Troubleshooting in amino acid column pico-tag

I have agilent 1260 infinity II instrument and i found that pressure is reaching 400 without installation of column , what is the problem ? and how to solve it ?

How to decide the known/unknown impurity limit in Related substance??

How injection volume effect on peak shape or results.?

Capacity factor calculation

Preparation of a sample of methylcobalamin in liquid form, in low concentration for analysis in HPLC. Any ideas?

How to calculate the S/N ratio?

Have you heard about capillary HPLC?

Capillary HPLC is an even more miniaturized form of HPLC, where the columns have internal diameters of less than 1 mm, typically in the range of 50 to 250 µm. Capillary HPLC systems are specifically designed for ultra-low sample volumes and are highly sensitive due to the very small column dimensions.

Column Size: Capillary columns are extremely narrow and often have lengths ranging from 10 to 100 cm. This provides a high surface area for interaction between the stationary phase and analytes, enhancing resolution and separation power.

Sample Volume: Due to the narrow diameter of the column, sample injections are even smaller than in micro HPLC, often in the nanoliter to microliter range. This makes capillary HPLC ideal for ultra-low-volume analysis, such as when working with highly valuable or limited samples.

Advantages:

1. Very High Sensitivity: Capillary HPLC offers the highest sensitivity due to the small sample size and the high surface area-to-volume ratio of the column.

2. Low Solvent Consumption: The smaller column dimensions mean that far less mobile phase is required, which is both economical and environmentally friendly.

3. Faster Analysis Time: With shorter, narrow columns, capillary HPLC can provide faster analysis times compared to traditional HPLC methods.

Challenges:

1. Higher Backpressure: Capillary HPLC systems generate very high backpressure due to the narrow column size, which can strain the system and require special equipment.

2. Injection Limitations: Injecting a very small volume accurately can be challenging, and special injectors or autosamplers may be required to handle such low volumes.

3. Technical Complexity: The setup and maintenance of capillary HPLC systems can be more complex compared to standard systems, requiring more precision and careful handling.

Applications:

1. Proteomics and Genomics: Used in applications where high sensitivity is crucial, such as in the analysis of peptides, proteins, or nucleic acids in biological research.

2. Trace Analysis: Ideal for the analysis of trace contaminants in highly sensitive environments, such as pharmaceuticals, forensic analysis, or environmental monitoring.

3. Microanalysis in Clinical Chemistry: Used for analyzing extremely small samples in clinical diagnostics, such as blood or urine samples.

Have you ever applied this approach when testing selectivity during analytical method validation? I've red about this sometimes, but I usually don't use this approach for selectivity testing while developing, I tend to use more they clasic PDA-assisted spectral evaluation of impurities profile.

For calculation of k prime and

Can you tell how can we calculate the peak height of any peak of hplc

How do i turn off the lamp automatically after my last peak of interest is eluted off, to save lamptime

High-Performance Liquid Chromatography (HPLC) is more than just a tool — it's the backbone of accurate pharmaceutical testing. But without proper calibration, even the best system can lead you astray. Here's why calibration matters, and how to do it right.

Why HPLC Calibration Is a Must
Trust Your Data
Imagine spending hours running tests—only to realize your results are off. Calibration ensures your data is accurate and consistent, every single time.
Stay on the Right Side of Regulations
The FDA and EMA aren’t just suggesting calibration—they require it. Validated, calibrated systems are a must for passing audits and staying compliant.
Protect Product Quality
In pharma, quality is everything. Calibration helps you avoid unexpected errors that could compromise patient safety and company reputation.
What Needs to Be Calibrated?
Pump Calibration
Flow Rate Accuracy: Use a certified flow meter to check that your flow rate matches the set value.
Pressure Consistency: Keep an eye on system pressure—unexpected changes might signal deeper issues.
Detector Calibration
Wavelength Accuracy: Verify with standard calibration filters to ensure your detector is locked onto the correct wavelength.
Linearity Check: Confirm your detector’s response is proportional across different concentrations. No surprises, no drifts.
Column Performance
Efficiency & Resolution: Use standard mixtures to check that your column is still separating compounds like a champ.
System Suitability Tests
Before running samples, confirm everything’s working as it should: retention time, peak area, theoretical plates, and more.
Best Practices to Keep Things Running Smoothly
Routine Maintenance = Peace of Mind
Don’t wait for a breakdown. Regular servicing keeps your system—and your confidence—running strong.
Use Certified Standards
Accuracy starts with the right tools. Always rely on certified reference materials for calibration.
Document Everything
Keep a detailed log of every calibration. If something goes wrong, you’ll have a clear trail to track it down.
Train Like It Matters (Because It Does)
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What will be the concentration of any API for LOD ,& LOQ estimation , if signal to noise ratio concentration is 0.001% ?

While running a long sequence, I started seeing ghost peaks appear randomly in blanks and sometimes in unrelated samples.

At first, I thought it was carryover, but after thorough needle washes and even changing vials, the issue persisted. I eventually traced it back to mobile phase contamination. I had reused an old acetonitrile bottle and later realized it had absorbed impurities from the environment.

Switching to freshly opened solvent bottles solved the issue. Now I always label solvent bottles with opening dates and discard them regularly.