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.
No answers yet.
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?
No answers yet.
No answers yet.
Can anyone help me with HPLC method for mannitol with UV or RID detector?
just stick to RID because mannitol can only absorb low wavelengths under UV especially above 200nm. But if you are demanded to use UV detector, you will need to react the mannitol with PMP
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
the compound i found is ciprofloxacin.
the peaks are not well separated
No answers yet.
How to calculate xrd quantification for tablet
No answers yet.
I wont method of assay the Enilcanzol in HPLC
1- Mobile phase
2- Parameter
3_ Colum
4- preparation of sample and standard
No answers yet.
Types of Band broadening in hplc
No answers yet.
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.
---
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.
---
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.
---
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).
No answers yet.
No answers yet.
No answers yet.
Hi there
We have shimdazu lab solution softwear . Where signal to ratio calculation is difficult.
Someone who can guide how to calculate ?
Activate QC parameters in METHOD drop down menù. Select noise and drift and set critiria. Normally i use ASTM for noise and save the method, then start a run without injection (-1 in the batch vial position). In the browser open the data file, drag over the method. In a drop down menù you find execute QC and noise drift report in created with resulta pass o fail. there are other method to obtail noise drift calculation in labsolutions software but this is the simplest
In Lab solutions to calculate signal by noise ratio first integrate blank and use the option in integration parameters select the option of signal and drift select the usp not astmand save.
after blank integrate the analyte and keep the spectral reference data of blank it automatically provide the s/N
Can we do custom calculations with Shimadzu Lab solutions software? If yes what are the steps to be followed?
No answers yet.
Someone has to work with condroitín and Glucosamine sulfato, HPLC PDA 195 nm ?
No answers yet.
What is the possible causes that affect peak to valley ratio?
Standard preparation? System? Column?
But I'm using a new column
mostly caused by poor column. maybe you probably injected too much sample which exceeded the capacity of the column.
if you r using new columns the wrong standard preparation and in few cases if system is not in good condition.