HPLC-Fluorescence Detection of Eosin Y in Groundwater Studies

Susanne Fabel and Holger Franz, Dionex Corporation

Eosin Y is an important tracer in groundwater studies for determining water migration paths and identifying the distribution of environmental pollutants. Spectrofluorimetry, capillary LC/fluorescence, and HPLC-fluorescence are approaches to sensitively detect Eosin Y (1, 2). HPLC provides a convenient way to separate eosin from humic substances and interfering tracers. Figure 1 shows the separation of an Eosin standard from impurities obtained with a binary gradient. A gradient is used to reduce peak broadening and improve signal-to-noise ratio. Zero carry-over is assured by wash cycles before and after the injection.

Figure 1

The variable emission filter of the UltiMate® 3000 RSLC fluorescence detector is a unique method development tool for real-time compound related sensitivity optimization. To find the best filter, the excitation wavelength at 511 nm and the emissions wavelength at 537 nm were kept constant. An Eosin Y standard was repeatedly injected with filter positions alternating between a cut-off at 280 nm, 370 nm, 435 nm, 530 nm, and no filter. The best signal-to-noise ratio was achieved with the filter wheel position at 435 nm.

The following LODs and LOQs are obtained injecting a 9.5 fg/ ?L Eosin Y standard (three replicates):

LOQ (S/N=10)=2.9 ng/L

LOD (S/N=3)=0.87 ng/L

Instrumentation

Binary UltiMate 3000 RSLC system with FLD-3400RS fluorescence detector. Filter water samples through 0.2 μm filter frit.

Chromatographic Conditions

Mobile Phase: Eluent A: 10 mM sodium acetate buffer, pH 5.5

Eluent B: Acetonitrile

Wash solvent: Acetonitrile/Water (5/95 % v/v)

Column: Acclaim RSLC 120 C18, 2.2 μm, 50 × 2.1 mm

(P/N: 068981)

Column Temp.: 30 °C

Flow rate: 0.7 mL/min

Gradient: 0–1.1 min 24–50% B, 1.1–1.3 min 50–100% B, 1.2–1.8 100% B, 1.8–2.017 100–24% B, 2.0–4.0 24% B.

Detection: Emission wavelength 511 nm; Excitation wavelength 537 nm, Filter Wheel 435 nm, Response Time: 2 s, Data collection rate: 100 Hz, Lamp Mode: High Power

Flow cell: analytical (8 μL)

References

(1) W. C. Brumley and J. W. Farley, Electrophoresis, 24, 2335–2339 (2003).

(2) R. E .J. Van Soest, J. P. Chervet, M. Ursem, J. M. Suijlen, LCGC International, 1996, September, 586–593.

Dionex Corporation

1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088

tel. (408)737-0700; fax (408)730-9403

Website: www.dionex.com.