Pollution, loss and other problems will inevitably appear during the use of HPLC columns, thus it is necessary to flush column for maintenance at that time. From the point of view of the bonded phase used, HPLC columns can be divided into reversed-phase columns and non-reversed phase columns. For the reversed-phase bonded phase, such as C18, C8, phenyl, etc., due to its wide application with complex and changeable mobile phase composition, it is difficult to have a fixed idea and mode for the maintenance of such columns. In most cases, specific analysis of specific issues is required. However, for packing materials that are not reversed-phase, such as silica gel, gel, amino group, cyano group and ion exchange, owning to the specific items used or the small span of their mobile phase composition, there is also a relatively fixed way to address problems.
Solutions and common maintenance for non-reversed phase columns are recommended as follows.
— G10 —
G-10 is a soft gel whose mechanical strength is obviously less strong than the silica gel, thus organic solvents should be avoided to add in mobile phase or samples during the process of using. Otherwise, the column bed will collapse easily. If there is any problem after using for a period of time, such as worse peak shape and decreased resolution, the column can be reactivated with distilled water according to the instructions. Flushing time can be a little longer, and then equilibrate the column with mobile phase at low flow rate overnight. If there is no obvious improvement, flushing can be carried out according to the regeneration flushing method in the care and use manual. The steps of regeneration flushing are illustrated in the following part.
If there is no obvious improvement in the above operation, the most likely reason is that packing materials were seriously polluted, or even packing materials collapsed. In this case, flushing again is of little significance, customers are suggested to purchase a new column.
Regeneration flushing of G-10 column:
1. Mix 0.5mol/L NaOH and 0.5mol/L NaCl as 1:1 (v/v), then flush 20-30 times the column volume.
2. Flush the column with distilled water until it is neutral.
3. Determine system applicability test of dextran 2000 reference material according to pharmacopoeia method.
— SEC —
SEC packing materials is hydrophilic globular protein silica gel, which is commonly used for molecular weight exclusion separation of polymer. The treatment method is similar to G-10. If there is a problem, reactivation is the common way to solve. Rinse with pure water for a long time, and then equilibrate with the mobile phase. If there is no obvious improvement, flush according to the abnormal flushing method in the care and use manual. The steps are illustrated in the following part. As for the resolution, if it decreases, that can be settled by adjusting the flow rate.
Steps of abnormal flushing for SEC column:
1. Flush with high concentration of neutral salt at low pH (such as 0.5M sodium sulfate solution, adjust pH to 3.0 with sulfuric acid) and wash 15 times the column volume, which is conducive to removing alkaline protein.
2. Buffer salt solution (such as 50 mM phosphate buffer, pH 7.0) containing organic solvent (such as 10%-20% methanol, acetonitrile) can be used to flush 15 times column volume, which is helpful to flush out hydrophobic proteins.
3. Adding the cosolvent helps remove strongly adsorbed substances on the stationary phase (such as adsorption by hydrogen bond, etc.).
4. Test column efficiency for reference substances according to COA.
Cosolvents (e.g. 6-8 mol/L of urea or 0.2-0.3% of sodium dodecyl sulfate) are recommended only if there is no significant improvement after both neutral saline and organic solvent flushing.
— Silica Gel Column —
The common problems of silica gel column in normal-phase mode are usually caused by insufficient equilibrium, insufficient transition, or containing water. Water content in normal-phase chromatography is an important parameter which affects retention and selectivity. Most solvents contain little dissolved water (such as the water content of n-hexane at 20℃ is 0.0111% w/w). Problems such as resolution and drift of retention time, which are common in normal-phase chromatography, can be attributed to changes in moisture of the stationary and mobile phase while the packing materials may remain intact.
The following methods are recommended:
1. 100% isopropanol — 100% methanol — 100% isopropanol, ensure the full transition of isopropanol (isopropanol has strong viscosity and high pressure, (adjust the flow rate in the appropriate range), and then equilibrate mobile phase overnight.
2. Remove the water on the stationary phase, flush column with 2.5% dimethoxypropane and 2.5% glacial acetic acid hexane of 30 times the column volume.
3. Use half saturated mobile phase to bisect the nonpolar mobile phase without water. Add a certain amount of water in one part and mix well for an hour. After stratification, remove aqueous phase and mix two parts of nonpolar solvents together, then “half saturated mobile phase” is prepared. (Choose one between method 2 and method 3).
4. Inject samples according to the sequence of empty injection — blank solvent — reference substance — test substance, then observe the appearance of peak.
— NH2 column —
NH2 column is one of the common columns which can be used in both normal-phase and reversed-phase. When using normal phase, the treatment and maintenance method of NH2 column is the same as that of silica gel column.
In condition of reversed-phase, especially when analysis sugar, peak broadening, tailing, short lifetime and other problems emerge frequently. First of all, it is necessary to understand that the aminopropyl group bonded by the NH2 column is easier to hydrolyse than the conventional C18, C8 and other reversed-phase groups. Therefore, when using the NH2 column, you need to prepare yourself for a shorter life of NH2 column than that of conventional column.
If any of the above problems occurs, it is recommended that:
1. 100% acetonitrile –100% methanol –100% isopropanol –100% acetonitrile, rinse for more than 40min (isopropanol has strong viscosity and high pressure, so please adjust the flow rate within the appropriate range).
2. After flushing according to method 1, equilibrate the mobile phase overnight. Prepare priming injections when necessary.
3. If there is still no obvious improvement, use pure methanol at 1.0 mL/min to normally flush for more than 3 hours. Then prepare the mobile phase, the solvent and the sample. Equilibrate the newly mixed mobile phase for 1 hour.
4. If the above treatment is not satisfying, it is most likely caused by the break of silica gel matrix during the process of using. At this time, continuing flushing costs time and solvent if the improvement is not obvious, it is recommended to directly purchase a new column.
— Cyano Column —
Cyano column is one of the common columns that can be used in both normal-phase and reversed-phase. The weakness of cyano column in reversed-phase condition is that it is very sensitive to medium polarity solvents, such as THF (tetrahydrofuran), EtOH (ethanol), etc. Therefore:
1. Avoid these medium polarity solvents while using and storing the column.
2. Cyano column phase should be through sufficient transition while switching, then prepare new mobile phase and mixed standards to avoid the impact of solvents. Rinse with each solvent for more than 40min according to the order of 100% methanol -100% acetonitrile -100% isopropanol -100% acetonitrile (isopropanol has strong viscosity and high pressure, so adjust the flow rate within an appropriate range).
3. The mobile phase shall be equilibrated for enough time (Prepare priming injections when necessary). After the baseline is stabilized, the sample shall be injected in the order of running with empty — blank solvent — reference substance — test substance.
— SCX/SAX —
High proportion of organic phase and high concentration of salt (>100 mM) should be avoided in the SCX/SAX columns, otherwise the bonded phase will easily fall off and the column may be blocked by salting out. High proportion of organic phase is not recommended to be used in storing the columns. In addition, if the column is kept for too long, the bonded phase is prone to fall off (it is generally recommended to store it in 10% methanol at 4℃, then take it out and rinse it every other day to ensure that the bonded phase is in an active state).
1. When the resolution decreases, retention time drift and other problems occur, flush the column with 10% methanol (1-2 hours) to remove all the salt and acids, then prepare new mobile phase to equilibrate the column. After that, inject samples according to the order of running with empty – blank solvent – reference substance – test substance.
2. If flushing with 10% methanol water is not effective, it is recommended to flush SCX column with 100mmol/L NaClO4 solution (adjust pH to 4.0) –10% methanol water for more than 60min.
3. If flushing with 10% methanol water is not effective, it is recommended to flush SAX column with 1M ammonium nitrate –40% methanol –100% isopropyl alcohol — water –10% methanol water for more than 60min.
— Sugar-H/Sugar-Ca —
The columns which take polymer as matrix are all packed tightly with swelling resins. If the sample is well pretreated, most of the problems are related to the column bed and packing materials. Control the column temperature and flow rate to avoid the damage of the column bed caused by temperature and pulse, and the maximum proportion of organic phase should not exceed 5%.
Sugar-H: when there are problems such as abnormal peak shape, decreased column efficiency and reduced resolution, the following methods can be used:
1. Flush with pH 2.5 acidic solution at low flow rate for 12 hours, acids can be sulfuric acid, hydrochloric acid, phosphoric acid, perchloric acid (avoid to use nitric acid and other strong oxidation acids), then adjust pH to 2.5. Set the temperature at 80℃ or 85℃ while flushing, and the flow rate is usually 0.5 mL/min. The water that used to prepare acidic solution should have no interference of other cations as far as possible.
2. After flushing, use mobile phase to equilibrate, and then inject samples in the order of running with empty – blank solvent – reference substance – test substance.
3. Check whether there is a temperature difference between the top and the end of the column in daily use, and try not to contain other cations in the water used to prepare the mobile phase (especially Na+, K+ and other monovalent ions).
Sugar-Ca: the problems of calcium type cation column are mostly caused by Ca2+ loss in the process of use, so it is necessary to replenish the lost Ca2+ in time (Generally, Ca2+ solution needs to be reactivated if the above problems occur or 5L mobile phase has been used).
Sugar-Ca: the problems of calcium type cation column are mostly caused by Ca2+ loss in the process of use, so it is necessary to replenish the lost Ca2+ in time (Generally, Ca2+ solution needs to be reactivated if the above problems occur or 5L mobile phase has been used) .
1. Flush with 0.5g/L EDTA Ca (CAS: 23411-34-9) solution at low flow rate for 12 hours. Set the temperature at 80℃ or 85℃ while flushing, and the flow rate is usually 0.5 mL/min. The water that used to prepare Ca2+ solution should have no interference of other cations as far as possible.
2. After flushing, use mobile phase to equilibrate, then inject samples according to the order of running with empty – blank solvent – reference substance – test substance.
3. Check whether there is a temperature difference between the top and the end of the column in daily use, and the water that used to prepare mobile phase needs to avoid interference of other cations as far as possible (especially Na+, K+ and other monovalent ions).
— Zn Powder Reducing Column —
The reducing column of Zn powder should not contact with water, or Zn powder will be deactivated after that. Therefore, when encountering problems, you can solve them as following methods:
1. Flush with 100% methanol, then start back flushing at flow rate of 1.0 ml/min for more than 3 hours.
2. After flushing, use mobile phase to equilibrate, then inject samples in the order of running with empty – blank solvent – reference substance – test substance.
3. If any white substance is flushed out, packing materials may have been damaged, thus it’s necessary to buy a new column.
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