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.

What should I do if my chromatogram encounters a leading edge peak?

 What should I do if my chromatogram encounters a leading edge peak?

✔ 01The influence of frontier peak

1️⃣ A. Calculation of impact peak height

When the peak areas are the same, their peak heights will differ greatly due to the peak front. This has an impact when we use peak height to calculate sample results, especially when calculating the minimum detection limit.

2️⃣ B. Calculation of peak area affected by

When calculating peak areas, the start and end points of the peak are generally determined by the slope of the chromatographic peak. At the front of the chromatographic peak, the flatter the front baseline, the more difficult it is to confirm the starting point, resulting in inaccurate peak area quantification.

3️⃣ C. Affects the confirmation of certain trace components

When the resolution of the previous peaks is not very good, the sample peaks will be easily hidden at the leading edge of the later peaks and cannot be calculated.

✔ 02. Cause analysis and solutions for frontier peaks

1️⃣ A. Column overload

Each chromatographic column has its maximum sample carrying capacity. When the sample amount is too large, overloading will occur, including mass overload and volume overload. We can see whether the peak shape is improved by reducing the sample amount or sample concentration. It can also be solved by increasing the column diameter and using a higher capacity stationary phase.

2️⃣ B. Improper selection of sample solvent

The leading edge peak occurs when the elution power of the sample solvent is much stronger than that of the mobile phase. Specifically, if the sample moves forward uniformly in the chromatographic column, we will usually get a normal distribution of peaks. When the sample solution reaches the chromatographic column in a relatively short time after injection and is not fully diluted by the mobile phase, due to the presence of a sample solvent with relatively strong elution ability, some samples are eluted faster and pass through the chromatographic column quickly, and finally Leading to peak front extension.

3️⃣ C. Chromatographic column damage

After long-term use of a chromatographic column, the silica gel packing will dissolve and the column bed will collapse, resulting in reduced column efficiency and leading to peak fronting. If it is found that the column is damaged, it is recommended to replace the column directly.

4️⃣ D. Peak interference

The two compounds co-elute, that is, a small peak appears before the large peak, and the chromatographic peaks are not separated. Interference can be reduced by adding sample purification procedures. The mobile phase can also be adjusted to improve resolution.