Separation of selenium species in plant tissues by high performance liquid chromatography-ultraviolet treatment-hydride generation atomic fluorescence spectrometry using various mobile phases

Figures & data

Table 1. Optimized operational conditions for HG-AFS.

Reducing solution 2.0% (m/v)
Currents 10% (v/v)
Negative high voltage 300
Lamp current 80
Carrier gas flow rate 400 mL min^–1
Make up gas flow rate 600 mL min^–1
HPLC SHIMADZU
Column Hamilton PRP-X100
Mobile phase 60 mmol L^–1 (NH4)2HPO4 pH 6.0
Flow rate 1.0 mL min^–1
Injection volume 100 μL
Column temperature 30 °C

Figure 1. Typical chromatogram obtained when using eluent containing 40 mmol L⁻¹ NH₄H₂PO₄ pH 4.48 (A), 40 mmol L⁻¹ NH₄Ac at pH 6.94 (B), 40 mmol L⁻¹ NH₄NO₃ at pH 5.18 (C) and 40 mmol L⁻¹ (NH₄)₂HPO₄ at pH 6.00 (D). The mixed standard concentration of SeCys 118.2 μg L⁻¹, Se(IV)85.8 μg L⁻¹, SeMet 88.7 μg L⁻¹, Se(VI) 83.0 μg L⁻¹ determined by HPLC-HG-UV-AFS. The optimized operational conditions for HG-AFS as described in Table 1. Note: HPLC, high-performance liquid chromatography; HG-AFS, hydride generation atomic fluorescence spectrometry.

Figure 2. Retention times of four Se species as a function of the concentration of mobile phase containing 30–70 mmol L⁻¹ (NH₄)₂HPO₄ at pH 6.00. Other HG-AFS conditions were as described in Table 1. Note: Each experiment was repeated four times.

Figure 3. Retention times of four Se species as a function of the pH of the mobile phase containing 60 mmol L⁻¹ (NH₄)₂HPO₄. Other HG-AFS conditions were as described in Table 1. Note: Each experiment was repeated four times.

Table 2. Standard curves of four Se species (n = 3).

project	SeCys		Se (IV)		SeMet		Se (VI)
	Concentration (μg L^–1)	Peak area	Concentration (μg L^–1)	Peak area	Concentration (μg L^–1)	Peak area	Concentration (μg L^–1)	Peak area
1	0	0	0	0	0	0	0	0
2	23.635	8043551	17.16	5583790	17.732	4193034	16.6	921679
3	47.27	16430408	34.32	11908256	35.464	8496419	33.2	1822900
4	70.905	26712402	51.48	18388717	53.196	12999655	49.8	2702454
5	94.54	35088087	68.64	25443995	70.928	17322109	66.4	3578569
6	118.175	44093230	85.8	32805157	88.66	22047269	83	4338503
7	236.35	90196554	171.6	67143562	177.32	45378878	166	8464516
R^2	0.9996		0.9991		0.9995		0.9993
Linear equation	y = 383796x-887357		y = 395357x-1E + 06		y = 256666x − 477540		y = 50756x + 109275

Figure 4. Typical chromatogram of the flue-cured tobacco roots treated with 22.2 mg kg⁻¹ Se obtained when using an eluent containing 60 mmol L⁻¹ (NH₄)₂HPO₄ at pH 6.00. The optimized operational conditions for HG-AFS as described in Table 1. The retention times of the unknown species were 135 s (2’15") and 492 s (8’12") in Figure 4. The unknown species were not identified due to the lack of Se standards in this method.

Table 3. Se species in the leaves and roots of flue-cured tobacco after applying the proposed extraction procedure and measurement with UG-AFS.

	Se treatment (mg kg^–1)	SeCys (μg g^–1)	Se(IV) (μg g^–1)	SeMet (μg g^–1)	Se(VI) (μg g^–1)	4 species sum(μg g^–1)
Leaves (n = 3)	2.2	0.02 ± 0.00	n.d.	n.d.	0.12 ± 0.02	0.14 ± 0.02
	22.2	0.04 ± 0.00	0.02 ± 0.00	0.09 ± 0.00	0.76 ± 0.00	0.91 ± 0.00
Roots (n = 3)	2.2	0.05 ± 0.01	0.03 ± 0.00	n.d.	n.d.	0.08 ± 0.01
	22.2	0.13 ± 0.02	0.15 ± 0.02	0.22 ± 0.02	0.26 ± 0.02	0.75 ± 0.07

Note: n.d. = not detected; Sum of species= [Se(IV)] + [Se(VI)]+ SeCys + SeMet.

Data availability

All data that support the findings reported in this study are available from the corresponding author upon reasonable request.