Various problems with the spectrum of LC troubleshooting
Many problems with liquid chromatography systems can be reflected in the spectra. Some of these problems can be solved by changing the device parameters; other problems must be solved by modifying the operating procedures. The right choice for the column and mobile phase is the key to getting a good chromatogram.
A, peak tailing
1, sieve plate blocking
a, backflushing column b, replacing the imported sieve plate c, replacing the column
2, column collapse
a, packed column
3. Interference peak
a, use a longer column b, change the mobile phase or replace the column
4, mobile phase PH selection error
a. Adjust the PH value. For basic compounds, low pH is more favorable for obtaining symmetrical peaks.
5. The sample reacts with the melting point on the surface of the filler.
a, add ion pair reagent or alkaline volatile modifier b, change the column
B. Possible reasons for peak delay
Before the big peak, there is a small peak out
Column dead volume
Sample solvent is not appropriate
Partially blocked filter
Column overload
1, the column temperature is low
a, increase the column temperature
2, sample solvent selection is not appropriate
a, using the mobile phase as a sample solvent
3, sample overload
a, reduce the sample content
4, column damage
a, see A1, A2
C, peak fork
1. Pollution column or analytical column contamination [dead volume in column]
a. Remove the guard column and analyze it. Replace the guard column if necessary. If the analytical column is clogged, remove it and wash it. If the problem persists, it may be that the column is contaminated with strong retention materials and appropriate regeneration measures are applied. If the problem persists, the inlet may be blocked, replace the sieve plate or replace the column.
2. The sample solvent is insoluble in the mobile phase.
a, change the sample solvent. If possible, take the mobile phase as a sample solvent.
D, peak deformation
1, sample overload
a, reduce the sample load
E, early peak distortion
1, the sample solvent selection is not appropriate
a, reduce injection volume b, use low polarity sample solvent
F, the early peak tail is greater than the late peak
1. Extra-column effect
a, adjust the system connection (use a shorter, smaller inner diameter pipeline) b, use a small volume of the flow cell
When G and K' increase, the tailing is more serious.
1, secondary retention effect, inverting 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 pillar
2, secondary retention effect, normal phase mode
a, add triethylamine (or alkaline sample) b, add acetic acid (or acidic sample) c, add water (or polyfunctional compounds)
d, try another method
3, secondary retention effect, ion pair
a, adding triethylamine (or alkaline sample)
H, peak tailing of acidic or basic compounds
1, the buffer is not suitable
a, using a concentration of 50-100 mM buffer b, using a buffer of Pka equal to the pH of the mobile phase
I, extra peak
1, there are other components in the sample
2. The 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 the mobile phase as the sample solvent c, reduce the injection volume
J, retention time fluctuations
1. Improper temperature control
a, adjust the column temperature
2. Changes in mobile phase composition
a, prevent changes (evaporation, reaction, etc.)
3, the column is not balanced
a. Give enough time to equilibrate the column before each run
K, retention time is constantly changing
1, the flow rate changes
a, reset the flow rate
2, there are bubbles in the pump
a, remove bubbles from the pump
3. The mobile phase is not properly selected
a, replace the appropriate mobile phase b, choose the appropriate mixed mobile phase
L, baseline drift
1. The column temperature fluctuates. (Even a small temperature change can cause fluctuations in the baseline. It usually affects the difference detector, conductivity detector, lower sensitivity UV detector or other optoelectronic detector.)
a, control the temperature of the column and mobile phase, use a heat exchanger before the detector
2. The mobile phase is not uniform. (The baseline drift caused by changes in mobile phase conditions is greater than the drift caused by temperature.)
a. Use HPLC grade solvents, high purity salts and additives. The mobile phase is degassed prior to use and helium is used during use.
3. The flow cell is polluted or has gas
a. Flush the flow cell with methanol or other strong polar solvent. If necessary, 1N nitric acid can be used. (Do not use hydrochloric acid)
4. The detector outlet is blocked. (High pressure causes the flow cell window to rupture, producing a noise baseline)
a. Remove the obstruction or replace the tube. Refer to the detector manual to replace the flow cell window.
5. Improper flow ratio or flow rate change
a. Change the ratio or flow rate. To avoid this problem, the mobile phase composition and flow rate can be checked regularly.
6. The column balance is slow, especially when the mobile phase changes.
a. Flush with a medium strength solvent. When changing the mobile phase, rinse with 10-20 volumes of new flow relative to the column before analysis.
7. Mobile phase contamination, deterioration or formulation from low quality solvents
a. Check the composition of the mobile phase. Use high quality chemical reagents and HPLC grade solvents
8. The strongly retained material in the sample (high K' value) is eluted as a peak of the taro, thus exhibiting a gradually increasing baseline.
a. Use a guard column and, if necessary, periodically flush the column with a strong solvent between injections or during the analysis.
9. The circulating solvent is used, but the detector is not adjusted.
a. Reset the baseline. When the detector dynamic range changes, a new mobile phase is used.
10. The detector is not set at the maximum absorption wavelength.
a, adjust the wavelength to the maximum absorption wavelength
M, baseline noise (regular)
1. Air in the mobile phase, detector or pump
a, the mobile phase is degassed. Flush the system to remove air from the detector or pump.
2, leakage
a. See the third part. Check for loose connections, pump leaks, salting and abnormal noise. Replace the pump seal if necessary.
3, the mobile phase is not completely mixed
a, shake by hand to make the mixture even or use a low viscosity solvent
4, temperature effects (column temperature is too high, the detector is not heated)
a, reduce the difference or add heat exchanger
5. There are other electronic devices on the same line.
a. Disconnect the LC, detector, and recorder to check if the interference is from the outside and correct it.
6, pump vibration
a, add a pulse damper to the system
N, baseline noise (irregular)
1, leakage
a. See the third part. Check if the joint is loose, if the pump leaks, if there is salt precipitation and abnormal noise. Replace the seal if necessary. Check the flow cell for leaks.
2. The mobile phase is contaminated, deteriorated or composed of low-quality solvents.
a. Check the composition of the mobile phase.
3. The mobile phase is incompatible with each solvent.
a, choose a mutually soluble mobile phase
4, the detector / recorder electronic components
a. Disconnect power from the detector and recorder, check and correct.
5, there are bubbles in the system
a, clean the system with a strong polar solution
6, there are bubbles in the detector
a, clean the detector, install the background pressure regulator behind the detector
7. Flow cell pollution (even very few pollutants can cause noise.)
a, use 1N nitric acid (can not use phosphoric acid) to clean the flow cell
8, the detector lamp energy is insufficient
a, replace the lamp
9. Column packing loss or blockage
a, replace the column
10. The mobile phase is not evenly mixed or the mixer is not working properly.
a. Repair or replace the mixer. When the mobile phase does not go through the gradient, it is recommended not to use the pump mixing device.
O, wide peak
1. Changes in mobile phase composition
a, re-prepare a new mobile phase
2. The mobile phase flow rate is too low
a, adjust the flow rate
3, leakage (especially between the column and the detector)
a. See section 3. Check for loose joints, leaking pumps, salt out and abnormal noise. Replace the seal if necessary.
4, the detector settings are incorrect
a, adjust the settings
5, the effect of extra-column effect
a, column overload b, detector response to reaction time or pool volume is too large c, the pipeline between the column and the detector is too long or the inner diameter of the pipeline is too large d, the response time of the recorder is too long
a, small volume injection (for example: 10ul instead of 100ul) dilute sample b at a ratio of 1:10 or 1:100, reduce response time or use a smaller flow cell c, use a short tube with an inner diameter of 0.007-0.01 d, reduce response time
6, the buffer concentration is too low
a, increase the concentration
7. Protection column pollution or failure
a, replace the guard column
8. The column is contaminated or invalid, and the number of plates is low.
a. Replace the same type of column. If the new column provides a symmetrical peak, flush the old column with a strong solvent.
9. Column entrance collapse
a, open the column entrance, fill the collapse or replace the column
10. A peak that exhibits two or more substances that are not completely separated
a, select other types of columns to improve separation
11, the column temperature is too low
a, increase the column temperature. Unless special circumstances, the temperature should not exceed 75 ° C
12, the detector time constant is too large
a, use a smaller time constant
P, reduced resolution
1. Mobile phase contamination or deterioration (causing retention time changes)
a, reconfigure the mobile phase
2, guard column or analytical column block
a. Remove the guard column for analysis. Replace the guard column if necessary. If the analytical column is blocked, a backflush can be performed. If the problem persists that the column may be damaged by strongly retained contaminants, it is recommended to use an appropriate regeneration procedure. If the problem persists, the inlet may be blocked, replace the screen at the inlet or replace the column.
Q, all peak areas are too small
1, the detector attenuation setting is too high
a, reduce the attenuation setting
2, the detector time constant setting is too large
a, set a small time constant
3, the injection volume is too small
a, increase the injection volume
R, all the peak areas are too large
1, the detector attenuation setting is too low
a, take greater attenuation
2, too much injection
a, reduce the amount of injection
3, the recorder is not connected correctly
a, correctly connected to the recorder
S, troubleshooting of high performance liquid chromatography HPLC retention time drift
The retention time is not as heavy as the existing two different cases: retention time drift and retention time fluctuations. The former means that the retention time changes only in one direction, while the latter refers to the fluctuation of the retention time without a fixed regularity. It is often helpful to distinguish between the two situations to find the cause of the problem. For example, the drift of retention time is often caused by column aging; while column aging is unlikely to cause irregular fluctuations in retention time. In fact, most of the retention time drift is due to column aging of different mechanisms, such as loss of stationary phase (eg by hydrolysis), column contamination (caused by sample or mobile phase). The most common reasons for retention time drift are as follows:
1: Column balance
If we observe retention time drift, we should first consider whether the column has been completely equilibrated with the mobile phase. Usually a balance of 10-20 column volumes of mobile phase is required, but if a small amount of additive (such as an ion pair reagent) is added to the mobile phase, it takes a considerable amount of time to equilibrate the column.
Mobile phase contamination may also be one of the reasons. A small amount of contaminants dissolved in the mobile phase may slowly enrich on the column, causing drift in retention time. It should be noted that water is a mobile phase component that is easily contaminated.
2: stationary phase stability
The stability of the stationary phase is limited and the stationary phase will slowly hydrolyze even when used within the recommended pH range. For example, the silica gel matrix has the best hydrolytic stability at pH 4. The rate of hydrolysis is related to the type of mobile phase and the ligand. The bifunctional ligand and the trifunctional ligand are more stable than the bonded phase of the monofunctional ligand; the long chain linkage is more stable than the short chain bonding phase; the alkyl linkage is much more stable than the cyano bonded phase.
Frequent cleaning of the column also accelerates the hydrolysis of the column stationary phase. Other silica matrix bonded phases may also undergo hydrolysis in aqueous solutions, such as amino bonding.
3: Column contamination
Another common cause of retention time drift is column contamination. The HPLC column is a very effective adsorptive filter that filters and adsorbs any material carried by the mobile phase. Sources of contamination can be: mobile phase itself, mobile phase vessels, connecting tubes, pumps, injectors and instrument gaskets, and samples. The source of the contamination can usually be determined experimentally.
If there is a strong component remaining on the column in the sample, it may be a potential source of drift in retention time. These roots are usually the sample matrix. Such as: excipients in the drug, protein and lipid compounds in biochemical samples (such as serum), starch in food samples, humic acid in environmental water samples, etc. Usually, the strongly retained component in the sample has a higher molecular weight, in which case the retention time drifts or there is an increase in back pressure. The effect of the sample matrix can be removed by using a sample preparation method such as solid phase extraction (SPE).
The easiest way to avoid column contamination is to prevent it from happening. In contrast, finding the problem and designing an effective cleaning step to remove contaminants is much more difficult. Strong solvents are usually used under the given chromatographic conditions, but not all contaminants can be dissolved in the mobile phase. For example, THF removes many of the contaminants in the reversed-phase column, but the protein does not dissolve in THF. DMSO is often used to remove proteins from reversed phase columns.
Using a guard column is a very effective method. Recoil columns are only a last resort.
4: Mobile phase composition
Slow changes in the composition of the mobile phase are also common causes of retention time drift. For example, the volatile components in the mobile phase are volatilized and the flow in circulation is equal.
5: Hydrophobic collapse
When a reverse-phase packed column with a small pore size and a good end-blocking seal uses nearly 100% water as the mobile phase, a sudden loss of separation and a significant decrease or no retention of the analyte retention sometimes occur, which is hydrophobic collapse. This phenomenon is caused by the fact that the mobile phase does not wet the surface of the stationary phase. The salvage method achieves infiltration of the stationary phase with a mobile phase containing a large amount of organic components, and then equilibration with a mobile phase having a high water content. This phenomenon can also occur due to long-term storage of the column. Collapsing can also be avoided by using a reversed-phase column with a non-polar group or a non-end-sealed column.
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