Chromium is widely used in various industries such as electroplating, leather tanning, chrome paint, dying, hardened steel, ceramic and glass industry. Chromium exists in two stable oxidation states, hexavalent Cr(VI) and trivalent Cr(III). Cr(VI) is produced solely by industrial processes, whereas in nature, chromium exists in its trivalent form. Cr(III) is generally regarded as nontoxic due to poor absorption. Cr(VI) is considered a pulmonary carcinogen and has tested positive in genotoxicity tests. It is one of the most serious pollutants in many water streams due to its carcinogenic potential. Most countries apply a legal limit of 50-100 ug/L Cr in drinking water
Chromium Assay Kit provides a simple one-step colorimetric means to directly measure Cr(VI) in a sample. In the assay, Cr(VI) forms a stable complex with a specific chromogenic dye. The optical density at 480nm is directly proportionate to the Cr(VI) concentration in the sample. Cr(III) can be converted to Cr(VI) with nitric acid/hydrochloric acid, thus allowing the determination of Cr(III) or total Cr [Cr(III)+Cr(VI)] in the sample. The assay is sensitive with a detection limit of 20 ug/L Cr.
Key Features:Sensitive and accurate. Linear detection range of 20-2000 ug/LChromium
Convenient and high-throughput. Homogeneous "mix-incubate-measure" type assay. No wash and reagent transfer steps are involved. Can be readily automated for processing thousands of samples per day.
Applications:Determination of chromium in biological (serum, plasma etc), environmental (water, soil etc), food and beverage samples.
Kit Contents: (sufficient for 250 tests in 96-well format)Reagent A: 300ul Reagent B: 20mlCr(VI) Standard: 300ul 40 mg/LStorage conditions: store Reagent A at -20°C and other reagents at 2-8°C. Shelf life of 6 months after receipt
Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.
96-Well Assay Procedure:Use clear flat-bottom plates. Prior to assay, bring all reagents to room temperature. Unused Reagent A should be stored at -20°C. Samples should be clear, colorless and free from particles or precipitates. Substances that may potentially interfere with the assay include: azide, Ba2+, Pb2+, Fe3+, Gold(III), Sn(II), Ti (IV). If necessary, water samples can be concentrated by evaporation. If determination of Cr(III) or total Cr [Cr(III)+Cr(VI)] is desired, please refer to the General Sample Treatment Procedure
1. Standards. Prepare 600ul 2000 ug/L Cr(VI) Standard Premix by mixing 30ul Standard and 570ul deionized water dH2O (>18 megaohm)
Dilute standard as follows
No Premix+dH2O Standard (ug/L)1 300ul+0ul 20002 150ul+150ul 10003 75ul+225ul 5004 0ul+300ul 0Transfer 250ul standards into separate wells of the plate
Samples: transfer 250ul of each sample into separate wells of the plate
2. Assay. Prepare enough Working Reagent, for each well, by mixing 1ul Reagent A and 55ul Reagent B. Add 50ul Working Reagent to each well. Tap plate to mix. Incubate for 20 min at room temperature. Read optical density at 480 nm (430-505nm).
Cuvette Assay Procedure:The cuvette Assay Procedure: is essentially the same as the 96-well plate assay. The Working Reagent is prepared by mixing 4ul Reagent A and 220ul Reagent B. Assay by mixing 1000ul Standard or Sample with 200ul Working Reagent.
Calculation:Subtract the blank control OD (#4) from the OD values of the standards. Plot the Standard Curve and determine its Slope. Cr(VI) concentration of a Sample is calculated as,[Cr(VI)] =ODSAMPLE–ODBLANKSlope× n (ug/L)where ODSAMPLE and ODBLANK are the OD480nm values of the Sample and Blank Control (#4), respectively. Slope is the slope of the standard curve. Note: if the Sample Cr(VI) concentration is higher than 2000 ug/L, dilute sample in deionized water and repeat the assay. Multiply result by the dilution factor n
Conversion factor: 1000 ug/L chromium is equivalent to 19.2uM or 1 ppb.
General Sample Treatment Precedure:The following procedure converts Cr(III) in a sample to Cr(VI) by oxidation with nitric acid. This experiment should be performed with special care in a chemical fume hood. Weigh 0.5 g solid sample (e.g. alloy, food, hair), or transfer 1-2ml blood or serum samples, into a 50ml beaker. Add 10ml concentrated HNO3 and 1ml concentrated HCl. Cover with a watch glass until the initial brisk reaction is subsided. Add another 5ml concentrated HNO3 and heat the solution gently until all carbides are decomposed. After cooling down to room temperature, neutralize the solution with 3% ammonia. Filter the solution with Whatman No. 42 and use the filtrate for assay.
Materials Required, But Not Provided:Pipetting devices, centrifuge tubes, clear flat bottom 96-well plates, plate reader, cuvette, spectrophotometer, concentrated HNO3, concentrated HCl, ammonia and chemical hume hood.
