Causes Of Pressure Fluctuations in HPLC

Pressure fluctuations in high-performance liquid chromatography (HPLC) can lead to issues with the system's stability and performance. Several factors can contribute to pressure fluctuations, and it's important to identify and address these issues to maintain reliable chromatographic results. Here are some common causes of pressure fluctuations in HPLC:

🏛 Mobile Phase Composition: Changes in the composition of the mobile phase, such as variations in solvent ratios, can lead to fluctuations in pressure. It's important to ensure that the mobile phase is properly prepared, filtered and maintained throughout the analysis.

🏨 Air Bubbles: Entrapped air bubbles in the mobile phase or within the HPLC system can cause pressure fluctuations. Bubbles can form in the solvent reservoir, tubing or in the injector. Proper degassing of solvents and careful handling of the mobile phase can help minimize the formation of air bubbles.

🏡 Leakages: Any leaks in the HPLC system, including in the tubing, fittings or the column, can lead to pressure fluctuations. Regular inspection of the system for leaks and prompt repairs are crucial to maintaining system integrity.

🏯 Column Issues: Problems with the chromatographic column, such as blockages, irregular packing, or a damaged frit, can cause pressure fluctuations. Regular column maintenance and inspection are essential to prevent these issues.

🏣 Pump Issues: Malfunctions or irregularities in the HPLC pump can lead to pressure variations. This may include issues such as pump pulsation, air in the pump, or problems with check valves. Regular maintenance and calibration of the pump are important for stable operation.

🏭 Detector Issues: Problems with the HPLC detector, such as air bubbles in the flow cell contamination, can lead to pressure fluctuations. Regular maintenance and troubleshooting of the detector is essential.

💒 Degraded or Contaminated Solvents: The use of degraded or contaminated solvents can impact system stability and cause pressure variations. Ensure that solvents are of high purity and properly stored to prevent degradation.

🏘 System Equilibration: Sudden changes in flow rate or mobile phase composition during method runs can lead to pressure fluctuations. Allow sufficient time for the system to equilibrate after making changes to prevent sudden pressure spikes.

🏪 Sample Matrix: Highly particulate or contaminated samples can cause blockages in the system, leading to pressure fluctuations. Proper sample preparation and filtration techniques should be employed.

How to avoid gas trouble

 How to avoid gas trouble … well, in your HPLC at least.

If you do not degas your mobile phase, then:

-  tiny bubbles in your HPLC’s fluidics may stop the pumps, or ...
-  micro-bubbles inside the detector will create really weird chromatograms.

Hence, thou shalt degas.

Five methods exist:

1)  Solvent refluxing. The best method.

It's been around since 1976. Tested on all HPLC solvents. And is almost 100% effective. An inexpensive reflux condenser will suffice.

The problem is, refluxing is a pain. Takes too much time. That's why no one uses it - but should.

2) Helium sparging:

In 1979, Spectra-Physics (now part of Thermo), obtained a patent for an in-line He-sparger. Their HPLC's were the first to use low-pressure solvent mixing, that I've discussed before.

Outgassing was a problem with these HPLC's. The in-line helium sparger was a clever solution.

The patent couldn't stop others from making their own He spargers, though!

Essentially, we bubble helium through the solvent in a controlled manner, for a few minutes.

It may seem odd to use a gas for degassing.

Helium has virtually no solubility in any solvent. Forcing helium through a solvent will displace other gases and reduce the partial pressure above the solvent. This will 'pull out'  gases, so to speak. The efficiency can be enhanced by applying a gentle vacuum.

It's based on Henry's Law. Do look it up.

He sparging is hardly seen in Indian labs. This is because Helium is very expensive.

3)  Ultrasonication:  As a cheap alternative, this method is not too bad.

The technique is not efficient by itself.  But when used in combination with in-line membrane degassers, ultrasonication will generally serve the purpose.

4)  In-line degassers.

Modern HPLC's come with in-line membrane degassers. These compact modules use a semi-permeable membrane across which solvents flow, on their way to the HPLC column. Some models have a vacuum option. The idea is that dissolved gases will diffuse out through the membrane.

Membrane degassers have improved considerably, but they're still not very efficient. They are convenient though, and quite reliable when used in combination with ultrasonication.

5) Vacuum filtration.

Filtering a solvent through a 0.45 or 0.2u membrane, under hard vacuum, does reduce dissolved gases. If you don't have an ultrasonicator, you can try vacuum filtration.

If you're using a buffer, then vacuum filtration is a must.

No matter which degassing method you use, degas your solvents separately - even for simple isocratic mobile phases.

Degassing can alter your mobile phase composition, if you first mix and then degas.

Therefore, always degas first and then mix.

Bottom-line:  For freedom from gas, use reflux. Or else, ultrasonicate -and use an in-line membrane degasser.

Never forget the law of GIGO - Garbage in, garbage out.

Cheers ... SK Srinivas, Chromatographer.
Bangalore.

Chromatography ... peaks for itself!

Performance Verification Test for Dissolution Apparatus (PVT)

A Performance Verification Test (PVT) is a comprehensive test outlined in the United States Pharmacopeia (USP) General Chapter <711>. It assesses the functionality of an entire dissolution apparatus setup, with a focus on demonstrating its suitability for use. The test employs Prednisone tablets as a reference standard. The primary objective of the PVT is to offer evidence of the instrument and apparatus being appropriate for their intended purpose.

The PVT evaluates various aspects, including measurable, dimensional, and operational parameters, which are crucial factors in determining the suitability of the dissolution apparatus. By conducting the PVT, it ensures that the entire dissolution system is functioning correctly and meets the necessary standards, thereby instilling confidence in its performance.

In recent developments, the USP has introduced a new 'Dissolution Performance Verification Standard' (DVPS) by reformulating Prednisone tablets. This change aims to enhance the reliability of performance qualification for both Dissolution Apparatus 1 (basket) and Dissolution Apparatus 2 (paddle). The new DVPS tablets feature a ball-shaped design, a departure from the previous convex tablet shape, and exhibit distinct behavioral characteristics.

The new formulation of Prednisone, represented by DVPS tablets, has been officially adopted as the standard for PVT as of May 1, 2023, coinciding with the revision of USP General Chapter <711> Dissolution. Extensive studies support the advantages of DVPS tablets, demonstrating their increased sensitivity to operational and mechanical variables within the instrument setup. Additionally, they are less sensitive to media degassing and yield more consistent results.

According to USP, the introduction of DVPS tablets is expected to provide greater accuracy, with a coefficient of variation (%CV) under 5% in both Dissolution Apparatus 1 and 2. These improvements contribute to a more reliable and precise performance verification process for dissolution apparatuses.