Quality Control in Chromatography: How to Prevent Bad Results from Bad Buffers

Accurate pH control is a critical quality control step in the biopharmaceutical industry. It is mandated under cGMP standards.
Drug formulation and manufacturing processes need to keep tight control on pH to prevent variability in molecule solubility, stability, sample precipitation, protein expression, and overall cell physiology. These issues can negatively impact chemical reactions and even affect liquid chromatography instruments.
An important part of this process is the preparation and use of quality buffers. There are several manual steps that take time to perform but are essential for accuracy. For better quality control and efficiency, many manufacturers today are automating the entire reagent preparation process.

How do pH changes affect the drug manufacturing process?

During research

R&D efforts will be inconsistent and inconclusive if a high-quality buffer is not used, and the pH changes. This is a drain on time, budgets, and the productivity of skilled resources.

During liquid chromatography

Bad buffers can hold up the production process during QC. Liquid chromatography is a gating factor. This step separates samples into their individual parts for analysis and verifies that drug formulations pass quality standards.
Variations in buffer preparation during chromatography can affect product recovery and purity. Without consistency, QC teams cannot release production batches. This can cause expensive delays.
pH can change after an injection is made – when the sample and the mobile phase mix, particularly if the sample’s pH is significantly different from the mobile phase’s pH. Additionally, a mobile phase can absorb carbon dioxide when left standing, making it more acidic.
pH changes also affect the retention time (RT). RT is the time taken for a solution to pass through a chromatography column. With ionizable or ionic samples, even a minor change of 0.1 pH units can change RT by as much as 10%. This affects throughput and the overall analysis.

Other reasons influencing buffer quality

Buffer stability

While some buffers like phosphate and acetate are more stable, others may be more volatile and experience loss over 24 hours.

Microbial growth

Buffers for HPLC (High-performance liquid chromatography) are breeding grounds for microbial growth at room temperature. Buffers must be checked for contaminants before use.


Biological buffers in an organic solvent eluent are susceptible to precipitation. Buffers must be inspected for particulate crystals and monitored for changes in concentration.

Color change

Buffers should be inspected for a change in color as it indicates degradation. Analysts must measure how long a specific buffer can sit before it changes color.


When preparing the mobile phase during chromatography, buffers can become turbid, i.e., increase opacity. Cloudy buffers must be discarded.

Impact of bad buffers on chromatography instruments

Using bad buffers can adversely impact the quality of instruments over time.

  • Without the right buffer quality, the entire system must be flushed.
  • If microbial contaminants are present, entire columns and many consumables like pump seals may need replacing. These costs can add up.
  • It can take anywhere from a few hours to days to reset the entire system before it can be used again.
  • If several consumables and components must be replaced, the entire qualification can be considered invalid and will have to be requalified. This process needs a validation engineer, a highly-skilled resource. Not every metrology team has a validation engineer onsite. Based on the engineer’s availability, requalification can take more than 2-3 days.

Once a system is back online, analysts will have to validate the entire system.

Challenges in validating buffer quality

Degradation, drifting base, and peak tailing in chroma peaks can offer insights into what aspects need adjustment. Overlapping peaks can indicate a loss of resolution. Analysts can use this information to increase column length, decrease particle size, adjust the temperature of the solution, and more. If the RT shifts, there may be an increase of back-pressure in the column from contamination or a clogged frit.

If a bad buffer has been used, the instrument must be tested and checked for trapped air or air bubbles. Leaks must be identified, and the system must maintain constant pressure. This process can take 15-20 minutes.

Next, a set of visual inspections are needed to confirm precipitation, color, and turbidity. Buffer density must be tested too.

Once these steps are done, analysts must verify lot numbers on specific chemicals to detect potential issues with an entire batch of chemicals being used. For UPLC (Ultra  Performance Liquid Chromatography) or LCMS (Liquid Chromatography-Mass Spectrometry), buffers should use ultra-pure chemicals.

Checking for buffer quality and the accurate functioning of the chromatography instrument can be cumbersome but it is critical.

Since many aspects of the buffer validation and QC process are manual, they rely on the skill and meticulousness of each team member conducting the test. There is no detailed end-to-end visibility or control of the entire process.

Automated reagent preparation offers quality and efficiency

Fortunately, the entire solution preparation workflow can be automated. Simple, automated reagent preparation solutions like Revo from LabMinds can help eliminate the risk, errors, stress, costs, and delays from the use of bad buffers. Powders and liquids are automatically mixed to create the perfect buffer formulation. Reagents can be prepared ahead of time, even before the team walks through the door.

The entire process reduces the risk of human error with quantities and types of chemicals being used. Chemical containers are bar-coded to prevent mix-ups. Solution creation and expiry dates are imported into ELN and LIMS and printed on the solution labels.

Additionally, the cloud-based platform means the Revo can be operated remotely. With digital inventory tracking and management, teams can automatically track expiration dates and reorder what they need, when they need it. This offers the flexibility to plan in-lab time effectively.

Want hassle-free, reproducible results every time? Learn more about what Revo can do for you.