Packing of HPLC Column
Hawach Scientific provides HPLC Columns with a variety of particle sizes and specifications to meet all separation requirements, including higher resolution, enhanced sensitivity, faster analysis, and consistent performance. All these are guaranteed by strictly selected and qualified materials such as spherical and pure silica gel, a highly cross-linked rigid spherical polystyrene/ divinylbenzene (PS/ DVB) non-porous scraping packing surface, a hydrophilic polymer layer, which can effectively eliminate the nonspecific absorption of antibody protein and improves the column efficiency and recovery.
Improvements of HPLC Columns
Hawach Scientific gains some highlights in capping, ultra-pure, silica gel chromatographic columns, which can significantly reduce peak trailing by means of universal gradient and C18 selectivity. With excellent resolution, efficiency and sensitivity, Hawach HPLC column can give the customer confidence in the accuracy and quality of the analysis data.
The highly homogeneous, dense-packed weak cationic exchange layer is chemically bonded to the hydrophilic polymer coating surface. And the complete characterization of monoclonal antibodies contains the identification and monitoring of acidic and alkaline subtypes, which is beneficial to provide a higher degree of separation and excellent column property in peak shape.
Experimental Matters Needing Attention
HPLC columns must undergo correct aging before the analysis and operation, and Hawach has tested and aging each column. The eluents recommended for use on such columns are low conductivity buffers, such as citric acid or tartaric acid buffers ( or their mixed solution), pH ranges from 2.5 to 6.5, in which ethylenediamine is used as eluting cations.
The salicylic acid buffer must not be used because salicylic acid decomposition products change the properties of the stationary phase. The eluent should be deaerated to avoid detection and pumping problems. Besides, be sure to check for microbial growth before you start using the system, or your column will clog and the column pressure will rise to an unacceptable level.
Parameters of HPLC Columns
In HPLC columns, silica gel is the most universal matrix, the strength is large, the chemical modification is easy, but the pH value is limited; the chemical properties of the polymer matrix are stable, and the pH range is wide, but the strength is not large enough, the batch repeatability is relatively poor, and the price is slightly higher. Additionally, the smaller the particle size, the faster the separation, the higher the column efficiency.
In HPLC, the chromatography packing you choose has a great influence on the separation effect. However, the range of chromatographic packing choices is huge. Consequently, if you want to make your best choice, you must have some understanding of chromatographic packing.
Silica gel for HPLC packing
Generally speaking, the stationary phase of normal-phase chromatography is silica and some other amine. Because the SiOH on the surface of silica and other polar groups have great polarity, so we separate according to the polarity of each component, which means the one with the weakest polarity would be flushed out of the column. Comparing to the stationary phase, the mobile phase has weaker polarity.
Inversed phase chromatography uses silica as a matrix, with weaker polarity surface bond. The mobile phase in Inversed Phase Chromatography has great polarity. However, the components with great polarity would be flushed out, and other components with small polarity would be reserved.
Polymer for HPLC packing
Usually, polymers for HPLC packing are polymethacrylate. One of its great advantages is that all PH ranges can use. Comparing to silica gel packing, the polymer has stronger hydrophobic properties and works well on separating the protein samples. However, the shortage is the column efficiency is too low.
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