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Top of Liquid Chromatography Q&A
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Q1
The pressure was low at first but gradually increased during use. What is the cause, and what is the solution?
A1
The filter or column inlet may be blocked. If the pressure exceeds 20 MPa, the inlet filter is probably blocked. Once the pressure has increased, attach the column in the reverse direction and let the liquid through without connection. If the problem persists, replace the column. Remove non-dissolved matter in a specimen in advance by filtration or other means.
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Q2
We are performing analysis using a guard column. The column pressure has increased and the column cannot be used under the same solvent conditions as before. What should we do?
A2
If the pressure of the analytical column is normal when the guard column is removed, replace the guard column only. If the pressure of the analytical column is also high, replace both the guard column and the analytical column.
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Q3
We are now performing analysis by the ion-pair method, which works quite well but slight leading occurs before the main peak on some days. When using a new column or a rinsed column, the original good peak is difficult to restore and may be split in the worst case. There is no problem with analysis after stabilization, but perhaps there is a problem with column conditioning or the instrument?
A3
The ion-pair method takes time for column equilibrating. When using a new column, be sure to perform full equilibrating. Usually, column rinsing is not recommended. Another possible cause is the influence of sample solvent. Prepare a sample (final dilution) in a mobile phase containing an ion-pair reagent. When using the autosampler, it is recommended to use a solution of organic solvent composition equal to the mobile phase minus salts for the rinsing liquid. If a solvent of extremely different composition (especially high elution power) is used, the sample may be diffused and the peak shape may be affected.
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Q4
When STD was prepared from methanol only, the peak was split. In this case, what kind of solvent should be used for preparation from STD or formulation?
A4
In principle, a sample is generally prepared in a mobile phase. Make the preparation such that the final sample solvent will be a mobile phase. In general, a lotion or powder sample is directly dissolved into a mobile phase. For an emulsified sample, methanol is added and ultrasonic dispersion is applied to destroy the emulsification. Then water is added to make the sample closer to the mobile phase in water content. The sample is ultrasonically dispersed and filtered.
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Q5
We are trying reproducing some application with an RI detector. Why is the solvent peak so large, and why the object peak small and having a bad shape?
A5
Regarding the peak intensity, the sample concentration may be lower than the application data. The sample solvent and the rinsing liquid of the autosampler may affect the peak shape. Change the sample solvent to a mobile phase and the rinsing liquid to a solvent of the mobile phase without salts.
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Q6
The chromatogram in the column report attached to the column cannot be reproduced. The peak becomes like a bar. Why? When was the data in the column report obtained?
A6
The sample may be too much for the packing materials. Adjust the sample concentration so that the peak height is 200 to 300 mV. The column report chart was obtained immediately after its packing process.
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Q7
Why does the retention time fluctuate?
A7
A gradual decrease of the retention time indicates that the column may have deteriorated by the dissociation of functional groups. Fluctuation of the retention time suggests a problem with the pumping stability of the mobile phase and the temperature stability of the column oven. Regarding the pumping stability, check the actual pumping rate of the mobile phase with a pipette, a volumetric cylinder, or other tools. Since the oven is affected by the room temperature, change the set temperature to at least +5°C higher than the room temperature if it is close to the room temperature. In addition, if a solution of higher elution power than the mobile phase is used for the sample solvent, the retention time may be affected. Dissolve or dilute a sample with the mobile phase.
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Q8
Why does the peak area change?
A8
A change of peak area may be attributable to the precision of the autosampler, the sample stability, or the conditions of the mobile phase. The precision may become low if air is trapped in the syringe of the autosampler or there is no rinsing liquid. Check the autosampler operation. The reproducibility is also low if the sample is unstable in the sample solvent or mobile phase or is adsorbed to the sample tube.
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Q9
What are the possible causes of unstable data?
A9
Unstable data may be due to the autosampler, the column, or the substance to be analyzed. The most probable cause is the autosampler. Is the injection precision of the autosampler ok? Is a solution of equivalent composition of mobile phase minus salts used for the rinsing liquid? If a solvent of extremely different composition (especially high elution power) is used, the sample may be diffused and the peak shape may be affected. If the column is deteriorated, the peak shape or the retention time may change. Is the chromatogram the same as before? In case of column deterioration, the peak area tends to become large or small gradually. If the target compound is unstable in the mobile phase, the precision may become low. For example, the result may vary at pH where both ionized and non-ionized forms coexist. Check the pKa of the target compound and pH of the mobile phase. For the pH of a mobile phase, usually set a value 2 away from the pKa value of the target compound (pKa±2).
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Q10
We are performing gradient analysis with acetonitrile but the baseline drift is too large for analysis. Why?
A10
There may be a problem with the grade of acetonitrile. The absorption of solvent itself at the UV region differs greatly between HPLC-grade acetonitrile and special-grade acetonitrile: absorption at the short wavelength is small for the HPLC grade but not for the special grade. Therefore, if gradient analysis by detection at a short wavelength (e.g. 215 nm) is performed with special-grade acetonitrile, the baseline may drift greatly. Be sure to use HPLC-grade acetonitrile. Also for methanol and THF, use HPLC-grade solvents. In particular, THF of other than the HPLC grade contains antioxidant and has high absorbance.
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Q11
When using the column for gradient analysis, we emptied the solution on one side by mistake. Is the column now unusable?
A11
In this kind of case, a liquid flow usually restores the column. Start the liquid flow as follows:
1. Remove the outlet of the column.
2. Let through the liquid of the initial concentration (initial composition) of gradient. Check that the mobile phase comes out for the column outlet. *If the flow of the initial composition is not possible, let through acetonitrile of 50% or less. If the aqueous mobile phase is buffer solution and causes precipitation in the column, let through water (if necessary, water of about 40°C). If a high pressure is imposed, reduce the flow rate.
3. Once the liquid flow has recovered, stop pumping once, connect the column outlet, and start pumping again.
4. After checking that the liquid flows to the last outlet of the tubing, set the analytical conditions to the highest acetonitrile concentration and purge air thoroughly. *Since air may be trapped also in the detector cell, purge air thoroughly.
5. Check that the baseline has become stable and then restart the analysis.
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