Leading C18 Reversed Phase HPLC Columns

Independently Tested at The University of Aberdeen, UK

Test Results with Acidic, Basic and
Neutral Molecules

An Independent Comparison of Leading C18 Column Brands

Introduction
In recent years significant improvements have been made in the quality of bonded silica particles used in HPLC. An increased understanding of the chemical nature of the silica surface and the manufacture of purer silicas have led to a tightening of physical specifications. Additional improvements have also been made in bonding and column packing techniques. Consequently, there has recently been a dramatic increase in the number of improved C18 reversed phase columns available to the chromatographer.

Problem
With so many different C18 column brands to choose from, finding the right column for a particular separation can be a very time consuming and expensive exercise. Comparison of manufacturers quoted results cannot always be relied upon to accurately predict column performance under searching test conditions.

Solution
In order to differentiate between columns and substantiate manufacturers claims, a number of well recognised tests have been performed on a selection of leading HPLC column brands by an independent test laboratory. The results reported are for previously unused columns of identical dimensions, purchased directly from either the manufacturer or their approved distributor and tested under highly controlled conditions.

Test Protocol
Three tests have been selected which accurately represent column performance under searching conditions. Many "validated" columns are evaluated using such tests, but comparison between columns is not normally possible due to the slightly different evaluation conditions employed. Columns have been ranked by efficiency on all three tests, measured as N 0.1. As shown by Figure 1, this calculation ensures both column efficiency and peak tailing are considered -factors which affect the final resolution obtained. An overall ranking has then been determined by averaging the ranking of each column on each test.

Figure 1 - Efficiency Measurement

Ranking - Neutral Molecules

Summary
Column efficiencies were seen to differ greatly. The use of high efficiency columns is recommended to reduce analysis time. Column length can be reduced without loss of resolution.

Ranking - Basic Molecules

Summary
C18 bonded columns show significant differences in chromatography for basic compounds. These variations are generally caused by undesirable secondary silanol interactions with the silica surface. Increasing silanol activity results in a deterioration of chromatographic performance. Highly inert "base deactivated" C18 columns with very low levels of silanol activity all exhibit similar selectivity, with differences limited to efficiency and peak shape.

Ranking - Acidic Molecules

Summary
Testing with acidic molecules shows similar trends to those seen with basic molecules. Smaller particle size, highly inert "base deactivated" phases are again seen to offer improved separations.

Final Ranking
The results from all three tests have been analysed and compared. As an aid to column comparison, a final ranking has been determined by averaging the test rankings for each

column (see Table 1). This final ranking of column performance is illustrated by Figure 2.

Figure 2 - Final Ranking

Conclusion
Significant differences in efficiency, peak shape and selectivity are seen with C18 bonded columns when evaluating acidic and basic compounds. These variations are caused by undesirable secondary silanol interactions. The selection of a high efficiency base deactivated phase with very low silanol activity will prove highly beneficial for the majority of analyses.

  Ace C18 materials were the highest performing 3 µm and 5 µm phases respectively. Superior column efficiency and peak shape are combined to provide excellent separations with acidic, basic and neutral molecules.

Trademarks and registered trademarks appearing in this catalogue are registered with the following companies;Ace -Advanced Chromatography Technologies;Genesis -Jones Chromatography;Hypersil and HyPurity -ThemoQuest Corporation;Prodigy -Phenomenex Inc;Spherisorb, Symmetry and XTerra -Waters Corporation;Zorbax -Agilent Technologies. Comparative separations may not be representative of all applications.

Further Information
A more comprehensive guide comparing over 100 commonly used C18 phases is now available. Subject areas discussed include:

• phase specifications
• efficiency and selectivity comparisons
• causes of peak tailing with acidic and basic molecules
• column reproducibility
• optimising selectivity
  
  Please contact your distributor now to request your free copy.