CX-1273C
`CX Page 1
`
`_
`
`V.
`
`2/5/99
`/in the margin/
`Erlen 250 mL
`
`with glass cap
`for 2 samples
`and for blank
`
`Iodine
`
`- Fraction 1 with pre—heated L-cysteine: 0.2030 g
`15 mL cyclohexane acetic acid 1:1 vol/vol poured into three 5 mL glass tubes to put a first one into oil,
`pipette this solution in erlen, then pour a second tube of cyclo—acetic acid into small glass tube which
`contained oil measured on a new scale, and pipette in erlen, and finally pour the small tube which
`contained the oil with 3” tube of cyclo—acetic acid, and pipette in erlen. Then oil is accurately weighed to
`minimize losses.
`
`The blank contains cyclo—acetic acid divided into 3 tubes, also to reproduce the loss of cyclo—acetic acid
`generated by these 3 tubes.
`
`1’I1L NagS2O3
`
`— Fraction 1 without pre—heated L—cystein: 0.2056 g (seems less dry)
`
`13.9 mL — 7.1 mL = 6.8 mL
`
`- Potassium dichromate: 0.1704 g
`/in the margin:
`to test Na2S;O3/
`Gives very very dark upon addition of K1 10% and no clear yellow color = light green
`went too far?
`
`After starch addition and titration, this gave light green and not colorless or yellow...
`23 H114 Na2S2O3
`
`N = 20.394 X 0.1704 g/23 mL = 0.15 N
`
`- The blank gives very dark upon addition of K1, and there was a fuschia layer at the surface 41.8 mL —
`l’I1L : Na2S203 -->
`l’1’1L
`
`Page 1
`
`NEP877|TC-00679852
`
`NEPN Ex. 2030
`
`Aker v. Neptune
`IPR2014-00003
`
`NEPN Ex. 2030
`Aker v. Neptune
`IPR2014-00003
`
`Page 1
`
`
`CX Page 1
`
`_
`
`V.
`
`2/5/99
`/in the margin/
`Erlen 250 mL
`
`with glass cap
`for 2 samples
`and for blank
`
`Iodine
`
`- Fraction 1 with pre—heated L-cysteine: 0.2030 g
`15 mL cyclohexane acetic acid 1:1 vol/vol poured into three 5 mL glass tubes to put a first one into oil,
`pipette this solution in erlen, then pour a second tube of cyclo—acetic acid into small glass tube which
`contained oil measured on a new scale, and pipette in erlen, and finally pour the small tube which
`contained the oil with 3” tube of cyclo—acetic acid, and pipette in erlen. Then oil is accurately weighed to
`minimize losses.
`
`The blank contains cyclo—acetic acid divided into 3 tubes, also to reproduce the loss of cyclo—acetic acid
`generated by these 3 tubes.
`
`1’I1L NagS2O3
`
`— Fraction 1 without pre—heated L—cystein: 0.2056 g (seems less dry)
`
`13.9 mL — 7.1 mL = 6.8 mL
`
`- Potassium dichromate: 0.1704 g
`/in the margin:
`to test Na2S;O3/
`Gives very very dark upon addition of K1 10% and no clear yellow color = light green
`went too far?
`
`After starch addition and titration, this gave light green and not colorless or yellow...
`23 H114 Na2S2O3
`
`N = 20.394 X 0.1704 g/23 mL = 0.15 N
`
`- The blank gives very dark upon addition of K1, and there was a fuschia layer at the surface 41.8 mL —
`l’I1L : Na2S203 -->
`l’1’1L
`
`Page 1
`
`NEP877|TC-00679852
`
`NEPN Ex. 2030
`
`Aker v. Neptune
`IPR2014-00003
`
`NEPN Ex. 2030
`Aker v. Neptune
`IPR2014-00003
`
`Page 1
`
`
`
`CX Page 2
`
`
`
`Iodine Fraction 1 with L-cystein
`(B-S) XN X 12.69
`=(26.8 mL— 7.1 mL) X 0.15 X 12.69
`mass (g)
`0.2030 g
`= 184.72
`
`Iodine Fraction 1 without L-cystein:
`126.8 mL— 6.8 mL) X0.15 X 12.69 = 185.16
`0.2056 g
`
`2/9/9
`
`2/8/99
`
`EXtraction Krill
`with L-cystein
`
`E. pacifica
`250 g fresh K.
`1,500 mL acetone
`0.1 g L-cystein
`20 min.
`Filtration
`Rince 500 ml acetone
`Filtration
`500 mL + butanol
`
`Determination of density
`
`Oil fraction 1 heated without L-cystein :
`1 empty cup 0.9989 g
`cup + oil
`(1 mL) 2.0554 g
`
`2 empty cup 0.9913 g
`cup + oil tip rinsing
`
`1.0126 g
`
`3 oil fraction heated + L-cystein:
`empty cup 0.9938 g
`cup + oil (1 mL) 2.0387 g
`
`4 empty cup 0.9966 g
`cup + tip rinsing 1.0410 g
`
`Page 2
`
`NEP877|TC-00679853
`
`Page 2
`
`
`
`CX Page 3
`
`Note: Do not pipette twice.
`Pipette only once the oil in the same tip. Do it again!
`
`M
`
`i
`
`69
`2/9/99
`
`Extraction of E—pacifica krill without L—cystein
`
`Batch evaporation on Friday 2/5/99
`
`heating bath does not heat at all (not at 100% in any case, the light does not go on)
`t-butanol
`Then rely on vacuum only
`not lab
`
`Volatiles + humidity
`Fraction 1 not heated without L-cysteine
`im with significantly the same starting weight, the weight goes down less rapidly than for oil with L-
`cysteine.
`Hypothesis: L-cysteine reduces, thus prevents oxidation, and oxidation produces volatiles (smell,
`L-cysteine seems to prevent the production of volatiles, thus the weight seems to go down more rapidly
`given the lack of these volatiles?
`
`/in the left margin:
`Can cysteine produce its 1/2 action (reduce) and then disappear from the medium and this action lasts?
`
`L-cysteine seems to remain at the bottom of the oil container fraction 1 (with L-cysteine). Could this be
`that cysteine goes into the filtrate by gravity and does not remain in the residue or in the t-butanol
`fraction?
`
`Try t-butanol and L-cysteine
`
`Page 3
`
`NEP877|TC-00679854
`
`Page 3
`
`
`
`CX Page 4
`
`
`5 2/9/99 0
`cmpty cup 6.5278 g
`oil only 5.0017 g
`cup + oil 11.5299 g
`l.817< 11.3213 g
`0.677 < 11.2456 g
`0.447 < 11.1958 g
`0.367< ll.l552g
`0.327<11.ll92g
`0.237 < 11.0940 g
`
`Time 0
`Time 30 minutes
`1 hour
`1.5 hours
`2hours
`2.5 hours
`3 hours
`
`“
`
`“
`
`Determination of density starting with oil from the volatiles + humidity test (fraction 1) without L-
`cysteine (2.8 /illegible
`11ot heated before the test)
`
`1 empty cup: 0.9969 g
`Cup + oil (1 mL): 1.8125 g
`
`2/10/99 1.8147g
`
`2 empty cup: 0.9971 g
`cup+ oil (tip rinsing): 1.0803 g 2/10/99 1.8000 g
`/ilZegible/ it nonetheless
`
`1 1.8125 g — 0.9969 g = 0.8156 g
`2 1.0803 g — 0.9971 g = 0.0832 g
`
`0.8156 g + 0.0832 g = 0.8988 g
`
`thus density of fraction 1: 0.8988 g/mL
`
`2/ 10/99 Extraction of krill
`without L-cysteine in acetone
`with 0.1 g L-cysteine in t-butanol
`Evaporation of t-butanol with cysteine
`
`E. pacifica
`
`2/ 1 1/99 Extraction of krill
`without L—cysteine in acetone
`without L-cysteine in t-butanol
`
`E. pacifica
`
`Page 4
`
`NEP877|TC-00679855
`
`Page 4
`
`
`
`CX Page 5
`
`2/11/99 Saponification
`1x fraction 1 with cysteine (one layer at the surface like softening butter)
`2x fraction 2 with cysteine (seems to have a melting point < 80°C)
`1X blank
`
`5” 1
`
`Heated for 15 minutes at 125°C for them to dry
`in desiccator to bring back at room temperature
`fraction 1 with cysteine 5.0041 g
`fraction 2 with cysteine 5.0212 g
`fraction 2 with cysteine 2.8323 g
`
`fraction 1 with cysteine 17.5 mL H Cl 1N
`fraction 2 with cysteine 42.0 mL — 17.5 mL = 24.5 mL HCl1N
`(2.8323 g) fraction 2 with cysteine
`See below
`
`10.7 + 20.4
`
`blank (23.1 mL -12.4 mL) + (49.3 mL — 28.9 mL) = 31.1 mL 1-1C1 /z'llegib[e/
`Note: small pieces which seem overly cooked adhering to the erlen bottom
`fraction 2 5.0212 g
`After heating, the blank has the same color and appearance as KOH + E70H left for a long time on the
`window sill
`
`(washing of burettes) = yellow and slightly turbid However, the solution was prepared on 2/3/99. Shall
`we prepare a fresh one each time?
`
`--> Fraction 2 (2.8323 g)
`28.4 mL pH 6
`
`-- 1 1nL KOH then pH 6.4
`-- 1 mL KOH “
`“
`6.8
`-- 1 mL KOH “
`“
`7.6
`
`-- 1 drop HCl
`-- 1 drop HCl
`-- 1 drop HCl
`-- 2 drops HCl
`-- 3 drops HCl
`-- 4 drops HCl
`
`7.2
`7.2
`7.2
`7.2
`7.2
`7.0
`
`28.9 mL — 28.4 mL = 0.5 mL
`
`Page 5
`
`NEP877lTC-00679856
`
`Page 5
`
`
`
`CX Page 6
`
`2/ 16/99 Determination of density
`Fraction 1 with cystcinc from Volatiles + humidity
`2““‘ test
`2/17/99
`
`empty cups: 1.0025 g
`1.9372 g
`
`1.9300 g (do with glass pipette)
`
`cup 5 empty: 1.0003 g
`1.0693 g
`1.0693 g
`rm: Pipette completely messed up
`~ 1.04 g weighed once and ~ 1.0034 g the other time
`
`2/16/99 Saponification (cont’d)
`44.5 mL -19.7 mL = 24.8 mL pH 6.8
`-- 1.0325 mL KOH pH 7.6
`—— (45-44.5) = 0.5 mL HCl pH 7
`
`° Fraction 1 without cysteine
`19.7 mL HCI thus 19.7 moq KOH
`blank 31.5 mL HCl thus 31.5 moq KOH
`-19.7 mog KOH
`11.8 moq KOH used
`
`1 moq = 1m mole KOH
`11.8 moq =11.8m moles KOH
`
`1 mole KOH: 56.1 g
`1 mmole = 56.1mg
`11.8 mmoles = (661.98 mg)
`
`661.98 mg --> 5.0077 g
`(132.2) <-— 1 g
`thus 132.2 g KOH for 1 g of oil
`
`Page 6
`
`NEP877|TC-00679857
`
`Page 6
`
`
`
`CX Page 7
`
`
`
`-Fraction 2 without cysteine
`31.5 moq KOH
`- 24.8 mog KOH
`6.7 moq KOH
`+ 0.65 moq KOH (addition of 1.0325 mL KOH because pH 6.8 went too far)
`1.0325 mL X 0.63N = 0.65
`
`7.35 moq KOH pH 7.6
`- 0.5 moq KOH (addition of 0.5 mL HCl because went too far and bring back to pH 7)
`
`6.85 moq KOH used
`
`1 meg = mmole
`6.85 meg = 6.85 mmoles KOH
`
`1 mole KOH = 56.1 g
`1mmo1e KOH = 56.1 mg
`6.85 mmoles = (384.285 mg) KOH
`
`384.285 mgKOH ——> 5.0099 g
`(76.7) 6 1g
`thus 76.7 g KOH for 1 g ofoil
`
`. Fraction 1 without cysteine paper pH 6, 7, 8 instead of 6.2, 6.4, 6.6,
`31.5 moq KOH
`- 20.0 moq KOH
`
`18/21
`
`11.5 moq KOH used
`
`1 moq = 1 mole
`11.5 moq =11.5 mmoles KOH
`
`1 mole KOH = 56.1 KOH
`
`1 mmole KOH = 56.1 mg KOH
`11.5 mmoles = (645.15 mg) KOH
`
`645.15 mg KOH ——> 5.0005 g
`(129.0) 6 1 g
`thus 129.0 g KOH for 1 g ofoil
`
`Page 7
`
`NEP877|TC-00679858
`
`Page 7
`
`
`
`CX Page 8
`
`Calculation of lipids in erlen
`cup 1: 2.2452 g — 0.9884 g = 1.2568
`cup 2: 2.2647 g — 0.9962 g = 1.2685
`cup 3: 2.2422 g — 0.9927 g = 1.2495
`
`X 1.2583 g
`
`V1.1! was
`
`1.2583 g =
`2 mL
`
`X
`63 mL
`
`X = 39.63645 g
`thus 39.63%
`
`Notebook p. 143:
`had 66.2 mL X 0.87 = 57.6% --> (41.65)
`and here I have 63 mL X 0.87 = (54.8%) --> 39.63
`
`Thus p. 143 ofnotebook 1 had 41.65% instead of 57.6%
`
`2/ 18/99 Extraction of krill Epacifica 0.1 g cysteine acetone
`0.1 g cysteine t-buta11ol
`
`Volatiles fraction 1 without cysteine 130°C
`empty cup 6.4982 g
`oil only 5.0050 g
`cup + oil 7.21%
`11.4993 g
`time 0
`0.78% 10.6696 g
`time 30 minutes
`0.42% 10.5858 g
`time 1 hour
`0.24% 10.5415 g
`time 1.5 hours
`0.10% 10.5158 g
`time 2 hours
`10.5050 g
`time 2.5 hours (eont’d p. 29)
`
`Determination of density
`with glass pipette + heXane rinsing and drying pipette tip
`- Fraction with cysteine
`Cup 1 empty 1.0050 g
`Cup 1 with oil 1.8142 g
`
`21“ Value
`(was affected by humidity)
`
`2“ Value
`Cup 2 empty 1.0058 g
`(was affected by humidity)
`Cup 2 with pipette rinsing 1.1770 g
`(L8142g——10050g)+(l1770g——10058g)=(l9804ghnL
`0.8092g+
`0.1712
`
`Page8
`
`NEP877|TC-00679859
`
`Page 8
`
`
`
`CX Page 9
`
`
`2/ 18/99 Determination of density
`- Fraction 1 without cystcinc
`Cup 3 empty 1.0030 g
`Cup 3 + oil 1.7195 g
`
`2nd Value
`(was affected by humidity)
`
`Cup 4 empty 0.9942 g
`Cup 4 + rinsing 1.2787 g
`(1.7195 g —1.0030 g) + (1.2787 g — 0.9942 g) = 1.001 g/mL
`0.7165 g
`0.2845 g
`
`(H!)
`
`2nd Value
`(was affected by humidity) 2/19/99
`
`IHL NEIZSZO3
`44.3 -23.4
`20.9 mL
`
`Iodine
`
`Fraction (small yellow layer at the surface then light) 2 without cysteine heated — 130 for 15 min. 0.2076
`
`g F
`
`raction 2 without cysteine heated 130°/15 min. 0.2073 g
`--> fuschia on the surface and yellow layer on the surface, later dark
`Grease came out of the drying oven because had to stay in there for a few minutes due to the fact that the
`solid was at room temperature, so was warmer than the first sample which had somewhat cooled down in
`the desiccator.
`
`Blank (fuschia layer on the top before and after): 270 mL Na2S2O3
`
`- Fraction 2 without cysteine 0.2076 g
`gB-S) XN X 12.69 = (27.0 + 23.4) X 0.15 X 12.69
`mass (g)
`0.2076
`
`:33.0
`
`- Fraction 2 without cysteine 0.2073 g
`(B-S) X N X 12.69 = (27.0 + 20.9) X 0.15 X 12.69
`mass (g)
`0.2073 g
`
`=5607
`
`5.06
`4.46
`
`/margin:/
`Less soluble because cooled down, so did not react as much and the indeX is lower.
`Better, more reliable
`
`Page 9
`
`NEP877lTC-00679860
`
`Page 9
`
`
`
`CX Page 10
`
`2/ 18/99 Volatiles (cont’d) Fraction 1 without cysteine
`Time 3 hours
`Cup + oil
`10.5039
`0.01%
`
`11.4993 g — 10.5039); 100 = 19.89%
`5.0050g
`
`2/19/99
`Volatiles
`
`Fraction 2 without cysteine
`Empty cup: 6.4389 g
`Cup + oil: 23% 11.2160 g
`1.11% 8.6384 g
`0.39% 8.5425 g
`0.19% 8.5092 g
`0.19% 8.4929 g
`like asphalt 0.35% 8.4630 g
`like asphalt 0.36% 8.4328%
`like asphalt 0.31% 8.4065 g
`
`improperly adjusted
`Oil only: 4.7830g
`Time 0
`Time 30 minutes
`Time 1 hour
`Time 1.5 hours
`Time 2 hours
`Time 2.5 hours
`Time 3 hours
`Time 3.5 hours
`
`Extraction of krill Epacifica
`0.1 g cysteine acetone
`0.1 g cysteine t—butanol
`
`instead of 6.2, 6.4, 6.6
`
`paper pH 6. 7, 8
`Saponification
`/in the left margin:
`5 .0 100 g
`4.4692 g/
`cc
`cc
`Fraction 1 with cysteine heated for 15 min. at 130°C
`Fraction 2 with cysteine
`desiccator to bring back to room temperature
`
`Fraction 2 25.6 mL HCl1N Very light green
`+1mL KOH
`pH7
`
`Fraction 1 44.1 mL — 25.6 mL = 18.5 mL HCl1N Very light green
`+1mL KOH
`pH7
`
`Blank 31.2 mL
`
`Page 10
`
`NEP877|TC-00679861
`
`Page 10
`
`
`
`CX Page 11
`
`4 5
`
`For white shit, this formed a pellet without oil after centrifugation. Oil removed and rendered soluble
`again in 0.5 mL SDS solution 1 hour at room temperature.
`
`For Bradford assay, having SDS S 0.05%, so to have SDS 0.05%:
`take 100 mL of supernatant
`— non trans. butter milk
`— extracted butter milk
`
`- Euphansia
`- Meganyc tiphanes
`- White shit
`
`and add 19.9 mL nano. This gives 0.05% because 10% X 100 mL = 0.05% x 20,000 mL. Take 10 mL of
`each for Bradford + 200 mL reactive 1/5.
`
`Costar plate on the left, triplicates
`1-2-3
`4-5-6
`
`White shit krill
`1 BSA 150 pg/mL nano
`duplicate aqueous phase krill
`2 blank SDS 0.05%11ano
`3 non trans. bab
`Maryse big
`4 Extracted buttermilk
`Maryse small
`'
`V
`..
`
`;
`
`.......
`
`I
`.z.
`
`1''
`
`...........
`
`.
`I
`
`1
`
`..............................................................
`
`.
`
`3
`
`5 Meganyc tiphanes
`6 Euphansia
`
`Bradford:
`
`3 x 10 = 30
`
`30 x 200 mL = 6,000 mL I need for 6 mL
`
`Make 10 mL
`thus 2 mL reactive + 8 mL nano
`
`.
`
`
`h
`i
`
`‘i
`"
`
`1%: based on calculations, there should be
`0.1 mg/ml (ofprot.) = 100 mg/mL:
`(expect to have 50% ofthe mass in prot. 20 mg x 50% = 10 /illegible/)
`10 mg/0.5 mL x 0.1 mL = C; x 20 mL
`2 mg = C; x 20 mL
`2 mg/20 mL = 0.0] mg/mL = 100 mg/mL
`and during Bradford all the samples and blank (except the 2 of Maryse and aqueous phase) were like
`Bradford = yellow-brown, thus no prot.
`
`Page 11
`
`NEP877lTC-00679862
`
`Page 11
`
`
`
`CX Page 12
`
`
`
`100 mg/mL, we should have seen a color like BSA 150 pg/mL. Triton must be used in Femand’s
`\lV
`opinion because SDS docs not solubilizc, only gives ncgativc loads.
`Start again with Triton 3%.
`
`lférr
`
`............................ ..
`
`_ Ml‘
`xi
`
`Triton 10% --> Triton 3%
`C1V1
`: C2 V2
`10% (
`= 3% X 20 1nL
`= 60/10
`V1
`2 6 mL
`V1
`thus 6 mL triton 10% + 14 mL nano =
`20 mL on
`
`)
`
`+ 0.5 mL trito11 3%
`
`centrifugate
`
`3% --> 0.05%
`
`3 divided by 0.05 = 60 so dilute 60 times:
`3% X l0O mL =0.05 X 6,000 mL
`100 mL triton 3% + 5,900 uL nano = 6 mL of prot. solution containing triton 0.05%
`
`If at 10 mg of prot./0.5 mL solution of triton + sample,
`10 ing/O.51nL X 0.1 mL = C2 X 6 111L
`2 mg = C2 X 6 mL
`2 mg/6 mL = C2
`03 mg/mL = C2
`= 300 pg/ml
`
`Make a blank with water nano to subtract from BSA in water
`
`blank 0.05% triton to subtract from sample
`Take BSA 150 pg/mL in water
`
`Page 12
`
`NEP877lTC-00679863
`
`Page 12
`
`
`
`CX Page 13
`
`47
`
`Note: In reality, we should have heated at 100°C for 5 min. in SDS
`So repeat as done with SDS on 4/16/99, but heat for 5 n1in. at 100°C to denature prot. (go get them). Do as usual for the assay and
`for the gel.
`
`Make a blank water nano to subtract from standard BSA.
`
`shredding Vs. non-shredding
`4/17/99 Emeu
`Calculations of shredded lipids
`Biovials
`1. 4m3
`2. 4m3
`3. 4m3
`4. 4m3
`5. 3.3 nL
`
`with lipids 4/18/99 4/19/99 4/20/99
`2.1515
`2.1326
`Cup 1: 0.9967
`2.1759
`2.1545
`Cup 2: 0.9915
`2.1772
`2.1594
`Cup 3:0.9911
`Cup 1: 2.1326 g — 0.9967 g =1.1369g
`Cup2: 2.1544g—0.9915g=1.1629g X=1.1560g
`Cup 3: 2.1544 g — 0.9911 g = 1.1683 g
`1.1560
`= X
`2n1L
`
`X=12.3114g
`
`21.3rnL
`
`2.1321
`2.1544
`2.1598
`
`2.1336
`2.1548
`2.1602
`
`
`= X
`12.3114 v
`100 g
`20g
`Calculations 11011-shredded --> lots of suspended crystals before evaporation
`balloon resolubilize by heating
`
`X = 61.56 thus 61.56%
`
`with lipids 4/18/99 4/19/99
`1.9068
`1.9250
`1.9299
`
`1.9073
`1.9246 1.9253 (4/20/99)
`1.9300
`
`1.9416
`1.9509
`1.9544
`
`x = 0.9259 g
`
`X=9.1664
`
`Biovials
`6. 41113
`7. 41113
`8. 41113
`9. 1.8 11L
`0.9908
`Cup 4:
`0.9942
`Cup 5:
`0.9985
`Cup 6
`Cup 4: 1.9068 g — 0.9908 g = 0.9160 g
`Cup 5: 1.9246 g — 0.9942 g = 0.9304 g
`Cup 6: 1.9299 g — 0.9985 g = 0.9314 g
`0.9259
`= X
`2n1L
`
`en1pty
`
`19.81nL
`
` _ = X
`20 g
`
`100 g
`
`X=45.8320g
`
`thus 45.83%
`
`1&1
`Then take shredded e111eu using a meat grinder.
`
`Page 13
`
`NEP877|TC-00679864
`
`Page 13
`
`
`
`CX Page 14
`
`
`
`4/17/99 Extraction of egg yolk chloroform-MeOH
`for TLC as standard
`
`1 g egg yolk centrif. tube
`5 mL chloroform
`5 mL MeOH
`Stir.
`2 mL saline 0.9%
`
`Centrifiigate 3000 RPM 5 min.
`
`Place 12 uL on TLC
`
`LN and P
`TLC
`10 uL buttermilk lipids
`8 uL yeasts as TLC of 4/13/99
`20 ],LL powdered butter milk (extract) 4/13/99 fraction 2E
`12 uL egg 4/17/99 chloro-MeOH
`39 11L std cholesterol
`10 uL shredded emeu
`10 ul non-shredded emeu
`
`° Buttermilk lipids
`1g+2mLMeOH
`1000mg = 0.780 mg
`2 mL
`(0.00156 mL)
`= 1.56 },LL
`= 0.780 mg
`(0.0078 mL)
`= 7.8 “L
`
`100 mg
`10 mL
`
`So take 1 g of buttermilk lipids + 10 mL MeOH
`and place 10 uL ofthis on TLC
`
`Shredded emeu
`
`1175.1 mg: 0.780 mg
`2 mL
`(00013 mL)=1.33 uL
`
`0.780 mg proposed
`
`
`
`""
`——————————————— --
`AAAAAAAAAA M
`F; :;g_..( 5
`
`W
`............... ..
`
`
`
`________
`
`”
`»»»»»»»»»»»»»»» -V
`............... ..
`
`5 --»
`
`
`
`Page 14
`
`NEP877lTC-00679865
`
`Page 14
`
`
`
`CX Page 15
`
`49
`
`- So take 2 mL of BIOVIAL and evaporate acetone in aluminum cup, and take 195 mL of lipids + 1805
`mL acetone for shredded emeu (cup 1) and non-shredded emeu (Cup 2).
`
`° Buttermilk powder (extract)
`78 mg in 2 mL of distilled H20 and 20 uL on TLC.
`
`/margin:
`After development: iodine/
`Neutral lipids as if there is cholesterol in egg, emeu (both), and std cholesterol (/illegible/) and yeasts.
`
`rinsing 10 mL pure acetone
`4/ 18/99 Emeu
`Incubation of shredded emeu 5 g in 45 mL pure acetone, stirring for 20 min.
`2h,4h, 8h, 24h
`started at 1:00 p.m. and 1:30 p.m.
`noon, 6:00 p.m., 8:00 p.m.
`started at 9:00 pm.
`
`4/18/99 and 4/19/99
`
`Separation of aqueous phase from krill oil by centrifugation, but pigmented interface and little oil which
`seems pure, so extraction with hexane. Volume of oil + interface ~ 10 mL
`So add 20 mL of hexane and add all the aqueous phase and place in ampulla. 10 ml rinse aqueous phase.
`Oil combination which goes through the cylinder which collects the aqueous phase and oil in ampulla +
`10 mL saline 20% (10 g in 50 mL). Evaporation ofhexane in oil.
`
`4/ 19/99
`
`TLC to do (do it again because photo film broken)
`°Place same samples on plate
`and develop phosphorus
`/iZlegz'ble/
`iodine
`
`by concealing /z'lIegz'bZe/ with iodine with cardboard, and do iodine last.
`
`with usual migration solvents.
`
`. Cause powder buttermilk dissolved in 50% MeOH, 50% H20 to migrate using migration solvent: 90%
`MeOH, 10% water
`. Dissolve buttermilk lipids in 10% chloro 90% MeOH, and migrate using migration solvents
`
`Page 15
`
`NEP877lTC-00679866
`
`Page 15
`
`
`
`CX Page 16
`
`(continued)
`
`. TCA aqueous phase Fl
`. std PM
`
`Prot. extraction by SDS:
`0.5 mL SDS 10% aqu.
`20 mg powder --> extracted buttermilk
`--> non-transf. buttermilk
`
`--> Euphausia
`--> Meganyctiphanes
`
`5 min. 100°C
`
`.,.€3:f::_ _
`
`
`
`3f?飒%}{iW
`fiiifgv an
`
`10 mL: 2 mL reactive + 8 mL nano
`
`Page 16
`
`NEP877lTC-00679867
`
`Page 16
`
`
`
`CX Page 17
`
`KS4
`
`Name:
`Reatieri
`WEHS:
`Lag Time:
`
`"""""""""""" -»-4:» ».=u
`NONAMEPRT
`fi,t§Q[_)j§'-'§EEj
`Powerwave 209
`A ‘I - H6
`00:00:09
`
`Shaking:
`
`No
`
`Wavelengif #1
`
`595 nm
`
`
`Aszsay Description
`
`Data Name:
`
`NQNAME, FLA
`
`Reading ‘fype:
`
`Reader
`
`Reading Date:
`
`99/0412? 12:36:42
`
`Report Bate:
`
`9e;o4;2g 12;3;?;4.3
`
`Prompt #1 2
`
`P73 "K35 #23
`P|’Dfi“i¥3t #31
`Ccmments:
`
`Pmmpt #4:
`
`Prompt #5:
`Prompt #3:
`
`Meas. OD
`
`0.249 9 50 Egg/mL
`
`Splashing on the side
`
`
`
`Mcganyctiphancs:
`
`Prot.
`D
`0064 ..> (3855 pg/ml) x1.5
`0.249 --> 150 pg/mL
`
`Euphausia:
`
`0.084 --> (50.60 pg/mL X 1.5
`0.249 --> 150 pg/mL
`
`Page 17
`
`NEP877lTC-00679868
`
`Page 17
`
`
`
`CX Page 18
`
`{:3
`
`(continued)
`. TCA aqueous phase Fl
`. std PM
`
`Prot. extraction by SDS:
`0.5 mL SDS 10% aqu.
`20 mg powder --> extracted buttermilk
`--> non-transf. buttermilk
`
`--> Euphausia
`--> Meganyctiphanes
`
`5 min. 100°C
`Microcentri 5 min.
`
`and
`
`0,5 mL SDS 10% aqu.
`0.5 mL solution --> aqu. phase
`--> shitty interface
`
`5 min. l00°C
`Microcentri 5 min
`
`For Bradford assay: incub. 5 min
`Having SDS S 0.05%
`10% x (l00 ul) = 0.05% x 20,000 uL (20 mL)
`
`595 nm
`
`Thus 100 uL of each supematant + 19.9 1nL nano
`
`Take 10 uL of each dilution and 200 uL rcactivc 1/5 (1 mL + 4 mL nano).
`
`COSTAR plate on the left, triplicates
`1-2-3
`
`1 BSA 150 pg/mL nano
`2 blank nano
`
`3 blank SDS 0.05%
`4 Non-trans buttermilk
`5 Extracted buttermilk
`
`6 Meganyctiphanes
`
`4 — 5 -6
`
`Euphausia
`Fl interface
`
`Fl aqu. phase
`
`Bradford quantity to prepare: 3 x ll = 33
`33 x 200 uL = 6,600 uL = need for 6.6 mL
`
`Prepare 10 mL: 2 1nL reactive + 8 1nL nano
`
`Page 18
`
`NEP877lTC-00679869
`
`Page 18
`
`
`
`CX Page 19
`
`;:
`":
`1
`1-Nu-uansforrned buttermilk
`
`DO: 0.016
`
`Extracted buttermilk
`Meganyctiphanes
`Euphausia
`
`DO: 0.002
`DO: 0.064 --> 38.55 pg/mL
`DO: 0.084 --> 50.60 pg/mL
`x 1.5 = 75.9 mg/mL
`
`X 1.5 = 57.82 mg/mL
`
`Assay Bradford nd
`1/5
`
`~ 750 mg/mL
`~ 150 mg/mL
`
`Page 19
`
`NEP877|TC-00679870
`
`Page 19
`
`
`
`CX Page 20
`
`
`
`E3’na§«::ing:'
`
`No
`
`§%.fiDmF!§ED
`
`
`
`1-
`
`A-F5
`
`........
`Name :
`Reader:
`'W‘e€1s::
`Lag Time:
`'*J‘a.‘éw'e:<i£24figt1”
`
`............................................................
`NONATME. PW’
`Pr:.w.!>t3m‘a~.»a:
`M*J—H’1 2
`mzvflrozfifl
`5% nm
`
`:
`
`
`NDNAMEFLA
`
`Reader
`91938412? 15-.:22:fl2
`
`Repmrfi Daie:
`
`$19354,-'22 $5.2-3:215
`
`Fromm #4:
`Prampi
`PE‘£.1§]”I;3i
`
`{$33.5 Mame:
`
`Reazéing Type:
`Reading Date‘
`
`Pmmpi‘: #1 :
`Pmrnpi #2:
`§7mn‘:_m. #3.:
`Sx'Jmméams':
`
`Meas. OD
`
`'1
`
`J
`
`3
`
`H
`
`:5
`
`E
`
`5‘
`
`n?
`
`3
`
`11)
`
`1‘:
`
`‘E2
`
`H 5
`
`
`
`
`
`Aqueous phase:
`
`Interface:
`
`DO
`0224 -->
`0.249 -->
`
`0.224 -->
`0.275 -->
`
`5
`
`Prot.
`150 mg/mL
`(166.74 mg/mL) X 1.5 = 250.1 mg/mL
`
`150 mg/mL
`(184.5 mg/1nL) X 1.5 = 276.2n1g/111L
`
`Page 20
`
`NEP877|TC-00679871
`
`Page 20
`
`
`
`CX Page 21
`
`
`Results:
`Non-transformed buttermilk
`Extracted buttermilk
`
`Meganyctiphanes
`Euphausia
`Fl interface
`
`F1 aqu. phase
`
`To do
`
`4/22/99
`
`DO: 0.016
`DO: 0.002
`
`X 1.5 = 57.82 pg/mL
`DO: 0.064 ——> 38.55 ug/mL
`DO: 0.084 ——> 50.60 pg/mL
`DO: 0.004
`
`x 1.5 = 75.9 pg/mL
`
`DO: 0.003
`
`15 mg ——> 20 uL
`(750 mg) --> 1000 uL
`
`Meganyctiphanes starting
`
`prot. at the end
`
`38.55 ug/mL
`20 mg ——>
`(389.11 mg) <—— 750 pg/mL (per gel well)
`
`So take 778.22 mg of Meganyctiphanes powder
`and place in 1 mL SDS 10%
`Heat for 5 min. at 100°C
`
`Place in microcentrifuge
`
`Take 100 uL of supernatant and place into 19.9 mL nao
`
`Perform a Bradford assay
`
`nd
`1/5
`
`~ 750 ug/mL
`~ 150 ug/mL
`
`Euphausia
`15 mg ——> 20 uL
`20 mg ——> 50.60 ug/mL
`(296.44 mg) <—— 750 ug/mL (per Well)
`
`So take 592.88 mg of Euphausia powder
`and place into 1 mL DSD 10%
`Heat for 5 min. at 100°C, microcentrifuge
`
`Take 100 uL of supernatant + 19.9 mL nano
`
`Perform a Bradford assay
`
`nd
`1/5
`
`~ 750 ug/mL
`~ 150 ug/mL
`
`Page 21
`
`NEP877|TC-00679872
`
`Page 21
`
`
`
`CX Page 22
`
`Non-transforrned buttermilk
`
`-->
`0.249
`-->
`0.016
`(per Well)
`
`150 pg/mL
`20 mg
`(9.64 pg/mL) -->
`750 pg/mL --> (1556.02 mg)
`= 1.556 g
`So take 6.24 of non-transformed buttermilk powder + 2 mL SDS 10%
`Heat for 5 min. at 100°C
`
`{'.}’‘.i
`
`‘$.33
`
`Microcentrifuge
`Take 100 mL of supernatant + 19.9 mL nano
`Perform a Bradford assay nd ~ 750 ug/mL
`1/5 ~ 150 pg/mL
`
`Meganyctiphanes Extracted buttermilk
`0.249
`--->
`150 pg/mL
`0.002
`-->
`(1.20 pg/n1L) --> 20 mg
`(per well)
`750 pg/mL --> (12.500 mg)
`= 12.5 g
`
`So take 125 g of extract powder + 0.5 mL SDS 10%
`Heat for 5 min. at 100°C
`
`Microcentrifuge
`Take 100 uL of supernatant + 19.9 mL nano
`Perform a Bradford assay nd
`~ 750 pg/mL
`1/5
`~ 150 pg/mL
`
`/margin: Interface/
`I do not have 125 g of extracted buttermilk...
`0.249
`--->
`150 pg/mL
`0004
`..>
`(2.41 pg/mL) --> 0.5 mL
`(per well)
`750 pg/mL --> (155.6 mL)
`
`So take 155.6 mL of interface but impossible as I do not have enough,
`so direct assay without SDS and nd same thing for aqu. phase
`
`Note: Euphausia does not resolubilize
`0.5929 g on 1 mL
`So I assume that the others do not either...
`
`/margin:
`1 1/ 1 0/ 12
`nano
`
`aqu. phase
`interface/
`
`Page 22
`
`NEP877|TC-00679873
`
`Page 22
`
`
`
`CX Page 23
`
`
`siée 57
`I9—de for SDS
`
`38é5 57.82 pg/mL after dilution
`Meganvctiphanes:
`10% ——> 0.05%
`57.82 pg/mL X 200 (dilution 200 times) = 1-1464 11,564 pg/mL
`= 7.—7—l— 11,564 mg/mL in the 100 pL taken for dilution
`
`100 mL ——> 7710 mg/mL
`(9.73 ml)<—— 750 mg/ml j
`Hé64
`11 564 pg ——> 1,000 pL
`15 pg ——> 1.30 pl.
`
`1.30+11.7
`+7
`
`
`
`Dilutc 1/10 and take 13 mL + 7 mL mcrcapto
`
`MegaH-yeti-pha.-‘&es-
`E1-,1«phasiaé0-.60 75.90 mg/mL alter dilution
`10% --> 0.05%
`
`(dilution 200 times)
`
`50-.60-75.90 mL X 200 = -l-0,—l-20—15,180 mg/mL in the 100 pl taken for dilution
`
`15,180 mg——>1000 pL
`15 pg ——> 0.99 pL
`
`0.99+12.01+7
`
`To do
`
`and place on RC non-transformed butteimilk
`extracted buttermilk
`
`and develop witl1/1'IIeg1'bIe/
`
`To do for SDS (continued)
`Aqu. Phase -1-0-p-L-9
`
`S0: Do ——> 166.74 X 1.5 = 250.1 pg/mL
`250.1 pg ——> 1000 mL
`(3.25 pg) <—— 13 pl,
`
`13 pl, + 7505 mercapto
`
`
`
`Page 23
`
`NEP877|TC-00679874
`
`Page 23
`
`
`
`CX Page 24
`
`Interface
` +
`S0: D0
`
`0.275 --> 184.15 X 1.5 = 276.2 mg/mL
`276.2 pg/mL --> 1000 pL
`(3.59 pg) <-—- 13 pL
`
`including X 1.5
`.»- ->“K’,
`2 :~_
`
`
`13 pL + 7 pL mercapto SDS
`
`4/23/99
`
`Precipitation of TCA for SDS-PAGE
`10% final
`20%X(V1)=10% X 1 mL
`Non—transf. buttermilk 20 mg
`V1 = 0.5 mL
`Extracted buttermilk 20 mg
`So 0.5 mL sample
`Aqu. phase
`+ 0.5 mL TCA 20%
`Interface
`Microcentrifuge
`
`Bradford assay of precipitate in
`Note: Only non-transf. buttermilk which precipitates with TCA
`
`Summary SDS-PAGE
`
`20 mg + 0.5 mL
`5 min. 100°C
`
`Microcentiifuge
`500 pL supernatant + 900 pL nano
`Meganyctiphanes --> (see p. 53) 57.82 pg/mL X 200 = 11,564 pg/1nL
`Euphausia
`--> 75.90 pg/mL X 200 = 15,180 pg/mL
`
`On gel: Mega 11,564 pg--> 1000 pL
`15 pg --> (1.30 pL)
`So dilute 1/10 supernatant
`1156.40 pg --> 1000 pL
`15 pg --> (12.97)
`and take 13 pL + 7 pL mercapto
`
`heat...)
`
`Euphau 15,180 pg --> 1000 pL
`15 pg --> (0.99 pL)
`So dilute 1/ 10 supernatant
`1,518 pg --> 1000 pL
`15 pg --> (9.88 pL)
`and take 10 pL + 3 pL nano + 7 pL mercapto... heat...
`
`Since aqu. phase, interface, and eXtracted buttermilk do not have any prot. which precipitate with TCA
`(there are no prot.), place on gel:
`
`Page 24
`
`NEP877|TC-00679875
`
`Page 24
`
`
`
`CX Page 25
`
`F2 (
`
`F2 (
`
`Test of volatiles
`
`Cup with an “X” underneath F2 (1)
`Other cup F2 (2)
`
`F2
`
`X F2 (1)
`Outlet of evaporator
`
`F2 1 empty cup 6.5259
`Oil only 3.1251 g
`Actual
`9.6443
`
`Cup + oil
`
`Should be
`9.6510 g
`9.4556 g
`9.4466
`9.4402
`
`0.025% 9.4378
`9.6510 g — 9.4378 g X 100 =
`3.1251 g
`
`6.82%
`
`Time 0
`30 minutes
`1 hour
`1.5 hours
`
`2 hours
`
`2é—heurs
`3—heurs
`3.6-heu-rs
`
`/margin:/
`Stored at 4°C for a while and solvent evaporated under N
`
`F2 (2) empty cup
`Oil only
`
`Actual
`
`6.4400 g
`3.1230 g
`Should be
`
`Cup + oil
`
`9.5518 g
`
`9.5630 g
`8.7822 g
`8.7699
`8.763 8
`0.01% 8.7629
`
`8.—7-62-9 cannot calculate
`
`due to loss because of overturned cup
`
`Note:
`
`F2 (2) cup overturned...
`
`/margin:/
`Iodine calculations
`
`Time 0
`30 minutes
`1 hour
`1.5 hours
`2 hours
`2%-hours
`3—heurs
`
`3.é—heurs
`
`0.2699 g
`Olive
`[B-S!XN X 12.69 =128.1mL— 16 mL) X 0.15 X 12.69
`mass (g)
`0.2699 g
`
`= 85.34
`
`81.1 in handbook, so the solutions are still good. Continue with F2): 2 ethyl acetate
`
`Page 25
`
`NEP877lTC-00679876
`
`Page 25
`
`
`
`CX Page 26
`
`F2 (1) 0.2051g
`
`F2 (2) 0.2094g
`
`F2 (-19- (2) 18.2 mL — 6 mL = 12.2 mL
`
`F2 (1) 30.71nL — 18.2 1nL = 12.5 mL
`
`Blank 34.2 mL — 8 mL = 26.2 mL
`
`Calculations:
`
`Iodine test
`
`(B-S) XN X1269 =126.2 mL— 12.5 mL) x 0.15 X1269
`mass (g)
`0.2051 g
`
`= 127.15
`
`Note: Was lacking 1 mL of cyclohexane, so evenly distributed for both F2s.
`
`24/6/99 Note: During chromato gaseous phase (GLC), F2
`ethyl acetate has peaks which hit a ceiling... However I had put a ~ qty of emeu and other F2s for
`methylation... Explanation: Ethyl acetate picks up a lower quantity of impurities than the other solvents,
`so there is more oil, and this why peaks hit a ceiling as if I had methylated twice as much oil.
`
`Iodine test
`F2 (2) Calculations:
`(B-S) XN X 12.69 =g26.2 mL—12.2 mL) X 0.15 X 12.69)
`mass (g)
`0.2094 g
`
`= 127.26
`
`29/6/99 Shark (liver)
`10:40 a.m.
`
`2 hrs 4°C
`Bottle 2 Aeterna
`
`Bottle (1A) + Bottle (1B) Aeter11a
`
`900 mL acetone
`Mr. Schmithi #7 100 g
`shredded ground meat stirred for 20 minutes
`followed by ethyl acetate 200 mL 30 min. 4°C
`10:45 a.m. Mr. Schmitthi #7 2§20g 132 264 mL chloro MeOH
`Bottle (4) Aeterna 0.9% Saline 80 mL Folch
`
`Page 26
`
`NEP877|TC-00679877
`
`Page 26
`
`
`
`CX Page 27
`
`30/6/99 5/7/99
`/margin:/
`Foleh
`17n1Ltota1
`
`Cllp 4
`5
`6
`/margin:z'llegzbZe/
`Acetone
`53 mL total
`
`Cup 7
`8
`9
`/margin:/
`Ethyl acetate
`:10?/1AL
`10-12 : 15.4 total
`
`Cup 10
`11
`12
`Shark acetone
`
`empty
`0.9808
`0.9761
`0.9815
`
`0.9774
`0.9782
`0.9768
`
`0.9717
`0.9746
`0.9785
`
`2.0403
`2.0233
`2.0268
`
`2.4335
`2.3777
`2.3833
`
`1.9663
`1.9621
`1.9646
`
`2.3813
`2.3341
`2.3367
`
`2.0416
`2.0186
`2.0213
`
`2.4283
`2.3764
`2.3796
`
`
`
`. 5:»?
`
`*9“ -*“-‘+-'
`
`1.5595
`1.5793
`1.5799
`
`1.5313-1.5558 200 mL 30 1I1in.4°C gravimetry et yl acetate
`1.5534
`- 1.5795
`1.5486 - 1.5683
`47 + 6 mL = 53 mL total
`
`Since water to separate 25 mL hexane
`rinse 10 mL hexane, re-suspended 10 mL hexane
`
`/margin:/
`11 (i11c1 1 mL of precipitate) + 6 mL : 17 mL total
`BIOVIAL #6-9
`Shark Foleh
`
`10 mL hexane --> gravimetry
`6/30/99
`
`Chicken
`
`100 g 900 mL pure acetone 3hrs 15 min 4°C
`Stirring for 20min.
`Rinsing 200 mL pure acetone
`
`Ampu11a+ 25 mL hexane
`/rnargin:illegz'ble/
`ethyl acetate 900 mL 100 g Bottle (3) Aeterna
`30/6/99 Shark
`shredded stirred 20min. 2 hrs 4“C
`
`1.0566 g = X X = 12.52
`21111.
`23.7 n1L
`thus 12.52%
`
`Page 27
`
`NEP877|TC-00679878
`
`1/7/99 at 4:35 p.m. Mr. Schmith
`Ampulla13
`0.9829
`Ampulla14
`0.9797
`Ampulla 15
`0.9845
`/margin:/
`49 + 6 =
`55 n1L total
`6+9+8.7
`= 23.7 rnL total
`
`Chicken
`0.9869
`Ampulla 16
`0.9840
`Ampulla 17
`0.9844
`Ampulla 18
`Calculations: % chicken of 30/6/99
`2.0346 —0.9869 = 1.0477
`2.0392 — 0.9840 = 1.0552 X = 1.0566 g
`2.0574 — 0.9844 = 1.0670
`
`2.4051
`2.3764
`2.3943
`
`2.0805
`2.0932
`2.1054
`
`
`
`7/5/99
`2.3137
`2.3113
`2.3240
`
`7/6/99
`2.3137
`2.3110
`2.3240
`
`7/5/99 (c0nl’d)
`2.0358
`2.0391
`2.0525
`
`7/7/99
`2.0350 2.0346
`2.0397 2.0392
`2.0520 2.0392
`
`Page 27
`
`
`
`CX Page 28
`
`-Q
`“
`
`Legend sample of shark liver oil
`sent to Aetema
`
`/margin:
`bottled without solvent
`
`after heating 125°C— 1 30°C
`15 min./
`
`6/3 0/99 IA --> Acetone fraction of beaudoin-Martin 3 A and B --> ethyl acetate only
`1B --> Acetone fraction of Beaudoin-Martin 4 --> Folch
`
`2 --> Ethyl acetate fraction of Beaudoin-Martin
`
`2/7/99
`
`Shark G.galeus 100 g 900 ml acetone
`shredded ground meat, stirred for 20 min. 2 hours 4°C
`followed by ethyl acetate 200 ml 30 min. 4°C
`+ 25 ml of hexane i11 ampulla
`Shark G.galeus 20 g Folch
`shredded
`
`264 mL chloroform
`132 mL MeOH
`80 mL saline 0.9%
`2 hrs
`
`Shark Ggaleus 100 g 900 ml ethyl acetate
`shredded ground meat, stirred for 20 min. 2 hours 4°C
`
`Calculations: % chicken lipids as of 6/ 17/99
`1.9574 g — 0.9855 g = 2-942-9-g 0.9719 g
`1.9701 g — 0.9825 g = 0.9876 g
`1.9822 g — 0.9869 g = 0.9953 g
`0.9849 g
`= x
`2mL
`20 mL
`
`x_= 9.85 g with 100 g
`:9.85%
`
`x = 0.9849 g
`
`5/7/99 Q: Friday (7/2/99) had forgotten to extract G. galeus with ethyl acetate after acetone, so we did
`that this morning.
`5/7/99 Shark Angel 100g 900mL acetone
`shredded with meat grinder, stirred for 20 min. 2 hrs 4°C
`monitoring ethyl acetate 200mL 30min 4°C
`+ 25 mL hexane in ampulla
`
`/margin:/
`(cont’d) error poured on top...
`2.0350 2.0346 204062-2604
`2.0397 2.0392 2.0396
`
`2.0520 2.0514 2,0517
`
`Shark Angel 20 g 264 mL chloroform
`shredded
`132 mL MeOH
`80 mL Saline 0.9%
`2 hrs
`
`Page 28
`
`NEP877|TC-00679879
`
`Page 28
`
`
`
`CX Page 29
`
`Sham Angel 100 g 200 mL ethyl acetate
`shredded with the meat grinder, stirred for 20 min. 2 hrs 4°C
`5/7/99 G.galeus
`re—suspended 10 mL hexane
`/margin:
`6+28 mL=34 mL total/
`
`empty with lipids
`2.2514 2.2502 2.2506
`0.9821 2.2529
`Cupl
`2.2328 2.2315 2.2316
`Cup2 0.9809 2.2332
`2.2307 2.2365 2.2372
`Cup 3
`0.9801 2.2296
`G.galeus 4
`0.9810 1.6578 1.6682 1.6814
`ethyl acetate which follows acetone
`5
`0.9783 1.6410 1.6533 1.6686
`6
`0.9793 1.6458 1.6579 1.6708
`
`/margin:
`6 mL+9.5 mL = 15.5 mL/
`
`/margin:
`6+10 mL=16 mL total/
`
`G.galeus Folch re—suspended 10 mL hexane
`7
`0.9815
`1.8842
`1.7808
`8
`0.9839
`1.8340
`1.7440
`9
`0.9854
`1.7899
`1.7252
`
`1.7411 1.7436
`1.7432 1.7568
`1.7159 1.7271
`
`/margin:
`re—suspended
`10 mL
`
`ethyl acetate
`6 mL+34 mL=40 mL total/
`
`G.galeus
`10
`0.9870
`11
`0.9853
`12
`0.9879
`
`ethyl acetate only
`2.2848
`2.3071
`2.2831
`
`2.2736
`2.2968
`2.2732
`
`2.2742
`2.2971
`2.2735
`
`2.1616
`2.2015
`2.1733
`
`10 mL hexane
`re—suspended
`with lipids
`2.1702
`2.2050
`2.1829
`
`6/7/99 Angel Acetone
`Empty
`Cup 13 0.9866
`Cup 14 0.9868
`Cup 15 0.9858
`/margin: 32.25 total/
`Angel Ethyl acetate after acetone re—suspended 10 mL acetate
`Cup 16 0.9811
`1.7306
`1.7379
`Cup 17 0.9848
`1.7551
`1.7775
`Cup 18 0.9783
`1.7522
`1.7606
`/margin: 23.5 mL total/
`Angel Folch re—suspended 10 mL hexane
`Cup 19 0.9779
`1.8838
`1.8942
`Cup 20 0.9816
`1.9025
`1.9181
`Cup 21 0.9844
`1.8868
`1.9009
`/margin: 17.25 mL total/
`
`Page 29
`
`NEP877|TC-00679880
`
`Page 29
`
`
`
`CX Page 30
`
`Like AOCS
`
`11/4/99 Index of refraction
`
`.3;
`
`L5’?
`
`-Sealskin oil extract meat grinder and centrifuged 4000 rpm 10 min (supernatant thus) and heated 125 °C 30 min
`
`‘? At 23°C 9 1.4755
`At 26°C 9 1.4750
`
`-Sealskin oil extract Ineat grinder and extract by acetone aI1d heated to 125°C 30 n1in
`
`‘? at 23°C 9 1.4750
`at 26°C 9 1.4745
`
`-Sealskin oil extract meat grinder and acetate ethyl extract and heated to 125"C 30 n1i11
`
`‘? at 23"C 9 1.4745
`at 26"C 9 1.4740
`
`Bertolli extra Virgin olive oil
`
`At 23°C 9 -174670 1.4670
`
`At 26°C 9 -1—.466—5- 1.4665
`
`Thus, refraction index of diverse seal oil analyzed above are Very similar and this shows that the oil extract by the meat grinder and ccntrifilge is
`like oil extract by meat grinder and acetone or acetate ethyl solvent. Thus claim extraction by press.
`
`To weigh gross 382.70 g quantity of fat and mince by meat grinder.
`
`Clean meat grinder with acetate ethyl to maximize recuperation
`
`Do gravimetric analysis
`
`Do not throw away residue (collagen)
`
`2 ml 9
`
`l150ml9()
`
`
`
`Page 30
`
`NEP877lTC-00679881
`
`Page 30
`
`
`
`
`
`CX Page 31
`
`6/ll/99
`
`
`*To do. "
`Characterization of seal oil — subcutaneous fat
`
`. Done
`
`Try to remove as much as possible of the mat.
`
`Volatile by putting Buchner oil a11d vacuum witl1 pump and put dryer i11 waiting
`
`test iode
`—
`(test with olive oil and compare with last time) p. 92
`
`
`
`‘Clean and dry dish
`
`-)New starch stio11
`
`l g starch paste store bought
`
`Cold distilled water
`
`+100 mL boiling water
`
`small glass flask on new balance
`-
`weigh oil: and recover as such:
`
`15 mL cyclohexane and acetic (l : 1) transfer into 3 5ml tubes equally
`
`
` ‘sample and white
`
`Put the content from the first tube of oil pipette. This solution of Erlenmeyer for iode.
`
`Transfer the content of the 2"“ tube into the glass flask in which the oil is weighed and pipette of Erlenmeyer for iode.
`
`Repeat with the content ofthe third tube.
`
`25 mL solution, 30 1nin to new piece
`
`20 mL KI + 100 mL distilled water
`
`titrate with sodium thiosu
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