Bridging Analytical Injections and Preparative Column Volumes: Alkaline pH Stability of NanoPak‑C All‑Carbon Columns
- 6 days ago
- 5 min read
A common question we receive when discussing our analytical NanoPak C All Carbon HPLC columns is: how many injections can they withstand at alkaline pH? A related question for our semi-preparative and preparative columns is: how many column volumes of alkaline mobile phase can they endure?
The stability of these columns at alkaline pH is important because a large share of current and future
drug candidates are ionizable and contain at least one basic center, including many peptides, proteins, oligonucleotides, and other biologics. These molecules often separate more effectively at pH values above 8. In some cases, pH > 8–9 is necessary to fully exploit their pKa profiles (see our blog on pKa to understand why) [1,2]. Conventional silica-based reversed-phase materials are known to suffer from silica dissolution and accelerated degradation under alkaline conditions, especially at elevated temperatures. So understandably, customers continue to ask about alkaline-pH stability [3].
As mentioned above, analytical scientists typically think about column lifetime in terms of number of injections. Still, at high pH, this can obscure the extent of chemical stress the stationary phase has actually experienced. In bioprocessing and prep LC, stress is often described in column volumes: how many times the column’s void volume has been flushed with mobile phase at the operating pH. This column volume view is central to our recent work.
What we did. In our latest application note, we evaluated 50 x 4.6 mm and 150 x 4.6 mm NanoPak C all-carbon analytical columns, both with the same chemistry, under alkaline conditions using GLP-1 analogs Semaglutide and Liraglutide at pH 8.5. We used the method from our white paper: a 17-minute gradient, 20 mM ammonium acetate buffer at pH 8.5 with acetonitrile, UV at 220 nm, and injections of 1 mg/mL solutions.
For each column, we ran 500 injections of Semaglutide and 500 injections of Liraglutide (1,000 injections total). At injections 0, 250, and 500 for each analyte, we monitored:
Retention time for Semaglutide and Liraglutide
Efficiency (theoretical plates)
System backpressure
Peak shape (tailing/asymmetry)
Across all checkpoints and for both column lengths:
Retention times remained stable and within method limits
Efficiency stayed within expected ranges
Pressure profiles were steady, with no backpressure drift
Peak shapes remained acceptable
Expressed as column volumes at pH 8.5, the cumulative injections represent substantial stress:
50 x 4.6 mm column
~0.5 mL void volume
~34 column volumes per run (17 mL / 0.5 mL)
~34,000 column volumes after 1,000 injections
150 x 4.6 mm column
~1.5 mL void volume
~11.3 column volumes per run
~11,334 column volumes after 1,000 injections
So, while both our columns received the same number of injections, the shorter 50 mm column experienced roughly three times the normalized exposure per column volume. Yet both remained chromatographically suitable at pH 8.5.
Extrapolating to semi prep and prep formats
Using the same run volume of 17 mL per injection, we extrapolated the column volume calculations to larger hardware typically used in GLP 1 purification. For 1,000 injections:
250 x 4.6 mm → ~6,800 column volumes
250 x 10 mm → ~1,414 column volumes
250 x 20 mm → ~362 column volumes
250 x 50 mm → ~58 column volumes
This means that for semi-preparative and preparative GLP 1 columns—which run at much larger IDs and column volumes—the same number of injections at pH 8.5 corresponds to hundreds to a few thousand column volumes, rather than tens of thousands. In other words, the chemical stress we have already tested on small analytical NanoPak C columns is significantly greater than that likely to be experienced by larger ID formats in normal GLP-1 campaigns, providing a strong high-pH stability margin.
Why this matters (and why we did it this way). Conducting long-term studies on the robustness of large-internal-diameter (ID) preparative columns at high pH levels requires substantial amounts of organic solvents and buffers. These tests can be both time-consuming and costly. Our analytical column-volume approach provides a practical and economical method to assess pH stability and scalability prior to committing to large-scale preparative studies.
The NanoPak-C all-carbon analytical columns have demonstrated stable performance after processing approximately 11,000 to 34,000 column volumes at a pH of 8.5. The extrapolated column-volume values for semi-preparative and preparative sizes indicate a significant margin of chemical stability for NanoPak-C media when purifying GLP-1. If the 4.6 mm inner diameter columns can withstand tens of thousands of column volumes under alkaline conditions without any loss of performance, then it stands to reason that the same media, when packed into larger inner diameter formats and subjected to only hundreds to a few thousand column volumes, will experience considerably less chemical stress per unit of packed bed at the same pH level.
Limitations and future work. Semi-preparative and preparative GLP-1 purification on 10–50 mm ID columns routinely use higher sample loads (for example, in the milligram-to-hundreds-of-milligrams-per-injection range). Our study design focused on resolution and robustness rather than high-throughput purification. Thus, we employed low-sample-load analytical conditions (1 mg/mL solutions, 10–20 µL injections, microgram-level loads). While our column volume analysis clearly demonstrates the NanoPak-C all-carbon column's chemical robustness at pH 8.5, it does not yet define maximum practical loading, fouling behavior, or recovery under high-load, crude-feed conditions.
We also used a single gradient program, mobile phase, and temperature on analytical-scale hardware, so mechanical factors such as higher flow rates, pressure cycles, and potential bed stress at larger IDs, as well as peptide or matrix buildup at higher loads, were not directly evaluated. Our ongoing work extends these studies to semi-prep and prep NanoPak C columns with realistic GLP-1 feed streams, gradually increasing injection mass and monitoring chromatographic performance, fouling, cleaning efficiency, and long-term pressure behavior. This will allow the high pH robustness observed at analytical scale to be fully quantified and confirmed under more realistic production conditions.
To download our application note on the Separation of Tirzepatide on Alkaline pH Stability of NanoPak‑C All‑Carbon Columns, please check here.
For inquiries or collaborations, or if you are interested in evaluating NanoPak-C all-carbon microbeads, please contact our technical team at inquiry@millennialscientific.com, call us at 855 388 2800, or fill out our online contact form at www.millennialscientific.com/contact.
[1] D.T. Manallack, The pK(a) Distribution of Drugs: Application to Drug Discovery, Perspectives in medicinal chemistry 1 (2007) 25-38.
[2] D. T Manallack, R. J. Prankerd, E. Yuriev, T. I. Oprea, and D. K. Chalmers, The Significance of Acid/Base Properties in Drug Discovery, Chem Soc Rev. 42(2) (2013) 485–496.
[3] R.E. Majors, Current Trends in HPLC Column Usage, LCGC Europe 25 (2012) 1-10.
