Self-designed experiment in instrumental analysis： online solid phase extraction-high performance liquid chromatography- post column derivatization for the determination of trace pesticide residues in vegetables

doi:10.3724/SP.J.1123.2025.10020

Abstract

Abstract:

In line with modern educational concepts and the demand for innovative talents， a course focused on self-designed experiments has been incorporated into the traditional instrumental analysis curriculum. This initiative aims to enhance students’ ability to apply knowledge effectively. While mastering instrument operation， students are encouraged to anticipate potential challenges during experiments. Under instructor guidance， students constructed an online system for solid-phase extraction coupled with liquid chromatography and post-column derivatization with fluorescence detection. They addressed real analytical needs such as improving sensitivity and precision， minimizing interference， and conducting rapid analyses of trace carbamate pesticides in vegetable samples. This self-designed liquid chromatography experiment course， tailored to students’ learning characteristics and interests， successfully engages their initiative and fosters independent innovation capabilities. As a result， it also improves teaching effectiveness and enhances learning efficiency.

Key words: self-designed experiment, online solid-phase extraction （online SPE）, high performance liquid chromatography （HPLC）, post-column derivatization, pesticide residue analysis

CLC Number:

O658

LI Yan, LIU Yuntao, HUANG Xien, ZHOU Ziyi, WANG Ziqiu, WANG Jing, DING Fei, NAN Jing, TANG Anna. Self-designed experiment in instrumental analysis： online solid phase extraction-high performance liquid chromatography- post column derivatization for the determination of trace pesticide residues in vegetables[J]. Chinese Journal of Chromatography, 2026, 44(9): 1074-1081.

Figures/Tables 10

Fig. 1 Online solid-phase extraction-high performance liquid chromatography-post-column derivatization-fluorescence detection （SPE-HPLC-PCD-FLD） systemA1： sample； B1： 10% methanol aqueous solution； A2： 60% methanol aqueous solution； B2： methanol； RT： room temperature； OPA： o-phthaldialdehyde derivatization solution.

Table 1 SPE-HPLC-PCD-FLD program and valve switching schedule

Stage	Sample time/min	Pump 1		Elution time/min	Pump 2		Valve state
Stage	Sample time/min	φ（A₁）/%	φ（B₁）/%	Elution time/min	φ（A₂）/%	φ（B₂）/%	Valve state
Balance	0	0	100	-	100	0	load
Balance	3.0	0	100	-	100	0	load
Sample loading	3.1	100	0	-	100	0	load
Sample loading	13.0	100	0	-	100	0	load
Washing	13.1	0	100	-	100	0	load
Washing	14.0	0	100	-	100	0	load
Elution & detection	-	0	0	0	100	0	inject
	-	0	0	8.0	100	0	inject
	-	0	0	12.0	25	75	inject
	-	0	0	24.0	25	75	inject
	-	0	0	25.0	100	0	inject

Fig. 2 Principle of derivatization

Fig. 3 Effect of pH in the sample solution on adsorption performance （n=3）

Fig. 4 Effect of eluent type on elution performance （n=3）

Fig. 5 Effect of the flow rate of the derivatization solution and the hydrolysate on the derivatization performance （n=3）

Fig. 6 Effect of hydrolysis temperature on derivatization performance （n=3）

Table 2 Linear equations， correlation coefficients （R2）， linear ranges， LODs， LOQs， intra-day RSDs （n=5） and inter-day RSDs （n=5） of the three target compounds

Compound	Linear equation	R²	Linear range/（μg/L）	LOD/（μg/L）	LOQ/（μg/L）	RSDs/% （n=5）
Compound	Linear equation	R²	Linear range/（μg/L）	LOD/（μg/L）	LOQ/（μg/L）	Intra-day	Inter-day
Methomyl	y=4.9x‒1.11	0.9979	2.0‒400	0.20	0.69	2.2	3.5
3-Hydroxycarbofuran	y=10.9x‒0.19	0.9692	1.0‒200	0.10	0.34	2.1	3.6
Carbaryl	y=21.7x‒1.07	0.9997	0.5‒100	0.05	0.15	2.3	4.0

Table 3 Spiked recoveries of carbamate in three real samples （n=5）

Sample	Analyte	Content/（μg/L）	Spiked/（ng/g）	Recovery/%	RSD/%
Cabbage	methomyl	ND	40	88.2	2.5
	methomyl	ND	200	92.1	3.4
	3-hydroxycarbofuran	ND	40	89.1	3.0
	3-hydroxycarbofuran	ND	200	96.0	3.1
	carbaryl	ND	40	87.9	2.9
	carbaryl	ND	200	91.3	3.5
Cucumber	methomyl	ND	40	89.8	1.9
	methomyl	ND	200	90.5	2.2
	3-hydroxycarbofuran	ND	40	88.9	2.8
	3-hydroxycarbofuran	ND	200	92.7	2.6
	carbaryl	ND	40	87.5	3.0
	carbaryl	ND	200	92.2	2.8
Celery	methomyl	ND	40	90.1	3.0
	methomyl	ND	200	95.5	3.4
	3-hydroxycarbofuran	ND	40	90.2	3.3
	3-hydroxycarbofuran	ND	200	94.3	2.8
	carbaryl	ND	40	88.2	3.3
	carbaryl	ND	200	93.0	3.5

Fig. 7 Chromatograms of （a） a blank cabbage sample and （b） a spiked （0.2 μg/g） cabbage sample1. methomyl； 2. 3-hydroxycarbofuran； 3. carbaryl.

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