HPLC failure and troubleshooting

Diagnosis

possible reason

solution Decision square law

( one )

Retention time change

1. Column temperature change

Column thermostat

2. The equivalence and the gradient are not fully balanced

Use at least 10 column volumes of mobile phase equilibration column

3. Buffer capacity is not enough

Use >25mmol/L buffer

4. Column contamination

Flush the column every day

5. Changes in column conditions

Stable injection conditions , conditioning the mobile phase

6. The column reaches life fast

Guard column

( two )

Shorter retention time

1. flow rate increase

Check the pump and reset the flow rate

2. Sample overload

Reduce sample size

3. Bond phase loss

The PH value of the mobile phase is maintained in the direction of the column from 3 to 7.5 .

4. Changes in mobile phase composition

Prevent mobile phase evaporation or precipitation

5. Temperature increase

Column thermostat

( three )

Extended retention time

1. Flow rate drop

Leakage of the pipeline , replace the pump seal and eliminate air bubbles in the pump

2. Change of active point on silica gel column

With mobile phase modifiers, such as triethylamine, or to use an alkali passivation column

3. Bond phase loss

Cit ( two ) 3

4. Changes in mobile phase composition

Cit ( two ) 4

5. Temperature reduction

Cit ( two ) 5

( four )

Shoulder or bifurcation

1. The sample volume is too large

With mobile phase , the total sample volume is less than 15% of the first peak

2. The sample solvent is too strong

Use weaker sample solvent

3. Column collapse or short circuit

Replace column, using less corrosive conditions

4. In- column sintered stainless steel failure

Replace sintered stainless steel , add in-line filter , filter sample

5. Injector damage

Replace the injector rotor

( Fives )

Ghost peak

1. Injecting valve residual peak

Purge valve after each use with a strong solvent, and the sample purge valve to improve

2. Unknown in the sample

Processing sample

3. The column is not balanced

Rebalance the column and use the mobile phase as the sample solvent ( Especially ion pair chromatography )

4. Trifluoroacetic acid ( TFA ) Oxidation ( Peptide spectrum )

New daily , with antioxidants

5. Water pollution ( Inversion )

Check the water quality by changing the equilibration time , using HPLC grade water

( six )

Baseline noise

1. Bubble ( Sharp peak )

Degassing the mobile phase , adding back pressure after column

2. Pollution ( Random noise )

Clean the column , purify the sample , 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

Degassing the mobile phase , adding back pressure after column

( Seven )

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. Silicic hydroxyl function

Add triethylamine , use a base-induced passivation column to increase the concentration of buffer or salt to lower the PH value of the mobile phase , passivate the sample

4. Same as before ( four ) 4

Cit ( four ) 4

5. Same as before ( four ) 3

5. Same as before ( four ) 3

6. Dead volume or extra-column volume is too large

The point of attachment to a minimum, make suitable adjustments for all connection points, as far as possible using the small inside diameter of the connection pipe

7. Reduced efficiency

With a lower etching conditions, the replacement column, guard column using

( Eight )

Peak broadening

1. The injection volume is too large

with ( four ) 1

2. Cause peak expansion in the injection valve

Bubbles are ejected 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 first half width of the first peak of interest

5. The mobile phase viscosity is too high

Increase column temperature , using low viscosity mobile phase

6. The detection pool is too large

Use a small volume pool to remove the heat exchanger

7. The retention time is too long

Increasing the solvent content during isocratic elution can also be eluted with a gradient

8. Extra-column volume is too large

Minimize the diameter of the connecting pipe and the length of the connecting pipe

9. Sample overload

Small concentration small sample