Various problems with the spectrum of LC troubleshooting
Many problems with liquid chromatography systems can be reflected on the spectrum. 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. Screen plate blockage a. Backflush column b, replace imported sieve plate c, replace column a. Adjust the PH value. For basic compounds, low pH is more favorable for obtaining symmetrical peaks. B. Possible reasons for peak delay Before the big peak, there is a small peak outflow column dead volume sample solvent inappropriate filter partially blocked column overload 1, low column temperature a, elevated column temperature 2, sample solvent selection is not appropriate a, use mobile phase as 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] 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 sample load E, early peak distortion 1, sample solvent selection is not appropriate a, reduce injection volume b, use low polarity sample solvent F. The peak of the early peak is greater than the peak of the late one. 1. Extra-column effect a. Adjust the system connection (use a shorter, smaller inner diameter pipeline) b. Use a small volume flow cell When G and K' increase, the tailing is more serious, the secondary retention effect, the reverse phase mode a, the addition of triethylamine (or alkaline sample) b, the addition of acetic acid (or acidic sample) c, the addition of salt or buffer ( Or ionized sample) H. Peak tailing of acidic or basic compounds 1. Buffer is not suitable a. Use buffer with concentration of 50-100 mM b. Use buffer with Pka equal to PH value of mobile phase. I. Additional peaks 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, mobile phase composition changes a, prevent changes (evaporation, reaction, etc.) K, constant retention time 1, flow rate change a, re-set flow rate 2, bubble a in the pump, remove air bubbles from the pump 3, improper selection of mobile phase a, replace the appropriate mobile phase b, select the appropriate mixing flow 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.) M, baseline noise (regular) N, baseline noise (irregular) O, broad peak 1, mobile phase composition change 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) There are two different cases of retention time: 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 law. 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 Equilibrium If we observe retention time drift, we should first consider whether the column is fully 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. 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. 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 contamination can usually be determined experimentally. 4: Mobile phase composition Slow changes in mobile phase composition 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 reversed-phase packing column with a small pore size and good end-end sealing uses nearly 100% water as the mobile phase, sudden loss of separation and significant or no retention of the analyte retention sometimes occur. It is a 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 of 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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2, column collapse
a, packed column
3. Interference peaks a. Use longer column b, change mobile phase or change column
4, mobile phase PH selection error
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.
a. Remove the guard column and analyze it. Replace the guard column if necessary. If the analytical column is blocked, remove it and clean 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.
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 compound)
d, try another method 3, secondary retention effect, ion pair a, add triethylamine (or alkaline sample)
3. The column is not balanced. a. Give enough time to equilibrate the column before each run.
a. Control the temperature of the column and the mobile phase, use the heat exchanger before the detector, and 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 contaminated or has a 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, resulting in 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. Using high-quality chemical reagents and HPLC-grade solvents 8. Strongly retained substances in the sample (high K' values) are eluted as a peak sample, showing a progressively elevated 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
1. There is 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, salt release and abnormal noise. Replace the pump seal if necessary.
3. The mobile phase is not completely mixed. a. Mix by hand to make the mixture even or use a solvent with low viscosity. 4. Temperature influence (column temperature is too high, the detector is not heated)
a, reduce the difference or add heat exchangers 5, there are other electronic devices on the same line a, disconnect the LC, detector and recorder, check whether the interference comes from the outside, and correct it.
6. Pump vibration a. Add pulse damper to the system.
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. Selecting the mutually soluble mobile phase. 4. Problems with the detector/recorder electronics. a. Disconnect the detector and recorder power, check and correct.
5, there are bubbles in the system a, clean the system with a strong polar solution 6, there are bubbles a in the detector, cleaning detector, install background pressure regulator 7 behind the detector, flow cell pollution (even very few pollutants) It also produces 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, the column packing loss or block a, replace the column 10, the mobile phase is not mixed uniformly or the mixer does not work Normal a, repair or replacement of the mixer, it is recommended not to use the pump mixing device when the mobile phase does not go through the gradient
a. See section 3. Check for loose joints, leaking pumps, salt out and abnormal noise. Replace the seal if necessary.
4. Incorrect detector setting a, adjustment setting 5, extra-column effect a, column overload b, detector response to reaction time or pool volume is too large c, pipe between column and detector is too long or The inner diameter of the pipe is too large d, the response time of the recorder is too long a, the small volume injection (for example: 10ul instead of 100ul) dilutes the sample b in a ratio of 1:10 or 1:100, reduces the response time or uses less circulation Pool c, use short pipe d with inner diameter of 0.007-0.01, reduce response time 6, buffer concentration is too low a, increase concentration 7, protect column contamination or failure a, replace guard column 8, column contamination or failure, tower Lower the number of plates a, replace the same type of column. If the new column provides a symmetrical peak, flush the old column with a strong solvent.
9. The column inlet collapses a, opens the column inlet, fills the collapse or replaces the column 10, presents the peaks of two or more substances that are not completely separated, selects other types of columns to improve the separation effect, and the column temperature is too low. a. Increase the column temperature. Unless otherwise specified, 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. Block the guard column or the analytical column. 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 sieve at the inlet or replace the column.
Q, all peak areas are too small 1, detector attenuation setting is too high a, attenuation reduction setting 2, detector time constant setting is too large a, setting a small time constant 3, injection amount too Less a, increase the injection volume R, all the peak areas are too large 1, the detector attenuation setting is too low a, take a large attenuation 2, too much injection a, reduce the injection volume 3, recorder connection Incorrect a, correctly connected to the recorder
S, troubleshooting of high performance liquid chromatography HPLC retention time drift
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.
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.
If there are strong components on the column in the sample, it may be a potential source of retention time drift. 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 used under 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. 