Study on Calibration of Chromlum( Ⅵ ) Analyzer

—This article presents a novel metrological calibration method for trace chromium(Ⅵ) analyzer. The study identifies all factors that affect the accuracy of chromium(Ⅵ) analyzers, and proposes calibration items and method for the analyzer based on its working principle. The calibration items include pump set flow error and pump flow stability, set temperature error and temperature control stability of column oven, baseline noise and drift, minimum detection concentration, linear range, and qualitative and quantitative repeatability. Experimental results shows that, the presented method can meet the requirements for the metrological performance evaluation of chromium(Ⅵ) analyzer. The calibration method is scientific and feasible.


Introduction
Chromium mainly exists in three forms in nature: metallic chromium, chromium(Ⅲ), and chromium(Ⅵ). Chromium(Ⅵ) combines with oxyacid, and is widely used in chemical production, metallurgy, electroplating, leather and textile industry, and other fields. Chromium(Ⅵ) can enter human body through skin, mucous membrane, respiratory system, etc. causing toxicity and carcinogenicity to multiple organs, including reproductive system, skeletal system, liver, and kidney, The International Cancer Research Structure of the World Health Organization explicitly lists chromium(Ⅵ) as a primary carcinogen. Therefore, China has promulgated standard limits and monitoring methods for chromium(Ⅵ) in surface water, groundwater, solid waste, food, electronic and electrical products, and other samples. The concentration of chromium(Ⅵ) in waste water should be no higher than 0.5 mg/L, and the concentration of chromium(Ⅵ) in ambient air should be no higher than 0.00025 mg/m 3 . [1][2][3][4] Currently, there are many methods for the measurement of chromium(Ⅵ), including 1, 5diphenylcarbazide photometric method [5] , atomic absorption spectrophotometry, chromatographInductively coupled plasma mass spectrometry, ion chromatography with post-column method, etc. Ion chromatography with post-column method is widely recognized as the standard method for the measurement of chromium(Ⅵ) in international or national standards, such as ISO 17075-2-2017 Leather -Chemical determination of chromium(VI) content in leather -Part 2: Chromatographic method [6] , SN/T 2210-2021 Determination of hexavalent chromium in foods for export [7] , and HJ 779-2015 Ambient Air -Determination of hexavalent chromium (VI) -Ion Chromatography with post -column method [8] .
Ultra-trace chromium(VI) analyzer is a specialized instrument for measuring chromium(VI) in samples. The sample is separated by chromatography and mixed with a derivative reagent (1, 5-diphenylcarbazide). Using the strong oxidizing property of chromate, 1, 5diphenylcarbazide is oxidized in an acidic environment and complexed to form a purplish red complex, whose absorbance can be measured at 540 nm by ultra-trace chromium(VI) analyzer. Ultra-trace chromium(VI) analyzer mainly consists of infusion system, sample injection system, separation system, detection system, and data processing system. Currently, there are no calibration specifications or verification regulations for ultra-trace chromium(VI) analyzer in China. This article discusses the calibration method of ultra-trace chromium(VI) analyzer based on the working principle of the analyzer, referring to JJG 705-2014 Liquid Chromatographs [9] and JJG 823-2014 Ion Chromatographs [10] .

Reference material
The reference material of chromium(VI) in water is used for calibration of minimum detection concentration, linear range, and qualitative and quantitative repeatability, and the relative expanded uncertainty of the reference material should not be greater than 0.2% (k=2).

Standard devices
Analytical balance with maximum weighing capacity of no less than 100 g, and definition of better than 1 mg, as well as stopwatch with definition of better than 0.1 s are used together to calibrated the pump flow set value error and pump flow stability. Pipet and volumetric flask of Class A are used to prepare standard solutions.

Pressure resistance performance of the pump
Connect all parts of the ultra-trace chromium(VI) analyzer, use pure water as the mobile phase, and set the flow rate at 1 mL/min. Start the instrument according to instruction manual. Use filter paper to check if there is leakage at each pipeline interface after the pressure is stable for 10 minutes. Remove the chromatographic column, block the pump outlet (below the pressure sensor), make the pressure reach 90% of the maximum allowable value of the analyzer, and maintain the status for 5 minutes to see if there is leakage.

Pump set flow error and pump flow stability
Start the ultra-trace chromium(VI) analyzer, use pure water as the mobile phase, and set the flow to 0.5 mL/min, 1.0 mL/min, and 2.0 mL/min in turn after the pressure is stabilized. Collect the mobile phase at the outlet of the separation column using volumetric flask whose weigh 1 W is already know. The time of collecting the mobile phase t is 20 min, 10 min, and 5 min respectively, at every set flow. Weigh the flask with mobile phase in it on the balance, and record the indication of the balance 2 W . The measured flow is calculated according to equation (1).
The pump set flow error is calculated according to equation (2).
Where m F is the average of m F at the same set flow, and s F is the set flow.
The pump flow stability is calculated according to equation (3), Where max F is the maximum of all measured value of flow under each set flow, while min F is the minimum.
The experimental results of pump set flow error and pump flow stability of a typical type of ultra-trace chromium(VI) analyzer is demonstrated in Table 1.
As we can see from Table 1, the maximum pump set flow error is -0.37%, and the maximum pump flow stability is 0.06%, both of which meet the requirements of JJG 705-2014 Liquid Chromatographs and JJG 823-2014 Ion Chromatographs.

Set temperature error and temperature control stability of column oven
Fix the probe of the digital thermometer as close as possible to the temperature sensor of the column oven or constant temperature block, set the calibration temperature S T , start the calibrated analyzer, record the indication of the thermometer every 10 minutes for 7 times after the temperature is stabilized. The set temperature error of column oven is calculated according to equation (4).
Where is the average of all 7 indications of the thermometer.
The temperature control stability of column oven is calculated according to equation (5). The experimental results of set temperature error and temperature control stability of column oven of the analyzer is demonstrated in Table 2.

Baseline noise and drift
Initialize and preheat the calibrated analyzer, monitor the baseline after the analyzer stabilizes, and record the baseline for 30 minutes. The baseline noise is the signal of the peak height of the peak of maximum noise in the 30 min baseline, and the baseline drift is the maximum signal of the baseline deviation from the starting point within 30 min.
The experimental result of the analyzer is 0.02mAU, and the baseline drift of the analyzer is 0.08mAU/30min, both of which meet the requirements of JJG 705-2014 Liquid Chromatographs and JJG 823-2014 Ion Chromatographs.

Minimum detection concentration
Analyze a chromium(VI) standard solution with the concentration of 1.0 ng/mL, inject 100 μL of solution, and the chromatogram is demonstrated in Fig. 1.

Linear range
Inject chromium(VI) standard solution with the concentration of 50 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, 400 ng/mL, 500 ng/mL, 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, and 1000 ng/mL in sequence, and the injection amount is 100 µL. Record the response signal value of each standard solution when it is stable. Repeat the measurement three times at each concentration point to get the average of measured values. Establish a linear regression curve with the measurement results of five concentrations (50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, and 400ng/mL). The experimental result of standard curve of the calibrated analyzer is demonstrated in Table 3.  (7) Where x is the theoretical concentration value of the standard solution calculated by linear regression equation (7), and y is the average of response signal values of the standard solution.
Calculate the linearity error according to equation (8).
is the standard concentration of the solution.
The experimental result of linearity error is demonstrated in Table 4. The upper detection limit C H is at the minimum concentration whose absolute value of linearity error is more than 5%, and the lower detection limit C L is the minimum detection concentration obtained from 3.4. The linear range is calculated according to equation (9). L H C C ge Linear ran = As we can see from Table 4, the upper detection limit of the calibrated analyzer is 900 ng/mL, and the minimum detection concentration of the analyzer is 0.03ng/mL, therefore, the linear range of the analyzer is 3×10 4 , which meets the requirement of JJG 705-2014 Liquid Chromatographs.

Qualitative and quantitative repeatability
Inject 200 μL of standard solution whose concentration is 10 ng/mL, repeat it 7 times, and record the retention time and peak area. Calculate the qualitative and quantitative repeatability RSD according to equation (10).
Where is the recorded retention time or peak area, and is the average of retention time or peak area. The experimental results of qualitative and quantitative repeatability is demonstrated in Table 5.