Liquid chromatography is a type of separation and analysis technique characterized by a liquid as the mobile phase. The stationary phase can take many forms, such as paper, sheets, and packed beds. In the development of chromatographic techniques, in order to distinguish between various methods, their respective names were generated according to the form of the stationary phase, such as paper chromatography, thin layer chromatography and column liquid chromatography.
First, the requirements of the liquid chromatograph
1. The mobile phase must be treated with HPLC grade reagents to remove particulate impurities and other materials (filtered using a 0.45 μm or finer membrane) prior to use.
2. After the mobile phase is filtered, it should be degassed by ultrasonic wave. After degassing, it should be used after returning to room temperature.
3. Pure acetonitrile cannot be used as the mobile phase, which will cause the check valve to stick and cause the pump to not enter the liquid.
4. When using the buffer solution, immediately rinse the tube and column with deionized water for one hour after the sample is finished, then rinse with methanol (or methanol aqueous solution) for more than 40 minutes to fully wash away the ions. For the outside of the plunger rod, it is necessary to rinse 20 mL or more with deionized water after the sample is finished.
5. If the instrument is not used for a long time, the column should be removed and sealed with a plug. Note that the column should not be stored with pure water, but the organic phase (eg, methanol, etc.) should be used because pure water is prone to mildew.
6. The sampler should be cleaned with solvent to dissolve the sample after each sample is completed.
7. C18 column must not enter protein samples, blood samples, biological samples.
8. The pressure caused by the blockage is too large. Check and clean according to the pre-column→filter in the mixer→line filter→check valve. Cleaning method:
1 rinse with isopropanol as solvent;
2 is placed in the middle of isopropyl alcohol and washed with ultrasonic waves;
3 Wash with 10% dilute nitric acid;
9. Air bubbles will cause unstable pressure and poor reproducibility, so try to avoid air bubbles during use.
10. If there is no liquid in the inlet tube, use a syringe to aspirate: usually the mobile phase is washed before the infusion.
11. Pay attention to the pH range of the column, and do not inject samples of strong acid and alkali, especially alkaline samples.
12. When replacing the mobile phase, the suction filter part should be placed in the beaker and vibrated while washing, and then inserted into the new mobile phase. Use isopropyl alcohol to change when replacing the immiscible mobile phase.
Second, common fault determination and solution
1. Change in retention time
(1) Column temperature change: The column temperature is constant, and an incubator is required if necessary.
(2) The equivalence and the gradient are not well balanced: at least 10 column volumes of the mobile phase equilibrium column.
(3) Buffer capacity is not enough: use "25mmol / L buffer.
(4) Column contamination: Flush the column every day.
(5) Change of conditions in the column: stable injection conditions and adjustment of the mobile phase.
(6) The column reaches the life quickly: a guard column is used.
2. Shorter retention time
(1) Increase in flow rate: Check the pump and reset the flow rate.
(2) Sample overload: reduce the amount of sample.
(3) Loss of bonded phase: The pH of the mobile phase is maintained in the direction of the column from 3 to 7.5.
(4) Change in mobile phase composition: Prevent evaporation or precipitation of the mobile phase.
(5) Temperature increase: The column is at a constant temperature.
3. Extended retention time
(1) Flow rate drop: The pipeline leaks, replace the pump seal, and eliminate the air bubbles in the pump.
(2) Change of active point on the silica gel column: using a mobile phase modifier such as triethylamine or a base to passivation column.
(3) Loss of bonded phase: The pH of the mobile phase is maintained in the direction of the column from 3 to 7.5.
(4) Change in mobile phase composition: Prevent evaporation or precipitation of the mobile phase.
(5) Temperature reduction: The column is at a constant temperature.
4. There is a shoulder or a point
(1) The sample volume is too large: with the mobile phase, the total sample volume is less than 15% of the first peak.
(2) The sample solvent is too strong: a weak sample solvent is used.
(3) Column collapse or short-circuit path: Replace the column with weaker corrosive conditions.
(4) Failure of sintering stainless steel in the column: replace the sintered stainless steel, add an in-line filter, and filter the sample.
(5) Injector damage: Replace the injector rotor.
5. Ghost Peak
(1) Residual peak of injection valve: Clean the valve with strong solvent after each use to improve the cleaning of the valve and sample.
(2) Unknown in the sample: Treat the sample.
(3) Column unbalanced: Re-equilibrate the column and use the mobile phase as the sample solvent (especially ion-pair chromatography).
(4) Trifluoroacetic acid (TFA) oxidation (peptide mapping): Newly formulated daily with antioxidants.
(5) Water pollution (reverse phase): The water quality was checked by changing the equilibrium time using HPLC grade water.
6. Baseline noise
(1) Bubbles (sharp peaks) Degassing of the mobile phase: back pressure after column addition.
(2) Contamination (random noise): Clean the column, purify the sample, and use HPLC grade reagents.
(3) Detector lamp continuous noise: replace the xenon lamp.
(4) Electrical interference (accidental noise): Use a regulated power supply to check the source of the interference (such as water bath, etc.).
(5) There are bubbles in the detector: the mobile phase is degassed, and the column is back pressed.
7. Peak tailing
(1) Column overload: reduce the sample volume and increase the column diameter with a higher capacity stationary phase.
(2) Peak interference: Clean the sample and adjust the mobile phase.
(3) Effect of silanol: Add triethylamine, increase the concentration of buffer or salt with alkali-induced passivation column to reduce the PH value of mobile phase, and passivate the sample.
(4) Failure of sintering stainless steel in the column: replace the sintered stainless steel, add an in-line filter, and filter the sample.
(5) Column collapse or short-circuit path: Replace the column and use weaker corrosive conditions.
(6) The dead volume or the extra-column volume is too large: the connection point is minimized, and all the connection points are properly adjusted, and a thin inner diameter connecting pipe is used as much as possible.
(7) Decrease in column efficiency: Replace the column with a lower corrosion condition and use a guard column.
8. Peak broadening
(1) The injection volume is too large: with the mobile phase, the total sample volume is less than 15% of the first peak.
(2) Causes peak expansion in the injection valve. Leakage of bubbles before and after injection to reduce diffusion.
(3) The sampling rate of the data system is too slow: The set rate should be greater than 10 points per peak.
(4) The detector time constant is too large: The set time constant is 10% of the first half width of the first peak of interest.
(5) The mobile phase viscosity is too high: Increase the column temperature and use a low viscosity mobile phase.
(6) The detection tank is too large: Use a small volume tank to remove the heat exchanger.
(7) The retention time is too long: Increasing the solvent content during isocratic elution can also be carried out by gradient.
(8) Excessive extra-column volume: Minimize the diameter of the connecting pipe and the length of the connecting pipe.
(9) Sample overload: Enter a small concentration of small sample.
The National Standard Material Resource Platform is mainly engaged in chemical reagents (high-end reagents, general reagents, analytical reagents), standard products, reference materials, experimental chromatographic consumables, instruments and other nearly 200,000 kinds of goods. Another biobw.com agent introduces well-known suppliers at home and abroad, from quality inspection, commodity storage management, professional preservation, logistics and transportation, after-sales service, etc., all the links are controlled by professionals. Through online operation and self-delivery logistics, the 3%-5% of the cost saved will be returned to the customer to ensure the high quality and low price. Imported brands include American ACC, US USP, German DR, German weitge, Canadian TRC, Canadian CDN, BEPURE and other well-known brands, with short delivery time and low price. Click on the online customer service to inquire about the purchase.
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First, the requirements of the liquid chromatograph
1. The mobile phase must be treated with HPLC grade reagents to remove particulate impurities and other materials (filtered using a 0.45 μm or finer membrane) prior to use.
2. After the mobile phase is filtered, it should be degassed by ultrasonic wave. After degassing, it should be used after returning to room temperature.
3. Pure acetonitrile cannot be used as the mobile phase, which will cause the check valve to stick and cause the pump to not enter the liquid.
4. When using the buffer solution, immediately rinse the tube and column with deionized water for one hour after the sample is finished, then rinse with methanol (or methanol aqueous solution) for more than 40 minutes to fully wash away the ions. For the outside of the plunger rod, it is necessary to rinse 20 mL or more with deionized water after the sample is finished.
5. If the instrument is not used for a long time, the column should be removed and sealed with a plug. Note that the column should not be stored with pure water, but the organic phase (eg, methanol, etc.) should be used because pure water is prone to mildew.
6. The sampler should be cleaned with solvent to dissolve the sample after each sample is completed.
7. C18 column must not enter protein samples, blood samples, biological samples.
8. The pressure caused by the blockage is too large. Check and clean according to the pre-column→filter in the mixer→line filter→check valve. Cleaning method:
1 rinse with isopropanol as solvent;
2 is placed in the middle of isopropyl alcohol and washed with ultrasonic waves;
3 Wash with 10% dilute nitric acid;
9. Air bubbles will cause unstable pressure and poor reproducibility, so try to avoid air bubbles during use.
10. If there is no liquid in the inlet tube, use a syringe to aspirate: usually the mobile phase is washed before the infusion.
11. Pay attention to the pH range of the column, and do not inject samples of strong acid and alkali, especially alkaline samples.
12. When replacing the mobile phase, the suction filter part should be placed in the beaker and vibrated while washing, and then inserted into the new mobile phase. Use isopropyl alcohol to change when replacing the immiscible mobile phase.
Second, common fault determination and solution
1. Change in retention time
(1) Column temperature change: The column temperature is constant, and an incubator is required if necessary.
(2) The equivalence and the gradient are not well balanced: at least 10 column volumes of the mobile phase equilibrium column.
(3) Buffer capacity is not enough: use "25mmol / L buffer.
(4) Column contamination: Flush the column every day.
(5) Change of conditions in the column: stable injection conditions and adjustment of the mobile phase.
(6) The column reaches the life quickly: a guard column is used.
2. Shorter retention time
(1) Increase in flow rate: Check the pump and reset the flow rate.
(2) Sample overload: reduce the amount of sample.
(3) Loss of bonded phase: The pH of the mobile phase is maintained in the direction of the column from 3 to 7.5.
(4) Change in mobile phase composition: Prevent evaporation or precipitation of the mobile phase.
(5) Temperature increase: The column is at a constant temperature.
3. Extended retention time
(1) Flow rate drop: The pipeline leaks, replace the pump seal, and eliminate the air bubbles in the pump.
(2) Change of active point on the silica gel column: using a mobile phase modifier such as triethylamine or a base to passivation column.
(3) Loss of bonded phase: The pH of the mobile phase is maintained in the direction of the column from 3 to 7.5.
(4) Change in mobile phase composition: Prevent evaporation or precipitation of the mobile phase.
(5) Temperature reduction: The column is at a constant temperature.
4. There is a shoulder or a point
(1) The sample volume is too large: with the mobile phase, the total sample volume is less than 15% of the first peak.
(2) The sample solvent is too strong: a weak sample solvent is used.
(3) Column collapse or short-circuit path: Replace the column with weaker corrosive conditions.
(4) Failure of sintering stainless steel in the column: replace the sintered stainless steel, add an in-line filter, and filter the sample.
(5) Injector damage: Replace the injector rotor.
5. Ghost Peak
(1) Residual peak of injection valve: Clean the valve with strong solvent after each use to improve the cleaning of the valve and sample.
(2) Unknown in the sample: Treat the sample.
(3) Column unbalanced: Re-equilibrate the column and use the mobile phase as the sample solvent (especially ion-pair chromatography).
(4) Trifluoroacetic acid (TFA) oxidation (peptide mapping): Newly formulated daily with antioxidants.
(5) Water pollution (reverse phase): The water quality was checked by changing the equilibrium time using HPLC grade water.
6. Baseline noise
(1) Bubbles (sharp peaks) Degassing of the mobile phase: back pressure after column addition.
(2) Contamination (random noise): Clean the column, purify the sample, and use HPLC grade reagents.
(3) Detector lamp continuous noise: replace the xenon lamp.
(4) Electrical interference (accidental noise): Use a regulated power supply to check the source of the interference (such as water bath, etc.).
(5) There are bubbles in the detector: the mobile phase is degassed, and the column is back pressed.
7. Peak tailing
(1) Column overload: reduce the sample volume and increase the column diameter with a higher capacity stationary phase.
(2) Peak interference: Clean the sample and adjust the mobile phase.
(3) Effect of silanol: Add triethylamine, increase the concentration of buffer or salt with alkali-induced passivation column to reduce the PH value of mobile phase, and passivate the sample.
(4) Failure of sintering stainless steel in the column: replace the sintered stainless steel, add an in-line filter, and filter the sample.
(5) Column collapse or short-circuit path: Replace the column and use weaker corrosive conditions.
(6) The dead volume or the extra-column volume is too large: the connection point is minimized, and all the connection points are properly adjusted, and a thin inner diameter connecting pipe is used as much as possible.
(7) Decrease in column efficiency: Replace the column with a lower corrosion condition and use a guard column.
8. Peak broadening
(1) The injection volume is too large: with the mobile phase, the total sample volume is less than 15% of the first peak.
(2) Causes peak expansion in the injection valve. Leakage of bubbles before and after injection to reduce diffusion.
(3) The sampling rate of the data system is too slow: The set rate should be greater than 10 points per peak.
(4) The detector time constant is too large: The set time constant is 10% of the first half width of the first peak of interest.
(5) The mobile phase viscosity is too high: Increase the column temperature and use a low viscosity mobile phase.
(6) The detection tank is too large: Use a small volume tank to remove the heat exchanger.
(7) The retention time is too long: Increasing the solvent content during isocratic elution can also be carried out by gradient.
(8) Excessive extra-column volume: Minimize the diameter of the connecting pipe and the length of the connecting pipe.
(9) Sample overload: Enter a small concentration of small sample.
The National Standard Material Resource Platform is mainly engaged in chemical reagents (high-end reagents, general reagents, analytical reagents), standard products, reference materials, experimental chromatographic consumables, instruments and other nearly 200,000 kinds of goods. Another biobw.com agent introduces well-known suppliers at home and abroad, from quality inspection, commodity storage management, professional preservation, logistics and transportation, after-sales service, etc., all the links are controlled by professionals. Through online operation and self-delivery logistics, the 3%-5% of the cost saved will be returned to the customer to ensure the high quality and low price. Imported brands include American ACC, US USP, German DR, German weitge, Canadian TRC, Canadian CDN, BEPURE and other well-known brands, with short delivery time and low price. Click on the online customer service to inquire about the purchase.
Http://
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