Quantifying citric acid using High Performance Liquid Chromatography (HPLC) involves several steps. Here's a general outline of the process:

1. Preparation of Standards:Prepare a series of standard solutions with known concentrations of citric acid. These solutions will be used to create a calibration curve, which relates the concentration of citric acid to the HPLC response.

2. Sample Preparation:If you're analyzing a sample containing citric acid, you'll need to prepare it for HPLC analysis. This might involve extraction, filtration, or other sample clean-up procedures.

3. HPLC Instrument Setup:Set up your HPLC instrument with the appropriate column, mobile phase, and detection method. In this case, a common detection method for citric acid is UV detection at around 210 nm.

4. Calibration Curve:Inject the standard solutions of citric acid into the HPLC instrument. Plot a calibration curve by plotting the peak area (or peak height) of the citric acid peaks against their corresponding known concentrations. This will help you establish a linear relationship between concentration and HPLC response.

5. Sample Analysis:Inject your prepared sample into the HPLC instrument. The chromatogram will show peaks corresponding to various compounds in the sample, including citric acid.

6. Quantification:Use the calibration curve to determine the concentration of citric acid in your sample. Identify the peak corresponding to citric acid in the chromatogram and measure its peak area. Then, use the calibration curve equation to calculate the concentration of citric acid in the sample.

7. Data Analysis:Calculate the average of multiple injections of your sample and report the concentration of citric acid based on the calibration curve.

Keep in mind that the specifics of the HPLC method, column type, mobile phase, and instrument settings will depend on the equipment you're using and the laboratory's protocols. It's essential to follow proper lab practices and consult with experts if needed.

Additionally, make sure to validate the method for accuracy, precision, and sensitivity to ensure reliable and accurate quantification of citric acid in your samples.

Specifically for your challenge, having a non-zero baseline or observing peaks in blank samples like methanol can be a common issue in HPLC analysis. Here are some potential causes and troubleshooting steps to address this problem:

1. Contaminated Mobile Phase or Solvent:Check the purity of the solvents you're using, especially the mobile phase. Contaminated solvents can introduce impurities and cause baseline issues. Use high-quality, HPLC-grade solvents and ensure proper storage.

2. Column Contamination:Contaminants from previous analyses can build up in the column and affect the baseline. Try flushing the column with a cleaning solution or performing a column wash according to the manufacturer's recommendations.

3. Sample Contamination:Ensure that your samples are properly filtered and free from particulates or contaminants that could contribute to unwanted peaks. Using syringe filters with appropriate pore sizes can help prevent sample contamination.

4. Detector Noise:Check the baseline noise level of your detector. If it's too high, it can create the appearance of peaks in blank samples. Consult the instrument manual to adjust the detector settings for optimal signal-to-noise ratio.

5. Injection Issues:Issues during sample injection, like air bubbles or uneven injection, can cause baseline disturbances. Ensure proper sample handling techniques and use a needle that doesn't introduce air into the system.

6. Column Degradation:If the column is old or has been used extensively, it might be degraded or contaminated. Consider replacing the column if it's past its recommended lifespan.

7. Detector Drift:Detectors can experience drift over time, leading to non-zero baselines. Perform regular calibration and validation checks on the detector to ensure accurate readings.

8. System Flushing:Sometimes, flushing the system with the mobile phase for an extended period can help remove any lingering contaminants or air bubbles causing baseline fluctuations.

9. Autosampler Issues:The autosampler can introduce issues if it's not properly cleaned or maintained. Check for leaks, sample carryover, and proper functioning of the autosampler.

10. Instrumental Issues:If you've ruled out all other possibilities, it's worth checking with the instrument manufacturer's support or a qualified service technician to diagnose and repair any potential hardware issues.

Remember that HPLC troubleshooting can be iterative and may involve testing various parameters. Document the steps you take and any changes you make so that you can track improvements or identify persistent issues. If the problem persists despite your troubleshooting efforts, it's advisable to seek assistance from experienced colleagues or the instrument manufacturer's technical support.

@Ambreen Latif

Dear Ambreen,

I always find the BioRad bulletins on HPLC very helpful:

https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1928.pdf

On page 12 you find a recommendation for organic acid separation and the influence of eluent acidity on retention time of citrate using an Aminex HPX-87H column. You can use isocratic elution and an slightly acidic eluant (3 to 12 mM H2SO4).

Good luck

Michael

This is easy (I think though,it's been >50 years!). We had an HPLC method for Citric Acid in fruit juices. If I remember it was 15% cold meta phosphoric acid (not the more common ortho!) in Methanol, PDA/DAD at 248 nm, and a common C18 ubondapak (Waters) HPLC column.

I forgot. I didn't have a C18 uBondapak but used a Spherasorb (I used the column for everything) instead.

H type ligand exchange column under isocratic flow of mild acidic D.I water and refractive index detector is advisable here...sample matrix has major effect to select the most versatile methodology.