Organic/inorganic solution for HPLC separation problems

1st April 2013

High-efficiency silica-based packings have been used in high performance liquid chromatography (HPLC) since 1973. Over the past 27 years, says Sylvie Mamon, many improvements have been made to the technology of these packings.

A notable development of recent years is the use of high-purity silica. Furthermore, much progress has been made in the reproducibility of the synthesis of packings.

A general problem of silica-based packings remains however: silica dissolves at alkaline pH values (above pH 8). On the other hand, packings made from organic polymers such as poly (divinylbenzene), are stable over a wide pH range, but suffer from low plate counts and are plagued from poor mechanical stability.

Hybrid organic-inorganic technology provides a novel solution to this dilemma and has resulted in new packing, XTerra.To design hybrid organic-inorganic particles, a path was followed that was first explored by Unger and co-workers. A tetraalkoxysilane and an alkyltrialkoxysilane are reacted with each other to form a precursor, and this precursor is then used to create the particles:

1. (RO)4Si + n (RO)3SiR* + (1.5 n + 2) H2O _ SiO2(R*SiO1.5)n + (3 n + 4) ROH

As one can see, the final particle contains an alkyl group (R*) that is incorporated into the matrix of the packings. This approach yields a material that contains both inorganic units (SiO2) and organic units (R*SiO1.5), combined at the molecular level. With this composition the concentration of residual silanols is significantly reduced compared to classical packings, and a large portion of the surface is occupied by methyl groups instead of silanols. The packing is as hard as silica. The pH stability of bonded phases based on these hybrid particles far exceeds that of silica-based bonded phases.

Two chemistry families are available. The RP ligand has a carbamate group inserted into the chain that will ashield' the residual silanol activity. The best peaks shapes and wettability in 100 per cent water are the key properties of the XTerra RP packings.

The other chemistry is the XTerra MS packings. It is made using a trifunctional silane (C8, C18). These packings are designed for LC/MS applications, where very low bleed is a must. Both XTerra RP and XTerra MS packings are fully endcapped.

XTerra packings reduce by one third the amount of unbonded silanols compared to silica-based packings. This reduced silanol activity results in improved peak shapes for basic analyses.

pH stability

XTerra packings have significantly improved pH stability compared to classical silica-based packings. While the recommended range for silica-based packings is pH 2 to pH 8, XTerra packings can be used from pH 1 to pH 12.

Now it is possible to run basic analytes under non-ionising conditions. This also provides new opportunities to manipulate selectivity of an assay. The reproducibility of an assay improves at high and low pH, and the packings give us this option.

The stability of these packings has been investigated at both acidic and basic pH. At acidic pH, the XTerra MS C18 packings compares favourably to the better silica-based packings that are commercially available. More important is the stability of XTerra packings at alkaline pH.

XTerra has been tested column lifetime at pH 11.5 using a pyrrolidine buffer. The column (4.6mm x 150mm, 5µm XTerra RP18) was run continuously at 30o C at 1ml/min, and a mixture of basic analytes, methamphetamine (pK 9.5), nortriptyline (pK 9.7), and propranolol (pK 9.5), was injected continuously. The column lasted for more than 45 days at pH 11.5 without deterioration of performance as measured by plate count and peak asymmetry (other than those due to changes in mobile phase composition).

The extended pH stability of XTerra allows for the first time the compromise-free chromatography of basic analytes under non-ionising conditions. This is a powerful tool for the manipulation of the selectivity of a separation. This can be demonstrated best by looking at the shifts in retention found for acidic and basic analytes as a function of pH. We have studied this using the XTerra RP18 packings. Strong shifts in retention are observed for the ionisable compounds.

The retention of the neutral analyte, toluamide, is not affected by the pH changes. The acidic analyte, ibuprofen, changes from high retention below pH 4 to a retention factor of less than 1 at pH 9.

Conversely, the basic analytes nortriptyline, doxepin, lidocaine and imipramine are weakly retained at acidic pH and reach high retention at pH 9 to pH 10. Obviously, a pH shift is a very powerful tool if analytes of different charge need to be separated. For example, for compounds of the same charge, pH shifts lead to shifts in elution order. At acidic pH, doxepin elutes before imipramine and nortriptyline. Conversely, at alkaline pH nortriptyline and doxepin elute very close to each other, and imipramine elutes last.

Stable, ie, pH independent retention is observed at both ends of the pH scale. At pH values above 9, basic analytes have lost the positive charge and their retention factor stabilises. Under these circumstances, the control of the mobile phase pH does not need to be as stringent as at intermediate pH values. This has advantages for the ruggedness of a separation.

The XTerra packings are based on a new hybrid technology that combines the advantages of silica-based packings with the extended pH range of polymeric packings. The use of these packings is as simple as the use of any other reversed-phase packings.

On the other hand, the pH stability of XTerra packings extends the pH range accessible for reversed-phase packings. This opens up new options in the manipulation of the selectivity of a separation.

Enquiry No 42

Sylvie Mamon is with Waters Corporation European Headquarters, Guyancourt, France.





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