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Mono and diallyl amines are silylated to protect them from reacting with high activity Ziegler-Natta catalysts so they may be copolymerized with propylene; also disclosed are copolymers of such amines with propylene and their hydrolyzed or alcoholyzed counterparts.
1. Method of making a functionalized copolymer of propylene comprising an allyl silyl amine with propylene in the presence of a supported high activity Ziegler-Natta catalyst to form a copolymer of propylene and said allyl silyl amine, followed by the step of solvolysis of the copolymer to remove the silyl groups whereby the resulting copolymer to propylene incorporates functional amine groups.
2. Method of making a copolymer of olefin monomers and amine-containing monomers comprising copolymerizing said olefin monomers with monomers of the formula CH2 ═CHCH2 NR4 SR1 R2 R3 wherein R1, R2, and R3 are independently selected from allyl and alkyl groups having one to about eight carbon atoms, and R4 is selected from hydrogen, CH2 ═CHCH2 --, saturated alkyl groups having from about one to about twenty carbon groups, and SiR1 R2 R3 in the presence of a supported Ziegler-Natta catalyst to make a copolymer thereof, and solvolyzing said copolymer to remove the silyl groups thereon.
3. Method of claim 1 wherein the allyl silyl amine is a diallyl silyl amine.
4. Method of claim 1 wherein the catalyst includes a titanium halide catalyst on a magnesium chloride support.
5. Method of claim 1 wherein the catalyst includes a cocatalyst of trialkyl aluminum.
6. Method of claim 1 wherein the amine monomer is about 0.001% to about 99.999% of the monomers present in the polymerization mixture.
7. Method of claim 1 wherein the solvolysis comprises washing away sily groups with an alcohol and replacing them with hydrogen.
8. Method of claim 2 wherein the olefin monomers comprise propylene.
9. Method of claim 2 wherein the solvolysis is conducted with an alcohol.
10. Method of claim 9 wherein the alcohol is methanol.
11. Method of claim 1 wherein the allyl silyl amine comprises diallyl trimethyl silyl amine.
12. Method of claim 1 wherein the allyl silyl amine comprises diallyl triethyl silyl amine.
13. Method of claim 1 wherein the allyl silyl amine comprises diallyl triisopropyl silyl amine.
14. Method of claim 2 wherein the silyl monomer comprises diallyl trimethyl silyl amine.
15. Method of claim 2 wherein the silyl monomer comprises diallyl triethyl silyl amine.
16. Method of claim 2 wherein the silyl monomer comprises diallyl triisopropyl silyl amine.
This is a division of application Ser. No. 422,275 filed Oct. 16, 1989, which is a continuation-in-part of Ser. No. 257,895 filed Oct. 14, 1988, by two of the inventors herein, which in turn is a continuation-in-part of application Ser. No. 047,960 filed May 8, 1987.
This invention relates to copolymers of propylene and other alpha olefins with certain amines having polymerizable unsaturated groups. The invention includes the use of supported Ziegler-Natta catalysts for the copolymerization and methods of inhibiting the attack of the catalysts by the amines during polymerization, through the use of a class of silyl groups attached to the amine nitrogen. Both mono-unsaturated and di-unsaturated polymerizable amines are used. The silyl-modified diallyl amines are believed to be novel.
Certain silylated unsaturated amines are known in the prior art. Giannini and his co-workers in 1967 and 1968 synthesized compounds of the structure CH2 ═CH(CH2)n NHSi(CH3)3 (Polymer Letters, 5, 527-533 (1967) J. Polymer Sci.: Part C, No. 22, pp. 157-175, 1968) and polymerized them using classical Ziegler-Natta catalysts. However, it has not been shown in the prior art (so far as we are aware) how to copolymerize propylene or other alpha olefins with such amines in the presence of highly active contemporary catalysts, i.e., titanium chlorides on magnesium chloride supports and having internal donors such as an alkyl phthalate or esters of aromatic carboxylic acids, to achieve the high molecular weights for which the lower olefin polymers are known. The copolymers so produced are useful for the presence of the amine functional groups covalently attached to or in the polymer backbone; various functions can be placed on the amine groups as will be discussed below.
Diallyl amines have been shown to form linear polymers in aqueous media in spite of their di-unsaturation; see Butler et al U.S. Pat. No. 2,926,161 and Hoover et al U.S. Pat. No. 3,412,019, for example. While diallyl amines and their quaternized counterparts are commonly commercially copolymerized with acrylamide, and are suggested for copolymerization with other acrylic monomers such as lower alkyl acrylates, acrylonitrile, various vinyl and styrenic monomers, it is not believed they have been successfully copolymerized with a lower olefin by use of a Ziegler-Natta catalyst system, which would be expected to be attacked by the amine.
In European Patent Application No. 88900936.1 (see corresponding PCT International Publication No. WO88/08856, Nov. 17, 1988), we (two of us) disclose methods of protecting the oxygen of hydroxyl groups in certain compounds having polymerizable unsaturated groups so the compounds can be used as comonomers with lower olefins. It is also suggested that a similar system of protection can be used for amines having polymerizable unsaturated groups. This application is particularly concerned with copolymerization of lower alpha olefins and amines having ethylenically unsaturated polymerizable groups and protected from reaction with highly active Ziegler-Natta catalysts by the silyl groups defined herein.
We have invented a series of copolymers of propylene and polymerizable unsaturated amines wherein the amine monomer content is about 0.001% to about 99.999% of the monomers, and methods of making such copolymers. In addition, our invention includes certain silylated di-unsaturated amines. Our invention also includes a method of copolymerizing propylene and amines of the formula CH2 ═CHCH2 NR4 SiR1 R2 R3 where R1, R2, and R3 are independently selected from alkyl and aryl groups having from one to about eight carbon atoms and R4 is selected from hydrogen, CH2 ═CHCH2 --, alkyl groups having from one to twenty carbon atoms and SiR1 R2 R3, wherein propylene and such comonomers are placed in the presence of a catalyst comprising a titanium halide catalyst on a magnesium chloride support and a co-catalyst of trialkyl aluminum (e.g., triethyl aluminum) optionally in the presence of an added external donor such as diphenyl dimethoxy silane, in a ratio of propylene to comonomer of about 0.001 to about 0.999, under conditions commonly used for the polymerization of propylene, i.e., in a hydrocarbon solvent (or liquid monomer) subject to temperatures of about 20° to 100° C. and pressures of 0.1 to 20 atmospheres. The invention includes the copolymers of propylene and amine comonomers having molar ratios of 0.001% to about 99.999% and the corresponding polymers from which the silyl groups have been removed by solvolysis. By solvolysis, we mean either a simple treatment with water or alcohol whereby the silyl group is washed away and replaced by a hydrogen or a more complex treatment with an aqueous, alcohol, or other solution containing a functional moiety other than hydrogen which may be desired on the amine, such as a dye group. In the latter type of treatment, the individual amine may pass through the coordinations with the silyl group, the hydrogen, and the dye or other moiety more or less simultaneously; in any event, the resulting copolymer incorporates functional amine groups. Our invention also includes the diallyl amine monomer of the formula (CH2 ═CHCH2)2 NSiR1 R2 R3, copolymers of it with propylene, and its polymeric derivatives from which the silyl groups have been removed and replaced with hydrogen.
While we include wide ratios of propylene to amine as stated above, for many purposes such as dying, a preferred range of copolymers has about 0.01% to about 50% amine monomers (as a percent of the monomer units in the polymer) and more preferably about 0.05% to about 5%.
Following is a description of the preparation of a copolymer of propylene and trimethylsilyl diallyl amine and propylene: a 100 ml Ar purged flask was sequentially charged with propylene (Ar purge replaced with propylene purge), 7.4 g trimethyl silyl diallyl amine, 5.0 ml of 25% TEA solution in heptane, 5.0 ml n-heptane and 772 mg of FT-1-SS. Within 2 minutes, the flask was hot to the touch and propylene consumption was rapid. Reaction temperature was moderated with a cool water bath. After 1.5 hours, another 322 mg of solid co-catalyst was added to the reaction mixture. After a further hour, 4.0 ml n-heptane were added. After a further 20 minutes, the reaction was quenched with acidified methanol. Solids were washed with methanol/water, methanol/water/HCl, methanol/water/HCl, methanol/water/KOH and finally methanol until the wash liquid tested pH 7-6. The reaction product was shown to be the desired copolymer by demonstrating its affinity for the acidic dye Acid Alizarin BB as shown by a permanent coloration after treatment with the dye by visual inspection and comparison with homopolypropylene which did not pick up the dye under the same conditions of simple contact. No attempt was made to determine the extent, if any, of cross-linking, or of unsaturation in the polymer.
In these examples, triethyl silyl diallyl amine was used as the comonomer under the conditions shown in Table I with propylene. Heptane was used as the solvent DPM5 as modifier; the reaction was conducted for two hours at 50° C. under an atmosphere of propylene. The molar concentrations of monomer, aluminum co-catalyst concentration, and yields of copolymer are shown in Table I, in which "M-1" is the amine comonomer and "M-2" is propylene. All analyses were performed after solvolysis.
TABLE I__________________________________________________________________________COPOLYMER POLYMERIZATION CONDITIONSCOMPOSITION TOTAL M-1 A1 MOD CATRUN MOLE % MOLE % VOL CONC CONC CONC CONC YIELD RATIOSNO. M-1 M-2 (ml) (m/l) (m/l) (m/l) (mg/ml) (g) A1/M-1 A1/mod A1/Si__________________________________________________________________________347 <0.3 99.5 78.18 0.68 0.31 0.02 2.62 7.83 0.45 19.6 0.44 0.5354 0.4 99.2 95.70 0.39 0.18 0.01 2.79 16.07 0.47 19.6 0.46 0.8351 0.5 99.5 116.87 0.27 0.12 0.01 1.99 13.82 0.46 19.5 0.45 0.5343 0.5 99.3 135.00 0.14 0.06 0.00 2.13 27.72 0.44 19.6 0.43 0.7359 <0.3 100.3 117.39 0.34 0.73 0.04 1.01 8.69 1.87 19.6 1.7 <0.3355 <0.3 99.1 103.63 0.39 0.28 0.01 2.23 13.00 0.71 19.6 0.68 0.9356 <0.3 99.3 81.16 0.27 0.49 0.02 2.98 11.45 1.79 19.6 1.64 0.7352 <0.3 99.2 122.76 0.27 0.18 0.01 0.75 11.92 0.69 19.6 0.67 0.8345 <0.3 98.9 131.85 0.14 0.03 0.00 2.86 22.59 0.22 19.5 0.22 1.1344 <0.3 99.4 137.75 0.14 0.09 0.00 1.78 18.98 0.66 19.5 0.63 0.6__________________________________________________________________________
In these examples, bis(trimethylsilyl) allyl amine was used as the comonomer under conditions similar to those of Examples 2-11. Results are shown in Table II in a format similar to that of Table I.
TABLE II__________________________________________________________________________COPOLYMER POLYMERIZATION CONDITIONSCOMPOSITION TOTAL M-1 A1 MOD CATRUN MOLE % MOLE % VOL CONC CONC CONC CONC YIELD RATIOSNO. M-1 M-2 (ml) (m/l) (m/l) (m/l) (mg/ml) (g) A1/M-1 A1/mod A1/Si__________________________________________________________________________385 <0.3 100.3 88.28 0.63 0.37 0.01 2.07 3.8 0.59 19.6 0.57 <0.3381 <0.3 100.3 135.09 0.13 0.06 0.00 1.89 13.7 0.48 19.5 0.47 0.3369 <0.1 100.3 135.35 0.12 0.06 0.00 1.04 20.0 0.49 19.5 0.48384 <0.4 100.34 71.30 0.50 0.24 0.01 3.49 5.5 0.48 19.6 0.47 <0.4372 <0.3 98.89 62.14 0.23 0.19 0.01 4.39 12.5 0.82 19.6 0.79 1.1370 <0.3 99.0 138.96 0.12 0.10 0.00 1.66 23.2 0.8 19.5 0.77 1.0__________________________________________________________________________
Because of the difficulty of elemental analysis for nitrogen, a second series of analyses were performed on the reaction products described in Tables I and II, with the results shown in Tables I and II as the second entries under Mole % M-1. For comparison purposes here and in the dyeing experiments to be mentioned, Table III ranks the copolymerizations in order of increasing comonomer concentration present during polymerization.
Our explanation/rationalization of these results is that the elemental analysis of nitrogen must indeed be defective/difficult.
TABLE III______________________________________RUN NO. COMONOMER CONCENTRATION______________________________________ ALLYL AMINE RUNS370 0.122369 0.123381 0.125372 0.229 384** 0.495385 0.628 DIALLYL AMINE RUNS345 0.137344 0.141343 0.143351 0.267352 0.268356 0.272354 0.387355 0.391359 0.391357 0.676______________________________________ **See the discussion of dyeing results below.
Dyeing was used as another method of characterizing these materials and distinguishing them from homopolypropylene.
Three groups of these copolymers were subjected to acid dyes under varying sets of conditions.
______________________________________Group 1:Polymers from 347 (homopolypropylene forrun nos.: comparison), 344, 345, 384, and 372.Conditions: 2% Acid Phloxine Green (CI No. 16570) in water; 1 g polymer in 25 ml dye bath; 2 h at 80-90° C.; filtered, washed with isopropanol (40 ml), 20 ml 2% soap solution at 70° C., 40 ml distilled water, vacuum dried.Results: Blind ranking of dyed polymer.Person least colored → most coloredRJS 347, 344 = 345, 372, 384MKH 347, 344 = 345, 372, 384AJS 347, 345 = 344, 372, 384Group 2:Polymers from 347 (homopolypropylene),run nos.: 343, 345, 384, and 370.Conditions: 2% Tartrazine in water; 1 g polymer in 20 ml dye bath and 5 ml isopropanol; 2 h at 75-90° C.; filtered, washed with 30 ml 2% soap solution at 70° C., 40 ml distilled water twice, vacuum dried.Results: Blind ranking of dyed polymer.Person least colored → most coloredRJS 347, 345 = 343, (=) 370, 384MKH 347, 344 = 345, 370, 384AJS 347, 345 = 344, (=) 370, 384Group 3:Polymers from 347 (homopolypropylene),run nos.: 343, 345, 381, 384, 370, 372, and 385.Conditions: 2% Acid Alizarin Blue in water; 1 g polymer in 10 ml dye bath and 10 ml isopropanol; 6 h at room temperature; filtered, washed with 50 ml 1% soap solution at 70° C., 40 ml distilled water twice, vacuum dried.Results: Blind ranking of dyed polymer.Person least colored → most coloredRJS 347, 381, 345 = 372, 370, 385, 343, 384MKH 347, 381, 345, 372 = 340, 385, (=) 343, 384AJS 347, 381, 345 = 372, 370, 385 = 343, 384______________________________________
The net results of ranking the dyed polymers from these three groups is the following:
Homopolypropylene was always ranked lowest in color uptake. It always appeared "undyed." If there was color present it was never the same color as the dye or other "dyed" polymer samples. This confirms our expectation that homopolymer and catalyst residue will not account for coloration if it is found.
This also implies that copoly(di)allyl amine/propylenes are formed and do take up acid dyes.
For the diallyl amine containing copolymers the order of ranking of dyed material is
where "<=" is read "less than or about equal." The procedure required the polymers to be ranked but if it was felt that there was no real difference in color, this was to be specified. This means that these three polymer samples were found to be all about equally dyed. The concentrations of amine during the copolymerizations were (from Table III)
0.141, 0.137, 0.143
molar, respectively. Thus, one should expect that the polymers should have about the same amount of comonomer incorporated which in turn would lead one to expect about the same degree of dye uptake. This was empirically observed.
For the allyl amine containing polymers, the order of ranking of the dyed material is
The concentrations of amine during the copolymerizations were (from Table III)
0.125, 0.229/0.122, 0.628, {0.48}, respectively.
Before further discussion, the case of polymer from run 384 must be commented upon. The behavior of this material was unique. The dyeing proceeded to 100% exhaustion of the dye bath with the result that all of the dye put into the dye bath went into the polymer. Without a doubt 384 polymer was the most intensely colored sample. The elemental analysis shows, however, that 15-20% of this material is unaccounted for by C, H, and N. One determination of ash after combustion found it to be 7.28%. At present, these anomalous results are unexplained and this sample may not be considered valid.
Though the results for the allyl amines are not as straightforward as in the diallyl amine case, it appears that dye uptake more or less follows expected comonomer incorporation as might be expected from comonomer concentration.
That amine comonomer was in fact incorporated into the copolymer was further demonstrated by ,Hnmr spectroscopy of the copolymer. Polymer from dianyl runs 344, 354, 355, and 357 were examined by "Hnmr at 300 MHz at 110° C. in C2 D2 Dl4 with HMD5 internal reference. Resonances absent in homopolypropylene were detected 5.79, 5.69, 5.15, 4.62, 4.20, and 3.97. Run 385, employing the monoallyl amine, showed a broad doublet at 7.28, and peaks at 5.25, 4.65, 4.18, and 3.85. The polymers are in fact amine containing copolymers.
The Ziegler-Natta catalysts we employ in our polymerization reactions are those utilizing components aluminum triethyl or other trialkyl aluminum, a titanium chloride on a magnesium chloride support, an internal donor which may typically be phthalate or monocarboxylic aromatic acid esters, and optionally an external donor such as diphenyl dimethoxy silane. Any other of the contemporary ("high yield") catalysts used commercially for the polymerization of propylene may be used, which is the significant point--such catalysts would normally be attacked by the allyl and diallyl amines. With our silyl protecting groups, however, they are inhibited from such attack and accordingly the amines are incorporated into a polymer otherwise similar to homopolymer polypropylenes.
Preferred silyl groups include trimethyl silyl, triethyl silyl and triisopropyl silyl. These and the other silyl groups included in the generic formula above may be attached to the allyl or diallyl amine by the usual methods.
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Cape turns tables on Yarmouth, wins quarterfinal
YARMOUTH—Three weeks ago, the proud Cape Elizabeth football team took it on the chin on its home field by 20 points in a first-ever meeting with Yarmouth.
Friday night, the Capers returned the favor.
On the Clippers' homefield.
In the biggest game of the year so far.
In a Western Class C quarterfinal, Yarmouth picked up where it left off last time by driving for a quick touchdown, courtesy the running of senior Matt Woodbury, but the game turned for good in the second period as Cape Elizabeth rode the running of junior Jack Drinan to a 14-6 halftime advantage.
The Clippers had their chances in the second half, but couldn't cut into their deficit and early in the final stanza, another Drinan TD run, followed by a short scoring run from senior Nick Moulton, put the game out of reach. Yarmouth got a cosmetic touchdown in the waning seconds, but Cape Elizabeth had the last laugh, prevailing, 27-14.
The Capers improved to 6-3, ended the Clippers' fine season at 6-3 and advanced to meet top-ranked Leavitt (8-0) Friday at 7 p.m., in Turner in the semifinals.
"We were still really mad from last time," said Drinan, who had 198 yards and two TDs on 28 carries. "We knew we could play better. We weren't going to go out in the first round this year. I love being the underdog, coming in and shutting the home crowd up, playing your game the way you know you can play it."
First of many
Prior to this season, Cape Elizabeth and Yarmouth, rivals in many sports over the years, had never met on the gridiron. That changed Oct. 11, when the Clippers went to Hannaford Field and beat the host Capers by a surprising margin of 27-7.
That was part of a superb 6-2 regular season by Yarmouth. The Clippers opened with a 36-6 "home" victory over Gray-New Gloucester in a game moved to the turf at Deering High due to issues with Yarmouth's new field. The Clippers then gave preseason favorite Leavitt fits before falling to the visiting Hornets, 14-7. Decisive victories at Lake Region (63-0) and Mountain Valley (34-0) followed, but on Oct. 4, special teams breakdowns led to a 22-15 home loss to Wells. After winning at Cape Elizabeth, Yarmouth closed with a 41-14 victory at Poland and a 31-0 home blanking of Freeport in the "Battle of the Bay."
Yarmouth finished tied for third with Spruce Mountain in the Western C Crabtree Points standings, necessitating a coin toss last weekend to determine who would be third and host Freeport and who would be fourth and host Cape Elizabeth.
The Clippers lost the toss.
"My wife was behind me calling, 'heads' and I called, 'tails,'" said Yarmouth coach Chris Pingitore. "I should have listened to my wife. It would have been an easier road, but to be the best we had to beat the best. You have to fight who's in the ring with you. It's too bad it came down to a coin flip. It might have been different."
The Capers opened with a 26-7 home loss to Leavitt that was closer than the final score indicated. A 31-0 victory at Poland and an impressive 26-12 home victory over Wells followed, but Cape Elizabeth then stumbled at Spruce Mountain, 36-14. After a 42-22 victory at Freeport, the Capers fell at home to Yarmouth. They then finished strong by romping at Gray-New Gloucester, 57-14, and at home over Mountain Valley, 64-24.
None of those previous eight games meant a thing for either squad when the playoffs commenced Friday night, where Cape Elizabeth reminded everyone it has to be taken serious when the calendar reads November.
The game began amid balmy 61 degree conditions and the Clippers were red-hot to start, taking the opening kickoff and marching 71 yards in nine plays in just under four minutes.
Woodbury set the tone on the first play, a 27-yard scamper to the Capers' 44. Yarmouth gradually moved the ball forward on the ground before Woodbury capped the drive with a 19-yard burst up the gut. The extra point try of sophomore Andrew Beatty was blocked, but with 7:55 to play in the first quarter, the Clippers had a 6-0 lead and appeared to be on their way to great things.
Cape Elizabeth's first drive was short-circuited by an illegal chop block penalty and Moulton's punt was partially blocked by Yarmouth sophomore Jack Venden, giving the hosts the ball at the Capers' 48, giving them a chance to really seize control.
Instead, on first down, Woodbury was stripped of the ball and Cape Elizabeth junior Devon Stanford recovered at midfield.
The visitors weren't able to capitalize, but the tide had turned.
The Capers got their initial first down on an 8-yard Drinan run, but a false start penalty set them back and on fourth-and-5 from the Yarmouth 34, Cape Elizabeth junior quarterback Noah Wolfinger threw incomplete.
The Capers' defense then forced a three-and-out, not allowing the Clippers a single yard, and with 1 second to play in the first quarter, Cape Elizabeth took over at its 34.
As the first period (in which Yarmouth had a 77-23 advantage in yardage) gave way to the second, the Capers began to exercise their will.
Two plays in, Drinan burst free down the right sideline and wasn't tackled until he reached the Clippers' 7, good for a gain of 48.
Junior Christian Lavallee then rumbled to the 1 and after Wolfinger was held no gain, Lavallee got another chance and bulled his way into the end zone, capping a six-play, 66-yard drive to tie the game. Freshman Ben Ekedahl added the extra point and Cape Elizabeth was on top to stay, 7-6.
After Yarmouth went three-and-out again, the Capers began at their 20 and after Wolfinger found junior Ethan Murphy for 28 yards with a perfectly thrown pass down the left sideline, Cape Elizabeth's hopes of extending its lead were dashed when Lavallee fumbled and Clippers' senior Matt Klepinger fell on the loose ball at the Yarmouth 38.
Again, the Clippers could do nothing on offense and after Capers senior Caelan Houle (remember that name) threw Klepinger for a 3-yard loss on third-and-13, Yarmouth had to punt. A 14-yard return by senior Tony Moulton set Cape Elizabeth up at its 40 with 4:38 left in the half and the Capers set out to extend their lead.
It took 11 plays and almost all of the remaining time to reach paydirt, but Cape Elizabeth got the job done.
A15-yard burst from Nick Moulton and a 14-yard run by Drinan got the visitors to the doorstep, but after Drinan appeared to score on the next play, an illegal procedure penalty negated it.
No problem.
After a Wolfinger incomplete pass, Drinan got the ball again and ran to his right, not stopping until he dove and barely got inside the pylon and his 10-yard scamper with 20 seconds showing, followed by a Ekedahl extra point, made it 14-6 Capers.
Yarmouth, which hadn't thrown a pass to that point, went for broke before halftime and almost was rewarded.
Starting at their 34 with 14.8 seconds to go, Clippers senior quarterback Brady Neujahr was sacked by Houle on first down, but he hit a pass to Woodbury for 23 yards to the Capers' 45 with 3.4 seconds left, giving Yarmouth one more play.
Neujahr dropped back and launched a pass deep down the right sideline to senior Race Morrison, who came down with the prayer at Cape Elizabeth's 10, but he was brought down at the 5 and the half ended before the hosts could run another play, preserving the Capers' 14-6 advantage.
"We had a young guy back there and he let the guy get behind him, but for once we got a break and he fell at the 5-yard line," said Capers coach Aaron Filieo.
"If we got that pass it might have been a different ballgame," lamented Pingitore. "It wasn't in the cards for us tonight. The last time we played, they blitzed linebackers and gave us problems in the pass game. We knew we could execute our running game. Our inside game chewed up yardage. There was a good flow. It wasn't until they went up 14-6 that we thought we had to pass the ball. Maybe if we did it earlier, it could have been effective. Who knows? Our game is running the ball."
When the Clippers defense forced a Cape Elizabeth three-and-out to start the second half, Yarmouth had a chance to cut into the deficit, but after Neujahr found senior Thomas Lord for 14 yards on first down, Woodbury was held to no gain, Klepinger was thrown for a 3-yard loss (by Houle, naturally) and Neujahr threw incomplete, forcing a punt.
Cape Elizabeth wasn't able to pick up a first down and had to punt, but after Woodbury ran for 26 yards to the Capers' 45 and Neujhar moved the chains again with a 12-yard bootleg to the 30, the threat fizzled, as Woodbury rushed for a yard and Klepinger gained two before Neujahr threw incomplete. On fourth down, Feenstra sacked Neujahr and Cape Elizabeth took over at its 33.
Again, the Capers offense couldn't move the ball and late in the third period, Yarmouth took over at its 37. Woodbury ran for 6 yards and Klepinger got almost 4, setting up third-and-inches, but Woodbury was thrown for a 1-yard loss on third down and on fourth-and-1, Woodbury got the ball again and was thrown backwards for a 2-yard loss by Stanford, giving Cape Elizabeth the football at the Clippers' 44.
It would take the final 58 seconds of the third quarter and the first 92 of the final period for the Capers to drive for some breathing room.
A 9-yard Drinan run on the final play of the third moved the chains. On third-and-8 from the 25, Wolfinger found Tony Moulton for 15 yards and a first down at the 10 and on the next snap, Drinan scored from 10 yards out up the gut and Ekedahl's extra point made it 21-6 Cape Elizabeth with 10:28 to play.
Yarmouth's next drive began auspiciously as Neujahr hit freshman Grant Tobias for 16 yards to the Capers' 44, but after an incomplete pass, Houle broke through the line once again and sacked Neujahr for a 2-yard loss.
"Caelan's a good player," Filieo said. "He's big and he's physical. He's pretty crafty. He's not just a big brute. He's pretty nimble in there."
Sophomore Cody Cook then ran for 6 yards, setting up fourth-and-6 at the 40.
Then, stunningly, the Clippers, despite trailing by 15 and having promising field position, lined up to punt and senior Nathaniel Shields' Auble's boot went into the end zone for a touchback and a net gain of 20 yards.
It was never supposed to happen.
"That was a mistake," Pingitore said. "I'd called a fake punt. Apparently, our punter went in and called a punt regular. We had Brady out there. It was supposed to be a direct snap to him and a pass. They weren't covering our gunners. We set it up. It was just a mistake."
With 8:52 remaining, Cape Elizabeth took over at its 20 and embarked on a textbook clock-draining drive to end all doubt, using up three seconds shy of eight minutes to march 80 yards on 13 plays.
A 10-yard run by Drinan got it started. Lavallee then ran for 11. Runs of 5-yards by Drinan and 6 from Lavallee moved the chains again. A holding penalty was overcome in part by a Clippers' offsides transgression and Drinan ran for 5 to pick up a first down. After Tony Moulton ran for 7, a chop block penalty moved the ball back to the 38, setting up second-and-18. After Moulton lost a yard, Drinan ran for 11, but it was still fourth-and-8.
No problem.
Wolfinger threw his final pass of the night, hitting Feenstra for 25 yards to the 3 and two plays later, Nick Moulton scored from a yard out. The extra point was no good, but just 55.3 seconds remained and the Capers led, 27-6, almost the identical score to their previous loss to the Clippers.
To its credit, Yarmouth, on the final drive of its accomplished seniors' careers, didn't quit and put up one final touchdown.
After Woodbury returned the kickoff 40 yards to the Cape Elizabeth 49, Neujahr hit Morrison over the middle for 23 yards and after three incomplete passes, Neujahr threw the ball into the end zone, where Woodbury came down with it for a 26-yard score with just 5.5 seconds remaining. The Clippers then faked the extra point, as Neujahr rolled right and hit Woodbury for the two-point conversion, cutting the deficit to 27-14.
Yarmouth wasn't able to recover the ensuing onsides kick, however, as Nick Moulton fell on it and one Wolfinger kneel-down later, the Capers put the finishing touches on their 27-14 victory.
"We probably played our best game of the season," Houle said. "They knocked us down early on, but we didn't give up. We played as hard as we could. We had a chip on our shoulder. We made sure we were ready for this. It's a huge step up from last season. We're moving in the right direction. We're under the radar, but we've gotten better as the season progressed. Our chemistry has gotten better."
"We'll definitely enjoy this for the time being because it's been awhile (since we've won a playoff game)," said Filieo. "These guys are just resilient. They don't get high or low and in this game, that's the best way to be. Our first game (against Yarmouth), I was more fired up than the players and it detracted from my focus and they were't really stoked. Coming in, we knew we had a good game plan and we executed it. We took the first shot, but we stuck to the plan. I like our offensive line. It's been getting better every week. We just wanted to pick our spots in our passing game. We wanted to control the line of scrimmage and set the tone. I thought our line did a nice job. It's about picking your spots."
Cape Elizabeth finished with a 321-256 edge in yardage, with a 258-130 bulge coming on the ground.
Drinan stole the show with his 198 yard, two TD performance.
"I didn't really expect to be such a big part of the offense," Drinan said. "Usually we split it between me, Christian and Nick. The fact that Coach trusted me with the ball so much made me feel great and made me rush better. The line was so good. They blocked their best. The holes were there because of great blocks."
"(Jack's) so good," Houle said. "He's just so dynamic. He runs hard all the time. He's a great running back."
"Jack's an outstanding football player," Filieo added. "He's been coming along and he had a breakout game."
Wolfinger threw just nine times, completing five attempts, good for 80 yards. Three of the passes went to Murphy, who ended up with 40 yards.
The Capers did have one turnover and were flagged seven times for 53 yards.
They also played a tremendous defensive game.
"We were playing vicious defense," Drinan said. "We were attacking the line, playing good coverage in the secondary. Their speed killed us last game, but we didn't let them take advantage this game."
"We just got after it, flew around and made gang tackles," said Feenstra. "This shows everybody that we're here and we want to win a Gold Ball."
"We had to contain them inside," said Houle. "They didn't have good off the edge running like last time."
End of an era
Yarmouth was paced by 88 yards and a TD on 16 carries from Woodbury. Neujahr, a four-year starter under center, completed 6-of-13 attempts for 142 yards and a score. Morrison had two catches for 63 yards and Woodbury caught two balls for 49 yards and a score.
The Clippers had a turnover and committed two penalties for 10 yards.
Yarmouth's seniors won state titles in 2010 and 2011, made the playoffs in dramatic fashion in 2012 and produced a better-than-expected record this fall, but it was still a tough way for the Clippers' season to conclude.
"It is disappointing," Pingitore said. "Since I saw Cape play Leavitt in the first game, I've said they're the most dangerous team in the conference. They're a lot like we are. They have athletes at skill positions. It was a role reversal of our last game. If they play the way they did tonight, they have a chance to beat Leavitt. It started well, but Cape came with a game plan and they executed it. They beat us at our own medicine, power football. Credit to them. They ran isos, got us on the edge and figured us out defensively after our first series. We couldn't get untracked after that. We had some good drives, but they stalled. We just haven't given up yardage like that all season. We've held everybody to under three yards a play. That got in the kids' heads a little bit.
"Going into the season, there weren't many expectations for us. We're still a fairly young team in a new conference. I had no doubt we'd compete. I've coached the seniors for four years. They're probably the best group of seniors in the conference. They were pretty special. You can compare them with anybody. They've accomplished a lot."
Yarmouth can't be overlooked going into 2014.
"I think we have a lot of potential," Pingitore said. "Unfortunately, we're low in the junior class. We have ebbs and tides. Our sophomore class is as talented as any class we've had. We should have a big freshman class coming in. We'll be young again, but we have talent to do some things."
A trip to Turner
Cape Elizabeth will be a decided underdog when it goes to Leavitt Friday evening for the semifinals. The Capers played the Hornets tough in the regular season, but doing it on the road in November will be a tall task.
Cape Elizabeth lost to Leavitt, 35-21, in the 2009 Class B state final, in the lone prior playoff meeting between the schools.
The Capers know they'll be the underdog, but will make the trip confident they can shock the world.
"The first time against Leavitt, we didn't have as good chemistry and we shot ourselves in the foot," Houle said. "We did play hard that game. Hopefully we'll play hard next week. We'll practice hard, work hard and be up for the challenge."
"We have to play even better," Drinan said. "Leavitt's a great team. We'll come out as hard as we possibly can, knowing it could be the end of the season or the beginning of something greater. No one is expecting a lot of us."
"We played them tough the first time and we know what to expect," Filieo added. "They're big, they're physical, they're fast. They've been there before. Our guys are playing with confidence right now. What the hell do we have to lose? We'll go up to Turner and have some fun."

subset: commoncrawl


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Hannah Gay
- Filed Under
University of Mississippi Medical Center pediatrician Dr. Hannah Gay, who recently helped cure an HIV-positive infant, has been named one of Time magazine's 100 most influential people in the world.
Gay, along with Dr. Katherine Luzuriaga, an immunologist from the University of Massachusetts, and Dr. Deborah Persaud, a virologist at Johns Hopkins Children's Center, were honored by the magazine for their participation in the HIV breakthrough case.
The magazine, which published this year's list Thursday online, compiles the list each year to include "artists and leaders to pioneers, ...


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A task is an activity that needs to be accomplished within a defined period of time. You need not juggle around with multiple tasks, instead Zoho Projects will prioritize and help you manage all the tasks.
While adding new tasks, you can now, attach and share screenshots of any webpage or screen related to the task using the screen-grabber option. In Drop files or add attachments here.., click the screen grab option and paste or attach the required image, and then click Done to upload the screenshot. An inbuilt draw tool lets you highlight what is important and blackout what you don't want in the screenshot.
In Task List choose a task list from the Internal option if you want to display the task only to your portal users. However, if you want to collaborate and display the task with client users, you must associate a task list from the External option.
In the selected project, in Tasks & Milestones, click open the required task, to edit. You can edit the task in Classic, Plain and Kanban view. You can modify task details such as task comments, sub tasks, log hours, documents, forums and so on.
While adding comments to a task, you can now, attach and share screenshots of any webpage or screen related to the task using the screen-grabber option. In Drop files or add attachments here.., click the screen grab option and paste or attach the required image, and then click Done to upload the screenshot. An inbuilt draw tool lets you highlight what is important and blackout what you don't want in the screenshot.
You can associate bug(s) to a task. There will be instances where you might wish to associate bug(s) to a task, so that it's easy to track and close the task as well as the associated bugs. While completing tasks, you can opt to enable or disable the alert message about the associated bugs in Org Settings.
In addition, you can also associate new bugs to the selected task. In the Bugs tab, click Associate Bugs, and then click the Click here option to associate new bugs to the task.
Specify the new Bug Title and Bug Description and then click Save to associate a new bug to the task.
Likewise, you can also delete a task . Click to edit the task and then click delete to delete the task.
This deletes the tasks along with the associated timesheets entries and delinks the set dependencies (if any).
Zoho Projects supports 4 different type of tasks view Classic, Plain, Kanban and Dependency.
1. Classic View
The classic view is the default view for task lists and tasks. You can customize your columns in classic view.
2. Plain View
This is a simple view sorted based on due date. The plain view displays all tasks and its dependencies with a tree icon.
3. Kanban View
Kanban cards are an easy, dynamic way to create and visualize your workflows. Projects and tasks are visually arranged by status...such as To-Do, In Progress, Complete...so every team member knows what's required to be done at any time.
4. Dependency View
In dependency view you can see those tasks that are dependent on other tasks. The predecessor tasks and successor tasks are displayed in an order.
You can now import XLS/JSON/CSV files into Zoho Projects and start collaborating.
A sample scheduled import and its XLS file is shown below:
While importing, if you map only the TASKLIST ID, the corresponding tasklist alone is imported, even if there are identical tasklist names. However, if you map the TASKLIST NAME, all the available tasklist with the given tasklist name are imported irrespective of their tasklist ID.
|Mapped Field Name
|Imported Tasklist
|Edit Template
|Tasklist ID
|Edit Template -1234
|Edit Template
|Tasklist Name
|Edit Template-1234 and Edit Template-5678
Sorting tasks helps you to focus on specific information for any desired column in project management. In Classic View, you can sort tasks based on its Priority, Start Date, Due Date, Created by and % Completed.
In Classic View, click Sorting, and then select the desired column criteria to sort tasks.
Tasklist | Subtasks | Copy / Move tasks | Task Dependency | Gantt View


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Quantity Available:
Marset is a Barcelona-based company dedicated to creating warm contemporary lighting with a underlying sense of comfort and character, enhancing otherwise impersonal residential and commercial spaces. The Marset mood is minimalist, subtle, with an aesthetic appeal that stems from an "illuminate, not dazzle" philosophy.
The Ledpipe C Wall Spot is available with the following:
- Injected aluminum head with stainless steel lid
- Metal base and joint
- Rectangular wall plate
- Switch on base
- Adjustable swivel head
- Surface mounting
- LED Color Temperature: 2700K
- 73 lumens
- UL Listed
- Made in Spain
- Designed by Joan Gaspar, 2005
Finish: Silver Grey, White
- One 3 Watt 120 Volt LED array (integrated).
- Fixture: Height 5.31", Width 1.97", Depth 6.5"
- Plate: Depth 1.18"
Manufacturer IDs: view
Need help with some of our terminology? Check out Lumens' Lighting & Design Glossary.


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Advocate Effectively for Students and Families
All young people need adults looking out for them, helping them find and take their individual paths toward academic success. IRRE's Family and Student Advocacy System provides every student with an advocate dedicated to his or her academic success. Teachers, administrators, or other staff may serve as advocates, each one usually working with 15 to 17 students for the entire time these young people are in the school. Advocates use both data and dialogue to track the progress of students, and continually follow up with the students in their advocacy group, teachers, and counselors. Advocates also bridge the gap between home and school, meeting with each student and family together at least once a year and keeping families apprised of their student's progress. IRRE's Family and Student Advocacy System currently supports almost 50,000 students and families in schools ranging from small rural to large urban secondary schools.
How IRRE supports the advocacy system
Schools implementing IRRE's advocacy system receive intensive support to:
- Create the time and space for advocates, students, and families to meet and do their work.
- Provide advocates with training and clarity about the boundaries and potential of their role.
- Equip advocates with techniques for working more effectively with students and parents/guardians and for carrying out their other responsibilities.
- Teach advocates to use professional learning communities of fellow advocates to build their repertoire of skills, monitor their own and other advocates' work, and use data to guide what they do.
To talk about advocating for students and families, contact:
An IRRE Partner Practitioner


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Site Council
Medford High School Site Council 2012-2013
What is the Site Council?
The Medford High School Site Council (MHSSC) is a representative advisory group (members include students, parents, educators, and community members) that works together in planning for and implementing school improvement. It is an important component of site-based management where a collaborative and deliberative team meets regularly with the principal to assist in planning for and implementing goals and practices that enhance the achievement of all students.
Stakeholders are needed to serve on this important team. In return, membership offers:
– an opportunity to help shape policy decisions
– increased public confidence and mutual support
– added insight regarding community perceptions and expectations
– a better understanding of the school's possibilities and challenges
The first meeting of the MHSSC will be on October 16th. If you think you might like to join this important group, or if you are just curious, please attend. If you would like more information but can't make it to the first meeting, please contact Dr. Perella.
The meetings will be held Tuesdays, 6-8 PM in the viewing room of the MHS library.
The following is the schedule for the 2012-2013 site council meetings:
– October 16, 2012
– November 20, 2012
– December 18, 2012
– January 22, 2012
– February 5, 2013
– March 19, 2013
– April 2, 2013
School Improvement Plan 2012-2013
Prefer a PDF? Please click here.


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MacKerron, Grant and McGlynn, K (2000) Self-assessment using the EFQM's excellence 2000 model: which method is best? Engineering Science and Education Journal, 9 (6). pp. 273-277. ISSN 0963-7346Full text not available from this repository. (Request a copy)
Official URL: http://dx.doi.org/10.1049/esej:20000608
The authors describe how many organisations, of all sizes, are adopting the European Foundation for Quality Management's (EFQM's) Excellence 2000 model as a means of assessing themselves to gain the multiple benefits of improved performance. Various approaches are available to do this-the question is which one to use.
|Item Type:
|Print ISSN:
|Uncontrolled Keywords:
|European Foundation for Quality Management; Excellence 2000 model; quality management self-assessment; organisations; performance;
|University Divisions/Research Centres:
|The Business School > School of Management
|Dewey Decimal Subjects:
|600 Technology > 650 Management & public relations > 658 General management
|Library of Congress Subjects:
|H Social Sciences > HD Industries. Land use. Labor > HD28 Management. Industrial Management
|Item ID:
|Depositing User:
|Mrs Lyn Gibson
|Date Deposited:
|30 Jan 2012 15:20
|Last Modified:
|12 Sep 2013 14:56
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"We rushed into this [Libya] without a plan," David Barno, a retired Army general who once commanded U.S. and NATO forces in Afghanistan, told the Los Angeles Times. "Now we're out in the middle, going in circles."
U.S. officials concede to the Times that some of their assumptions before they intervened in the Libyan conflict may have been "faulty." Among them was (a) the notion that air power alone would degrade Muammar Qaddafi's military to the point where he would be forced to halt his attacks and (b) the U.S. could leave the airstrikes primarily to warplanes from Britain, France, and other European countries.
"By the U.S. taking a back-seat role, it has a psychological effect on the mission," said Dan Fata, a former Defense Department official who was responsible for overseeing NATO issues during the George W. Bush administration. "If I'm Qaddafi, I'm thinking I can probably wait the Europeans out."
That would be a reasonable surmise.
If the United States had acted quickly and forcefully in the early days of the uprising, it's quite likely that Qaddafi could have been removed from power, which would have been a good thing. But President Obama delayed, sent conflicting signals and then decided to intervene only at the 11th hour. That was bad enough; that Obama did so in a way that was irresolute and radiated weakness made things worse. And now we are where we are.
Obama himself has conceded we now "have a stalemate on the ground militarily." But the president assures us that Qaddafi is still getting squeezed in all kinds of other ways. He's "becoming more and more isolated," which I'm sure troubles Qaddafi to no end. "The "noose is tightening," the president assures us (it's been tightening for more than a month now). And Obama's expectation is that "if we continue to apply that pressure and continue to protect civilians, which NATO is doing very capably" — a dubious claim — "then I think over the long term, Qaddafi will go and we will be successful."
Over the long term, Qaddafi will go — because like all of us, in the long term Qaddafi will die. But whether this intervention will be deemed to be successful is another matter entirely. If we do have a satisfactory outcome in Libya — which looks increasingly unlikely at this juncture — then the credit will go to the French and the British, not to us.
Mr. Obama has mishandled this situation in almost every way imaginable — and right now, Qaddafi is humbling America and NATO, both of which look impotent. It turns out a community organizer leaves something to be desired as commander-in-chief.
Who knew?


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A cool new Figo
The Figo concept showcased by Ford at the 2014 Indian Auto Expo will soon see the light of the day. The vehicle will go on sale in Brazil in the coming months, while India will get it by early next year.
With this launch, Ford India will enter the hot-selling, entry-level, sub-4m-sedan category, which is presently dominated by Maruti Suzuki's Dzire, Hyundai's Xcent and Honda's Amaze.
The next generation Figo is christened as KA in Brazil. The compact sedan's name, which is still a mystery in India, is known as KA+ in Brazil. So, will the sedan in India be known as Figo+?
The major difference between the KA+ and compact sedan that the company showcased at the Expo this year, is that the KA+ will be a tad over 4m while the Indian version will be under 4m.
Recently, Ford also revealed pictures of its next generation Figo, which is scheduled to be launched along with the sedan.
Text: Charu Kishnani, Car Dekho


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CE Certification
Getting CE certification can be a long and sometimes frustrating process. Once you go through the long road of getting certification, you need to make it worth your while. A listing in the CE Directory can mean the difference between a product making a splash or not selling at all in the European market. First, you need to get CE certification and here are a few basics.
What is CE Certification?
Each product will have a different series of tests--for example medical equipment has a different quality assurance guideline than household electrical equipment. High risk products will have to conform to a longer list of directives. It is very beneficial for manufacturers to obtain CE certification since it ensures that once the standards are met, they are free to distribute goods throughout the EU.
Once the testing process is complete, a manufacturer needs to reach buyers who are looking for legal CE marked products. Without a service like the CE directory, you might not even have a chance to export your products. The CE Directory is known to only list high-quality products that, of course, already tout the appropriate certification.
CE Certification
Web Design in Vancouver by Medora Solutions


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Some things can only be learned with hands-on experience. Walbridge's internship and cooperative programs offer on-the-job training and mentoring that can be your first step toward a great career. For a list of available intern and cooperative positions, please check our current openings.
Our Intern Program
Walbridge interns can work in a variety of departments in our corporate and regional offices or venture into the field and work on projects. Several of our senior executives and general managers began their careers working as interns at Walbridge. For example, Mike Haller is President of the company's Industrial Group and a member of the Walbridge board of directors. He began his career with the company in 1973 as an intern from the University of Detroit.
Value Proposition
Meet with senior executives and managers, get immersed in 100 years of history with our company and get acquainted with our culture, policies and procedures.
Project Life Cycle
This interactive 'game' brings light to the entire construction process by illustrating the interdependence of Business Development, Estimating and Operations.
Peer Group Discussion Series
Lunch meetings every other week include discussions on relevant topics.
Walbridge Passport
Interactive exercises broaden an intern's view. Learn about Walbridge cross-functional operations, network and build relationships by meeting with senior staff and subject matter experts.
SharePoint Blog
An interactive blog where interns share their work experiences. Interns post something about what they're working on and are encouraged to respond to one another.
Innovation Workshop
Our innovation manager leads a discussion and a group activity to help interns critically think about innovation and why it is necessary to Walbridge.


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Earlier this week, the folks of Yogscast debuted a perplexing new revenue sharing system, one that revolves around taking a cut of the sales of some games featured on their massively popular YouTube show. Specifically, the model is set up so that if a game in the new "YogDiscovery" program sees a sales spike after its appearance on the show, the creator of the video in question gets a percent of the revenue from all new copies sold.
This sparked a new debate about this specific program (How can some indies afford this? How do they know all new sales are directly attributable to Yogscast?), but it also brought an old debate back to life again regarding YouTubers being paid for coverage of specific games.
For all the rumors of games journalists being paid to give certain titles higher or lower scores with every new release, when it comes to the YouTube gaming scene, it's not just a ghost story. The practice of bought coverage is widespread, and while you may have some YouTube stars like the reputable Total Biscuit trying to be upfront about what's paid content and what isn't, others aren't so forthcoming. There's often no telling what's a paid campaign and what's free coverage, or whether the deal had stipulations attached to it, like the publisher saying the game can't be portrayed in a negative light.
I wanted to comment on this issue specifically, because I know what it's like to try and build up credibility in the games media industry. I've suffered countless accusations of bias for or against every platform or publisher out there during my time writing for Forbes and other outlets. Rarely a day goes by where I'm not accused of being paid on the sly by Sony Sony or Activision to plug them, or to trash their rivals like Nintendo Nintendo or EA. I'm told I'm being paid to praise the Xbox One one day, then I'm a PS4 shill the next. I've been referred to as a "so-called journalist" so many times at this point, I'm tempted to make it my official job title.
The Fight For The Future Of Video Games is a warts-and-all look at the clashes between the video game business and its passionate fans.
It's hard. I've made mistakes. I've written stupid things before, and will again. But no matter if I'm praising or criticizing a game, developer, publisher or platform, I'm always doing it because it's simply how I feel. I may have innate biases, who doesn't? But they're not formed as a result of being paid. I've never taken money to cover a game, and the idea would be laughable if it wouldn't immediately get me fired and ostracized from the industry forever.
YouTube is different. Most of the time, these channels are owned by individuals who don't answer to anyone but their own fans. They have no code of conduct policy in place that will literally get them fired if they don't comply. As such, they are within their rights to take deals like these when they're offered (though Eurogamer believes that by UK law, if advertorials aren't clearly enough marked, it could be illegal).
But while these YouTubers can do this if they like, I don't understand how they're willing to pay the price in the trust of their fans. It's taken me years to assemble even a moderate collection of readers who believe I'm telling the truth when I write. I would never do anything to intentionally jeopardize that, as I know everything I've worked for could crumble as a result.
But take Yogscast, who now seem intent on telegraphing the message to their fans that they can no longer trust them. In the interest of "transparency," they explained how YogDiscovery will work, but all it did was confuse and upset fans further.
"No. Just no," one fan said on the Yogscast Reddit. "This is wrong on too many levels and you know it. I like your content, I like what you do, but you've managed to get this far without it. Doing something like this now, looks greedy and pointless to me."
YouTube is a place of shifting sands when it comes to the intent of the video maker. Lets Play videos, the main focus of complaints like this, can range from more or less comedy sketches (see #1 YouTuber in the world, PewDiePie) to videos that are closer to actual reviews and recommendations. While if your channel is meant entirely to do nothing but entertain, I suppose I can understand how these kind of deals could benefit both parties. But if you're saying that you're actually attempting to inform and educate and give your genuine opinion of a game, I'm not sure how you can do that with a "This video produced in partnership with XYZ Games" banner at the bottom. In this specific case, Yogscast may fall into that comedy category, but not enough so where their fans aren't outraged about this idea all the same, it seems.
Games journalism, the written kind, is not without its moral grey areas. I can see both sides of the debate about whether or not receiving free review copies of games is an acceptable practice. There are unspoken pressures to maintain good relations with publishers than can be strained if you constantly trash their titles, but most good journalists will push past it and remain unbiased, and most good publishers will continue to provide review copies anyway, even if they risk bad reviews.
But this YouTube debate? I don't see all that much grey about it. This is literally a transaction. A physical check, written from publisher to content creator, sometimes with stipulations attached to it about what can and can't be said. What if I posted my thoughts on the Destiny beta next week, but was paid $500 to write that article by Activision? If I kept it a secret, that would be downright deceitful, but even if I publicly shared the details of the deal, why would anyone take what I had to say seriously? One path would cause me to get fired and crucified if it came to light, and the other would simply make me irrelevant as a commentator or critic. Neither seems worth it for a quick buck.
And yet, this is the YouTube situation. It's in video form, it's sometimes with smaller publishers, but it's the same deal. It's just that the YouTuber has no one to answer to but fans, and with fans expressing extreme unease and discontent with this practice, I don't see how this is a viable way forward for many YouTubers still hoping to have their judgment or opinions be considered trustworthy.
Follow me on Twitter, and on Facebook, and pick up a copy of my sci-fi novel, The Last Exodus, and its sequel, The Exiled Earthborn, along with my new Forbes book, Fanboy Wars.
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Join The 7th Generation Project
Here's a link to it:
- worth a look definitely; thanks Rachel!
Welcome toThe 7th Generation Project
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Posted by Rachel Craig on January 16, 2011 at 5:32
Are Journalists & Activists Safe Anywhere in Mexico? Protests Erupt over Killing of 5 in Mexico City
Naomi Klein on Visiting the Vatican & the Radical Economic Message Behind Papal Climate Encyclical
Naomi Klein: Obama Is Beginning to Sound Like a Climate Leader, When Will He Act Like One?
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Cissy Houston opened up to Oprah Winfrey in an intimate interview that touched on her famous daughter, Whitney Houston's sexuality. Cissy revealed that if Whitney had came out as being gay, she would have not been okay with it.
The famous singer in her own right sat down with Oprah on the Own Network for the one on one interview about her late daughter and also promoting her new book, "Remembering Whitney."
In the interview, which aired on Monday, the 79-year-old shared an excerpt from her book:
I knew I didn't want Robyn around my daughter, and I told [Whitney] that. There wasn't much I could do though. [Whitney] liked Robyn. She was past the age when I could forbid her from seeing someone. Kids have a mind of their own when they get older. They want to experiment with all kinds of things. And I don't know if it was more than that.
After reading the excerpt, Winfrey asks Cissy, "Would it have bothered you if your daughter, Whitney, was gay?"
"Absolutely," Cissy said.
"You would not have liked that?"
"Not at all," she responds.
"You wouldn't have condoned it?"
Watch clips from the interview below and be sure to leave your thoughts in the comment section.


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(excerpt from Little Shakers)
As a pastor I watch people move from church to church for many different reasons. Some moves are worthwhile, such as relocations for career advancement. However, many moves are because of disappointment or bitterness toward the church. We all face these times of disappointment, and we do not want to instill these messages of bitterness or disillusionment into our children. Many parents would say that they want to raise children that love God and His church, but by moving from church to church they send a message to their children that church commitment really isn't a priority. Their love becomes conditional and intolerant. They simply forget that the church is truly God's people – His ecclesia – His called out ones who are not perfect, but human and flawed just like everyone else.


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Lady Vikings go to 4-0 atop conference
Southern Lee's girls basketball squad had its starting five back together again as Cape Fear Valley Conference play resumed Friday night. It didn't make much of a difference to Union Pines (11-2, 4-0) as two unbeaten teams in the CFVC standings became just the Lady Vikings as they got a 63-40 win at home.
The league foes met in a non-conference game last week, with Union Pines winning 65-30 in the Yellow Jacket Holiday Basketball Classic.
Southern Lee (7-6, 2-1) played all three games in the tournament without starting point guard Jasmine McIver and starting forward Erin Pickens. Getting the two Lady Cavs back helped, but the Lady Vikings still made things tough on Southern Lee, forcing 24 turnovers on the night and pulling away in the second quarter.
After a back-door lay-up by Pickens started the scoring, the Lady Vikings rolled off the next 11 points, with five, including a three-pointer, by Nyona Shields leading the way.
Southern Lee worked back within an 11-7 deficit after a quarter and to within 13-11 shortly into the second period.
Shields, a freshman point guard, took over in a 16-3 Lady Viking run which spanned about four minutes. Her high-arcing running shot along the baseline made it 18-12 Lady Vikings, followed by Rachel Breassaeale laying in a Shields dish on a break.
Shields hit two more jumpers, then another triple. A bucket in the paint by Johnson made it 29-14 Union Pines.
The Lady Vikings led 33-21 at halftime and by as much as 42-21 in the third quarter.
Southern Lee chipped the lead in half and had chances to get within single digits in the fourth quarter. Makenzie Cicogna made two free throws and Southern Lee pressing led to Jordanna Kalkhof's steal and lay-up getting the Lady Cavs to a 47-36 margin with more than six minutes to play.
The Lady Viking defense picked up its intensity from there and Johnson, a 6-foot sophomore, helped her side put the game away. She finished with 21 points, nine rebounds and six steals. Tiffany Hicks, a 6-foot senior, had four points and 16 rebounds.
Shields had 18 points and four assists. Breassaeale collected six points, five assists, three blocks and three steals.
Cicogna led the Lady Cavaliers with 19 points, nine rebounds and three blocks. Kalkhof and Pickens added six points each. Rachel Wilson led the Lady Cav rebounding with 11 boards.
Southern Lee is off for a week before hosting Westover at 6 p.m. Friday.
Union Pines travels to Western Harnett for a 6 p.m. tip on Monday.


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1. An improved method of preparing Factor VIII procoagulant activity protein comprising the steps of
(a) adsorbing a VIII:C/VIII:RP complex from a plasma or commercial concentrate source onto particles bound to a monoclonal antibody specific to VIII:RP,
(b) eluting the VIII:C,
(c) adsorbing the VIII:C obtained in step (b) in another adsorption to concentrate and further purify same,
(d) eluting the adsorbed VIII:C, and
(e) recovering highly purified and concentrated VIII:C.
2. A method according to claim 1, wherein the elutant used in each of steps (b) and (d) is a saline solution.
3. The method according to claim 2, wherein the saline solution is calcium chloride.
4. The method according to claim 3, wherein the concentration of said calcium chloride solution used in steps (b) and (d) ranges from about 0.25 M to about 0.5 M.
5. The method according to claim 1, wherein said adsorbent particles in step (a) are agarose.
6. The method according to claim 1, wherein aminohexyl agarose is employed in step (c) as the adsorbent.
7. The method according to claim 6, wherein calcium chloride solution is employed as the elutant in steps (b) and (d), concentration of said calcium chloride solution ranging from about 0.25 M to about 0.5 M in step (b) and from about 0.25 M to about 0.5 M in step (d).
8. An improved immunoadsorbent for isolation and purification of VIII:C from VIII:C/VIII:RP comprising a monoclonal antibody specific to VIII:RP bound to solid particles.
9. The improved immunoadsorbent of claim 8, wherein said solid particles comprise a resin.
10. The improved immunoadsorbent of claim 9, wherein said resin comprises agarose.
11. The improved immunoadsorbent of claim 10, wherein said agarose is cross-linked agarose.
12. The improved immunoadsorbent of claim 11, wherein said immunoadsorbent has a coupling density of 3 to 4 g of monoclonal antibody per liter of agarose.
13. Highly purified and concentrated .Iadd.human or porcine .Iaddend.VIII:C prepared in accordance with the method of claim 1.
14. Highly purified and concentrated .Iadd.human or porcine .Iaddend.VIII:C prepared in accordance with the method of claim 6.
15. In a method for purifying Factor VIII procoagulant activity protein from plasma or concentrate, the improvement comprising the step of passing said plasma or concentrate through a chromatographic type column having adsorbent to which is bound monoclonal antibodies which is specific to VIII:RP and eluting the VIII-C therefrom.
16. The method according to claim 15, wherein said adsorbent is agarose and said elutant is a saline solution. .Iadd.
17. A VIII:C of claim 13, wherein said VIII:C is human VIII:C. .Iaddend. .Iadd.18. A VIII:C of claim 14, wherein said VIII:C is human VIII:C. .Iaddend. .Iadd.19. An improved immunoadsorbent of claim 8 wherein said monoclonal antibody remains bound to VIII:RP during elution of VIII:C. .Iaddend. .Iadd.20. An improved immunoadsorbent of claim 19, wherein said elution is carried out with a saline solution. .Iaddend. .Iadd.21. An improved immunoadsorbent of claim 20 wherein said solution contains calcium ions. .Iaddend. .Iadd.22. An improved immunoadsorbent of claim 21, wherein said solution is calcium chloride. .Iaddend. .Iadd.23. An improved immunoadsorbent of claim 22, wherein the concentration of said calcium chloride is from 0.25 M to about 0.5 M. .Iaddend. .Iadd.24. A human VIII:C preparation having a potency in the range of 134 to 1172 units per ml, and
being substantially free of VIII:RP. .Iaddend. .Iadd.25. A human VIII:C preparation of claim 24, wherein the VIII:C concentration is at least 160,000 fold purified relative to VIII:C in plasma. .Iaddend. .Iadd.26. A human VIII:C preparation of claim 24, wherein the ratio of VIII:C to VIII:RP is greater than 100,000 times the ratio in plasma. .Iaddend. .Iadd.27. A human VIII:C preparation of claim 24, wherein said VIII:C is isolated from VIII:C/VIII:RP and 90-100 percent of the VIII:RP has been
removed. .Iaddend. .Iadd.28. A human VIII:C preparation having a specific activity greater than 2240 units/mg. .Iaddend. .Iadd.29. A human VIII:C preparation of claim 28 wherein the potency is in the range of 134 to 1172 units/ml. .Iaddend. .Iadd.30. An improved method of preparing Factor VIII procoagulant activity protein comprising the steps of
(a) adsorbing a VIII:C/VIII:RP complex from a plasma or commercial concentrate source onto a substrate to which is bound a monoclonal antibody specific to VIII:RP,
(b) eluting the VIII:C,
(c) adsorbing the VIII:C obtained in step (b) in another adsorption to concentrate and further purify same,
(d) eluting the adsorbed VIII:C, and
(e) recovering highly purified and concentrated VIII:C. .Iaddend.
.Iadd. A method according to claim 30, wherein said substrate is a resin. .Iaddend. .Iadd.32. A method according to claim 30, wherein said substrate is comprised of particles. .Iaddend. .Iadd.33. An improved immunoadsorbent for isolation and purification of VIII:C from VIII:C/VIII:RP comprising a monoclonal antibody specific to VIII:RP bound to a substrate. .Iaddend. .Iadd.34. Highly purified and concentrated human or porcine VIII:C prepared in accordance with the method of claim 30. .Iaddend. .Iadd.35. In a method for purifying Factor VIII procoagulant activity protein from plasma or concentrate, the improvement comprising the step of passing said plasma or concentrate over a substrate to which is bound monoclonal antibodies which are specific to VIII:RP and eluting the VIII:C therefrom. .Iaddend. .Iadd.36. The method according to claim 35, wherein said substrate is agarose and said elutant is a saline solution. .Iaddend.
10-20 units of VIII:C per ml of eluant are obtained with the present invention, in contract to 0.5-1.25 units per ml of eluant with the Tuddenham et al method.
The present method also permits the selection of a monoclonal antibody having a high affinity for VIII:RP; however, the use of polyclonal antibodies results in varying affinities. It should be realized that there is an indirect relationship between the affinity of the bound antibody for VIII:RP and the elution of VIII:RP. Thus, the higher the affinity of the antibody for VIII:RP, the less VIII:RP will be present with VIII:C in the eluant. The present invention also makes it possible to produce an unlimited supply of the specified monoclonal antibody, thus eliminating variations among different batches.
Although Austen, as earlier described, has reported the use of aminohexyl-agarose to separate VIII:C from VIII:RP, such a material has not heretofore been used to concentrate VIII:C following a separation and purification step. Heretofore, the highest VIII:C concentrations achieved by using aminohexyl agarose in chromatography were 0.53 units per ml of eluant for human protein and 2.38 per ml of eluant for porcine VIII:C. The present method permits concentrations several orders of magnitude greater than these. Perhaps of even greater significance, is the fact that the present invention provides for a greater purification of human VIII:C than has ever been reported (164,000 vs 17,000 fold over plasma). The present method, which is described in more detail hereinafter, yields VIII:C with a specific activity of 2,300 units/mg when commercial concentrate is used. This corresponds to a 164,000 fold purification from plasma. The ratio of VIII:C to VIII:RP is greater than 105 as compared to the ratio in plasma.
The following description provides details of the manner in which the embodiments of the present invention may be made and used in order to achieve the separation, purification and concentration of VIII:C to a degree of purity and concentration not known heretofore. This description, while exemplary of the present invention, is not to be construed as specifically limiting the invention and such variations which would be within the purview of one skilled in this art are to be considered to fall within the scope of this invention.
The monoclonal antibody to VIII:RP which is subsequently bound to the separation substrate may be prepared in a stepwise procedure starting with a highly purified preparation of factor VIII/von Willebrand factor (VIII:C/VIII:RP complex). The purification for immunization is accomplished with material obtained from a plasma source. Less highly purified material for coating polyvinyl plates is obtained in higher concentration from commercial extracts such as FACTORATE (trademark of Armour Pharmaceutical Co., Tuckahoe, N.Y.) or Hemophil (trademark of Hyland Laboratories, Costa Mesa, California). Purification is performed by a standard agarose-gel filtration of cryoprecipitate, such as that described by Zimmerman and Roberts, "Factor VIII Related Antigen", appearing in IMMUNOASSAYS: CLINICAL LABORATORY TECHNIQUES FOR THE 1980's, R. M. Nakamura et al, eds., Alan R. Liss, Inc., New York, pp. 339-349 (1980). Mice were injected with highly purified factor VIII/von Willebrand factor obtained from plasma according to the following procedure. On day zero, the mice are injected intraperitoneally with a composition prepared by dissolving (or suspending) 10 Mg of the protein in 0.1 ml of buffer containing 0.05 M Tris, 0.15 M sodium chloride, 0.02 % sodium azide, 1 mM phenyl methyl sulfonyl fluoride, traysylol 10 units/ml at pH7.3. and shaking with an equal volume of complete Freund's adjuvant. On day 14, the mice are again injected with the same material except that incomplete Freund's adjuvant is substituted for complete Freund's adjuvant. On day 21, the injection of day 14 is repeated. On day 38, the mice are injected with purified VIII:C/VIII:RP only. On day 42, the spleens of the mice are removed and fused according to a standard procedure, of the type described by J. P. Brown et al "Protein Antigens of Normal and Malignant Human Cells Identified by Immunoprecipitation with Monoclonal Antibodies", JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 225, pp. 4980-4983 (1980). The standard technique is varied only to the extent that 35% polyethylene glycol 1000 is substituted for 50% polyethylene glycol. A radioimmunoassay method for clones producing antibody to VIII:RP is performed according to the following procedure. Polyvinyl plates with a "V" bottom, flexible type are coated with 0.1 ml of factor VIII purified from commercial extract according to the procedure indicated above and having a concentration of 0.125 mg/ml of protein. The plates are blocked with albumin, washed with buffer and incubated with the culture fluids from the clones to be tested. The plates are then washed and reacted with rabbit anti-mouse IgG antiserum, washed a second time and 125 I labeled goat anti-rabbit IgG antiserum is added to the wells and incubated. The plates are again washed, then dried and the wells cut-out and counted. After determining the clones which are positive they are subcloned at least twice and stable clones producing antibody to VIII:RP are then injected into the peritoneal cavities of Balb/C mice which have been pretreated intraperitoneally with 0.5 ml of pristane at least four days prior to injection of cells. Hybridoma cells are injected at concentrations of approximately 5�106 cells per mouse in 0.5 ml of Delbecco's modified Eagle's medium without fetal bovine serum. The mice are tapped when bloated and ascites fluid is collected in heparin at approximately 10 units/ml. Ascites fluid from multiple mice is pooled to provide a convenient volume for subsequent isolation of the monoclonal IgG. If the heparinized ascites fluid is not used immediately, it may be stored at -70� C. and thawed just prior to use. The final yield of IgG from the ascites fluid is approximately 1 g of IgG per 100 ml of ascites fluid.
The specificity of the monoclonal IgG for the purpose of purifying VIII:C may be assessed by coupling the IgG to a separation substrate medium, in the manner described hereinafter, and demonstrating that the bound IgG removes from VIII:RP and VIII:C from plasma and that the VIII:C may be subsequently eluted with a solution containing calcium ions while the VIII:RP remains complexed to the monoclonal IgG which is bound to the solid-state substrate.
The monoclonal IgG, which is to be used subsequently to prepare the immunoadsorbent, may be isolated from heparinized pooled ascites fluid immediately after collection or a frozen portion of the stored solution may be thawed. Regardless of whether fresh or frozen material is used, the solution is brought to 4� C. and treated with an equal volume of phosphate buffered saline solution (PBS), the composition of which is set forth below. The diluted ascites is precipitated by dropwise addition with stirring at 4� C. of an equal volume of saturated ammonium sulfate (SAS); prepared by boiling an excess of ammonium sulfate in water, cooling to 4� C., filtering undissolved crystals and adjusting the pH to 7.0 with ammonium hydroxide. The precipitate and its supernatant liquid are stirred for at least 2 hours and centrifuged at 4� C. Centrifugations are preferably carried out at 14,000 rpm for 60 minutes (30,000�g). The supernatant solution of ascites is precipitated twice more with SAS and the mixture of precipitate and supernatant liquid stirred and centrifuged in the same manner as in the first cycle. The pellets resulting from the third precipitation are resuspended in a volume of PBS equal to that of the diluted ascites fluid and then disalyzed exhaustively against PBS. Clots appearing in the dialysis bags are removed by centrifugation at 20� C. The dialyzed IgG is adsorbed by stirring it with a 5% aqueous solution of aluminum hydroxide at room temperature and centrifuging at 20� C. after adsorption. The adsorption treatment is repeated at least three more times using 2.5% aluminum hydroxide solution for each treatment after the first. The adsorbed IgG is brought to 4� C. and reprecipitated once with SAS as described above. The precipitated pellets may be stored at -20� C. until used.
The immunoadsorbent is prepared by suitably preparing the monoclonal IgG for coupling, preparing the solid substrate for coupling and reacting the two components to bind the former to the later.
(i) Preparation of IgG for Coupling
Either freshly precipitated IgG may be used or previously frozen precipitate may be thawed for use. The material is then dialyzed against PBS, and while still in the PBS, the volume and IgG concentration (A280 /1.4=mg/ml IgG) are determined. The IgG is then treated with between 10 and 30 microliters, preferably 20 microliters, or diisopropylfluorophosphate per 50 ml of IgG solution. The resulting solution is stirred at room temperature in a hood for 30 minutes and the treated IgG, immediately prior to use, is dialyzed overnight against coupling buffer. The coupling buffer found most suitable is a 0.25 M sodium bicarbonate solution adjusted to a pH of 9, preferably with sodium hydroxide.
(ii) Preparation of Solid Substrate for Coupling
Although the monoclonal antibody may be bound to any material which does not have a high affinity for protein, particularly factor VIII itself, such materials as glass beads, agarose and derivatives thereof are preferred. Most preferred is a crosslinked agarose available commercially as a gel known as Sepharose CL2B (trademark of Pharmacia Fine Chemicals, Piscataway, N.J.).
The method of preparing the preferred immunoadsorbent resin is generally the same as that disclosed in the literature, such as the method of J. Porath et al, JOURNAL OF CHROMATOGRAPHY, Vol. 86, pp. 53-56 (1973). The method found most suitable is as follows: a volume of about 2 liters of Sepharose CL2B is placed in an acid-cleaned 2 liter sintered glass filter funnel. The resin is washed with water and filtered to a moist cake. The washed resin is placed in a large (approximately 4 liter) glass beaker equipped with a magnetic stirring bar. To the resin is then added 750 ml of cold potassium phosphate buffer solution, prepared by mixing one part of a 5 M dibasic potassium phosphate solution with two parts of 5 M tribasic potassium phosphate solution. Sufficient cold water is added to bring the final volume to 3 liters. The mixture is then chilled to 4� C. and maintained at between 4�-10� C. in an ice-water bath placed on a magnetic stirring plate. In a hood, cyanogen bromide is added to 300 ml of water in a stoppered glass bottle containing a magnetic stirring bar. The mixture is rapidly stirred until solution results. The cyanogen bromide solution is then added with stirring over a 2 minute period to the cold Sepharose mixture. Stirring is continued for an additional 8 minutes and then transferred to a chilled 2 liter sintered glass filter funnel supported in a 4 liter vacuum flask. The cyanogen bromide treated resin is then washed with approximately 20 liters of cold water or until the pH of the filtrate is neutral. The washed resin is then quickly equilibrated with cold coupling buffer and then transferred to a 4 liter plastic beaker equipped with a large magnetic stirring bar.
(iii) Coupling the Monoclonal Antibody to the Solid Substrate
The solid substrate resin, prepared as indicated above, is ready to be used when it is equilbrated with coupling buffer and should not be stored thereafter. Accordingly, the resin mixture is combined with the IgG which was previously dialyzed overnight against coupling buffer. The combined resin/IgG suspended mixture is stirred at 4� C. for a period of about 24 hours. The A280 of an undiluted sample of the supernatant coupling liquid may be determined using bovine serum albumin (BSA) as a standard or Bio-Rad protein assay (Bradford reagent) with BSA as standard. The percentage ligand which is coupled may then be calculated. When the above described procedure is followed, this is usually about 95%. Any remaining active sites on the resin not coupled to antibody may be blocked by washing the resin on a sintered glass filter funnel with cold coupling buffer containing 0.1 M glycine. The resin is then resuspended in this solution to a final volume equal to that when the resin and antibody, each in coupling buffer, were combined. The suspension is stirred slowly overnight at 4� C. The resin is then washed thoroughly with VIII:C-buffer, the composition of which is given below. The coupled, blocked resin is then pre-eluted with VIII:C-buffer additionally containing 0.5 M calcium ions, preferably calcium chloride. The resin is again washed with VIII:C buffer alone and stored at 4� C. or in a continuously pumped column at room temperature until ready for use. The coupling density of IgG to SEPHAROSE should be 2-5 g, preferably 3-4 g IgG/liter of SEPHAROSE.
(i) Sample preparation of factor VIII, such as human and animal plasmas and commercial concentrates of factor VIII, may be employed in the present invention and the method is not limited as to a particular type of material. Preferred materials, and those which have demonstrated successful results, are porcine and human plasmas and commercially available concentrates of human factor VIII, such as FACTORATE available from Armour Pharmaceutical Co. The following description provides details for using both porcine plasma or commercial human concentrate such as FACTORATE:
FACTORATE is reconstituted by adding 25 ml portions of VIII:C-buffer to the contents of each of 20 bottles containing 400-500 VIIIC units per bottle (25 ml per bottle). The mixture is adjusted to a final volume of 1 liter with VIII:C-buffer. A sample aliquot of 0.5 ml may be removed for assay and the remaining material applied to the immunoadsorbent column overnight at a rate of approximately 60 ml/hour.
Porcine plasma, when not freshly drawn, is citrated by conventional means and stored frozen. When ready to be used it is thawed at a temperature of between 35�-40� C., preferably 37� C. and applied directly to the column at 60 ml/hour.
It should be noted that while the description of the present invention refers, and is directed primarily, to the use of immunoadsorbent coupled particles in a chromatography column, it is within the scope of this invention to perform batchwise separations by placing the antibody-bound resin particles in a suitable container and after adding reconstituted concentrate or plasma, VIII:C as outlined above and described in more detail below.
When the process is carried out in a chromatography process, the following embodiments are preferred:
The resin is placed in a column, such as an Amicon 86001, (trademark of Amicon Corp., Lexington, Mass.), equipped with a peristaltic pump and a high flow head. When concentrate is used as the source of factor VIII, for 20 bottles of diluted concentrate, approximately 1.5 liters of resin, prepared as indicated above, is used. When porcine plasma is used, 150 ml of resin is used for each liter of plasma.
After the sample is applied to the column, it is washed with 1 liter of VIII:C-buffer, followed by a second washing with VIII:C-buffer which additionally contains 0.5 M NaCl. Approximately 20 liters of saline-buffer is used when factor VIII is applied as concentrate and 20 bed volumes when porcine plasma is employed. Optimum results are obtained with a flow rate of 1 liter/hour.
Elution of purified VIII:C is accomplished with VIII:C-buffer containing calcium ions. Although a linear gradient, as taught by Tuddenham et al, supra, works well, it is not required in order to accomplish the object of this invention; a solution having a fixed calcium ion concentration is quite adequate. Thus, when VIII:C derived from concentrate is being eluted, VIII:C-buffer, 0.25 to 0.5 M with respect to calcium chloride, preferably 0.35 M, is used advantageously as a flow rate of between 450 to 750 ml/hour and preferably 600 ml/hour. When the VIII:C is obtained from porcine plasma, elution is performed with VIII:C-buffer being a calcium chloride concentration of between 0.35 and 0.7 M, preferably 0.5 M and at a flow rate of between 10 and 30 ml/hour, preferably 20 ml/hour. Fractions of 12 ml and 3 ml are collected for VIII:C originating from concentrate and porcine plasma, respectively. Those fractions containing at least 1.0 unit/ml of VIII:C activity are pooled and the total volume and activity of the pool determined.
The VIII:C pool is initially concentrated to 10-20 ml by a standard procedure such as pressure ultrafiltration. For this purpose, Amicon stirred cell in which a YM-10 membrane under 50 psi of nitrogen pressure has been found to work well. Slow stirring is continued for 30 minutes after nitrogen pressure is released, and the volume and activity of the concentrated pool are determined. The pool may be stored for a brief period, that is, overnight for example, if a temperature of 4� C. is maintained.
It may be noted that the immunoadsorbent column described above may be regenerated by treatment of the column with 2 bed volumes of 3 M aqueous sodium thiocyanate solution run at a flow rate of about 0.5-1 liter/hour to elute VIII:RP.
Although the VIII:C recovered from the separation from VIII:RP by means of the immunoadsorbent column is highly purified, it is still too dilute to be therapeutically useful. Further concentration and a four fold increase in purification when porcine plasma is used is accomplished by use of an aminohexyl agarose column which is prepared and used in the following manner:
(i) Preparation and/or Conditioning of a Aminohexyl Agarose Column:
Aminohexyl agarose is agarose which has been reacted with 1,6-diaminohexane to yield an agarose resin having a number of 6 carbon atom chains, each of which has a terminal amino group. It may be prepared according to the method described by Austen, supra, or acquired from a commercial supplier. One such material which has been used successfully in the present invention is available under the name of AH-SEPHAROSE 4B (trademark of Pharmacia Fine Chemicals, Piscataway, N.J.).
Whether prepared or purchased, the resin should be conditioned prior to use. This may be accomplished as follows, the volumes, amounts and dimensions being adjusted in proportion to the amount of material to be concentrated:
Approximately 1 gram of aminohexyl agarose (AH-SEPHAROSE 4B) is placed in a sintered glass filter funnel and washed with at least 200 ml of 0.5 M sodium chloride, while stirring. The resin is then equilibrated with VIII:C-buffer and packed in a column of approximately 0.9 cm diameter. A Bio-Rad Econo-Column with flow adapters has proven quite suitable for the type of use considered here. The bed volume of the packed column is approximately 4 ml.
(ii) Application to and Use of the Aminohexyl Agarose Column
The concentrated pool, prepared as described above, is diluted 1:10 in VIII:C-buffer to a final concentration of 100-200 ml when using the amounts of resin and column size as described in the immediately preceding section. The diluted pool is applied to the column at a flow rate of 200 ml/hour.
The column is then washed with VIII:C-buffer which contains calcium ions, preferably from calcium chloride. The solution should be between 0.01 M to 0.03 M, preferably 0.025 M with respect to calcium ions.
Elution of the concentrated VIII:C is achieved at a flow rate of between 5 to 20 ml/hour, preferably 10 ml/hour with VIII:C-buffer containing a higher concentration of calcium ions than was employed with the preceding washing step. Again, calcium chloride is the preferred source of calcium ions in a concentration of between 0.25 to 0.5 M, preferably 0.3 M. Fractions of 1 ml volume are collected and assayed as described below. Collected fractions may be stored at 4� C. or frozen. Preparations of VIII:C obtained from a porcine plasma source should be stabilized within 5 to 10% human serum albumin prior to storage.
Assays may be performed by diluting the fractions with VIII-C buffer if necessary and further diluting the fraction 1:100 in assay buffer prior to addition to the substrate. A standard partial thromboplastin time assay is employed.
The composition of the buffer solutions is as follows:
1.6 g sodium phosphate, monobasic monohydrate
8.4 g sodium phosphate, dibasic anhydrous
61.4 sodium chloride
Water to 7 liters
pH of buffer is 7.2
ml 0.02 M imidazole
ml 0.15 M sodium chloride
ml 0.10 M lysine
ml 0.02% sodium azide
pH of buffer is adjusted with concentrated hydrochloric acid to 6.8.
The data listed hereinafter in Tables I and II are representative of that obtained according to the present invention, as described above.
TABLE I__________________________________________________________________________VIII:C Obtained From FACTORATE Concentrate as the Source ofVIII:C/VIII:RP Specific From VIIIC VIIIC Activity Plasma Volume (Units/ (Total Protein Protein Recovery (Units/ (Fold (ml) ml)* Units) (mg/ml) (Total mg) (%) mg) Purif.)__________________________________________________________________________Sample Applied to 500 18.8 9400 29 14.500 -- 0.7 50ImmunoadsorbentPool resulting from 1020 4.6 4692 -- -- 50 -- --ImmunoadsorbentPool After Initial 20 134 2680 -- -- 29(57) -- --ConcentrationSum Resulting from -- -- 1576 -- -- 17(59) -- --Aminohexyl ColumnAminohexyl 0.95 1172 1112 0.51 0.48 12 2294 163.857Fraction #3Aminohexyl -- 545 -- 0.23 -- -- 2370 169.285Fraction #4__________________________________________________________________________ *A frozen human plasma pool used as the standard for VIIIC assays and assigned the value of 1 human unit per ml.
TABLE II__________________________________________________________________________VIII:C Obtained From Citrated Porcine Plasma Specific From VIIIC VIIIC Activity Plasma Volume (Units/ (Total Protein Protein Recovery (Units/ (Fold (ml) ml) Units) (mg/ml) (Total mg) (%) mg) Purif.)__________________________________________________________________________Sample Applied to 1000 1* 1000 76 76.000 100 0.013 --ImmunoadsorbentPool Resulting from 70 8.8 613 -- -- 61 -- --ImmunoadsorbentPool After Initial 5.76 88 494.5 0.242 1.355 49.5 364 28.000ConcentrationSum Resulting from 5.0 49 247 0.035 0.175 25 1413 109.000Aminohexyl Column__________________________________________________________________________ *Porcine plasma used as the standard for VIIIC assays and assigned the value of 1 porcine VIIIC unit per ml.
Although only preferred embodiments are specifically illustrated and described herein, it will be appreciated that many modifications and variations of the present invention are possible in light of the above teachings and within the purview of the appended claims without departing from the spirit and intended scope of the invention.
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The first drop from the Liam Spring '12 range is now available at RUBY stores and online. Pop into any of our stores tomorrow morning for mimosas to celebrate!
The first drop includes THE DECIDED SHIRT in the Jacaranda Peony print, designed exclusively for this Liam range featuring broken gemstones and peonies.
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$3,699.99 After $200 OFF
Urban Islands 4-burner IOutdoor Kitchen Island by Bull Outdoor Products
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