GC Temperature Programming—10 Things You Absolutely Need to Know
LCGC North America
Temperature affects not only retention but also relative retention in gas chromatography (GC) and therefore, when we change temperature, we also change the selectivity of the separation. This is true as we alter the isothermal separation temperature, but also as we change the slope of the temperature program gradient.
1. Temperature affects not only retention but also relative retention in gas chromatography (GC) and therefore, when we change temperature, we also change the selectivity of the separation. This is true as we alter the isothermal separation temperature, but also as we change the slope of the temperature program gradient.
2. An increase of around 30 °C in oven temperature will reduce retention time by 50%.
3. Use the method shown in Figure 1 to "screen" samples.
Figure 1: Extract of chromatogram showing all pesticide analyte peaks eluted from a river water extract, analyzed under the screening conditions shown.
4. If the peaks are eluted within a "window" of less than 7 min (more accurately tg/4) then isocratic analysis may be possible (6.97 in our example, so isothermal analysis may be possible).
5. To approximate the required isothermal temperature for the separation, calculate the temperature at which the last analyte of interest is eluted and subtract 45 °C (239 °C in our example).
Figure 2: Optimum separations derived from a thermal gradient analysis (top) and an isothermal analysis (bottom).
6. To optimize the separation, alter the isothermal temperature in steps of 10 °C, within a range of ±50 °C. If a suitable separation is not obtained a temperature gradient should be used.
7. For splitless injection, the initial oven temperature should be 20 °C below the boiling point of the sample solvent and an initial hold time of 30 s should be used initially. For split injection, start with an oven temperature 45 °C lower than the elution temperature of the first peak from the screening chromatogram (149 °C in our exercise). For poorly resolved, early eluted peaks, decrease initial temperature rather than adding an initial isothermal hold unless the initial oven temperature is more than 30 °C below the boiling point of the sample solvent.
8. The optimum ramp rate for any separation can be estimated as 10 °C per hold-up time (7 °C/min in our screening example).
9. If a suitable gradient slope cannot be obtained to separate compounds eluted in the middle of the temperature gradient, insert a mid-ramp isothermal section at 45 °C below the elution temperature of the critical pair (203 °C to help separate peaks 8 and 9 in our screening example). Empirically determine the length required for the hold (start with a 1-min hold) and then resume the gradient at the same slope as before.
10. Set the final temperature at 20 °C above the elution temperature of the last analyte in the screen, but bear in mind that a higher temperature "burn" period may be required to elute high-boiling matrix components.
Liquid Chromatography to Analyze Vitamin D Proteins in Psoriasis Patients
January 21st 2025Can a protein involved in delivering Vitamin D to target tissues have an altered serum profile in psoriasis patients with cardiovascular disease? Researchers used liquid chromatography (LC) to help find out.
The Next Frontier for Mass Spectrometry: Maximizing Ion Utilization
January 20th 2025In this podcast, Daniel DeBord, CTO of MOBILion Systems, describes a new high resolution mass spectrometry approach that promises to increase speed and sensitivity in omics applications. MOBILion recently introduced the PAMAF mode of operation, which stands for parallel accumulation with mobility aligned fragmentation. It substantially increases the fraction of ions used for mass spectrometry analysis by replacing the functionality of the quadrupole with high resolution ion mobility. Listen to learn more about this exciting new development.
The Complexity of Oligonucleotide Separations
January 9th 2025Peter Pellegrinelli, Applications Specialist at Advanced Materials Technology (AMT) explains the complexity of oligonucleotide separations due to the unique chemical properties of these molecules. Issues such as varying length, sequence complexity, and hydrophilic-hydrophobic characteristics make efficient separations difficult. Separation scientists are addressing these challenges by modifying mobile phase compositions, using varying ion-pairing reagents, and exploring alternative separation modes like HILIC and ion-exchange chromatography. Due to these complexities, AMT has introduced the HALO® OLIGO column, which offers high-resolution, fast separations through its innovative Fused-Core® technology and high pH stability. Alongside explaining the new column, Peter looks to the future of these separations and what is next to come.
Oasis or Sand Dune? Isolation of Psychedelic Compounds
January 20th 2025Magic mushrooms, once taboo, have recently experienced a renaissance. This new awakening is partially due to new findings that indicate the effects of psilocybin, and its dephosphorylated cousin psilocin may produce long lasting results for patients who might be struggling with anxiety, depression, alcohol and drug abuse, and post-traumatic stress disorder. Hamilton Company has developed a methodology for the isolation and identification of 5 common psychedelic compounds used in the potential treatment of disease. The PRP-1 HPLC column resin remains stable in the harsh alkaline conditions ideal for better separations.
