Applications

Determination of Total Selenium in Satsuma Mandarin (Hydride Generation Atomic Fluorescence Spectrometry)

September 9, 2025

1. Method Overview

After the sample is digested by heating with acid, hexavalent selenium in the sample is reduced to tetravalent selenium in a 6 mol/L hydrochloric acid medium. Potassium borohydride is used as a reducing agent to reduce tetravalent selenium to hydrogen selenide in the hydrochloric acid medium. The hydrogen selenide is carried into the atomizer by the carrier gas (argon) for atomization. Under the irradiation of a selenium hollow cathode lamp, ground-state selenium atoms are excited to a high-energy state. When deactivated and returning to the ground state, they emit fluorescence with a characteristic wavelength. The fluorescence intensity is proportional to the selenium content, and quantification is performed by comparison with the standard series.

2. Instruments and Reagents

2.1 Instruments and Equipment

2.1.1 Testing Instruments

Serial No.	Name	Quantity	Technical Requirements	Accessories
1	Atomic Fluorescence Spectrophotometer	1 set	–	Selenium Hollow Cathode Lamp
2	Argon Gas	1 cylinder	Purity ≥ 99.999%	–

2.1.2 Sample Pretreatment Equipment

Serial No.	Name	Quantity	Technical Requirements	Accessories
1	Electronic Balance	1 set	Sensitivity: 1 mg	–
2	Microwave Digestion System	1 set	–	Polytetrafluoroethylene (PTFE) digestion inner tank, digestion acid-driving device
3	Adjustable Hot Plate	1 set	–	–
4	Micropipette	1 each (100 μL ~ 1000 μL, 1000 μL ~ 5000 μL)	–	–
5	Colorimetric Tube	Several (10 mL)	–	–

2.2 Reagents

2.2.1 Raw Reagents

Serial No.	Name	Technical Requirements	Remarks
1	Nitric Acid	Guaranteed Reagent (GR)	–
2	Hydrogen Peroxide	Guaranteed Reagent (GR)	–
3	Hydrochloric Acid	Guaranteed Reagent (GR)	–
4	Potassium Hydroxide	Analytical Reagent (AR)	–
5	Potassium Borohydride	Analytical Reagent (AR)	–

2.2.2 Prepared Reagents

Serial No.	Name	Preparation Method	Remarks
1	Hydrochloric Acid Solution (6 mol/L)	Measure 50 mL of hydrochloric acid, slowly add it to 40 mL of water, cool, then dilute to 100 mL with water and mix well.	–
2	Potassium Hydroxide Solution (5 g/L)	Weigh 5 g of potassium hydroxide, dissolve it in 1000 mL of water, and mix well.	–
3	Potassium Borohydride Alkaline Solution (15 g/L)	Weigh 15 g of potassium borohydride, dissolve it in potassium hydroxide solution (5 g/L), and mix well.	Prepare freshly before use
4	Hydrochloric Acid (10+90)	Measure 50 mL of hydrochloric acid, slowly add it to 450 mL of water, and mix well.

3. Operation Procedure

3.1 Sample Preparation

3.1.1 Preparation of Test Solution

Crush and homogenize the sample, and store it in a clean sample bag.
Weigh 0.2 g – 0.3 g of the sample (accurate to 0.001 g), place it in a digestion tube, add 8 mL of nitric acid and 1 mL of hydrogen peroxide, shake and mix well, then digest in a microwave digestion instrument. The recommended microwave digestion conditions are shown in the table below (digestion conditions can be set independently according to different instruments). After digestion, cool the digestion tube, then place it in the digestion acid-driving device and heat continuously at 140 °C until the volume is about 1 mL (do not evaporate to dryness). Remove the tube, add 5 mL of hydrochloric acid solution (6 mol/L), continue heating until the solution becomes clear and colorless with white fumes, then remove and cool. Transfer the solution to a 10 mL volumetric flask with deionized water, dilute to the marked volume with water, mix well, and set aside for testing. Conduct a reagent blank test simultaneously.

Microwave Digestion Temperature-Raising Program

Step	Temperature / °C	Holding Time / min
1	80	3
2	120	5
3	140	5
4	160	5
5	180	30

3.1.2 Preparation of Standard Solutions

Preparation of Selenium Standard Intermediate Solution (1 μg/mL):
Accurately pipette 100 μL of selenium standard solution (1000 μg/mL) into a 100 mL volumetric flask, add 20 mL of hydrochloric acid, dilute to the marked volume with water, and mix well.
Preparation of Selenium Standard Working Solution (10 ng/mL):
Accurately pipette 1.0 mL of selenium standard intermediate solution (1 μg/mL) into a 100 mL volumetric flask, add 20 mL of hydrochloric acid, dilute to the marked volume with water, and mix well for testing.
Preparation of Selenium Standard Series:
The instrument automatically dilutes to prepare a standard series with concentrations of 0.00 ng/mL, 1.00 ng/mL, 2.00 ng/mL, 4.00 ng/mL, 8.00 ng/mL, and 10.00 ng/mL.

3.2 Sample Testing

Testing Conditions
Reference Conditions for Atomic Fluorescence Spectrophotometer

Negative High Voltage (V)	280
Lamp Current (mA)	50/50
Atomization Temperature (°C)	200
Carrier Gas Flow Rate (mL/min)	300
Shielding Gas Flow Rate (mL/min)	600
Measurement Method	Standard Curve Method
Reading Mode	Peak Area
Delay Time (s)	4.0
Reading Time (s)	18
Sample Injection Volume (mL)	1.0

Sample Testing
Adjust the instrument to the optimal state. Use hydrochloric acid (10+90) as the carrier stream and potassium borohydride alkaline solution (15 g/L) as the reducing agent. After the blank discrimination value of the carrier stream is less than 10.0, inject the selenium standard working solution to determine its fluorescence intensity. Plot the standard curve with mass concentration as the abscissa and fluorescence intensity as the ordinate.

Under the same experimental conditions as those for determining the standard series solution, introduce the blank solution and the sample solution into the instrument respectively, determine their fluorescence intensities, and perform quantification by comparison with the standard series.