Analysis and Countermeasures of common problems in

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Analysis and Countermeasures of common problems in high performance liquid chromatography

many problems of liquid chromatography system can be reflected on the spectrum. Some of these problems can be solved by changing equipment parameters; Other problems must be solved by modifying the operating procedures. The correct choice of chromatographic column and mobile phase is the key to get a good chromatogram

a, peak tailing



1, sieve plate blockage

a, recoil chromatographic column B, replace imported sieve plate C, replace chromatographic column

2, chromatographic column collapse

fill chromatographic column

3, interference peak

a, use longer chromatographic column B, change the mobile phase or replace chromatographic column

4, mobile phase pH selection error

adjust pH value. For alkaline compounds, low pH value is more conducive to obtain symmetrical peaks

5, the sample reacts with the melting point on the surface of the filler

a, adds ion pair reagent or alkaline volatile modifier B, changes the chromatographic column

b, peak pre delay



1, low column temperature

increases the column temperature

2, improper selection of sample solvent

use mobile phase as sample solvent

3 Sample overload

reduce sample content

4. Chromatographic column damage

see A1, A2

c, peak bifurcation



1. Protective column or analytical column pollution

remove the protective column for analysis. Replace the protective column if necessary. If the analytical column is blocked, remove it for cleaning. If the problem still exists, it may be that the column is polluted by strongly retained substances, and appropriate regeneration measures should be taken. If the problem persists, the inlet may be blocked. Replace the sieve plate or replace the chromatographic column

2. The sample solvent is insoluble in the mobile phase

change the sample solvent. If possible, use the mobile phase as the sample solvent

d, peak deformation



1, sample overload

reduce sample load

e, early peak deformation



1, improper selection of sample solvent

A, reduce injection volume B, use low polarity sample solvent

F, the tailing degree of early peak is greater than that of late peak



1, out of column effect

a Adjust the system connection (use a shorter pipe with a smaller inner diameter)

B. when using a small flow cell

G, K 'increases, tailing is more serious



1. Secondary retention effect, reverse phase mode

A, add triethylamine (or alkaline sample) B, add acetic acid (or acidic sample) C, add salt or buffer (or ionized sample) d, replace a column

2 Secondary retention effect, normal phase mode

a, adding triethylamine (or alkaline sample) B, adding acetic acid (or acidic sample) C, adding water (or multi-functional compound) d, trying another method

3, secondary retention effect, ion pair

adding triethylamine (or alkaline sample)

H, peak tailing of acidic or alkaline compounds



1, improper buffer

a Use buffer with concentration of MM B, use buffer with PKA equal to the pH value of the mobile phase

i, additional peak



1, other components in the sample


2, elution peak of the previous injection

a, increase the running time or gradient slope B, increase the flow rate

3, vacancy or ghost peak

a, check whether the mobile phase is pure B, use mobile phase as sample solvent C Reduce the injection volume

j, retention time fluctuation



1, improper temperature control

adjust the column temperature

2, mobile phase component changes

prevent changes (evaporation, reaction, etc.)

3, chromatographic column is not balanced

give sufficient time to balance the chromatographic column before each operation

k, retention time changes



1 Flow rate change

reset the flow rate

2, there are bubbles in the pump

remove bubbles from the pump

3, the selection of mobile phase is inappropriate

a, replace the appropriate mobile phase B, select the appropriate mixed mobile phase

l, baseline drift



1, column temperature fluctuation. (even a small temperature change will cause the fluctuation of the baseline. It usually affects the differential detector, conductivity detector, UV detector with low sensitivity or other photoelectric detectors.)

control the temperature of the column and the mobile phase, and use the heat exchanger diagram

2 before the detector. The mobile phase is uneven. (the baseline drift caused by the change of mobile phase conditions is greater than that caused by temperature.)

use HPLC grade solvents, high-purity salts and additives. The mobile phase is degassed before use, and helium is used during use

3. The circulation tank is polluted or there is gas

wash the circulation tank with methanol or other strong polar solvents. If necessary, 1n nitric acid can be used. (do not use hydrochloric acid)

4. At the same time, the detector outlet can also be blocked by strain or other more advanced test methods. (the window of the flow cell is broken due to high pressure, generating a noise baseline)

remove the blockage or replace the pipe. Refer to the detector manual to replace the flow cell window

5. Improper flow matching ratio or flow rate change

change the ratio or flow rate. To avoid this problem, the composition and flow rate of mobile phase can be checked regularly

6. The column balance is slow, especially when the mobile phase changes

wash it with medium strength solvent. When changing the mobile phase, wash it with a new flow of times the volume relative to the column before analysis

7. The mobile phase is polluted, deteriorated or mixed with low-quality solvent

check the composition of the mobile phase. Using high-quality chemical reagents and HPLC grade solvents

8, substances with strong retention in the sample (high K 'value) were washed out as steamed bread peak samples, thus showing a gradually rising baseline

use a protective column. If necessary, wash the column regularly with strong solvent between injections or during analysis

9. Use circulating solvent, but the detector is not adjusted

reset the baseline. When the dynamic range of the detector changes, a new mobile phase is used

10. The detector is not set at the maximum absorption wavelength

adjust the wavelength to the maximum absorption wavelength

m, baseline noise (regular)



1, there is air in the mobile phase, detector or pump

mobile phase degassing. Flush the system to remove air from the detector or pump

2. Leakage

see part III. Check whether the pipe joints are loose, whether the pump leaks, whether there is salting out and abnormal noise. If necessary, replace the pump seal

3. Incomplete mixing of mobile phase

shake it evenly by hand or use low viscosity solvent

4. Temperature effect (column temperature is too high, detector is not heated)

reduce the difference or add heat exchanger

5. There are other electronic devices on the same line

disconnect LC, detector and recorder, check whether the interference comes from the outside and correct it

6, pump vibration

add pulse damper in the system

n, baseline noise (irregular)



1, leakage

see part III. Check whether the connector is loose, whether the pump leaks liquid, whether there is salting out and abnormal noise. Replace the seal if necessary. Check whether the circulating pool leaks liquid

2. The mobile phase is polluted, deteriorated or mixed with low-quality solvent

check the composition of the mobile phase

3. The solvents of the mobile phase are insoluble

select the mutually soluble mobile phase

4. The problem of the electronic components of the detector/recorder

disconnect the power supply of the detector and recorder, check and correct it

5. There are bubbles in the system

clean the system with strong polar solution

6. There are bubbles in the detector

clean the detector, install the background pressure regulator behind the detector

7. The circulation pool is polluted (even a few pollutants will produce noise.)

use 1n nitric acid (not phosphoric acid) to clean the flow cell

8, the energy of the detector lamp is insufficient

replace the lamp

9, the chromatographic column packing is lost or blocked

replace the chromatographic column

10, the mobile phase is mixed unevenly or the mixer works abnormally

repair or replace the mixer. When the mobile phase does not follow the gradient, it is recommended not to use the mixing device of the pump

o, wide peak



1 Mobile phase composition change

re prepare a new mobile phase

2, the flow rate of the mobile phase is too low

adjust the flow rate

3, leakage (especially between the column and the detector)

see Section 3. Check whether the connector is loose, whether the pump leaks liquid, whether there is salting out and abnormal noise. Replace the seal if necessary

4. Incorrect detector setting

adjustment setting

5. Effect of out of column effect

a, column overload B, detector response to reaction time or cell volume is too large C, pipeline between column and detector is too long or pipeline inner diameter is too large D, recorder response time is too long 5, a, small volume injection (for example, 10ul instead of 100ul) dilutes sample B in the ratio of 1:10 or 1:100 Reduce the response time or use a smaller flow cell C, and use an inner diameter of 0 01 short pipeline D, reduce the response time

6, the buffer concentration is too low

increase the concentration

7, the protective column is polluted or invalid

replace the protective column

8, the chromatographic column is polluted or invalid, and the number of trays is low

replace the same type of chromatographic column. If the new column can provide symmetrical chromatographic peaks, wash the old column with strong solvent

9, column inlet collapse

open the column inlet, fill the collapse or replace the column

10, present two or more peaks of substances that have not been completely separated

select other types of chromatographic columns to improve the separation effect

11, the column temperature is too low

increase the column temperature. Unless in special circumstances, the temperature should not exceed 75 ℃

12, the detector time constant is too large

use a smaller time constant

P, the resolution is reduced



1, the mobile phase is polluted or deteriorated (causing changes in retention time)

reconfigure the mobile phase

2, remove the protective column or the blocking diagram of the analytical column

for analysis. Replace the protective column if necessary. If the analytical column is blocked, recoil can be carried out. If the problem still exists, the chromatographic column may be damaged by strongly retained pollutants, it is recommended to use appropriate regeneration procedures. If the problem still exists, the inlet may be blocked. Replace the sieve plate at the inlet or replace the chromatographic column because the shrinkage is still large due to early solidification of the gate

q, all peak areas are too small



1, the detector attenuation setting is too high

the setting of reducing attenuation

2, the detector time constant setting is too large

set a smaller time constant

3, the injection volume is too small

increase the injection volume

4, the recorder connection is improper

use the correct connection

r All peak areas are too large



1. The attenuation setting of the detector is too low

adopt a larger attenuation

2. Too much sample injection

reduce the amount of sample injection

3. The recorder is not connected correctly

correctly connect the recorder

the determination and solution of common faults of HPLC


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