Column Selection in HPLC

Chromalytic has adopted the approach of providing a wide range of columns from a select number of suppliers and manufacturers

The general customer rationale seems to be - stick with proven historical based support materials; column selection is via libraries of chromatograms or based on long established methods eg USP for pharmaceuticals
For small neutral or moderately polar molecules a generic C18 column with 100A spherical particles is often suitable for a spectrum of sample types and often is the column of first choice in developing a new method.

There has evolved a need to extend the range of sample types to more acidic and basic compounds and to extend the MW range into proteins and biomolecules generally. This has been generally limited by . . .
Support Residual Activity
2 approaches . . .
1 Silica particle technology
There is also a demand for high speed, fast sample screening ( eg drugs and metabolites )

The advent of SPE has indeed simplified many analytical separations so that shorter smaller bore columns can be used with a dramatic saving in expensive solvent use

Direct LC-MS via ESI hasalso overcome many of the detection problems in terms of sensitivity and dead volume effects of detectoe cells when using fast HPLC columns and sharp peaks

Si-OH groups

Analysis of more polar material requires a variety of methods used by different manufacturers to shield the effect of active silanol groups left unreacted due to steric effects of different silane reagents
Various end-capping techniques may or may not have been used
Recent technolohy has evolved into the use of short chain polar embedded linking molecules ( to C18)

Support Purity

The underlying cause of column activity for many polar materials and subsequent non-elution or peak tailing is caused by metallic ion impurities in the silica particles

Modern state-of-the-art silica particles are of high purity silica ( for maximum inertnes )
spherical particles with as narrow a spread in size as possible for the maximum column efficiency and to minimise pressure drop

LC-MS columns in particular are unique as a limitinh factor of ESI-MS is to be able to use a high organic content mobile phase to enhance the MS sensitivity. From a design perspective obviously the purity of the silica and its inertness is of paramount importance but the phase itself requires a high degree of hydophobicity ( ie max C% )

Biochromatography columns

genrally use silica high pore size 300 to 600A or higher low surface area 80 to 120 m2/g

Polymer based supports - to eliminate the effects of silica impurities and Si-OH groups there is a trend to use polymer based particles ( eg DVB ) and cross-link C18 and other moeities via C-C bonds rather than Si-O-C. This enables the use of highly polar solvent and a much wider range of pH solvents, buffers and additives withouit the risk of hydrolysis and "bleed" associated with the use of silica based phases

The more recent advances by MicroSOLV enables them to be used over a pH range 1 to 13, pressures up to 9000psi and temperatures up to 150 degC ( mind you there are NO HPLC instruments yet capable of using these extremes - but the possibilities are there !

Modern materials can be judged on the relevant application chromatograms and comparative chromatograms of standard acid/base test mixtures one manufacturer column vs another.

HPLC Column Selection 102-108

Chromalytic PRODUCTS - OEMs

HPLC Columns - Column Selection ?

Restek

• PINNACLE - full range of packings

• NEW PINNACLE11 - reduced price C8, C18, CN, phenyl more inert, higher efficiency.

• Ultra and Allure ( in Pinnacle catalog ) range for LC-MS

BioCHROM Labs

• BioCell range of porous polymer-type supprts for biomolecules; proteins

ChiralSEP

• Chiral Columns

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