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Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000
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Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Dec 15, 2015

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Page 1: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Dyed Sand Lab Book

Donna S. Lutz

Iowa State University

2000

Page 2: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

COE Dyed Sand StudyMajor Objectives

Use dyed sand to trace dredge material movement in river system

Find appropriate dye to dye sand particles Develop fluorometry procedure to identify

presence of dyed sand Evaluate the background interference

caused by chlorophyll

10/3/00

Page 3: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Dayglo dyed sand

Supplied by the COE 0.6 mm sand, 227g w/ 3.2

g dye

Signal Green SG (534 nm) Saturn Yellow SY(563 nm) Rocket Red RR (614 nm) Horizon Blue HB(477 nm)

nm are dominant %transmittance /

reflectance supplied by Dayglo

10/3/00

Page 4: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

10/3/00

Page 5: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 6: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Testing Solvents

Prepared samples by dissolving 10 grains of 4 dyed sands in about 4.5 ml petroleum ether, 90% acetone, methylene chloride and water in 16x125mm test tube. Each tube was inverted 50 times, then vortex mixed for 15 sec.

Results, dye was soluble in acetone and methylene chloride but not in water or petroleum ether (These results were later confirmed by Dayglo)

10/3/00

Page 7: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 8: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Interference w/ chlorophyll

Chlorophyll pigment extractions (90% actetone) were examined under blue filter black light BLB (St 1, 10 and 7 from Sayl 1248)

BLB wavelength 365nm (345-400nm) It was determined that chlorophyll does indeed fluoresce in

the red light spectrum Main wavelength for chlorophyll is 630-665nm Recommend using dye outside this range

10/3/00

Page 9: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 10: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Preparation of Dyed Sand/Sand samples

2g 0.6mm sand with 0.1g of each of four dyes added Photo was taken under BLB of dry mixture Samples were dissolved in about 4.5 ml 90% acetone,

inverted and mixed BLB photo taken

10/3/00

Page 11: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 12: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 13: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Notes from 10/3/00 HB dye may be a good choice, however trace fibers also

fluoresce blue wavelength 477 is outside chlorophyll range it appears to fluoresce well Solvent could be acetone or methylene chloride MeCl Have ordered cells for Turner machine, should arrive in 2-

3 days

Page 14: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

To do next

Will prepare chlorophyll sample using MeCl to compare Dyed Sand/Sand samples could be centrifuged to remove

turbidity Will prepare Dyed Sand samples without the 2g sand to

compare Will arrange to use spectrofluorometer to define excitation

and emmission filter selection for Turner fluorometer

Page 15: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Labwork 10/9/00

Document lighting arrangement - COE’s results are brighter, either it’s our lighting or imaging

Investigate effect of centrifugation on turbidity Prepare samples of only 0.1g dyed sand with 90% acetone

to compare to COE samples

Page 16: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 17: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Log Spectral Irradiance of Blacklight and Blacklight Blue at 20 cm

1.0E-10

1.0E-09

1.0E-08

1.0E-07

1.0E-06

1.0E-05

1.0E-04

250 300 350 400 450 500 550 600 650 700 750 800

Wavelength [nm]

Irra

dia

nc

e [

W/(

cm

2 nm

)] BL

BLB

Page 18: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 19: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
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Page 22: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 23: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 24: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 25: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 26: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Notes from 10/9/00

COE lighting system is better Centrifuging at 3000 rpm for 10 min is

adequate to remove turbidity

Page 27: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Work done 10/18/00 at Dr Jenks Lab

5 dye samples, chorophyll (St 7 from Week 1248), acetone blank and sand/acetone blank were run

Scanning spectrophotometer was used to determine excitation wavelength

Using determined excitation wavelength samples were run on scanning spectrofluorometer

Dyed samples were dilute (20 drops from 0.1g dye to 4.5ml acetone samples diluted to 3 ml)

Chlorophyll sample was not diluted

Page 28: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 29: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
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Page 32: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 33: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Initial Data 10/18/00

SampleISU Excitation

WavelengthISU EmissionPeaks (nm)

DayGlo%Transmittance

/ReflectanceDominant

WavelengthSignal Green 450 nm 514, 628 534 nmSaturn Yellow 440 nm 540, 651 563 nm ??Rocket Red 527 nm 676 614 nm ??Horizon Blue 370 nm 735 477 nmAurora Pink 555 nm 692 493 nm ??Chlorophyll 433 nm 671, 765 --Acetone 419 nm ? 433, 474, 676, 793 --Acetone + Undyed sand 450 nm ? 454, 584, 658, 791 --

? just picked a wavelength, no peak?? doesn't seem to match graph

Page 34: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Questions to Ask

1. Overall, what do the ISU spectrograms tell us?

2. What are the spectrograms from DayGlo and why don't they match any of our results?

3. Using a fluorometer, which dye would be the most detectable (and distinguishable from backgound, i.e. acetone, chlorophyll and sand)?

Page 35: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

1. What do the spectrograms tell us?

The spectrophotometer output shows us what the excitation wavelength is for each sample. It is assumed that if we excite the sample at it’s peak excitation wavelength we will see the greatest peaks in the spectrofluorometer output.

The scanning spectrofluorometer shows us at what wavelength the sample fluoresces at, given that specific excitation wavelength

The peaks seen in the photometry output are mirrored with peaks in the fluorometry output, as expected

Page 36: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

1. What do the spectrograms tell us? (cont’d)

Acetone gave a huge absorbance between 210 and 345 nm which was mirrored in the spectrofluorometer results at 850 nm and above

Chlorophyll had two distinct peaks in absorbance at 433 and 663 nm

Turbidity needs to be eliminated to reduce noise/interference (see undyed sand-acetone results)

Acetone blank will always have to be used and acetone subtracted from results

Page 37: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

2. What are the spectrograms from DayGlo and why don’t they match our data?

Conference call 10/19 with N McVay, Corps and Dick Bonsutton, DayGlo

DayGlo graphs are from a spectrophotometer using dried paint samples

Measured parameter was reflectance Not considered strange that our dyed sand-

acetone samples would have different optical characteristics

Page 38: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

3. Using a fluorometer, which dye would be the most detectable (and distinguishable from backgound, i.e. acetone, chlorophyll and sand)?

For best detection, the selected dye should have an excitation wavelength outside of the range 210-345 nm, 620-675 nm and 400-500nm. Thus, HB, RR and AP are candidates, however, HB may be a little close to acetone.

HB has the sharpest fluorometer peak but it is excitated near chlorophyll’s excitation wavelength.

AP and RR appear to be good choices at this time.

Page 39: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Comparison of Excitation Wavelengths

RR and AP may be the best dyes to detect by flluorometry because they excite at different wavelengths than either acetone or cholorphyll

Page 40: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

What to do next?

Return to Dr Jenk’s lab and

– Excite AP, chlorophyll and acetone at AP’s excitation wavelength (555nm) and compare spectrofluorograms

– Excite RR, chlorophyll and acetone at RR’s excitation wavelength (527nm) and compare spectrofluorograms

– Excite HB, chlorophyll and acetone at HB’s excitation wavelength (365nm) and compare spectrofluorograms

– Explore if it is possible to select the emission wavelength and scan the excitation wavelengths

Page 41: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

2nd trip Dr Jenk’s Lab 10/23/00

Samples were 10 dyed sand grains dissolved in 4.5 ml 90% acetone, acetone blank and chlorophyll sample from St 1 for Sayl 1248 (undiluted)

Redid scans with spectrophotometer to verify excitation wavelength for the dyes, AP, RR, and HB.

Optical density from these samples was below the optimum 0.1 to 0.3 range.

AP had the highest peaks on the spectrophotometer scans, it was difficult to discern significant peaks for HB.

Spectrophotometer scans revealed the same excitation wavelength for AP (555 nm) and RR (530 nm).

Page 42: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Spectrofluorograms from 10/23/00

– Excited AP, chlorophyll and acetone at AP’s excitation wavelength (555nm) and compared spectrofluorograms; AP peaks are different than acetone but are masked by chlorophyll

– Excited RR, chlorophyll and acetone at RR’s excitation wavelength (530nm) and compared spectrofluorograms; RR peaks are different than acetone but are masked by chlorophyll

– Excite HB, chlorophyll and acetone at HB’s possible excitation wavelengths (370nm and 730 nm) and compared spectrofluorograms; no good HB detection at 730nm, but there could be far end (>790nm) peak for HB that would be distinguishable.

– Was told it was not advisable to select the emission wavelength and scan the excitation wavelengths on the fluorometer; that the spectrophotometer would give better results.

Page 43: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 44: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.
Page 45: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

Conclusions from 2nd lab Jenks lab study

AP has highest optical density of the samples, which may mean greater detection and it is easily distinguished from acetone

However, chlorophyll can block all dyes at their excitation wavelengths.

Will need to minimize chlorophyll contamination in the sample before dissolving dye in order to measure spectrally.

Page 46: Dyed Sand Lab Book Donna S. Lutz Iowa State University 2000.

What to do next?

Might be worth taking one more look at HB and extend the scan to the maximum 880 nm

Then the focus should be on how to reduce the effect of chlorophyll– physically remove chlorophyll through sample preparation

– mechanically shake, rinse samples, dry, centrifuge and filter?

If the Corps wants to pursue this avenue further, we’d want “real” samples from the river with undyed sand and dyed sand (AP or RR).