method for iron determination by luminol, version 2...trace-level iron determination – fia-120...

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Trace-Level Iron Determination – FIA-120

PRINCIPLE

This method is designed for the determination of iron in water. The sample is prepared offline to ensure

that all iron is present in trivalent form and then introduced to the FIAlab analyzer. A chelating resin is

used for in-line preconcentration of iron, followed by elution with acid. Iron generates a

chemiluminescence signal when mixed with luminol and hydrogen peroxide.

This method is designed to be run on the FIAlyzer-1000.

SUMMARY

This method is designed for the determination of iron in water. The method is capable of detecting iron

in the range of 1-20 nmol / L. Note: range is highly dependent on preconcentration time. For more

information, see the section on performance metrics.

SAFETY

The toxicity or carcinogenicity of all reagents used in this method must be taken into account and

therefore each chemical listed below should be handled accordingly.

Each laboratory is responsible for maintaining compliance with OSHA regulations regarding the safe

handling of the chemicals specified in this method. Material Safety Data Sheets (MSDS) should be made

available to all personnel using the method.

All waste materials should be disposed of in a responsible manner, in accordance with federal, state,

local, and any other applicable regulations.

The following chemicals have the potential to be highly toxic or highly hazardous; for detailed

explanations consult the MSDS:

o Hydrogen peroxide o Hydrochloric acid o Acetic acid o Ammonia o Triethylenetetramine

Method for Iron Determination by Luminol, Version 2.1


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EQUIPMENT AND SUPPLIES

Equipment: o Balance, analytical, with a 0.01 g resolution o Pipettes for making standards

Glassware: o Acid-cleaned PP (polypropylene) volumetric flasks or measuring cylinders. o Pipettors with plastic trace metal clean tips o Acid-cleaned LDPE (low density polyethylene) or PP (polypropylene) storage containers o Note: acid cleaning = soak in ~1 M trace metal grade HCl for at least 7 days. Then rinse

with copious amounts of deionized water.

Autosampler (for high sample loads):

o Cetac ASX-280/560 (recommended) or AIM-3200/3300


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REAGENTS AND STANDARDS

Chemical part numbers refer to Sigma-Aldrich unless noted otherwise. Note: (T) = TOSOH Bioscience PN

Chemical Chem. Formula CAS# Supplier PN

Deionized Water. a H2O 7732-18-5

25% Ammonia. b (NH3) 1336-21-6 105432

Acetic acid, glacial c (CH3CO2H) 64-19-7 33209

Luminol (C8H7N3O2) 521-31-3 A8511

Sodium carbonate (Na2CO3) 497-19-8 106395

30% Hydrogen peroxide (H2O2) 7722-84-1 95321

30% Hydrochloric acid d (HCl) 7647-01-0 100318

Triethylenetetramine (TETA) (NH2CH2CH2(NHCH2CH2)2NH2) 112-24-3 90460

Toyopearl AF chelate-650M IDA resin

0014475 (T)

1000 ppm Fe standard solution

(H₄NFeO₈S₂ * 12 H₂O) 7783-83-7 1197810100

a For ultralow-level work, deionized water should be purified further with polishing cartridges for metal removal.

b For lowest level detection, it is recommended that ammonium hydroxide is purified by distillation (isothermal or elevated temperature). c For lowest level detection, it is recommended that acetic acid is purified by distillation (isothermal or elevated temperature). d For lowest level detection, it is recommended that hydrochloric acid is purified by distillation (isothermal or elevated temperature). Distillation is typically carried out after diluting HCl to ~6M strength.

Preparation of Reagents

o Carrier: 0.23 M hydrochloric acid (1L) □ Dilute 23mL of 30% hydrochloric acid to 1L with DI water.

o Luminol Stock Solution: 0.1M luminol (15mL)

□ Dissolve 0.27g of luminol together with 0.50g of sodium carbonate in 15mL of deionized water.

□ Keep shaking periodically until fully dissolved. □ Store in refrigerator. Prepare fresh weekly.

o Reagent 1: 0.24 mM luminol / 0.7 mM TETA solution (1 L)

□ Add 2.4mL of luminol stock solution and 40μL of TETA to 0.8L of deionized water.


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□ Add deionized water to a total volume of 1L.

o Reagent 2: 0.44M ammonia (1 L) □ Add 30mL of 25% ammonia to 0.8L of deionized water. □ Add deionized water to a total volume of 1L.

Reagent 3: 0.3M hydrogen peroxide (1 L)

□ Add 30mL of 30% hydrogen peroxide to 0.8L of deionized water. □ Add deionized water to a total volume of 1L.

o Sample oxidation solution: 0.06% hydrogen peroxide (10mL)

□ Add 20 μL of 30% hydrogen peroxide to 10mL of deionized water.

o Sample buffer: 0.35M CH3COOH – 0.11M NH3, pH 4.5 (1L) □ Add 20mL of glacial acetic acid to 600mL of deionized water and mix. □ Add 7.6mL of 25% ammonia and mix. □ Add deionized water to a total volume of 1L.

o Probe Wash Solution: Water with 0.1% Brij L23 (1L)

□ Add 3.3mL Brij L23 to 800mL of deionized water, dilute to 1L. □ Mix well and store in a polyethylene bottle. □ The wash solution should be clear with no particles in it.

Notes on reagent shelf life:

o Use of high quality laboratory glass bottles is important. o Carrier should be prepared every month. o Luminol stock solution should be prepared every week. Store in refrigerator. o Reagent 1 should be prepared fresh daily. o Reagent 2 should be prepared every 3 months. o Reagent 3 should be prepared fresh daily. o Sample oxidation solution should be prepared fresh daily. o Sample buffer should be prepared fresh every month.

Preparation of Standards

o Dilute 1000 mg/L (18mM) Fe stock solution with deionized water to the desired range of iron standards.

o Acidify to pH ~ 1.8 by adding 1 part of 11 M HCl to 500 parts of standard solution (e.g. 120µL HCl to 60mL standard).

o Add 1 part of sample oxidation solution to 1000 parts standard solution (e.g. 60μL sample oxidation solution to 60mL standard).


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SAMPLE COLLECTION AND PRETREATMENT

Sample collection and filtering depends on practices and apparatus in each individual laboratory

and is not described in this document.

Acidify filtered water samples to pH ~1.8 by adding 1 part of 11 M hydrochloric acid to 500 parts

of sample (e.g. 120µL HCl to 60mL sample).

One day before analysis, add 1 part of sample oxidation solution to 1000 parts sample (e.g. 60μL

sample oxidation solution to 60mL sample). This is to ensure that all iron is in the Fe(III) state.

INSTRUMENT SETUP – MID-RANGE

Flow injection analysis apparatus parameters:

Component Specifications FIAlab PN

Analyzer FIAlyzer-1000 flow injection analyzer. *Inquire

PMT Detector PMT-FL chemiluminescence detector. 77035

External Valve External injection/switching valve 51015

External Pump Ismatec Pump, Reglo ICC, 4 Channel, 12 roller 41029

Peristaltic pump tubing 0.76mm ID (black/black) *Inquire

Peristaltic pump tubing 1.02mm ID (white/white) *Inquire

Sample Injection loop 2x10mm cartridge. Filled with Toyopearl AF chelate-650M IDA resin.

*Inquire

Reaction coil 1 (heated) 800µL - 69.0in/175cm of Teflon capillary tubing with 0.03 in/0.75 mm ID

77030

Software parameters:

Parameter Value

Pump Speed 50%

External Pump Speed Variable

Heater Temperature 30°C


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Fig. 1- Flow schematic for trace-level iron determination.

FIASOFT METHOD

The program script using an autosampler is shown below. For manual sample introduction see the FIAsoft manual.

' Iron test script.

' RegloICC Line 1 = column conditioning solution ' RegloICC Line 2 = sample ' Reglo ICC Line 3 = buffer soln ' Reglo ICC Line 4 = not used FIAlab Injection Valve Sample Load


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Serial Command (?) GOB ' Prime lines & stabilize baseline FIAlab Peristaltic Pump CounterClockwise(%) 50 RegloICC Clockwise (Channel, mL/min) 1, 1.7 RegloICC Clockwise (Channel, mL/min) 2, 1.7 RegloICC Clockwise (Channel, mL/min) 3, 0.7 RegloICC Off (Channel) 4 Next Sample Delay (sec) 20 ' Condition column Serial Command (?) GOB Delay (sec) 10 ' Column conditioning time. ' Sample preconcentration Serial Command (?) GOA Delay (sec) 60 ' Preconcentration time. Serial Command (?) GOB Delay (sec) 20 ' Column rinse time. Loop Start Analyte New Sample FIAlab Injection Valve Sample Inject PMT Start Scanning Serial Command (?) GOB ' External valve to conditioning pos. Autosampler Wash Autosampler Probe Down Delay (sec) 5 ' Sample line rinse time. Next Sample Delay (sec) 55 ' Column elution time. ' Condition column FIAlab Injection Valve Sample Load Delay (sec) 10 ' Column conditioning time. ' Sample preconcentration Serial Command (?) GOA ' External valve to sample feed pos. Delay (sec) 60 ' Preconcentration time. Serial Command (?) GOB ' External valve to conditioning pos. Delay (sec) 20 ' Column rinse time. PMT Stop Scanning Autosave Data C:\fialabdata\DateTime\DateTime Loop End RegloICC Off (Channel) 1 RegloICC Off (Channel) 2 RegloICC Off (Channel) 3 FIAlab Peristaltic Pump Off


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PERFORMANCE METRICS

Fig. 2 – Example plot and calibration data for trace-level iron.

Lower limit of detection: 0.056µg Fe/L –1nmol Fe/L Upper limit of detection: 1.1µg Fe/L – 20nmol Fe/L Sample throughput: 20 samples / hour Startup + Calibration: 5 minutes Shutdown: 5 minutes


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REFERENCES

M.B. Klunder, P. Laan, R. Middag, H.J.W. De Baar, J.C. van Ooijen, Deep-Sea Research II 58 (2011) 2678–

2694

R. Clough, H. Sela, A. Milne, M.C. Lohan, S. Tokalioglu, P.J. Worsfold, Talanta 133 (2015) 162–169

G.H. Floor, R. Clough, M.C. Lohan, S.J. Ussher, P.J. Worsfold, C.R. Quetel, Limnol. Oceanogr.: Methods 13

(2015) 673–686

method for iron determination by luminol, version 2...trace-level iron determination – fia-120...

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