Dyed Sand Lab Book

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Dyed Sand Lab Book

• Donna S. Lutz
• Iowa State University
• 2000

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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
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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
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10/3/00
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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
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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
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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
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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

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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

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Labwork 10/9/00

• Document lighting arrangement - COEs results are
  brighter, either its 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

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Notes from 10/9/00

• COE lighting system is better
• Centrifuging at 3000 rpm for 10 min is adequate
  to remove turbidity

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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

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Initial Data 10/18/00
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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)?

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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 its 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

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1. What do the spectrograms tell us? (contd)

• 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

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2. What are the spectrograms from DayGlo and why
dont 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

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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 chlorophylls excitation
  wavelength.
• AP and RR appear to be good choices at this time.

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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

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What to do next?

• Return to Dr Jenks lab and
• Excite AP, chlorophyll and acetone at APs
  excitation wavelength (555nm) and compare
  spectrofluorograms
• Excite RR, chlorophyll and acetone at RRs
  excitation wavelength (527nm) and compare
  spectrofluorograms
• Excite HB, chlorophyll and acetone at HBs
  excitation wavelength (365nm) and compare
  spectrofluorograms
• Explore if it is possible to select the emission
  wavelength and scan the excitation wavelengths

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2nd trip Dr Jenks 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).

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Spectrofluorograms from 10/23/00

• Excited AP, chlorophyll and acetone at APs
  excitation wavelength (555nm) and compared
  spectrofluorograms AP peaks are different than
  acetone but are masked by chlorophyll
• Excited RR, chlorophyll and acetone at RRs
  excitation wavelength (530nm) and compared
  spectrofluorograms RR peaks are different than
  acetone but are masked by chlorophyll
• Excite HB, chlorophyll and acetone at HBs
  possible excitation wavelengths (370nm and 730
  nm) and compared spectrofluorograms no good HB
  detection at 730nm, but there could be far end
  (gt790nm) 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.

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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.

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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,
  wed want real samples from the river with
  undyed sand and dyed sand (AP or RR).