Comparison of Binary and Ternary Gradients to Separate Stage 1 Illicit Drugs

September 1, 2008
Guifeng Jiang

The Application Notebook

The Application Notebook, The Application Notebook-09-01-2008, Volume 0, Issue 0

Improve the separation of 14 drugs of abuse in a complex mixture by employing a ternary solvent gradient.

Guifeng Jiang, Thermo Fisher Scientific

Improve the separation of 14 drugs of abuse in a complex mixture by employing a ternary solvent gradient.

Abinary solvent gradient won't always resolve every analyte in a complex mixture in the short run time of ultra-high performance liquid chromatograph (UHPLC) methods. But the usual solution – manipulating selectivity with a third solvent – is not possible with high pressure binary solvent mixing equipment.

Figure 1

The Accela™ High Speed LC oversteps this limitation by using a quaternary solvent gradient pump. Developing methods is simpler because four solvent channels are available to modify mobile phase solvent strength, pH, and ionic strength. Methods perform better because the Accela pump blends up to four solvents to deliver optimized mobile phase gradients, rather than compromised binary gradients.

We show that a ternary gradient performs better than a binary gradient in the determination of 14 drugs/metabolites by UHPLC/mass spectrometry (UHPLC–MS).

Table I

Experimental Conditions

Instrument: Thermo Scientific Accela UHPLC system

with MSQ Plus™ Detector and

Xcalibur™ 2.05

Column: Hypersil Gold PFP (perfluorinated phenyl),

1.9 μm, 100 × 2.1 mm

Flow Rate: 1 mL/min

Mobile phase: A: Water with 0.06 % (v/v) acetic acid

B: Acetonitrile with 0.06% (v/v) acetic acid

C: Methanol with 0.06% (v/v) acetic acid

Injection: 1 μL partial loop injection, 25 μL loop size

Column Temp: 45°C


We compared two UHPLC–MS methods for the separation and detection of 14 illicit drugs.

Table II

Figure 1(a) shows the separation achieved by using the binary gradient. While some of the analytes separate with adequate resolution, several pairs are not baseline resolved. As shown in Figure 1 (b), the ternary gradient provides a better separation and all 14 drugs are baseline resolved.


UHPLC–MS employing a ternary gradient delivered by a quaternary mixing pump provides baseline resolution of 14 illicit drugs in 10 min.


(1) Application Note 423, Document 62751, 2008, Thermo Fisher Scientific.

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