solution), it is advisable to wash the chromatographic column (20 column volumes) with water instead of buffer solution and organic phase, and then wash with 100% organic phase If washing directly with organic phase, the deposition and precipitation of buffer solution will be caused and the column will be damaged When acid-base solution is added to the mobile phase, 20 column volumes can also be rinsed with high proportion of water (water: methanol = 90:10, V / V) according to the above method to prevent the dissolution of filler caused by strong acid and strong alkali solution If the column is not used for a long time, it should be filled with pure methanol or acetonitrile in the column after the completion of the washing of the chromatography, and sealed for preservation The state of chromatographic column can be determined by regular test of column efficiency See Section 2.4 for column efficiency test method If the column efficiency is significantly reduced, the following washing methods can be selected: wash in sequence with 100% methanol → 100% acetonitrile → acetonitrile: isopropanol (75:25, V / V) → 100% isopropanol For the pollutants that cannot be eluted by organic solvent, the dilute acid or alkali with lower concentration can be used for washing 2.3.2 Preservation of chromatographic column the chromatographic column shall be preserved in accordance with the solvent specified in the instruction manual and stored in 90-95% organic solvent as far as possible The chromatographic column can not be stored in water or solvent with high water content, otherwise it is easy to cause the growth of microorganisms and affect the life of chromatographic column When stored in 90-95% organic solvent, care should be taken to prevent the packing at both ends of the chromatographic column from drying up and faulting due to poor sealing, thus shortening the service life of the chromatographic column 2.4 Column efficiency test method the evaluation method of column efficiency is mainly based on the calculation of theoretical plate number of column The specific test method is detailed as follows: take the methanol solution of caffeine, acetophenone and toluene mixture as the sample, use the syringe liquid to inject the sample and use the water methanol system for gradient elution to obtain the separation map as shown in Figure 2 Fig 2 Separation Atlas of standards for evaluation of column efficiency According to the standard separation spectrum shown in Figure 2, the calculation formula of theoretical plate number of chromatographic column is as follows: or: n is the theoretical plate number; TR is the retention time of components; W1 / 2 is the half height width of chromatographic peak; W is the bottom width of chromatographic peak
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