Your graduation is small (10mAU) and the peaks are not very high.

I advise you to store your HPLC System in ETOH 20%, rinse with at least 30mL milliQWater and the same with your buffer. You must observe a stable baseline before starting your purification program with the column connected. Pass as much buffer as necessary to obtain it.

I am not so clear on this before getting more information from you. It may be the apendorf/vessel used was of plastic and glass.

If the repeatation of these splitting is uniform then it may be of plasticizers. Please check how to overcome the plasticizer repeated peaks in hplc, lcms

Sasha Nealand: Your irrational question/observation is attracting irrational responses on RG.

You are not "getting spiit peaks" at all. That is most likely the injector pulse. Using HPLC requires an understanding of the subject.

• In your example where no column is inline with the detector (which is an example of what we refer to as HPLC "Flow Injection"), no chromatography of any kind is taking place. *A slug of liquid is simply being moved through the tubing into the detector and out to waste. The first "peak" observed is the pressure pulse from the injector, not your sample.

You are injecting 100ul of a sample into the flow cell of the UV detector (the flow cell probably has a volume of around 10 ul so it fills the cell slowly over time). You are doing this with little to no system back-pressure, since no COLUMN is installed. Therefore the sample is being pushed through the flow cell as a liquid, which also contains gas, generating a signal. You may be viewing the refractive differences between the sample diluent and the mobile phase, but nothing else. No useful troubleshooting can be conducted w/o a column or restriction in place.

Please contact someone at your school who has formal training in chromatography for assistance (industrial experience > 5-years) and read one of the many classic texts on "Liquid Chromatography" for more information on the basic principles.

I think you are using the HPLC as a automatic sample injection system instead of a syringe pump. This can be useful when you are building an spectral library at home.

The flow through several meters of tubing can explain the split.

If you want to reduce the splitting, check the length and ID of the liquid lines.

I agree very much with William Letter flow injections are possible, if You know the spectroscopy of a) Your eluent, Your analyte, and the solvent You used to dissolve the sample well.

If You do not, You better do some real chromatography with a column in. Indeed You will observe that in nearly all systems You will see an injection peak at the void time of Your system.

Split peaks in HPLC analysis of a pure sample can arise from various factors. Here's a structured approach to diagnose and resolve the issue:

1. Column Issues

• Column Damage/Packing: Check for voids or channels in the column. Replace with a new column if necessary.
• Overloading: Reduce injection volume or sample concentration to ensure the column isn't overloaded.

2. Solvent Mismatch

• Mobile Phase vs. Sample Solvent: Ensure the sample solvent is weaker or similar in strength to the mobile phase. Dilute the sample in mobile phase if needed.

3. Instrumental Problems

• Injector Issues: Inspect the syringe needle and seals for damage or leaks. Perform a system suitability test.
• Detector Flow Cell: Check for air bubbles or contamination in the flow cell.

4. Chemical Degradation

• Stability: Assess compound stability under HPLC conditions (pH, temperature, light). Adjust pH, lower column temperature, or protect from light if degradation is suspected.

5. Secondary Interactions

• Silanol Activity: Use a buffer to control pH or add a silanol blocker (e.g., triethylamine) for basic compounds.
• Ion Pairing: Optimize buffer concentration or pH to minimize ionization effects.

6. Diluent Incompatibility

• Miscibility: Ensure the sample solvent is fully miscible with the mobile phase. Avoid strong solvents (e.g., DMSO) with high aqueous mobile phases.

7. Pressure Fluctuations

• Pump Issues: Inspect for blockages, worn pump seals, or faulty check valves. Ensure consistent flow rate.

8. Conformational Isomers/Tautomer's

• Column Chemistry/Temperature: Test different columns (e.g., C18 vs. phenyl) or adjust temperature to resolve interconverting forms.

9. Data Processing

• Integration Settings: Review software parameters to ensure peaks aren't being split artificially.

Troubleshooting Steps

• Test Injection Volume: Reduce to check for overloading.
• Column Swap: Use a new or different column.
• Solvent Adjustment: Match sample solvent to mobile phase.
• Stability Test: Analyze fresh vs. aged samples.
• Pressure Monitoring: Record pressure during runs to identify fluctuations.

By systematically addressing each potential cause, you can identify and resolve the split peak issue, ensuring accurate HPLC analysis.

Mohammad Asaad Mousa is good at ChatGPT.

Respected Sir,

With utmost respect, when I write something, I am confident in it. I have worked in the field of HPLC for over 35 years and have conducted training courses on this equipment. You may review the references I have studied to ensure that my previous response was not an error. I am seeking DeepSeek's assistance to coordinate and condense the reply for clarity and brevity.

1) https://www.agilent.com/cs/library/posters/public/Final%20TIPS%20and%20Tricks%20HPLC%20Troubleshooting%20(2).pdf

Please read Page 11-13

2) https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/128/929/4497.pdf?srsltid=AfmBOoqzZtEy1AvqwtFU6w7BPtcBWFIW_n_9YFH5BHZEoN0Fl9vsKRrC

Please read Page 8

3) https://www.agilent.com/Library/eseminars/Public/HPLC%20Column%20and%20System%20Troubleshooting.pdf

Please read Page 16-20

4) https://www.hplc.eu/Downloads/ACE_Guide_TroubleshootingHPLC.pdf

Please read Page 2

5) https://www.bio-works.com/hubfs/Documents/TN10000001BA_Split%20peaks_in_liquid_chromatography.pdf?hsLang=en#:~:text=Common%20causes%20for%20peak%2Dsplitting,1).

Please Read Page 2

6) https://uhplcs.com/how-to-deal-with-peak-splitting-in-hplc/#:~:text=Solution%3A%20For%20optimal%20results%2C%20it,separation%20and%20minimize%20peak%20splitting.

Causes and Solutions for Peak Splitting Please read

7) https://www.chromatographytoday.com/news/hplc-uhplc/31/breaking-news/what-is-peak-splitting/31445#:~:text=Frits%20and%20Voids,the%20head%20of%20the%20column

What is Peak Splitting Please Read

8) https://www.acdlabs.com/blog/an-introduction-to-peak-tailing-fronting-and-splitting-in-chromatography/#:~:text=The%20two%20common%20causes%20of,all%20the%20peaks%20to%20split.

Please Read What is Peak Splitting in Chromatography

9) https://uhplcs.com/how-to-deal-with-peak-splitting-in-hplc/#:~:text=Solution%3A%20For%20optimal%20results%2C%20it,separation%20and%20minimize%20peak%20splitting.

Please Read From Typical Examples of Peak Splitting to theConclusion

10) https://www.linkedin.com/pulse/causes-splitting-peaks-hplc-analysis-some-possible-sanghavi-shah-/

Please Read Causes of Splitting Peaks in HPLC analysis and some possible ways to fix it.

11) https://support.waters.com/KB_Chem/Columns/WKB194672_What_are_common_cause_of_peak_splitting_when_running_an_LC_column

Please Read What are common causes of peak splitting when running an LC column

12) https://support.waters.com/KB_Chem/Columns/WKB28044_How_to_troubleshoot_split_peaks_on_ACQUITY_UPLC_column

Please Read How to troubleshoot split peaks on an ACQUITY UPLC column

13) https://www.sepscience.com/peak-splitting-in-hplc-causes-and-solutions/

Please Read Peak Splitting in HPLC: Causes and Solutions

14) https://scioninstruments.com/us/blog/hplc-troubleshooting-guide-2/

Please Read Split peaks

Mohammad Asaad Mousa: Your original reply does not address the original poster's actual question. The student poster, Sasha Nealand, who is not knowledgeable about liquid chromatography, stated that they ARE NOT PERFORMING ANY TYPE of CHROMATOGRAPHY at all, so comments regarding how to address a chromatography issue, such as 'Peak Splitting' are not relevant. No chromatography is present when running a sample through empty tubing to waste.

If HPLC without Column , the sample Pure , Mobile Phase and solvent have no Absorbance , The pure sample give response If enter through the detector in this wave length

The pure sample can give Pure beak from zero time to end the beak

William Letter I am not a student and I am very knowledgeable about chromatography. I think you are not very knowledgeable about science as you do not observe even the obvious. Obviously without a column there is no separation, for a pure sample of a single compound no separation is needed, I am using this approach to test the HPLC system without a column to try to diagnose the instrument producing poor chromatography and noisy baseline. I am using this approach of shooting a pure compound without a column to remove the factor of any of the poor chromatography I am seeing on this particular instrument coming from a column. I hope to have useful suggestions as to troubleshooting either detector, mixing valves, seals, needle seat, etc ....., not irrational dogma from inexperienced users such as yourself,

Sasha Nealand: You are most certainly inexperienced and lack a basic understanding of the technique or principles involved. I have been professionally teaching and developing commercial grade HPLC and LC-MS instruments for 30-years so see no value in your rude comments. Your profound ignorance is easily ignored. - For an HPLC pump to operate properly, a sufficient amount of system back-pressure must exist in the system for the pump to deliver a smooth flow rate. Your "approach" to troubleshoot the issue demonstrates a lack of training, understanding and experience. A restriction capillary must be installed when NO COLUMN is placed in line to produce the required system backpressure (normally you will need >40 bars). Failure to install such a restrictive device in place of the column (which normally provides the needed back-pressure) results in invalid data being collected. No accurate measurements of baseline noise can be made without such a restriction in place. We use these restrictors as tools routinely to perform PV's on instrument modules (e.g. HPLC A/S, Detectors etc). They are part of most SOP's used commercially for troubleshooting and instrument qualification or validation. BTW: Baseline noise can be sourced to many areas, including a fouled HPLC column. If you are interested in learning about HPLC operation and troubleshooting, then you may wish to invest some time reading a few authoritative articles on the topic (I will post links in follow-up posts here). I am sure that will help you, but only if you work with an experienced user locally. Please obtain the help of an experienced and professional chromatographer if you wish to solve the issue you have.

"Common Causes of Baseline Noise in HPLC, UHPLC"; https://hplctips.blogspot.com/2014/09/common-causes-of-baseline-noise.html

"The HPLC Restriction Capillary; Troubleshooting, Qualification and Running Without A Column"; https://hplctips.blogspot.com/2018/04/the-hplc-restriction-capillary.html

"HPLC Retention Time Drift, Change, Area Variability or Poor Reproducibility. Common Reasons for it"; https://hplctips.blogspot.com/2015/11/hplc-retention-time-drift-change.html