HIC Standard for Column Performance

Product Description

CellMosaic’s Hydrophobic Interaction chromatography (HIC) standard is designed for day-to-day use to check HPLC column performance. The product consists of three proteins and one amino acid with varying surface hydrophobicity. The broad range of hydrophobic coverage allows one to test the performance of HIC column at high range of salt concentration. CellMosaic routinely uses this product for its internal bioconjugation-related research and services.

This product is packaged in a lyophilized form to allow more stable storage and shipment at ambient temperature. The lyophilized protein mixture can be quickly reconstituted in water without any insoluble particles. A total of two micro-centrifuge tubes are in a package. The product is ready for use after reconstituting in 120 µL of deionized water.

Application of the product:

• Hydrophobic Interaction column performance testing
• HIC HPLC Method development

Key Features of the product:

• Lyophilized solid. Ready to use for HPLC analysis after dissolving in 120 µL of deionized water.
• Mixture of 3 high purity proteins and 1 amino acid of varying hydrophobic behavior with broad MW coverage: 155 to 25,600 Da

SEC Standard Composition

Application Note 

The protein standard mixture can be used to assess HIC column performance. The HIC profile of the protein standards may differ from column to column after some use. The quality of the HIC profile will indicate whether the column is good. For example, split peaks may indicate column resin-bound hydrophobic molecules and that the column needs cleaning following column manufacturer recommendations.

Example 1: Separation of HIC protein standard using a cleaned Waters Protein-Pak™ Hi Res HIC column (4.6 mm x 100 mm, 2.5 µm, Part No. 186007583). HPLC condition: CellMosaic’s HIC Buffer set - CM02025 with 15 min gradient at a flow rate of 0.6 mL/min. Instrument: Agilent 1100 HPLC series. HPLC profile is shown in Figure 1 below.

Note: Self digested fragments from Chymotrypisinogen A are observed at 9.691 and 10.158 min.

Example 2: Separation of HIC protein standard using a new shorter Waters Protein-Pak™ Hi Res HIC column (4.6 mm x 35 mm, 2.5 µm, Part No. 186007582). HPLC condition: CellMosaic’s HIC Buffer set - CM02025 with 10 min gradient at a flow rate of 0.8 mL/min. Instrument: Agilent 1100 HPLC series. HPLC profile is shown in Figure 3 below.

  Note: Self digested fragments from Chymotrypisinogen A are observed at 5.968 min.