laboratory measurement protocols for:

Laboratory measurement protocols for:Laboratory measurement protocols for:

1.1. Particulate, Particulate, aapp((, and Detrital, , and Detrital, aadd(()), ,

absorption spectraabsorption spectra

2.2. Fluorometric chlorophyll Fluorometric chlorophyll aa concentrations concentrations [Chl [Chl aa]]

3.3. Colored dissolved organic matter (CDOM) Colored dissolved organic matter (CDOM) absorption spectra, absorption spectra, aaCDOMCDOM(() )

(or gelbstoff/gilvin, a(or gelbstoff/gilvin, agg((), or yellow substance, a), or yellow substance, ayy(())))

Contact info:Jen Cannizzaro [email protected] ext. 3-3954

Phytoplankton

H

O

H

Water

Detritus

CDOM

Particulate (>0.2μm) Dissolved (>0.2μm) Molecular

)(

)()(

a

bR b

rs

Review:

a() = ap() + aCDOM() + aw()

where ap() = aph() + ad()

=marine/terrestrial plant decay composed of humic and fulvic acids

=fecal pellets, dead phytoplankton, minerals, etc.

Review:

Detritus

CDOM

PhytoplanktonReview:

Longitude (oW)

84 83 82

Lat

itu

de

(oN

)

26

27

28

EcoHABHyCODE

Gulf ofMexico

Sarasota

Fort

Myers

Tampa

Tampa Bay

CharlotteHarbor

Example spectra…..

NEXT…..1. Particulate, ap(, and Detrital, ad(), absorption spectra

2. Fluorometric chlorophyll a concentrations [Chl a]

3. Colored dissolved organic matter (CDOM) absorption spectra, aCDOM() (or gelbstoff, ag(), or yellow substance, ay())

1. ap() and ad()…..Technique for measuring ap() and ad() is known as the “Quantitative Filter Technique” (QFT) (Mitchell, 1990)

Basic steps:

1.Filter seawater onto a filter to concentrate particles

2.Measure the optical density (or absorbance) spectra of the total particulate matter (phytoplankton + detritus), ODp()

3.Extract the phytoplankton pigments from the filter using hot methanol leaving only the detrital matter on the filter

4.Re-measure the optical density (or absorbance) spectra of the detrital particles on the filter, ODd()

5.Calculate ap() and ad() from ODp() and ODd(), respectively

Filtration set-up…

Vacuum pump

Trap (protects

pump from overfill)

Dessicant (protects pump from excess moisture)

Filtration apparatus (diameter=25mm)

Flask for collecting dissolved material

1. ap() and ad()…..

Sample filter preparation:

-Collect water sample in dark bottle and maintain at in situ temperature

-Filter sample immediately, or if not possible within a few hours of collection

-Use Whatman’s GF/F (25mm diameter) filters- made from glass fibers- nominal pore size = 0.7μm

-Filter samples under low vacuum pressure (~125mm Hg or ~5 in Hg) to prevent cells from bursting

-Protect sample from light to minimize photodegradation

1. ap() and ad()…..

http://www.usplastic.com/catalog/ImageViewer.aspx?description=Nalgene&

1. ap() and ad()…..


1. ap() and ad()…..

Record volume seawater

filtered here

Record ancillary info

here

Station

Logsh

eet


-Place sample filter in petri dish on drop of filtered seawater. Do not allow filters to run dry!

-Cover petri dish with aluminum foil to prevent photodegradation

-If long-term storage (>24hr) is necessary, then place filter in a Fisher Histoprep tissue capsule and store in a deep freezer (- 20oC) (< 1 week) or liquid nitrogen (> 1 week)

-Remember to keep filter flat (never fold)and do not touch colored surface with forceps

1. ap() and ad()…..

Reference filter preparation:

-Prepare reference filter same way as sample filter (e.g. low vacuum pressure), except use ~250ml of 0.2μm filtered seawater

-Prepare reference filter same day as measurements are to be taken

-Store pad in petri dish on drop of filtered seawater whenever not in use

-Do not allow reference filter to dry out

1. ap() and ad()…..

1. ap() and ad()…..Measurement of ap(λ): “Spectrix”

512-channel spectral radiometer(~350-850nm)

lamp (tungsten-halogen)

Computer for running DOS program “get_hex.exe”

“padbox”

Reference filter (on left)Sample filter (on right)

1. ap() and ad()…..Measurement of ap(λ):

-Allow Spectrix and lamp to warm up (~5-10 minutes)

-Record Specrix ID (e.g. 300, 303, 306) and spectral calibration coefficients (a, b, + c) on log sheet

λ = a + b [channel] + c [channel]2

(where channel = 0-511)

-Place reference and sample filters on plate inside “padbox” with the reference filter on the left and the sample filter on the right, and then close the door

Log sheet

1. ap() and ad()…..Measurement of ap(λ):

-Run “get_hex.exe” in c:\spectrix\- Choose filename (max. 9 characters; use *.ap extension)- Choose serial port #1

-Measure transmittance (T) of filters in the following sequence:[Treference(λ), Tsample(λ)] * 3 = 6 scans

-Press ‘ESC’ → ‘Y’ →’Enter’ to escape program

-Place both filters back in respective petri dishes (NOTE: again, do not allow reference filter to dry out in padbox while pigments are being extracted)

1. ap() and ad()…..Pigment extraction (Kishino et al., 1985):

-~10 minutes prior to measuring transmittance of filters for ap(λ), prepare hot methanol for pigment extraction

- fill tin cup ~1/2 to 2/3 full with tap water and place on hot plate

- turn hot plate on (~150-200oC)

- fill 4oz amber glass bottle (with handle) ~2/3 to 3/4 full with HPLC-grade methanol

- begin heating methanol in water bath ~2-3 minutes just prior to extraction

- heat methanol until hot (but NOT boiling)


-Label clean 4oz amber bottle with sample date, station ID, and sample depth and place uncapped inside plexiglass extraction rig

-Place sample filter on filtration apparatus, tighten the funnel securely, but not so tight that you rip the filter

-Pour ~10-15ml of hot methanol directly onto the filter(ALWAYS POUR AWAY FROM FACE!)

-Draw methanol initially through the filter by pumping hand vacuum pump once, then release the vacuum by pulling up on black rubber stopper

-Cover filter funnel with aluminum foil during extraction to prevent photodegradation


-Once all methanol has been drawn through filter or after ~5 minutes, add another ~15-20ml aliquot of hot methanol

-Repeat one more time allowing pigments to extract for a total of ~15-20 minutes using ~60ml of methanol (or until bottle is ~1/2 full)

-Once extraction is complete, draw any remaining methanol through filter using hand pump

-Remove amber bottle, cap it, and set aside for [Chl a] determination

-Replace with a designated “waste” bottle

-Add ~5ml of filtered seawater to re-moisten filter; draw water through filter using hand pump

-Place sample filter back in petri dish on drop of filtered seawater

-Rinse filtration apparatus with methanol prior to next extraction


DANGER! METHANOL IS HIGHLY FLAMMABLE!

THEREFORE,

•NEVER FILL AMBER BOTTLE ALL THE WAY FULL!

•NEVER FULLY TIGHTEN CAP WHILE HEATING!

•NEVER LEAVE HOT PLATE UNATTENDED!

•ALWAYS POINT BOTTLE AWAY FROM YOU WHEN POURING HOT METHANOL!

•WHENEVER POSSIBLE, USE FUME HOOD

1. ap() and ad()…..Measurement of ad(λ):

-Place reference and extracted sample filters back onto the plate inside the “padbox” in same order as before, and then close padbox door

-Re-run “get_hex.exe”- Choose same filename as for ‘ap’ filter, except this time use *.ad extension- Choose serial port #1

-Re-measure transmittance (T) of filters in the same sequence as before:[Treference(λ), Tsample(λ)] * 3 = 6 scans

-Press ‘ESC’ → ‘Y’ →’Enter’ to escape program

-Place reference filter on drop of filtered seawater in petri dish to re-moisten (if future scans are necessary)

-Sample filter can now be discarded

1. ap() and ad()…..

reference

sample

T

TLogOD 10

Calculation of ap(λ) and ad(λ) :

-Before calculating ap(λ) and ad(λ), one must know the geometric pathlength of the filtered material in suspension, lS

(units = m)

where

Vf is the volume of seawater filtered (units = m3)(Note: 1 cubic meter = 1,000 liters)

Af is the clearance area of the filter (units = m2)(Note: Af = π r2)

1. ap() and ad()…..

f

fS A

Vl

r

1. ap() and ad()…..Calculation of ap(λ) and ad(λ) :

-Then,

(units = m-1)

where

λnull is a near-infrared wavelength where absorption due to particles is negligible

(NOTE: many folks use 750nm as λnull , but we use the average between

780-800nm since coastal ODp(750) is often non-negligible)

β is the so-called “Beta factor” (or pathlength elongation factor) that corrects for pathlength increases due to multiple scattering in the filter

S

nulldpdpdp l

ODODa

,,

,

303.2


-Which β-factor should you use?

…changes depending on species composition …

Cultures… Field samples/ phytodetritus…

β = 1.0 + 0.6 * ODf(λ)-0.5

ODODfilterfilter

____________________________

ODODsuspensionsuspension

ββ = =


-Ideally, for the particulate filter you want:

- ODf(675) ≥ 0.04 to minimize uncertainty in β-factor (see below)

- ODf(440) ≤ 0.40 to prevent errors due to self-shading

-Must adjust volumes of seawater filtered if values fall out of range!

0.04

0.40

1. ap() and ad()…..Calculation of ap(λ) and ad(λ) :-Review…

TransmittanceParticulate filter: Treference

Tsample

Detrital filter: Treference

Tsample

Optical DensityParticulate filterDetrital filter

AbsorptionParticulate filterDetrital filter

-------- aph() = ap() - ad()

2. [Chl a]…..Measurement of [Chl a]:

Chlorophyll a concentrations, [Chl a], can be measured in three ways:

1. Spectrophotometrically

2. Fluorometrically

3. High-performance liquid chromotography (HPLC)

Here , we calculate [Chl a] fluorometrically

Advantages:- more sensitive then spectrophotometry- faster and simpler than HPLC

Disadvantages: - overestimations/underestimations due to accessory chlorophyll’s and chlorophyll degradation products (e.g. pheopigments)

2. [Chl a]…..Measurement of [Chl a]:Instruments: Turner Designs 10-AU Field Fluorometers (2 set-up’s)

Holm-Hansen (“acid”) technique Welschmeyer (“no acid”) technique

- Excitation filter: 340-500 nm - Excitation filter: 436 nm- Emission filter: >665 nm - Emission filter: 680 nm- Light source: Daylight white lamp - Light source: Blue lamp

-Calibration is done ~2 times/year using pure chlorophyll a (Sigma Aldrich)

-Secondary Solid Standard used to track and correct for instrument drift

Secondary solid standard

References: Holm-Hansen and Riemann (1978) Welschmeyer (1994)

Measurement of [Chl a]:

So why do we measure [Chl a] in two ways?. . . .

2. [Chl a]…..

from Welschmeyer, 1994

NOTE: overlapping fluorescence emission spectra for chlorophyll’s a, b, and c2 along with chlorophyll degradation products pheophytin a, b, and c2

[Chl a]’s measured using the “acid” technique are overestimated when Chl b: Chl a ratio is high and underestimated when Chl c: Chl a ratio is high


• difference between “acid” and “no acid” [Chl a]’s is low for Chl’s > 0.3 mg m-3

• “acid” [Chl a]’s are overestimated for Chl’s < 0.3 mg m-3 due to interference by Chl b

2001 ECOHAB DATA

acid [Chl a] (mg m-3)


-Remember the methanol that was used to extract the pigments from the total particulate filter pad?

-This is the sample that you’ll use to measure [Chl a]

-These samples should be stored in a cool, dark place until measurements are made (ideally, within a few hours

of extraction since pigments are unstable in methanol)


STEPS:

-Warm up fluorometer ~1-2 hours prior to measurement

-Record ‘low’ and ‘high’ readings for both fluorometers using secondary solid standard on logsheet

-Record volume of methanol using 100ml graduated cylinder on logsheet

-Remove glass-fiber particles using a 0.2μm syringe filter (discard first ~5-10ml of sample into waste container)

60ml syringe and 0.2μm syringe filter


Log sheet:

volume of seawater filtered (ml)

volume of methanol used to extract the pigments (ml)

Secondary solid standard low and high values for both fluorometers

date sample is processed

Sample info: station ID, sample date, sample depth, etc.


STEPS (continued):-Fill test tube ~3/4 full (Note: Never hold tube in middle and minimize light exposure to prevent photodegradation)

-Wipe test tube clean using dry Kimwipe

-Place tube in “no acid” fluorometer

-Press ‘*’ button (15 sec. delay, 10 sec. average)

-Record ‘No acid Rb’ value on logsheet

-Place tube in “acid” fluorometer

-Press ‘*’ button (same 15 sec. delay, 10 sec. average)

-Record ‘Acid Rb’ value on logsheet

-Add two drops of 10% HCl to sample

-Press ‘*’ button again

-Record ‘Acid Ra’ value on logsheet

-Discard methanol into waste container


Log sheet:

“No Acid” fluorometer reading (Rb) measured BEFORE the sample is acidified

“Acid” fluorometer readings measured BEFORE (Rb) and AFTER (Ra) the sample is acidified

Calculation of [Chl a]:

In order to calculate [Chl a] and [Pheo], you will need the following values measured during instrument calibration that can be found on the front face of each fluorometer:- ratio, ‘r’, of fluorescence of the pure chlorophyll a standard measured before and

after acidification (“acid” fluorometer only)

- ‘low’ and ‘high’ secondary solid standard values (both fluorometers)

For the “no acid” fluorometer…

[Chl a]no acid = (Rbcorr) (volume MeOH/volume seawater)

For the “acid” fluorometer…

[Chl a]acid = (r/r-1) (Rbcorr – Racorr

) (volume MeOH/volume

seawater)

[Pheo]acid = (r/r-1) (Racorr * r - Rbcorr

) (volume MeOH/volume

seawater)

2. [Chl a]…..

2. [Chl a]…..Calculation of [Chl a]:

where…

Rbcorr and Racorr

are the Rb and Ra values, respectively, corrected for

changes in the secondary solid standard measured since calibration….

Rbcorr = Rb * Secondary_Solidcorrection_value

Racorr = Ra * Secondary_Solidcorrection_value

where

Secondary_Solidcorrection_value =

[(Low_valuecal./Low_value) + (High_valuecal./High_value)] / 2----------------------------------------------------------------------------------------------------------------

Note: Secondary_Solidcorrection_value will differ for the two fluorometers and usually ranges between 0.9 - 1.2

[Chl a] and [Pheo] units: mg m-3 or μg l-1

Sample preparation:Recall our filtration set-up…

For ap(λ), ad(λ), and [Chl a], we cared only about the particles collected on top of the filter

For aCDOM(λ), we care about the dissolved material that passes through the filter

3. aCDOM()…..

Sample preparation is much the same as before….except you must also filter the water through a 0.2μm filter and

this time you save the FILTRATE and not the filter

Sample preparation:

-Collect water sample in dark bottle and maintain at in situ temperature

-Filter sample immediately, or if not possible within a few hours of collection

-Filter samples under low vacuum pressure (~125mm or ~5 in Hg) to prevent cells from bursting

-Protect sample from light to minimize photodegradation

-Use Whatman’s GF/F (25mm or 47mm diameter) filter to pre-filter your seawater sample (Note: this is especially

necessary for coastal waters where 0.2μm filters can clog easily)

•filter ~50ml of sample to rinse filter and flask; dispose of filtrate

•filter ~200ml of sample through cleaned filter

•to avoid an extra step, feel free to use some of the filtrate from the ap(λ) filtration

3. aCDOM() ...

↑ 47mm diameter filtration

apparatus

3. aCDOM()…..Sample preparation:

-Using 47mm glass filtration apparatus, rinse a 0.2μm filter (nylon membrane or Nuclepore polycarbonate) with ~50ml of the GF/F filtrate; shake flask and then dispose water

-Filter the remaining GF/F filtrate through the rinsed 0.2μm filter

-Rinse a clean 4oz amber glass sample bottle (labeled with date, station ID, and sample depth) twice with ~10-20ml of 0.2μm filtrate

-Then, fill sample bottle until ~2/3 to 3/4 full

-Process sample immediately, or if not possible place sample in refrigerator (<24 hrs) or freezer (>24 hrs) depending on length of storage time required

DO NOT overfill bottle or else bottle may crack during freezing!!!

3. aCDOM()…..Measurement of aCDOM(λ)

-Instrument: Perkin-Elmer Lambda 18 UV/VIS spectrometer- wavelength range: 185-900nm- photometric accuracy: 0.002 Absorbance units (AU)

Sample compartment (fits 1 cm or 10 cm cells)

Tungsten-halogen and deuterium lamps


-Instrument warm-up time ~1 hr

-If samples are frozen, place bottles in refrigerator ~24 hours prior to processing. Remove samples from refrigerator ~1 hr prior to measurement and allow to warm up to room temperature

- NOTE: thawing samples slowly helps minimize the creation of new particles via aggregation

-Collect ~2 liters of clean water from a water purification system (e.g. Milli Q) to use for cleaning cuvettes and for your reference

3. aCDOM()…..Measurement of aCDOM(λ)-Computer set-up:

- Open ‘UV Winlab’ software (be patient, takes time!)

- Choose Scan method ‘AG.MSC’ which contains parameters:Start λ = 800 nm End λ = 200 nmData Interval = 1 nmAutosave = ONAutoprint = OFF # Cycles = 1Ordinate Mode = A Scan Speed = 240 nm/minSmooth = 0 Lamp change = 319.2 nm

- Click ‘Utilities’→’Configuration’ to change the data storage path (typical path: c:\uvwinlab\data\YYMMMDD, e.g. \10mar01\)

- Click ‘Sample’ tab and change first filename to ‘DDMMM001’ (example: for 3/1/2010 the filename would be ’01mar001’; next scans will automatically be saved as ‘002’, ‘003’, etc.)


-Clean 10cm spectrophotometric cuvettes alternatively with 10% HCl, methanol, and then lots of pure water

NOTE: never touch optical windows and handle only from sides

-Carefully dry the outside of the cuvettes with Kimwipes

-Fill cuvettes slowly with pure water making sure not to create bubbles

-Hold cuvettes up to light and check for stray particles and smudges on optical windows

-Place cuvettes carefully in sample compartment. The cuvette in the back will be your ‘reference’ cuvette, the one in front is your ‘sample’ cuvette

-Allow water to settle for a few minutes prior to running baseline scan

10cm


- Run baseline scan by clicking on ‘Autozero’ (NOTE: info is stored internally)

-Check quality of baseline scan by scanning this same pure water

- Click the ‘Sample’ tab and type ‘10cm Milli Q baseline’ in the ‘Sample Info’ section

- Click ‘Start’ to measure the absorbance spectra (A()) of the pure water(Ideally, absorbance values should be 0.001 AU centered around zero. If not, repeat autozero and baseline check)

-Fill out logsheet…

Processing date

When instrument turned on and off

Operator’s initials

Filename (e.g. 01mar001)

Scan information: if baseline: ‘10cm Milli Q baseline’ if sample: cruise, sample date, sample depth, station ID, etc.


- Remove both cuvettes from sample compartment and discard water

- If CDOM sample was frozen, then re-filter sample through a 0.2μm syringe filter directly into the ‘sample’ cuvette after first discarding ~10-15ml sample to rinse the filter and cuvette (NOTE: 2nd filtration is necessary since particles often form during the thawing process)

- After wiping the cuvette dry with a Kimwipe and checking it for particles/smudges, place cuvette in sample compartment and allow to settle for a few minutes

- Meanwhile,- replace pure water in ‘reference’ cuvette, wipe cuvette and check

for particles/smudges, and then place back into sample compartment

- Click ‘Sample’ tab and enter Sample Info (cruise, sample date, sample depth, station ID, etc.) in data file and on logsheet


- Click ‘Start’ and monitor the red portion of the spectra:

• If A(800) > 0.001 AU, then re-check ‘sample’ cuvette for particles/smudges

• If A(800) < -0.001 AU, then re-check ‘reference’ cuvette for particles/smudges

- Repeat scan to collect duplicate measurement. Record sample info in data file and on logsheet

- Once both scans are complete, pour sample back into sample bottle and store in refrigerator until after data processing is complete

- Measure next sample making sure to rinse ‘sample’ cuvette and to replace pure water in ‘reference’ cuvette

- After the 4th or 5th sample, clean both cuvettes (10% HCl, methanol and pure water), fill cells with pure water, and then scan to check for drifts in baseline


- Final scan should also be a baseline check using pure water. Prior to this scan, clean both cuvettes with 10% HCl, methanol, and water

- Leave cuvettes filled with water inside spectrometer with caps on

- Exit software and download *.sp files (ASCII) in c:\uvwinlab\data\(data directory)\ to floppy disk

3. aCDOM()…..Calculation of aCDOM(λ)

- To calculate CDOM absorption spectra, aCDOM(λ), from CDOM absorbance spectra, ACDOM(λ)….

(units = m-1)

where

l = cuvette pathlength (10cm = 0.1 m)

and

λnull is a near-infrared wavelength where absorption due to CDOM is negligible (NOTE: This wavelength will depend on the type of

water being sampled. For oligotrophic samples, λnull = 550nm may be appropriate. For coastal samples, λnull =780nm may be appropriate)

nullCDOMCDOM AAl

a 303.2

- To reduce noise, spectra are smoothed using IDL ‘smooth’ function

3. aCDOM()…..Calculation of aCDOM(λ)

Temperature/ salinity artifact

Order of magnitude difference

null = 550nm null = 780nm

REFERENCES:

•Ocean Optics Protocols For Satellite Ocean Color Sensor Validation, Revision 4, Volume IV: Inherent Optical Properties: Instruments, Characterizations, Field Measurements and Data Analysis Protocols (http://oceancolor.gsfc.nasa.gov/DOCS/Protocols_Ver4_VolIV.pdf)

•Mitchell, B.G. 1990: Algorithms for determining the absorption coefficient of aquatic particulates using the quantitative filter technique (QFT). Ocean Optics X. 137-148

•Kishino, M., Takahashi, M., Okami, N., Ichimura, S., 1985. Estimation of the spectral absorption coefficients of phytoplankton in the sea. Bulletin of Marine Science 37, 634-642.

•Holm-Hansen, O., Riemann, B., 1978. Chlorophyll a determination: improvements in methodology. Oikos 30, 438–447.

•Welschmeyer, N. A. (1994). Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments. Limnology and Oceanography 39(8): 1985-1992.

laboratory measurement protocols for:

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