The failure of HPLC is a situation often encountered by staff engaged in chromatographic analysis. Hangzhou Saiertai Technology Co., Ltd. () is a company specializing in the production of HPLC and professionally serving the chromatographic industry. We recommend that you first understand the common fault conditions of HPLC and know some simple treatment methods.
(1) Change in retention time
1. Column temperature change
2. The isocratic and gradient are not fully balanced. At least 10 times the column volume of the mobile phase to balance the column
3. The buffer capacity is not enough for buffers> 25 mmol / L
4. Column contamination Rinse the column daily
5. Changes in column conditions Stable injection conditions and adjust mobile phase
6. The column reaches its life soon
(2) Shortened retention time
1. Increased flow rate Check the pump and reset the flow rate
2. Overload of sample Reduce sample size
3. Loss of bonded phase PH value of the mobile phase is maintained at 3 ~ 7.5 Check the direction of the column
4. Changes in mobile phase composition prevent mobile phase evaporation or precipitation
5. Temperature increase Column constant temperature
(3) Retention time extension
1. The flow rate drops, the pipeline leaks, replace the pump sealing ring, and eliminate the air bubbles in the pump
2. Change of active point on silica gel column Use mobile phase modifier, such as adding triethylamine, or use alkali to passivate the column
3. Loss of bonded phase Ibid. (2) 3
4. The composition of the mobile phase changes as before (2) 4
5. Lower temperature Same as above (2) 5
(4) Shoulder peaks or bifurcations
1. If the sample volume is too large, use the mobile phase to prepare the sample. The total sample volume is less than 15% of the first peak.
2. The sample solvent is too strong, the weaker sample solvent is used
3. The column collapses or forms a short-circuit channel to replace the chromatographic column, using weaker corrosive conditions
4. Failure of the sintered stainless steel in the column Replace the sintered stainless steel, add an online filter to filter the sample
5. The injector is damaged. Replace the injector rotor.
(5) Ghost Peak
1. The residual peak of the injection valve is washed with a strong solvent after each use to improve the cleaning of the valve and the sample
2. Samples from unknown samples
3. The column is not equilibrated. 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 prepared every day, with antioxidants
5. Water pollution (reverse phase)
Check the water quality by changing the equilibrium time, use HPLC grade water
(6) Baseline noise
1. Bubbles (sharp peaks)
Mobile phase degassing, back pressure after column addition
2. Pollution (random noise)
Clean the column, clean the sample, and use HPLC grade reagents
3. Detector lamp continuous noise replacement deuterium lamp
4. Electrical interference (accidental noise)
Use regulated power supply, check the source of interference (such as water bath, etc.)
5. Degas the mobile phase with bubbles in the detector, and add back pressure after adding the column
(7) Peak tailing
1. Column overload reduces the sample volume, increases the column diameter and uses a higher capacity stationary phase
2. Peak interference cleans the sample and adjusts the mobile phase
3. Add triethylamine to silanol, increase the buffer or salt concentration with alkali-induced passivation column, lower the PH value of mobile phase, passivate the sample
4. Same as before (4) 4
Same as (4) 4
5. Same as before (4) 3
5. Same as before (4) 3
6. The dead volume or the volume outside the column is too large. The connection points are reduced to a minimum. All connection points are adjusted appropriately, and the connection tubes with a thin inner diameter are used as much as possible.
7. Reduce the column efficiency, use lower corrosion conditions, replace the column, use a protective column
(8) Peak broadening
1. The injection volume is too large (4) 1
2. Cause peak expansion in the injection valve to discharge bubbles before and after injection to reduce diffusion
3. The data system sampling rate is too slow. The set rate should be greater than 10 points per peak.
4. The detector time constant is too large. Set the time constant to 10% of the half width of the first peak of interest.
5. The viscosity of the mobile phase is too high to increase the column temperature, using a low viscosity mobile phase
6. If the volume of the detection cell is too large, use a small volume cell and remove the heat exchanger
7. When the retention time is too long and the solvent content is increased when isocratic elution is performed, gradient elution can also be used
8. The volume outside the column is too large to minimize the connecting pipe diameter and connecting pipe length
9. Sample overload into small concentration and small volume sample
* Related products: syltech 500 high-performance liquid chromatograph (single pump), VERTEX STI 5000 isocratic (single pump) system standard configuration, N2000 chromatography data workstation (SP1 version)
Consultation hotline for liquid chromatograph; please download and reference for more instrument information.
Hangzhou Celtech Technology Co., Ltd. 2009-02
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