UHPLC: Reliable Solvent Mixing
Typically, poor mixing of solvent provides poor chromatography. In the first few years of high performance liquid chromatography (HPLC), solvent mixing and gradient delay volume (GDV) were rarely discussed because with packing materials of 5 μm to 10 μm and run times of >30 min, the GDV was of minor importance. This started to change with the introduction of columns that used sub-3 μm packing material. The smaller particle size provided higher efficiency; hence, it was possible to use shorter columns and significantly reduce run times by a factor of 5 or more. Also, smaller column i.d.s of 3.0 mm and 2.1 mm became standard, resulting in up to 5× smaller flow rates at equal linear velocity. This provided the advantage of lower solvent consumption and higher mass sensitivity. This innovation arrived when HPLC analysts needed higher sample throughput and lower costs per sample to increase productivity in a competitive marketplace, and to cope with the growing need for testing due to steadily increasing regulations (e.g., in drug development).
With typical pump dwell volumes of 2 mL to 6 mL,1 and the increased system backpressure required for running small particle size columns, a standard HPLC system is not well suited to run fast LC methods. For example, in gradient elution at a flow rate of 1 mL/min, the gradient will need several minutes after the injection to arrive on the column. With run times of <10 min, this is far too slow for fast LC applications. The same is also true for peptide mapping, as there is a strong interest in accelerating existing methods with common run times of <40 min.
In addition, with the commonly used UV-active ion-pairing agent trifluoroacetic acid (TFA), a considerable amplification of baseline ripples is observed. These ripples are caused by slight fluctuations in organic solvent concentration. For this kind of application, highly efficient solvent mixing is essential to obtain a smooth baseline even at small GDVs. To deal with these challenges, the main focus of attention is on reduction of the mixer dwell volume while at the same time maintaining (or even improving) the mixing performance. In addition, the flow paths of recent ultra HPLC (UHPLC) instruments are highly optimized using shorter capillaries with smaller internal diameters.
The solution we offer is our mixing technology (Thermo Scientific Dionex SpinFlow mixing design) which uses a unique two-step mixing process of radial mixing followed by longitudinal mixing resulting in the best results in attenuating baseline ripples.
Here are some general guidelines for selecting the right mixer for your application:
• For fast separations where the mixing ripple does not interfere with the detection (e.g., Thermo Scientific ESA Corona CAD Charged Aerosol Detectors or MS detectors), use low mixer volumes (35 μL and 100 μL).
• Use the medium sized mixers (200 μL and 400 μL) for the best balance between fast separation and low mixing ripple in UV detection.
• For highest sensitivity and when mixing ripples interfere with the detection (e.g., due to use of UV-absorbing solvents), use a larger mixer volume (400 μL and 800 μL).
• For UV-absorbing solvent additives that amplify the mixing ripples by interaction with the stationary phase (e.g., TFA applications), use the largest mixer volumes (800 μL and 1550 μL) in order to achieve the highest sensitivity.
For more details, we offer a poster and a technical note (downloadable PDFs):
Poster: Impact of Solvent Mixing on Liquid Chromatographic Performance describes an accurate way to experimentally determine the mixing performance using a sinus-like gradient.
Technical 108: Reliable Solvent Mixing in UHPLC describes the performance of mixing technology and the chromatographic impact of insufficient mixing on a TFA application example.
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