HPLC: Use and Maintenance of Chromatography Columns

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This post was last edited by JonhNashnh on 2010-6-28 at 11:01. High-performance liquid chromatography (HPLC) is a separation and analysis technique that was developed in the late 1960s, and it represents an important method for modern separation and determination tasks. Since its introduction, it has been widely used in biochemistry, pharmaceuticals, and clinical analysis due to its high separation efficiency, fast analysis speed, good detection sensitivity, and the ability to analyze thermally unstable bioactive substances with high boiling points that cannot be vaporized. High-performance liquid chromatography involves using a high-pressure pump to feed a mobile phase, which can be a single solvent with different polarities or a mixture of solvents in various proportions, as well as analysis buffers, into a chromatographic column containing a stationary phase. The sample to be analyzed is injected through an injection valve; the mobile phase carries it into the column, where the various components are separated from one another before reaching the detector in sequence. The chromatographic signals are then recorded by a recorder, integrator, or chromatography workstation. 　　The chromatography column is the heart of high-performance liquid chromatography; in the use of high-performance liquid chromatographs, it is very important to maintain the column’s efficiency, capacity, and permeability, as well as to extend its service life. Therefore, the maintenance of high-performance liquid chromatography columns is *critical. 　　1 Structure of the chromatography column The chromatography column consists of a column tube, a pressure cap, a collar (sealing ring), a sieve plate (filter), connectors, screws, and other components. The column tubes are usually made of stainless steel; when the pressure is not higher than 70 kg/cm2, thick-walled glass or quartz tubes can also be used, and the inner surface of these tubes must have a very high level of smoothness. To improve column efficiency and reduce wall effects, the inner surfaces of stainless steel columns are often polished. There are also those using molten silicon or glass linings, for fine column tubes. The column joints at both ends of the chromatography column used in instrumental analysis are equipped with sieve plates made of sintered stainless steel or titanium alloys, with pore sizes ranging from 0.2 to 20 μm (5–10 μm), depending on the particle size of the packing; these sieve plates serve to prevent the packing from leaking out. 　　Chromatography columns can be divided into two categories based on their purpose: analytical and preparative, and they also differ in terms of size specifications: (I) Conventional analytical columns (regular-sized columns), with an inner diameter of 2–5 mm (4.6 mm is commonly used; in China, 4 mm and 5 mm are also used), and a column length of 10–30 cm; (2) Narrow-bore columns, also known as fine-bore or semi-micro columns, with an inner diameter of 1–2 mm and a column length of 10–20 cm; (3) Capillary columns (also known as micro columns), with an inner diameter of 0.2–0.5 mm; (4) Semi-preparative columns, with an inner diameter greater than 5 mm; (5) Laboratory preparative columns, with an inner diameter of 20–40 mm and a column length of 10–30 cm; (6) Industrial preparative columns, whose inner diameter can reach several dozen millimeters. The inner diameter of the column is generally determined based on the column length, particle size of the packing, and equivalent flow velocity, with the aim of avoiding wall effects in the analysis. 　　2 Use and Maintenance of Chromatography Columns In daily separation and analysis tasks, the proper use and maintenance of chromatography columns are extremely important. The way in which these columns are used directly affects their lifespan; even minor carelessness can reduce their efficiency, shorten their useful life, or even cause damage to them. During chromatography operations, the following points should be noted to maintain the chromatography column. 　　(1) When loading, unloading, or replacing columns, handle them gently and tighten the joints moderately. Strong mechanical vibrations must be prevented to avoid gaps forming in the column bed. 　　(2) If the instrument is used for instrumental analysis, the types of samples are limited but the number of analyses is high, it is advisable to assign a dedicated column to each type of routine analysis, as this helps to extend the column’s lifespan. 　　(3) Avoid sudden changes in pressure and temperature as well as any mechanical vibrations. Sudden changes in temperature or dropping the chromatography column from a height can affect the packing condition inside the column; sudden increases or decreases in column pressure can also disturb the packing material within the column. Therefore, flow rates should be adjusted slowly, and the valve should not rotate too slowly when sampling with a valve. 　　(4) The composition of the solvent should be changed gradually; especially in reverse-phase chromatography, it should not be changed directly from an organic solvent to one that is entirely water, and vice versa. 　　(5) When using a column temperature control device, care should be taken to raise the temperature only after the mobile phase has been introduced. 　　(6) Generally, chromatography columns should not be backflushed; backflushing can only be done to remove impurities remaining at the column head when the manufacturer specifies that such backflushing is permissible. Otherwise, the backflow will rapidly reduce column efficiency. 　　(7) Select an appropriate mobile phase to avoid damage to the stationary phase. Sometimes a pre-column can be connected in front of the injector. When the analysis column is made of bonded silica, the pre-column is made of silica as well; this allows the mobile phase to be \"saturated\" by the silica before it enters the analysis column, thereby preventing the silica matrix in the analysis column from being dissolved. 　　(8) To avoid directly injecting samples with complex matrices, especially biological samples, into the column, it is necessary to pre-treat the samples or connect a guard column between the injector and the chromatographic column. Protection columns are generally short columns filled with a similar stationary phase. The protection column can and should be replaced frequently. 　　(9) The chromatography column is frequently rinsed with strong solvents to remove impurities remaining inside the column. During cleaning, the replacement of the mobile phase in the flow path system should be carried out gradually using solvents that are miscible with each other; the volume of each mobile phase should be approximately 20 times the volume of the column, meaning 50–75 mL is required for conventional analysis. 　　(10) When storing the chromatography column, it should be filled with acetonitrile or methanol, and the column connections must be tightened to prevent the solvent from evaporating and drying out. It is strictly prohibited to leave the buffer solution in the column overnight or for any longer period of time. 　　(11) During the use of the chromatography column, if the pressure increases, one possible cause is that the sintered filter is blocked; in such cases, the filter should be replaced or removed for cleaning. Another possibility is that large molecules have entered the column, causing contamination of the column head. If the column efficiency decreases or the chromatographic peaks become distorted, it may be that the column head has collapsed, resulting in an increased dead volume. 　　(12) After completing the separation analysis, the system should not be shut down immediately; instead, the chromatography analysis system needs to be flushed for at least 0.5 hours in order to remove impurities from the chromatography column. Two articles that seem quite good and worth reading: “Internal Structure and Working Principle of the Liquid Phase UV Detector” and “Sharing: Experience in Sample Preparation for Infrared Spectroscopy!” 》 3 Regeneration of the chromatography column 　　Since high-performance liquid chromatography columns are consumable items, as usage time or the number of injections increases, if the height of the chromatographic peaks decreases, their width increases, or shoulder peaks appear, it is generally indicative of a decline in column efficiency. 　　(1) For the regeneration of the reverse-phase column, solvents such as methanol:water = 95:5 (V/V), pure methanol, and dichloromethane are used as the mobile phases; the column is flushed sequentially, with each mobile phase flowing through the column for a volume equal to 20 to 30 times the column volume, after which the column is flushed in the reverse order. 　　(2) Normal-phase column regeneration. Flush the chromatography column sequentially with n-hexane, isopropanol, dichloromethane, and methanol as the mobile phases. Pay attention to maintaining the correct order of the solvents used. For instrumental analysis, do not reverse the order in which each mobile phase passes through the chromatography column; the volume of each fluid flowing through the column should be 20–30 times the volume of the column itself. (Due to the higher viscosity of isopropanol, adjust the flushing flow rate as needed during this process.) After flushing with methanol, flush the column in the reverse order. It should be noted that the aforementioned solvents must be strictly dehydrated. 　　(3) Regeneration of the ion exchange column. Prolonged use in buffer solutions with high pH and high ionic strength will lead to a decrease in the ion-exchange capacity of the chromatography column. Rinsing with a dilute acid buffer solution can regenerate cation columns; conversely, rinsing with a dilute base buffer solution can regenerate anion columns. The regeneration methods for the chromatography columns mentioned above are not absolute standard methods; users can, based on their own practical experience and objectives, select a suitable solvent that can dissolve the contaminants within the column as the mobile phase, and carry out flushing in either the forward or reverse direction. 　　It should be noted, however, that no matter what method is used to regenerate the chromatography column, it is impossible to fully restore the column efficiency or other parameters to those of a new column. 　　4 Development trends of chromatography columns Short columns have been developed to emphasize analysis speed; their length is 3–10 cm, with particle sizes of the packing material ranging from 2–3 μm. To improve analytical sensitivity, narrow-bore columns, capillary columns, and microbore columns with an inner diameter of less than 0.2 mm have been developed in conjunction with mass spectrometry (MS). The advantages of columns with small tube diameters are: reduced consumption of mobile phase, increased sensitivity, lower sample requirements, the ability to achieve high separation efficiency using longer columns, easier control of column temperature, and ease of integration with LC-MS. 　　However, as the column volume becomes smaller, the influence of off-column effects becomes more significant, requiring detectors with a smaller sample volume (even on-column detection), as well as column connectors and fittings with a smaller dead volume. The equipment used in conjunction should possess the following properties: the infusion pump should be able to deliver low flow rates of 1–100 μL/min, while the sampling valve should be capable of accurately and reproducibly sampling small volumes of sample. Furthermore, due to the small amount of sample applied, a highly sensitive detector is required, and electrochemical detectors and mass spectrometers have outstanding advantages in this regard.