Kromasil^® 100 Å

The well-known Kromasil^® Classic 100 Å family of products is used to separate and purify molecules of up to about 10 000 Da. In fact, drug candidates for the pharmaceutical, natural products and API industries are separated and purified using Kromasil^® Classic 100 Å columns and bulk material.

Derivatized products based on Kromasil^® 100 Å silica are developed and manufactured at Nouryon to achieve high reproducibility and chemical stability. The narrow and consistent particle size distribution of Kromasil^® 100 Å silica and its derivatizations lead to chromatographic columns with outstanding efficiency and bed stability.

Particle size distribution showing the dv₉₀ / dv₁₀ ratio.

Kromasil^® media in small particle sizes for UHPLC and HPLC

Kromasil^® bulk media is available in a variety of standard particle sizes from 1.8 to 16 μm (larger particles are available upon request). All particle sizes are based on the same Kromasil^® silica technology. Therefore, scientists can now employ the same quality products as their counterparts across the organization, making it easier, faster and more cost-effective for a drug to reach market.

Kromasil^® UHPLC columns with 1.8 μm particles are specifically targeted for fast chromatography to screen samples under UHPLC conditions. In this case, the chromatographic results show a separation in slightly more than a minute with significant baseline resolution.

The Kromasil^® 2.5 μm columns are intended for laboratory flexibility, maintaining exceptional performance. These columns are packed for UHPLC conditions giving users the option to run Kromasil^® 2.5 μm particle-based columns under UHPLC or HPLC conditions. Scientists can choose the scale that works best in their laboratory environment, and develop and adapt methods for fast turnaround under HPLC conditions or go one step further to UHPLC methods. As with all Kromasil^® particle sizes, these Kromasil^® 2.5 μm particles are based on very narrow specification ranges, resulting in columns with excellent performance and backed by the well-known Kromasil^® column-to-column reproducibility.

Kromasil^® columns allows easy transfer of methods developed on 2.5 μm particles to other departments, such as method validation and quality control. Kromasil^® 2.5 μm columns can also be a good start in open access screening by synthetic or medicinal chemists in the step before purification of key compounds of interest.

• Conditions
• Column: Kromasil 100-1.8-C18 2.1 × 50 mm
• Mobile phase: acetonitrile / water (65/35)
• Sample: 1 = dimethyl phthalate, 2 = toluene, 3 = biphenyl, 4 = phenanthrene
• Flow rate: 0.6 ml/min
• Temperature: 35 °C
• Detection: UV @ 254 nm

Separation within 2 minutes

• Conditions
• Column: Kromasil 100-2.5-C18 4.6 × 50 mm
• Mobile phase A: acetonitrile + 0.1% TFA
• Mobile phase B: water + 0.1% TFA
• Gradient: 0 min: 5%, 2.7 min: 70% acetonitrile
• Sample: 1 = sotalol, 2 = nadolol, 3 = timolol, 4 = metoprolol, 5 = alprenolol
• Flow rate: 0.6 ml/min
• Temperature: 35 °C
• Detection: UV @ 254 nm

Seamless scalability

Considering a project starts in R&D, scientists can develop a Kromasil^® based UHPLC method in the early stages, validate the corresponding conditions of analysis and transfer the method to HPLC scale for other departments. Being able to use the same type of stationary phase throughout discovery, development and production is a unique opportunity for chromatographic users not only due to the extent of the Kromasil^® phases, but also the quality and reproducibility of the materials, which is second to none.

Same selectivity in a fraction of the time

• Conditions
• Columns: Kromasil 100-1.8-C4 2.1 × 50 mm
•   Kromasil 100-2.5-C4 4.6 × 50 mm
•   Kromasil 100-3.5-C4 4.6 × 50 mm
•   Kromasil 100-5-C4 4.6 × 50 mm
• Substances: Vitamins E & D

•  
• Mobile phase: acetonitrile
• Flow rate: 0.6 ml/min (1.8 µm), 2.0 ml/min (2.5 µm), 1.5 ml/min (3.5 µm), 1.0 ml/min (5 µm)
• Temperature: 20 °C
• Detection: UV @ 215 nm

Kromasil^® columns for HPLC

Kromasil^® Classic HPLC columns based on 5 μm particle technology are the workhorse in analytical laboratories.

QC test, tricyclic antidepressants

• Conditions
• Column: Kromasil 100-5-C18 4.6 × 250 mm
• Mobile phase: methanol / potassium phosphate, 25 mM, pH 6.0 (80/20)
• Flow rate: 1 ml/min
• Temperature: ambient
• Detection: UV @ 215 nm
• Sample: 1 = phenylpropanolamine,
•   2 = nortriptyline,
•   3 = toluene,
•   4 = imipramine,
•   5 = amitriptyline

Pesticides

Lately, 3.5 μm particle columns are also becoming the standard for many laboratories in several sectors within pharmaceutical, food and beverage, natural products, clinical and industrial applications.

• Conditions
• Columns: Kromasil 100-3.5-C18 4.6 × 150 mm
• Mobile phase: acetonitrile/water
• Gradient: 0 - 1.5 min: 40%, 10 min: 90% acetonitrile
• Flow rate: 1.5 ml/min
• Temperature: 30 °C
• Detection: UV @ 254 nm

•  
• Substances: 1 = uracil
•   2 = fenuron,
•   3 = monuron,
•   4 = diuron,
•   5 = linuron,
•   6 = neburon,

A disruptive technology in purification

Independent of the chromatographer’s need for isolation and purification, Kromasil^® products are delivered both in slurry-packed columns for development and pilot laboratory isolation as well as bulk material for larger purifications.

One of the main distinguishing aspects of Kromasil^® qualities is that it is possible to use the same quality product whatever the scale required. This comprises the isolation and purification of compounds and their impurities for carrying out material characterization, pilot runs for campaigns in the pharmaceutical industry and full production purification including the latest polishing steps for delivery to patients.

The following examples illustrate the consistency of Kromasil^® silica across column dimensions.

Scalability

• Conditions
• Stationary phase: Kromasil 100-10-C18
• Column size: length: 250 mm
•   diameter as stated in figures
• Substances: uracil and toluene

•  
• Mobile phase: acetonitrile / water (30/70)
• Linear velocity: 0.66 mm/s
•   (equivalent flow rate as stated in figures)
• Detection: UV @ 254 nm

Wettability for selectivity

In cases where there is a need to use a completely wettable phase, or when the compounds in the sample have aromatic structures requiring unique selectivity for π-π interactions between the phenyl bonded phase and the solute, Kromasil^® Phenyl phase can be used. Kromasil^® Phenyl is derivatized using a mono-functional silane, followed by an extensive endcapping.

The result is a stationary phase with high stability, high reproducibility, and symmetrical peaks for basic compounds.

• Conditions
• Column 1: Kromasil 100-5-C18 4.6 × 250 mm
• Mobile phase: acetonitrile / 20 mM ammonium phosphate (12/88)
• Flow rate: 1 ml/min

•  
• Column 2: Kromasil 100-5-Phenyl 4.6 × 250 mm
• Temperature: 30 °C
• Detection: UV @ 254 nm
•  

Kromasil^® phases are also available in a dedicated wettable version of the C18 phase called Kromasil^® C18(w). Learn more about C18(w).

Consistency from batch to batch

Another important aspect in preparative chromatography is the stationary phase batch-to-batch consistency. A vast number of tests are performed in the quality assurance and control of Kromasil. In the figure below, batch-to-batch reproducibility of Kromasil^®, measured as selectivity and retention factor over time, is shown for particle sizes from 7 μm to 16 μm.

From analytical to prep chromatography - scalability with insulin

• Analytical conditions
• Column: Kromasil 100-3.5-C4 4.6 × 120 mm
• Mobile phase: acetonitrile / 0.05 M sodium phosphate, 0.1 M sodium chlorate, pH 2.5
• Gradient: 0 min: 30%, 55 min: 36% acetonitrile
• Flow rate: 1 ml/min

• Preparative conditions
• Stationary phase: Kromasil 100-10-C8
• Column size (DAC): 50 × 250 mm
• Crude loading: 6 g/l column volume
• Flow rate: 60 ml/min
•  

• Common conditions
• Crude purity: 90%
• Detection: UV @ 214 nm

The need for a strong material explained

Mechanical strength is required to withstand mechanical stress in an analytical or purification column. A silica packing is also often exposed to high mechanical stress when unpacked and packed again in production. Frequent packing and unpacking requires very stable packing material where no fines can be created.

The formation of fines in any part of the process leads to increasing backpressure. Eventually the pressure limit for the system is reached, and the column has to be repacked with new material. The Kromasil^® particles are essentially perfectly spherical. In addition, the pore shape and structure are more regular than other materials. The result is mechanical strength that allows extremely high piston pressure in columns.

Many Kromasil^® customers perform cleaning-in-place (CIP) using highly alkaline conditions to remove adsorbed polypeptide impurities, especially in insulin purification. Such conditions will quickly break down less stable materials mechanically. But with Kromasil^® packing medias, you can apply CIP over and over again.