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The Kromasil® wettable phase is a 100 Å HPLC phase with C18 derivatization that has been specifically designed for polar compounds. It permits loading and run start in 100% aqueous, enabling new opportunities for engineers and scientists working with HPLC.
For polar compounds
Whether you are performing separation or purification of APIs, your facility may have to deal with an increasingly number of complex mixtures also containing more polar compounds. Kromasil’s expanded portfolio now includes the new wettable C18 phase precisely manufactured for separating and purifying more polar compounds, amino acids and peptides.
Independently, if you work in development or screening laboratories, pilot facilities or manufacturing, the alternative selectivity offered by Kromasil® 100 Å C18 (w) can be of significant benefit for impurity isolation and API production.
Phase characteristics
Comparison of phase properties between Kromasil® wettable 100-5-C18(w) and the classic 100-5-C18 according to the Tanaka tests set.
A phase that will withstands dewetting
When comparing a separation of pyrimidine derivatives on Kromasil® C18(w) and regular C18 under fully aqueous conditions, different behavior can be observed.
Figures A and C illustrate the difference in selectivity before anything unexpected has happened to the system, such a stop flow situation. The chromatographic result with the wettable phase in figure A shows better retention and selectivity compared to the more hydrophobic C18 in figure C.
The chromatogram in figure B illustrates that if flow stops and pressure drops the wettable C18(w) will not be affected, continuing to perform just as expected. This is one of the advantages of the wettable phase when dealing with samples that need to be injected under 100% aqueous conditions compared to traditional C18 phases where the regular C18 surface will undergo dewetting, resulting in loss of separation efficiency, as seen in figure D.
Chemical stability
The new Kromasil® C18(w) will under tough conditions and over 300 column volumes clearly maintain its retentivity with time. This performance assures engineers and researchers of the long term usability of this wettable phase.
Reliable quality
Extensive quality control on every raw material together with several in process controls (IPC) ensures Kromasil® 100 Å C18(w) to be a high-quality product.
Totally aqueous conditions
When separating organic acids on the Kromasil® wettable phase, these small polar compounds show complete resolution even when run under fully aqueous conditions.
A C18, but with alternative selectivity
Since C18(w) has slightly different surface characteristics from a classic C18 as illustrated in the Tanaka test, some differences in elution can be expected. Shown here is the separation of a mix of various polar compounds on the wettable C18(w) versus classic C18, showing quite similar elution, but for a small order switch between phenol and caffeine.
- Mobile phase: methanol / water (70/30)
- Substances: 1: uracil, 2: phenol, 3: caffeine,
- 4: acetophenone, 5: nitrobenzene,
- 6: methyl-benzoate, 7: toluene
Aqueous versus HILIC conditions
The diversity of available Kromasil® phases gives the opportunity for choices best suited for you. Here is a separation example of serotonin and derivatives in highly aqueous versus HILIC conditions. In these conditions, the elution is nearly reversed.
- Substances: 1: 5-hydroxy-L-tryptophan,
- 2: serotonin, 3: L-tryptophan,
- 4: 5-hydroxyindoleacetic acid
- Flow rate: 1.0 ml/min
- Temperature: ambient
- Detection: UV @ 280 nm
Scalability
Good selectivity and reproducibility enable easy scale-up of analytical methods to preparative methods. This example shows the separation of a peptide mix on Kromasil® C18(w) columns with 5 and 10 µm particles. Conditions are adapted to respective particle size optimum and column length.
- Substances: 1: gly-tyr, 2: val-tyr-val, 3: met-enkephalin,
- 4: angiotensin II, 5: leu-enkephalin
- Flow rates: (5 µm): 1.0 ml/min
- (10 µm): 0.7 ml/min
- Temperature: 25 °C
- Detection: UV @ 220 nm
Best in prep-class
We benchmarked the Kromasil® C18(w) phase against other wettable preparative phases on the market.
- Analysis
- Mobile phase: acetonitrile / water (70/30)
- Flow rates 1.0 ml/min
- Temperature: 25 °C
- Detection: UV @ 254 nm
- Substances: 1: uracil, 2: 2-phenylethanol,
- 3: butyl-4-hydroxybenzoate, 4: naphtalene
Fully aqueous conditions when you need it
With Kromasil® C18(w), you can load your preparative samples under fully aqueous conditions, increasingly important benefit for researchers today as more polar structures are being considered, reduces organic solvent consumption, cuts costs and address sustainability goals.
Kromasil® C18(w) implementation can also be of benefit for facilities that have not fully implemented explosion proof requirements to meet industry standards.
When comparing analytical results between Kromasil® C18(w) (figure A) and Kromasil® Classic C18 (figure C), retention times are noticeably similar for both stationary phases. Also, when proceeding to an overloaded step, retention pattern for the main peak and the impurities are comparable as seen in figures B and D, respectively.
- Mobile phase: acetonitrile / ammonium acetate, 0.2 M
- Gradient: 0 min: 10%, 60 min: 30% acetonitrile
- Flow rates 0.7 ml/min
- Temperature: 25 °C
- Detection: UV @ 280 nm
Follow the standards
In this preparative example, we purify a peptide following Chinese Pharmacopeia ChP 2015 standard for wetted stationary phases. The results achieved is shown in figure A, with high purity of 96.4% and yield of 83.9%.
By using the ChP 2015, the wanted displace-ment effect is noticeable, see red and green lines in figure B, where Kromasil® C18(w) first presses out impurities from the main peak at a 20 mg sample load.
- Gradient:0 min: 0%, 5.1 min: 16.3%, 40.1 min: 24.4%,
- 40.2 min: 80%, 50.2 min: 80% acetonitrile
- Flow rate: 0.7 ml/min
- Temperature: 25 °C
- Detection: UV @ 235 nm
Availability of Kromasil® wettable C18(w) columns
| Column diameters, [mm] | |||||||
|---|---|---|---|---|---|---|---|
| Family | Phase | dp [μm] | 4.0 | 4.6 | 10 | 20 | 30 |
| 100 Å | C18(w) | 5 | M05WLB__ | M05WLA__ | M05WLP__ | M05WLO__ | M05WLV__ |
| 100 Å | C18(w) | 10 | M10WLA__ | M10WLP__ | M10WLO__ | M10WLR__ | |