OCN633 Fall 2013 maxime@hawaii.edu

Shipboard analysis of trace metals:

Flow Injection Analysis (FIA)

Sequential Injection Analysis (SIA)

Trace Metals in Seawater: The Analytical Challenge

Fe, Al, Mn, Zn, Cu, Co, Cd…

[pM-nM] Mg2+ Ca2+..

[mM]

• Sensitive (ppt/nM) • Selective • Blank Free • Robust

Some trace elements (Fe, Zn, Co) regulate marine productivity Others can be used as tracers of processes (Al: dust, Cd: paleoproductivty)

Analysis of Metals…

The standard approach:

• Collect water in acid cleaned bottles,

add acid, return to shore lab for processing (ship tons of water back to lab…$$$).

• Pre-concentrate on resin or other material return columns to shore (saves shipping water!)

• Better yet… do the analysis at sea…

Why measure metals at sea?

• Obtain immediate feedback: • Identify and rectify blank problems

• Identify unexpected features and alter sampling strategy accordingly

• Process larger number of samples

• Get off the ship with data rather than water…

• BUT needs lots of equipment and must be able to operate on a rolling ship

Dissolved Al [nM]

Shipboard Method Requirements

• Equipment must not be too large (hard to get more than 20 ft of bench space on most ships)

• Equipment not overly sensitive to power fluctuations

• Equipment should be reliable (no service calls at sea)

• Must be able to handle samples without contamination (ships are extremely dirty, very strong air blowers for air conditioning systems keep dust in air)

• Develop methods that do not require reactions or steps in open container (e.g. evaporations)

• Should not require large volumes of water (availability of samples is inversely proportional to sample size)

• Should be rapid allowing fast throughput of samples. (Hydrographic programs are capable of obtaining 36 samples during a 4 hour CTD cast)

Atomic Spectrometry/Electrochemistry/GC

• FAAS, ICPMS: take lots of space, need clean room to operate, stable power and large amounts of gas (ICPMS: 1 cylinder of Ar/day). Not suitable for shipboard work.

• Electrochemical Methods (ASV, CSV): good sensitivity, allows looking at speciation (including ligands) but poor sample throughput (equilibration time ~10-15min). Not suitable for high resolution sampling.

• Gas Chromatography of volatile metal chelates: has been used for several metals and metalloids (Se, Be, Al and Cr). Extremely sensitive with ECD

• Form an organic chelate

• Separate it from water with organic solvent

• Inject solvent metal chelate in column

• Organic solvent separation&GC=preconcentration

• Fast and sensitive

Flow Injection Analysis

• A means to automate batch chemistry:

SAMPLE SAMPLE

ADD REAGENT

By replicating wet chemistry steps in a tube….

MONITOR PRODUCT BY

SPECTROSCOPY

Absorbance

Fluorescence

Chemiluminescence

WAIT….

A typical (and very simple) FIA setup

1. Sample is held in a sample loop

2. At determined moment the valve

is turned and sample is

propelled into the flowing stream

of carrier

3. Reagent(s) are added

downstream by teeing into the

line

4. As the injected zones move

downstream, the sample

disperses into the reagent(s) and

the product forms at the

interface.

5. Detector placed at the end of the

line monitors absorbance,

fluorescence or

chemiluminescence.

Flow Injection Analysis: Benefits

• Samples maintain their integrity from preparation to detection by

remaining in closed containers (less prone to contamination)

• Small equipment footprint: a pump, some tubing, a valve, a detector

and laptop computer.

• Can perform all steps required for analysis: sample buffering,

extraction, elution, mixing with reagents and monitoring…

• FIA automates processes and yields higher reproducibility (hence low

detection limits)

• Very fast sample throughput (min to sec per sample!)

•Ideal for hydrographic surveys when large amounts of samples are

produced (e.g., 36 samples every 4 hours on CLIVAR)

A more complicated FIA setup…

The Measure’s lab van setup:

Shipboard determination of

dissolved Fe, Al and Mn by FIA.

Shipboard Determination of Fe by FIA

Measures et al. (1995). Mar. Chem. 59:3-12.

1. Raise pH of seawater sample to

pH>5.0

2. Preconcentrate Fe in buffered sample

on a resin functionalized with 8HQ

groups (A)

3. Elute preconcentrated Fe plug with

HCl

4. At pH>5 Fe will catalyze oxidation of

DPD in the presence of peroxide. It

yields a pink product (DPDQ)

monitored at 514nm (B)

5. The intensity of the color is

proportional to the concentration of Fe

present in the sample

The chemistry:

Shipboard Determination of Fe by FIA

Modified from Measures et al. (1995). Mar. Chem. 59:3-12.

1. Raise pH of seawater sample to

pH>5.0

2. Preconcentrate buffered sample on a

resin functionalized with 8HQ groups

(A)

3. Elute preconcentrated Fe plug with HCl

4. At pH>5 Fe will catalyze oxidation of DPD

in the presence of peroxide. It yields a pink

product (DPDQ) monitored at 514nm (B)

FIA: A Myriad of Applications

• One of the most popular shipboard analytical methods.

• Some example analytes: NO3-, PO4

3-, H4SiO4, NH3,TIC,

Fe, Al, Mn, Zn, Co….

•Also used as a sample preparation tool:

• Sorbent extraction (SPE)

• Membrane separation

• Gas/liquid separation

• Solvent extraction (hyphenated to FAAS

and ICPMS)

•More info: www.flowinjectiontutorial.com

Current state of the art for metals…

Miniaturized Flow Analysis:

micro-Sequential Injection Lab-on-Valve (μSI-LOV)

Lab this Friday: Dissolved Zn2+ by μSI-LOV

•The field of chemical oceanography is, for some key

elements, slowly moving towards in situ monitoring

• FIA is not well suited for this purpose because:

• Reagent consumption is too high (mLs/sample)

• Frequent user maintenance required (tubing)

• Lots of moving parts

• Sequential Injection Analysis (SIA) overcomes many of

the issues of FIA and brings in miniaturization.

• This lab this Friday will use a fluorometric SIA method

(called micro-Sequential Injection Lab-on-Valve: μSI-LOV)

to determine Zn2+ in samples collected during the field trip.

μSI-LOV and in situ monitoring

• Compactness & Automation

• Low Reagent consumption

• Minimal maintenance

• Low power needs

• Stability of system & reagents

In Situ Requirements:

What is μSI-LOV and how does it work?

μSI-LOV: latest generation of FIA techniques

• Like FIA, SIA is based upon the principle of partial dispersion (mixing) of

sample and reagents and the propulsion of resulting product into a detector

• BUT SIA operates on programmable rather than continuous forward flow

bringing considerable reagent savings, better control and full automation of any

assay

microSequential-Injection analyzer with

Lab-on Valve module Close up of the Lab-on-Valve

μSI-LOV Flow cell configurations Can do all absorbance, fluorescence and chemiluminescence detection with one fluidic

device by moving optical fibers in appropriate slots

Flow Programming: the basis of all SIA

1. Fill Holding Coil (HC) with

carrier solution

2. Aspirate reagent (25-

100μL) in HC

3. Aspirate sample (25-

100μL) in HC

4. Deliver mixture to flow cell

5. Monitor product.

Movie of a simple μSI-LOV Assay

LIGHT

from Ruzicka, 2009

Fluorometric Determination of Zn by μSI-LOV

Grand et al. Analyst, 2011, DOI: 10.1039/C1AN15033B

LOD ~ 0.1-0.5nM Zn2+

• < 1min per sample

PMT

REAGENT

SEAWATER

CARRIER

LED

470nm

Y

HC 500μL

MONITORING

REAGENT HC

50μL 75μL

SAMPLE

FC

SEAWATER REFERENCE SAMPLES

[nM] Consensus Measured % Recovery

GD 1.6 ± 0.2 1.8 110

SAFeD 7.2 ± 0.5 7.5 104

Steps towards a new μSI-LOV method

1. Select a reagent suitable to the task (sensitivity&selectivity)

2. Optimize reaction parameters (pH, reagent concentration, stability)

3. Determine reagent selectivity (seawater contains all stable elements of

PT)

4. Optimize fluidic protocol (volumes, aspiration sequence and flow rates)

5. Validate the method using available CRMs

6. Bring the instrument at sea and put it to the test

1. Write a methods paper that fellow nerds may read

Sensitivity and pH

From Grand et al. Analyst, 2011, 136, 2747

FluoZinTM -3 Ex-Em=494-516 nm; Kd=15nM

RhodZinTM -3 Ex-Em=550-575 nm; Kd=65nM

Reagent Concentration/Stability

From Grand et al. Analyst, 2011, 136, 2747

REAGENT

CONCENTRATION

REAGENT STABILITY @

22°C

Lab this Friday: Dissolved Zn2+ by SIA

• Include in your report (no more than 3 pages single spaced with figs and

refs):

• A brief description of the method and solutions used to perform the

analysis (calibration, acidity of samples, carrier solution used etc etc…

Remember, we may have to dilute the samples…)

• A calibration curve with its equation

•Precision of the method (hint: use replicate samples for this purpose.

e.g., precision was less than 5% @ 25nM Zn)

• Detection limit of the method in nM (3 times the standard deviation of

the +0 standard divided by slope of calibration curve)

• A brief comparison of your data with typical estuarine and open ocean

Zn levels.

• A brief interpretation of the trends seen along the transect. What are

potential sources of the Zn? Does Zn correlate with any other

parameter?

•If you claim that there is a gradient in Zn concentrations, back it up

with appropriate statistics!

Some Useful Resources

1.) Flow Injection Analysis Online Tutorial by Jarda Ruzicka, 2013.

www.flowinjectiontutorial.com

2.) Advances in flow injection analysis and related techniques. Kolev, S., McKelvie, I.D.,

eds. Elsevier, 2008

3.) A very good review on trace elements in seawater: Bruland and Lohan (2003).

Controls of Trace Metals in Seawater. Treatise on Geochemistry, 6: 23-47. Email me for

PDF.

A MUCH LESS SERIOUS RESOURCE:

Want to know what it is like to do TM analysis onboard the icebreaker RV

Palmer in the Southern Ocean???

check out http://vimeo.com/31474058

1. Select Fluorescent Indicator

Gee et al., Cell Calcium, 2002, 31

,245

MOLECULAR PROBES ®

FluoZinTM -1 Ex-Em=495-515 nm

Kd= 8000nM

FluoZinTM -3 Ex-Em=494-516 nm; Kd=15nM

NewPort GreenTM DCF Ex-Em=505-535 nm

Kd=1000nM

RhodZinTM -3 Ex-Em=550-575 nm; Kd=65nM

3. Indicator selection: selectivity

FluoZinTM -3 Ex-Em=494-516 nm; Kd=15nM

From Grand et al. Analyst, 2011, 136, 2747

For a 5nM Zn sample:

•[Hg]~ 1pM (0.01% error)

• [Cd]~5nM (~3% error)

4. Optimize Fluidic Protocol

Grand et al. Analyst, 2011, 136, 2747

FLOW CELL

HOLDING COIL

SAMP.

MONITORING

50μL 75μL

REAG. FLOW CELL

REAG.

MONITORING

HOLDING COIL

50μL 75μL

SAMP.

HOLDING COIL

FLOW CELL SAMP.

35μL 50μL 35μL

REAG REAG REAG. FLOW CELL

HOLDING COIL

50μL 35μL 35μL

SAMP SAMP

From: Grand et al. Analyst, 2011, DOI: 10.1039/C1AN15033B

5&6. Application and Validation

DI WATER

LOD=0.3nM

Zn2+ (3σ)