The Kromasil new wettable phase is the latest innovation of Nouryon, an HPLC phase that has been specifically designed for polar compounds. This Kromasil 100 Å C18(w) 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.

Tanataka tests results sumarized in spiderdiagram
The main differences between wettable C18(w) and classic C18 shows in silanol capacity and ion exchange capacity at pH 7. This is mainly due to the polar embedded end-capping of C18(w).

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.

  • Conditions
  • Column: Kromasil 100-10-C18(w) 4.6 x 250 mm
  •   versus regular C18
  • Flow rate: 1.0 ml/min
  • Temperature: ambient
  • Detection: UV @ 254 nm
  • Mobile phase: 20 mM potassium phosphate pH 2.5
  • Substances: 1: cytosine,
  •   2: fluorocytosine,
  •   3: uracil,
  •   4: fluorouracil

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.

  • Conditions
  • Columns: Kromasil 100-10-C18(w) 4.6 x 250 mm
  • Substances: caffeine and 2,7-dihydroxynaftalene

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.

  • Conditions
  • Column: Kromasil 100-10-C18(w) 4.6 × 250 mm
  • Mobile phase: acetonitrile / water (70/30)
  • Sample: 1: sodiumnitrite, 2: benzamide,
  •   3: methylbenzoate, 4: toluene,
  •   5: propylbenzene, 6: butylbenzene
  • Flow rate: 2.0 ml/min
  • Temperature: 20 °C
  • Detection: UV @ 254 nm
QC test of four batches of Kromasil 100-10-C18(w) showing excellent reproducibility.

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.

  • Conditions
  • Column: Kromasil 100-5-C18(w) 4.6 × 150 mm
  • Mobile phase: 25 mM potassium phosphate pH 2.5
  • Sample: 1: oxalic acid, 2: tartaric acid,
  •   3: ascorbic acid, 4: acetic acid,
  •   5: maleic acid, 6: citric acid,
  •   7: fumaric acid
  • Flow rate: 1.0 ml/min
  • Temperature: 25 °C
  • Detection: UV @ 210 nm

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.

  • Conditions
  • Columns: Kromasil 100-5-C18(w) 4.6 x 150 mm
  •   Kromasil 100-5-C18 4.6 x 150 mm
  • Flow rate: 1.0 ml/min
  • Temperature: 25 °C
  • Detection: UV @ 254 nm
  • 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.

  • Conditions
  • Columns: Kromasil 100-5-C18(w) 4.6 x 150 mm
  •   Kromasil 60-5-HILIC-D 4.6 x 150 mm
  • Aqueous mobile phase:
  • acetonitrile / ammonium acetate, 50 mM, pH 4 (10/90)
  • HILIC mobile phase:
  • acetonitrile / ammonium acetate, 5 mM, pH 4 (80/20)
  • 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.

  • Conditions
  • Columns: Kromasil 100-5-C18(w) 4.6 x 150 mm
  •   Kromasil 100-10-C18(w) 4.6 x 250 mm
  • Mobile phase: acetonitrile / water / 0.1% TFA
  • Acetonitrile gradients:
  •  (5 µm): 0 min: 12%, 3 min: 26%, 13 min: 28%
  •  (10 µm): 0 min: 12%, 4.3 min: 26%, 18.6 min: 28%
  • 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.

  • Conditions
  • Column: Kromasil 100-10-C18(w) 4.6 x 250 mm
  •   and other preparative wettable C18 phases
  • Hydrolysis
  • Mobile phase: methanol / water / TFA (5/95/0.1)
  •  120 column volumes
  • Temperature: 80 °C
  • 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.

Loading of peptide sample on Kromasil C18 and Kromasil C18(w).
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.
The scale-up result of the purification on Kromasil C18(w), for this sample, is shown in figure E, where the fractions pooled provide very high purity and the given yield. If the purity requirements were lower, then more fractions could be pooled and yield increased accordingly.
  • Conditions
  • Columns: Kromasil 100-10-C18(w) 4.6 x 250 mm
  •   Kromasil 100-10-C18 4.6 x 250 mm
  • Substance: crude of bivalirudin in feed solution
  • Equilibrium and feed solutions:
  •  C18(w): ammonium acetate, 0.2 M
  •  C18: acetonitrile / ammonium acetate, 0.2 M (5/95)
  • 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.

  • Conditions
  • Column: Kromasil 100-10-C18(w) 4.6 x 250 mm
  • Sample: thymalfasin (crude)
  •   in 20 mM potassium phosphate
  • Sample load: 20 mg
  • Mobile phase: acetonitrile / water / 0.1 % TFA
  • 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

Click/touch the dot for the combination of stationary phase and column diameter of your interest to access column length availability and further options.
Column diameters, [mm]
FamilyPhasedp [μm]4.04.61021.2
100 ÅC18(w)5 M05WLB__ M05WLA__
100 ÅC18(w)10 M10WLA__ M10WLP__ M10WLQ__
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