Possible Reasons and the Solutions of the Abnormality of the Liquid Chromatogram

Possible reasons and the solutions of the abnormality of the liquid chromatogram

1. There is no peak in the chromatogram.

Solution: The system does not inject or the sample is decomposed. The pump is not infused or the mobile phase is used incorrectly. The detector setting is incorrect. Make the adjustment according to above reasons.

2. A peak or several peaks are negative.

Solution: The mobile phase absorbs the background high. The air enters during the injection. The absorption of the sample components is lower than the mobile phase.

3. All peaks are negative peaks.

Solution: The signal cable is reversed or the settings of the detector output polarity is reversed. The optical device has not yet reached balance.

4. All peaks are wide peaks.

Solution: The system is not balanced. The solvent polarity of the dissolved sample is much different than the mobile phase. The chromatography column size and type are not correctly selected. The column or guard column is contaminated or the column efficiency is reduced. Temperature changes.

5. The peak is smaller than expected.

Solution: The sample viscosity is too high. The sample injection failure or injection volume error. The detector settings are incorrect. The volume of the loop is incorrect. The detect pool is contaminated. The detector lamp failure.

6. Retention time changes

a. Column temperature changes: keep temperature
b. There is insufficient balance between isocraticity and gradient: at least 10 column volumes of mobile phase equilibrium column
c. Buffer solution capacity is not enough: use >25mmol/L buffer
d. Column contamination: Flush the column everyday
e. Column condition changes: stable injection conditions, adjust mobile phase
f. The column reaches the life end: adopt guard column

7. The retention time is shortened

a. Increase in flow rate: check the pump and reset flow rate
b. Sample overload: reduce sample volume
c. Bonded phase loss: Keep the pH value of the mobile phase between 3-7.5, inspect the direction of column
d. Change in mobile phase composition: Prevent evaporation or precipitation of mobile phase
e. Temperature ascend: keep temperature

8. Retention time is extended

a. Flow rate drop: The pipeline leaks, replace the pump seal, and eliminate the bubble inside the pump.
b. Change of active point on silica gel column: use mobile phase modifier, such as triethylamine
c. Bonded phase loss: Keep the pH value of the mobile phase between 3-7.5, inspect the direction of column
d. Change in mobile phase composition: Prevent evaporation or precipitation of mobile phase
e. Temperature drop: keep temperature

9. Shoulder Peak

a. The sample volume is too more: use the mobile phase to matching the sample, the total sample volume is less than 15% of the first peak.
b. The sample solvent is too strong: use a weaker sample solvent
c. Column collapse or short circuit formed: Replace the column with weaker corrosive conditions
d. In-column sintered stainless steel failure: Replace sintered stainless steel, add in-line filter for sample filtration
e. Injector damage: Replace the injector rotor

10. ghost peak

a. Injection valve residual peak: Clean the valve with strong solvent after each use to improve the cleaning of the valve and sample
b. Unknown stuff in the sample: Processing sample
c. Column unbalanced: Re-equilibrate the column and use the mobile phase as the sample solvent (especially ion-pair chromatography)
d. Trifluoroacetic acid (TFA) oxidation (peptide mapping): mix daily, with antioxidants
5. Water pollution (reverse phase): Check the water quality by changing the equilibration time, using HPLC grade water

11. Baseline noise
a. Bubbles (sharp peaks): Degassing of the mobile phase, back pressure after column addition
b. Contamination (random noise): Clean the column, purify the sample, use HPLC grade reagent
c. Detector lamp continuous noise: Replace the xenon lamp
d. Electrical interference (accidental noise): Use a regulated power supply to check the source of the interference (such as water bath, etc.)
e. There are bubbles in the detector: the mobile phase is degassed, and the back pressure is added after the column is added.

12. Peak tailing

a. Column overload: reduce the sample volume and increase the column diameter, utilize a higher capacity stationary phase
b. Peak interference: Clean the sample, adjust the mobile phase, use a longer column
c. Silanol effect, Add triethylamine, use alkali-induced passivation column to increase the concentration of buffer or salt, reduce the PH value of mobile phase, passivate sample
d. In-column sintered stainless steel failure: Replace sintered stainless steel, add in-line filter to filter sample
e. Column collapse or short circuit formed: Replace the column, weaker corrosive conditions
f. Excessive dead volume or extra-column volume: The connection point is minimized, and all connection points are adjusted appropriately.
g. Column efficiency reduced: Replace the column in lower corrosion conditions, use a guard column, activate the column

13. Peak broadening

a. The injection volume is too large: With the mobile phase, the total sample volume is less than 15% of the first peak.
b. Peak expansion in the injection valve: Discharge air bubbles before and after injection to reduce diffusion
c. The data system sampling rate is too slow: the set rate should be greater than 10 points per peak.
d. The detector time constant is too large: Set the time constant to be 10% of the half width of the first peak of interest.
e. The mobile phase viscosity is too high: increase the column temperature, using a low viscosity mobile phase
f. The volume of detection tank is too large: use a small volume pool and remove the heat exchanger
g. The retention time is too long: the solvent content can be increased by isocratic elution.
h. Extra-column volume is too large: Minimize the diameter and length of the connecting pipe
i. Sample overload: inject small concentration small volume ample

14. Peak fork

a. Protection column or analytical column contamination: Remove the guard column for analysis. Replace the guard column if necessary. If the analytical column is blocked, remove it and clean. If the problem persists, it may be that the column is contaminated with strong retained materials. utilize appropriate regenerative measures. If the problem persists, the inlet may be blocked, replace the sieve plate or replace the column.
b. The sample solvent is insoluble in the mobile phase: change the sample solvent. If possible, take the mobile phase as a sample solvent.

15. baseline drift

a. The column temperature fluctuates. (Even a small temperature change can cause fluctuations in the baseline. Usually affects the difference detector, conductivity detector, lower sensitivity UV detector or other photoelectric detectors): control the temperature of the column and mobile phase, use the heat exchanger before the detector
b. The mobile phase is uneven. (Baseline drift caused by changes in mobile phase conditions is greater than that caused by temperature): use HPLC grade solvents, high purity salts and additives. The mobile phase is degassed prior to use and helium is used during use.
c. The flow tank is contaminated or has gas: flush the flow tank with methanol or other strong polar solvents. If necessary, 1N nitric acid can be used. (do not use hydrochloric acid)
d. The detector outlet is blocked. (High pressure causes the flow tank window broken, creating a noise baseline): Remove the obstruction or replace the tube. Refer to the detector manual to replace the flow tank window.
e. Improper mix ratio of mobile phase or flow rate change: change the ratio or flow rate. To avoid this problem, regularly check the mobile phase composition and flow rate.
f. The column balance is slow, especially when the mobile phase changes: flush with medium-strength solvent. When changing the mobile phase, rinse with 10-20 volumes of new flow relative column before analysis.
g. Mobile phase contamination, deterioration or formed from low quality solvents: check the composition of the mobile phase. Use high quality chemical reagents and HPLC grade solvents.
h. Use recycled solvent, but the detector is not adjusted: reset the baseline. When the detector dynamic range changes, a new mobile phase is used.
i. The detector is not set at the maximum absorption wavelength: the wavelength is adjusted to the maximum absorption wavelength.

2018-08-08T00:45:20+00:00 August 8th, 2018|