Baseline hump and ghost peak are two irregularities seen in chromatograms in HPLC (High-Performance Liquid Chromatography).

Baseline Hump:
A baseline hump is a broad, gradual rise in the baseline of the chromatogram, often appearing like a "hill" or "wave".

Causes:
1. Gradient changes (especially in gradient elution).
2. Temperature fluctuations.
3. Contaminants in solvent or glassware.
4. Detector noise or response to solvent mismatch.

Possible Solution:
1. Use fresh, high-purity solvents (HPLC grade), filter & degas all mobile phases.
2. Ensure proper re-equilibration between runs; optimize gradient program.
3. Use a column oven to maintain constant temperature.
4. Flush the system with strong solvents (e.g., methanol, acetonitrile).


Ghost Peak:
A ghost peak is a sudden, unexpected peak that appears intermittently in the chromatogram, without a corresponding analyte or injection.

Causes:
1. Carryover from previous sample.
2. Contamination in injector, vial, or column.
3. Air bubbles or particulate matter.
4. Mobile phase contamination.

Possible Solution:
1. Rinse injection needle and loop thoroughly; use needle wash step.
2. Clean injector thoroughly.
3. Prepare fresh sample; use light-protected or cold storage if needed.
4. Replace with freshly prepared, high-purity solvent.

After completing your HPLC runs—especially when using salt-containing mobile phases—taking the right steps in column care is key to preserving performance and extending the column’s lifespan.

This visual outlines the best practices for column storage:
✔️ Using salt-containing mobile phases? Always flush with a salt-free solvent before storage.
✔️ Using non-salt mobile phases? You can go straight to the wash step.
✅ In both cases, finish with a wash using 100% organic solvent (e.g., acetonitrile), then seal the column with end plugs.

? The second part highlights how column quality dramatically affects chromatographic performance:
• Well-end-capped, inert columns deliver sharp, symmetrical peaks.
• Poorly end-capped columns result in distorted peaks, even after flushing.

? Column chemistry, cleaning protocols, and storage conditions all play a vital role in achieving high-resolution, reproducible results and optimal sensitivity.

? What’s your post-analysis storage routine? Do you follow a flushing protocol every time?

Can anyone help me with HPLC method for mannitol with UV or RID detector?

can yoi help me to be sure that this is the real retention time of this compound? if not? what is the real retention time.
Colun: 4,6 x 150mm
mobile phase : A) TFA 0,025 % + water, and B) Acetonitril + TFA
Time: 12 min
inj: 10 ųl
debit: 0,5 ml/min
267 nm
temperature: 30°c

How to calculate xrd quantification for tablet

I wont method of assay the Enilcanzol in HPLC
1- Mobile phase
2- Parameter
3_ Colum
4- preparation of sample and standard

Types of Band broadening in hplc

1. Pump Calibration

Q1. What is the acceptance criterion for flow rate accuracy?
A1. The flow rate should be within ±2.0% of the set value.

Q2. How is flow rate accuracy measured in HPLC?
A2. By collecting HPLC grade water in a calibrated volumetric flask and recording the time to fill a known volume using a stopwatch. Flow rate = Volume (mL) / Time (min).

Q3. What is the acceptance criterion for flow rate consistency?
A3. The %RSD of retention time of caffeine should be NMT 1.0%.

Q4. How is flow rate consistency evaluated?
A4. By injecting 10 ppm caffeine solution six times and calculating the %RSD of the retention times.

Q5. What is meant by compositional accuracy?
A5. It refers to the accuracy of gradient composition delivery. Measured using 0.25% acetone in water and recording absorbance changes.

Q6. What is the delay volume limit for the pump system?
A6. The delay volume should be NMT 1.0 mL.


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2. Autosampler Calibration

Q7. What is the acceptable range for injection volume accuracy?
A7. The average volume for 10 injections should be 20 ± 0.4 µL.

Q8. How is injection volume accuracy determined?
A8. By weighing the HPLC vial before and after 10 injections and using the difference to calculate average injection volume.

Q9. What is the limit for injection volume precision?
A9. %RSD should not be more than 1.0%.

Q10. How is injection volume linearity evaluated?
A10. By injecting 5, 10, 20, 50, and 100 µL and plotting the peak area vs. volume. R-square should be NLT 0.9990.

Q11. What is the acceptance criterion for autosampler temperature?
A11. The measured temperature should be within ±2°C of the set temperature.


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3. Column Oven Calibration

Q12. What are the required set temperatures for column oven calibration?
A12. 60°C, 50°C, 30°C, 20°C, and 10°C.

Q13. What is the acceptance limit for column oven temperature accuracy?
A13. The measured temperature should be within ±2°C of the set temperature.

Q14. How is the temperature measured?
A14. Using a calibrated probe with a digital thermometer.


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4. Detector Calibration

Q15. How is wavelength accuracy evaluated?
A15. Using known UV maxima and minima, such as Maxima 273 ± 2 nm, Maxima 205 ± 2 nm, and Minima 245 ± 2 nm.

Q16. What is the acceptance criterion for detector linearity?
A16. R-square (correlation coefficient) should be NLT 0.9990.

Q17. What is the typical injection volume used for detector calibration?
A17. 10 µL.

Q18. What is the standard mobile phase composition for calibration?
A18. Methanol:Water (50:50 v/v).

Hi there
We have shimdazu lab solution softwear . Where signal to ratio calculation is difficult.
Someone who can guide how to calculate ?

Can we do custom calculations with Shimadzu Lab solutions software? If yes what are the steps to be followed?

Someone has to work with condroitín and Glucosamine sulfato, HPLC PDA 195 nm ?

What is the possible causes that affect peak to valley ratio?

Standard preparation? System? Column?

But I'm using a new column