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Chiral GC – Using Cyclodextrin Derivatization to Create Ultimate Selectivity

By: Denise Wallworth, Reporter EU Volume 27

Denise Wallworth

denise.wallworth@sial.com

As a fast, high efficiency and high sensitivity analytical technique, chiral GC has probably the most to offer out of all chromatographic possibilities. Although the choice of separation technique can, of course, be solely driven by the solute properties – and for GC, compounds need to be volatile and thermally stable – chiral GC has wide appeal, especially for complex matrixes in environmental, biological, agricultural, food, and essential oil applications. The high efficiencies in GC have the additional benefit of providing lower limits of detection for many applications.

Cyclodextrins have developed a dominant role in chiral GC since 1985 as a result of the ability to derivatise the three cyclydextrins (CD), alpha, beta and gamma, altering the mechanism and ultimate selectivity towards chiral molecules. Hydroxyl groups in the 2- and 3-position around the rim, and at the 6-position at the base are used to create derivatised CDs and a series of GC phases that offer an extremely broad range of chiral separations. Enantioselectivity occurs by either an inclusion mechanism or a surface interaction (such as dipole-dipole) - or by a combination of both – and is therefore determined by both the position and type of the derivative.

Figure 1: Application: Range of polar chiral compounds separated on Chiraldex G-TA

The first generation of chiral GC phases have all CD hydroxyls methylated and are solubilised (typically 10- 20%) in a polysilixane carrier. There is a large number of such permethylated phases available today – the Supelco DEX-110 and 120 (α, β,and γ) and the Chiraldex B-PM being key phases in this area. They are highly selective and versatile in a wide range of applications, especially for aliphatic compounds, with some acids and bases requiring derivatisation. A second generation of non-polar permethylated phases changed the chemistry of the 6- position of the CD ring from methyl to tertiary butyl silyl, a group hydrophobic enough to block residual surface activity of the capillary and, more importantly, to increase the solubility of the CD in the carrier, resulting in higher selectivity at lower retention times. Aromatics and cyclics in general now show greater separation than on the traditional permethylated. The two important phases here are the Supelco DEX 325 and Chiraldex B-DM and these differ in the type of carrier used and the concentration of CD (25 and 50% respectively). The effect of this is that the latter column gives higher efficiency for those separations that occur at lower temperatures.

Polar Derivatives

Once you start to substitute polar functional groups around the CD rim, versatility increases even further and selectivity broadens as you change the type and position of the derivative. Dipole-dipole interactions are created and the mechanism becomes a very effi cient surface interaction. This allows the efficient use of gamma CD with a larger surface to cover a broader range of molecular analogs.

The Chiraldex range provides a selection of such phases, two of which are the subject of global patents. The Chiraldex G-TA has probably one of the broadest range of applications in this series from diols and lactones to alcohols and amines (Figure 1 gives just one example), while the B-DP separates aromatic and aliphatic amines well. Changing the derivative to hydroxypropyl (B-PH) extends the range of separation further to include sugars, bicyclics and haloalkanes Additionally, the Supelco DEX225 phase uses a novel 2- and 3-acetyl derivative that provides unique selectivity. Together, this group offers some of the most effi cient separations available. The complete range is summarised in Table 1.

Coated or Bonded?

The majority of chiral GC columns in use today are coated CD capillaries that provide the widest scope of chiral applications. A further increased degree of inertness can be introduced by bonding the CD. A new bonding chemistry recently developed for the Chiraldex permethylated phase has resulted in a very low bleed that is ideal for MS applications. This new column, the Chiraldex Bonded B-PM, is effective in separating complex mixtures including underivatised volatile chiral acids, alcohols, lactones and diols. Figure 2 shows one application for the separation of a mixture of polar and non-polar molecules, including an underivatized alcohol.

Method Development Techniques

In chiral GC method development, the temperature window of separation is first found for the solute by running a temperature gradient to 150oC at 1-5oC/minute (or to the maximum allowable operating temperature (MAOT) of the column if the volatility of the sample requires it – using 5-10oC/minute). A sample solvent is chosen that volatilises at least 40oC below the elution temperature – often methylene chloride is a good choice – and a 30:1 or 40:1 split ratio used. Unlike achiral GC, temperature is not subsequently used as an optimising parameter. Instead, velocity is used to increase peak efficiency and reduce retention times. Most enantioselective separations are optimal at much lower temperatures than the MAOT of the column.

Figure 2: Separation of non-polar and underivatised polar solutes using the new CHIRALDEX Bonded B-PM

Derivatisation in chiral GC is far more common than in LC. Where a compound is insufficiently volatile, or is temperature labile at higher temperatures, derivatisation with a suitable achiral reagent is used. Creating a derivative can also introduce different chiral interactions, resulting in a faster analysis times and more efficient separations, and can even effect a separation where one was not possible before. Generally, it is very polar molecules, such as alcohols, amines, acids, amino alcohols that are derivatised prior to chiral separation The Chiraldex Handbook gives a very useful guide to the techniques used. All derivatization reagents used are available from Sigma-Aldrich.

Reversal of Elution Order

In trace analysis, elution order is critical for optimal quantification. Derivatisation becomes extremely useful in this case, and simply changing the type of derivative made can reverse elution order. In summary, elution order reversal can occur by:

  • Changing from one type of cyclodextrin to another (e.g. G-TA to B-TA)
  • Changing from one phase type to another (e.g. B-PH to B-DA)
  • Changing from one %CD to another (e.g. β-DEX 110 to 120)
  • Changing of derivative (e.g. trifl uoroacetyl to acetyl)
  • Operating below ambient temperatures

Applications

The list of application areas for each column type is too long to present here. For a comprehensive guide, see the table in the Astec Product Guide, or consult the Chiraldex GC Handbook. Table 1 shows the complete range of chiral GC capillary columns available; a special development kit is available that includes three Chiraldex columns for the broadest range of applications: – the Chiraldex G-TA, BDM and B-DA.

Chiraldex column kit



Table 1: Supelco Chiraldex polar and non-polar substituted chiral GC phases from-Sigma Aldrich.

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