High‑Temperature HPLC with All‑Carbon Columns for Difficult Separations

What is High-Temperature HPLC? High-temperature liquid chromatography (HPLC) uses elevated column temperatures to improve analyte separation. Most high‑temperature applications run between roughly 50 °C and 150 °C.

The factors affecting standard room temperature liquid chromatography are:

• The mobile phase's constituents (e.g., water, acetonitrile, methanol), pH, and ionic strength (buffers)

• The stationary phase's composition and size

• The column's dimensions

Temperature, often treated as a minor (secondary or niche) factor, can play a significant role in difficult separations by:

• Improving mass transfer

• Decreasing retention times

• Controlling problematic analyte behavior that can arise at lower temperatures

For labs working in demanding conditions, it is important to know not only whether higher temperatures improve separation but also whether the stationary phase performs well under these conditions.

How temperature changes the separation. The following mobile phase properties play important roles in analyte retention on stationary phases:

• Dielectric constant

• Surface tension

• Viscosity

For certain analytes, such as oligonucleotides, secondary structure also affects retention.

Dielectric constant is one way to describe the polarity of a mobile phase and its ability to reduce electrostatic interactions between charged species. It helps indicate how "strong" a solvent will be in a given mode of separation. For instance, in both normal- and reversed-phase HPLC, elution strength is primarily controlled by varying the mobile-phase polarity, typically by mixing water with organic modifiers such as acetonitrile or methanol. However, elution strength and solubility also depend on other properties such as hydrogen bonding, specific interactions with the stationary phase, and the chromatographic mode.

Surface tension is the cohesive force between molecules of the mobile phase. It contributes to how well the mobile phase wets the stationary phase and enters its pores. It can be changed by temperature and solvent composition. Surface tension, together with viscosity and other solvent properties, affects column backpressure and, in some cases, peak shape.

Viscosity tells you how "thick" the mobile phase is. A more viscous solvent makes it harder to push the liquid through the column, so backpressure increases and peaks can become broader unless you adjust conditions (for example, by lowering flow or increasing temperature).

In general, the dielectric constant, surface tension, and viscosity of mobile phase mixtures decrease as temperature increases.

Additionally, for analytes such as oligonucleotides, higher column temperatures help “unfold” the strands (their secondary structure), so they behave more like single, flexible molecules. This reduces extra interactions that can broaden peaks and make retention less predictable at lower temperatures.

Thus, high-temperature HPLC repeatedly appears in demanding applications. It not only allows faster runs but also facilitates predictable analyte behavior, making the method more usable.

What users need from a high-temperature column. When analysts develop a high-temperature method, they are usually trying to solve more than one problem at once [1].

They may want:

• Shorter retention

• Better peak shape

• Improved transfer of hydrophobic or structurally complex compounds

• Run a pharmacopoeial-style method without sacrificing reliability

In practice, that means a useful high-temperature stationary phase should support:

• Stable retention and peak shape under sustained heat

• Compatibility with demanding mobile phases and established analytical methods

• Flexibility across analytical, method-development, and purification workflows

• A credible path from feasibility work to routine use

These needs are especially clear when a lab is already working at elevated temperatures and has seen practical limitations with a conventional phase.

Importance of the stationary phase. High-temperature HPLC depends critically on the stability of the stationary phase. Conventional silica C18 phases can exhibit limited lifetime with increasing temperature, and pure silica dissolves more rapidly at elevated temperatures, especially at pH above 7.

To mitigate these issues, silica-hybrid particles have been developed for improved high-temperature stability. Additionally, other alternative inorganic (zirconia bonded with polybutadiene), carbon (porous graphitic carbon (PGC)), and polymeric phases have been developed that exhibit high-temperature stability. Porous graphitic carbon in particular has been used successfully in quantitative LC methods for drug candidates and their metabolites, demonstrating robust retention and MS‑compatible operation for polar analytes [2, 3].

Why All-Carbon media enter the discussion. NanoPak-C porous graphitic carbon microbeads (All-Carbon microbeads) represent a new class of porous graphitic media synthesized by a microfluidic-based method [4]. Their composition and synthesis method, key differentiators compared to other porous graphitic carbon materials, have enabled evaluation under high-temperature conditions where conventional silica phases may be less durable.

Below is an overview of completed, ongoing, and planned case studies.

A proven example: cyclosporine at 80°C. One of the clearest internal proof points for All-Carbon columns

(Top) Depiction of an All-Carbon HPLC column at thermal equilibrium at 80 °C. (Bottom) Representative chromatogram of cyclosporine on the NanoPak‑C 250 × 4.6 mm column. The result reproduces the target pharmacopoeial retention window while maintaining acceptable peak symmetry at 80 °C.

is the cyclosporine analysis under high-temperature, pharmacopoeial-style conditions, published as an application note. Cyclosporine (also known as cyclosporine A or CyA) is an immunosuppressive drug routinely used to prevent graft rejection during organ transplantation [5]. CyA is a cyclic undecapeptide analyzed via RP-HPLC, typically on C18 columns, under high-temperature conditions to minimize peak broadening caused by conformational isomerism. Cyclosporine analysis under pharmacopoeial conditions is commonly performed at 80 °C, but conventional silica C18 columns can show limited lifetime under these aggressive operating conditions [6]. This issue prompted the evaluation of NanoPak-C All-Carbon stationary phases as an alternative. The results are summarized below.

• All-Carbon 250 × 4.6 mm column showed cyclosporine peak elution at 26.95 min. This retention time matched the expected 25-30 min window.

• Seven consecutive cyclosporine injections on the 250 mm column showed consistent retention time, peak width, tailing factor, and area, supporting good repeatability at 80 °C.

• Shorter All-Carbon column formats (150 × 4.6 mm and 50 × 4.6 mm) predictably reduced the retention time, with 19.08 min on the 150 mm column and 6.38 min on the 50 mm column. These reduced retention times indicate room for faster methods when monograph-style retention is not required (for example, in-house QC or screening workflows).

This is an important type of case study because it not only shows that a peak can be detected. It shows that a high-temperature method can be tuned into the target window and then run repeatedly with stable chromatographic behavior, which is the practical threshold for real laboratory use.

Another strong fit: Illicit drug salt identification. Illicit drug salt identification is another application area where carbon-based chromatography has external precedent under demanding analytical conditions [7]. Illicit cocaine, amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA) are often encountered in the form of salts. Identification of specific counterions present in these salts may provide valuable information about the source and manufacturing pathway. For comprehensive characterization, it is especially useful to have an LC/MS method that detects both the drug cation and its associated counterion in a single workflow.

Operating the carbon column at an elevated temperature, for example, around 60 °C, can improve mass transfer and reduce mobile-phase viscosity, thereby supporting sharper peaks, shorter run times, and more robust separation of both species. A recent evaluation by a pharma customer of our NanoPak-C All Carbon column examined this application at 60 °C. The customer had issues with robustness and repeatability with other commercially available columns. Early feedback suggested that the NanoPak-C column was performing well in their method-development workflow. These results, together with published work on porous graphitic carbon phases, suggest that All‑Carbon columns can support separation of both cationic and anionic species in demanding LC–MS applications [2, 3].

Their experience highlights ongoing interest in all-carbon LC columns for challenging LC-MS separations where robustness, repeatability, and temperature tolerance are important considerations.

Oligonucleotides are a natural future application. High-temperature oligonucleotide chromatography is a future direction for this platform story. Increased temperature can enhance oligonucleotide mass transfer and improve resolution. As stated above, elevated temperature is also important for reducing the influence of secondary structure during ion-pair reversed-phase oligonucleotide analysis. Thus, for oligonucleotides, the column temperature is a core part of the method, not just a fine‑tuning setting.

In summary, NanoPak-C All-Carbon HPLC columns are an excellent choice for labs working with high-temperature methods. These columns are designed for reversed-phase applications that extend beyond standard ambient-temperature and solvent conditions in HPLC. Our research on cyclosporine at 80 °C, initial studies on identifying illicit drug salts, and future investigations into oligonucleotide separations highlight the significance of this high-temperature functionality.

Download our application note: High-Temperature HPLC Analysis of Cyclosporine on NanoPak-C All-Carbon Columns.

For more information on our NanoPak-C All-Carbon suite of products or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.

References

[1] L.C. Chen, High-Temperature Liquid Chromatography And The Hyphenation With Mass Spectrometry Using High-Pressure Electrospray Ionization, Mass Spectrometry 8(2) (2019) S0079-S0079.

[2] Y.-Q. Xia, M. Jemal, N. Zheng, X. Shen, Utility Of Porous Graphitic Carbon Stationary Phase In Quantitative Liquid Chromatography/Tandem Mass Spectrometry Bioanalysis: Quantitation Of Diastereomers In Plasma, Rapid Communications In Mass Spectrometry 20(12) (2006) 1831-1837.

[3] T.E. Bapiro, F.M. Richards, D.I. Jodrell, Understanding The Complexity Of Porous Graphitic Carbon (PGC) Chromatography: Modulation Of Mobile-Stationary Phase Interactions Overcomes Loss Of Retention And Reduces Variability, Analytical Chemistry 88(12) (2016) 6190-6194.

[4] M.J. Parente, B. Sitharaman, Synthesis And Characterization Of Carbon Microbeads, ACS Omega 8(37) (2023) 34034-34043.

[5] N. Talal, Cyclosporine As An Immunosuppressive Agent For Autoimmune Disease: Theoretical Concepts And Therapeutic Strategies, Transplant Proc 20(3 Suppl 4) (1988) 11-5.

[6] H.A. Claessens, M.A. Van Straten, Review On The Chemical And Thermal Stability Of Stationary Phases For Reversed-Phase Liquid Chromatography, Journal Of Chromatography A 1060(1) (2004) 23-41.

[7] J. Kochana, W. Tomaszewski, T. Moszczyński, A. Zakrzewska, A. Parczewski, Application Of Carbon Adsorbents For Extraction Of MDMA Impurities In TLC Drug Profiling, Journal Of Liquid Chromatography & Related Technologies 31(6) (2008) 819-827.