Determination and Resolution of Common Faults in High Performance Liquid Chromatograph

High performance liquid chromatography has been favored by the majority of laboratory enthusiasts for a long time. The operation is simple, efficient and results are perfect. Here's how to solve some of the common problems with HPLC. (Zhengzhou Zhongpai Instrument Equipment Co., Ltd. 0371-6379187063791871)

(I) Change in retention time of HPLC

1. Column temperature change Column thermostat, if necessary, need to be equipped with an incubator 2. The equilibrium between isocratic and gradient cannot be fully balanced with at least 10 column volumes of mobile phase equilibration column 3. Buffer capacity is not sufficient with >25mmol/L buffer 4.Column contamination wash column every day 5. Column condition changes stable injection conditions, adjust the mobile phase 6. The column will soon reach the end of life using guard column (b) High performance liquid chromatograph retention time shortened

1. Increase the flow rate Check the pump and reset the flow rate 2. Sample overload reduces sample volume 3. Loss of bonded phase Mobile phase pH remains in the range of 3 to 7.5 Check column 4. Changes in mobile phase composition Prevent evaporation or sedimentation of mobile phase 5 Temperature increase column thermostat (C) HPLC retention time extension

1. Flow rate drop Pipe leakage, replace the pump seal ring, eliminate air bubbles in the pump 2. Change the active point on the silica gel column with a mobile phase modifier such as triethylamine, or use alkali to passivate the column 3. Bonding phase Loss with the former (b) 3

4. Changes in mobile phase composition with the previous (2) 4

5. Temperature decrease with the previous (b) 5

(d) High-performance liquid chromatograph shoulder or fork

1. The sample volume is too large. With the mobile phase sample, the total sample volume is less than 15% of the first peak.

2. The sample solvent is too strong. Use a weak sample solvent. 3. The column collapses or forms a short-circuit channel. Replace the column. Use a weaker corrosive condition. 4. The stainless steel in the column fails. Replace the sintered stainless steel, add an in-line filter, and filter the sample. Injector Damaged Replacement Sampler Rotor (5) HPLC Ghost Peak

1. Retaining peak of the injection valve Clean the valve with strong solvent after each use to improve the cleaning of the valve and sample 2. Unknown sample in the sample processing sample 3. The column is not equilibrated Rebalance the column and use the mobile phase as sample solvent (especially ion Chromatography

4. Trifluoroacetic Acid (TFA) Oxidation (Peptide Mapping) Newly formulated daily, with antioxidants 5. Water contamination (reverse phase) Check the water quality by changing the equilibrium time, using HPLC-grade water (VI) HPLC baseline noise

1. Bubbles (sharp peaks) Degas mobile phase, back pressure after column addition 2. Contamination (random noise) cleaning column, purify sample, use HPLC grade reagent 3. Detector lamp continuous noise Replace neon lamp 4. Electrical interference (incidental Noise) Use a regulated power supply to check the source of the interference (eg water bath, etc.)

5. Degassing of bubble mobile phase in the detector, back pressure after column addition (7) Peak tailing by HPLC

1.Column overload reduces sample volume, increases column diameter using higher-capacity stationary phase 2. Peak interference cleans sample, adjusts mobile phase 3. Silicon hydroxyl effect plus triethylamine, use alkali to passivate column to increase buffer or salt Concentration decrease PH value of mobile phase, passivation sample 4. Same as before (D) 4 with before (D) 4

5. With the former (four) 35. With the former (four) 3

6. The outer volume or dead volume through the column to minimize the contact Dalian, make appropriate adjustments for all connection points, as far as possible small inside diameter tube connected to a lower column degradation 7. etching conditions, the replacement column, using guard column (eight HPLC peak broadening

1. The injection volume is too large with (4) 1

2. Causes peaks in the injection valve to cause air bubbles to escape before and after the sample injection to reduce diffusion 3. The sampling rate of the data system is too slow The setting rate should be greater than 10 points per peak 4. The detector time constant is too large Set the time constant for the sense 10% of the half width of the first peak of interest

5. The viscosity of the mobile phase is too high. Increase the column temperature and use a low-viscosity mobile phase. 6. Excessive volume of the detection cell. Use a small volume reservoir to remove the heat exchanger. 7. Excessive retention time. Elution 8. Excess volume outside the column minimizes the connecting tube diameter and connecting tube length. 9. Sample overload to small concentrations. Small sample volume