103.
REFERENCES
1. Bache, C. A., Hardee, D. D., Holland, R. F., and Lisk, D. J. Absence
of Phenoxyacid Herbicide Residues i n t h e Milk of Dairy Cows a t
High Feeding Levels. J. Dairy S c i . 47: 298 (1964).
2.
Bathe, C. A., Lisk, D. J., Wagner, D. G . , and Warner, R. G. Elmininat i o n o f MCP and MCPB i n t h e Urine fmm Cows. J. Dairy S c i . 47 :
93 (1964).
3.
Fisher, D. E., St. John, L. E., Jr., Gutenmann, W. H., Wagner, D , G.
and Lisk, D. J. Fate of Banvel-T, Ioxynil, Tordon, and T r i f l u o r i l i n i n t h e B i r y Cow. J. Dairy S c i . 48: 1 7 1 1 (1965).
4.
Gutenmann, W. E., Hardee, D. D., Holland, R. F., and L i s k , D. J.
Disappearance of 4(2,4-DB) Herbicide i n t h e Dairy Cow. 46:
991 (1963).
5.
Gutenmann, W. H.,
6.
Gutenmann, W. H., Wagner, D. G., and Liks, D. J. Gas Chromatographic
Analysis of the Fate of MC-A-600 i n t h e Dairy Cow. J. Dairy Sci.
47: 8 2 1 (1964).
7.
Hardee, D. D., Gutenmann, W. R., Keenan, G. I., Gyrisco, G. G.,
F. H., Trimberger, G. W., and Holland, R. F. J. Ec. Ent.
404 (1964).
8.
Lisk, D. J., Gutenmann, W. H., Bache, C . A., Warner, R. G., and Wagner,
D. G. Elimination of 2,4-D i n t h e Urine of S t e e r s Fed 4-(2,4-DB)
o r 2,4-D. J. Dairy S c i . 46: 1435 (1963).
9.
S t . John, L. E. Jr., Ammering, J. W., Wagner, D. G., Warner, R. G.,
and Lisk, D. J. Fate of DNOSBP, Simazine, and PCN'B i n t h e D a i r y
Cow. J. Dairy Sci. 48: 502 (1965).
-
Hardee, D. D., Holland, R. F, and Lisk, D. J.
Residue Studies with 2,4-D Herbicide i n t h e Dairy Cow and i n a
Natural and A r t i f i c i a l €lumen. J. Dairy Sci. 46: 1287 (1963).
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Fox,
57:
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10. S t . John, L. E. Jr., Wagner, D. G., and Lisk, D. J. Fate of Atrazine,
Kuron, Silvex, and 2,4,5-T in t h e h i r y Cow. J. Dairy S c i . 47:
1 2 6 7 (1964).
DR. CARPENTER: I would l i k e f o r you t o remain f o r a
e have no formal program f o r lunch and so I
b r i e f discussion. W
would l i k e t o c a l l on D r . A. M. Pearson t o e n t e r t a i n any questions
f o r LO o r 15 minutes. D r . Pearson.
DR. A. M. PEARSON: Thank you, M r . Chairman. In going
back t o some o f o u r experiences i n gas chromatography, I t h i n k they
began i n about 1956. So we have been doing work in t h i s area f o r
104.
q u i t e some time although o u r emphasis has been somewhat d i f f e r e n t from t h e speakers today because we have been primarily i n t e r e s t e d i n f l a v o r compounds. Flavor chemistry is much more d i f f i c u l t perhaps than where you know t h e compound t h a t you a r e working
with. In f l a v o r chemistry we know l i t t l e about t h e compounds we
a r e working w i t h and it i s mch more d i f f i c u l t t o i s o l a t e and
characterize compounds with which you a r e not acquainted. We had
f o u r e x c e l l e n t papers here dealing with gas chromatography. I
t h i n k t h e paper given by John Sink would i n d i c a t e t h a t it i s a
u s e f u l instrument. But I t h i n k w e have a problem here which we
do need t o d i s c u s s . Oftentimes, t h e most d i f f i c u l t thing i s
i s o l a t i o n of t h e sample which you wish t o analyze. I t h i n k t h i s
i s an a r e a that could stand some discussion today. The paper by
D r . S t a l l i n g c e r t a i n l y made an elegant one f o r discussion. In
f a c t , we have had two papers today which showed methods of d e t e r mining compounds which a t one time were thought impossible t o be
determined by gas chromatography; namely, t h e amino acids and t h e
t r i g l y c e r i d e s . We have made t h e statement a t one time t h a t they
could not be analyzed by gas chromatography. Now the methods have
been developed whereby you can use gas chromatography and they
have been very e l e g a n t l y presented t o us today by D r . S t a l l i n g and
by D r . Kukis. D r . Lisk, of course, gave us a very i n t e r e s t i n g
t a l k dealing w i t h gas chromatography of t h e p e s t i c i d e s and I was
p a r t i c u l a r l y i n t e r e s t e d i n t h e instrument which he described. I
t h i n k it would be of some i n t e r e s t if he would give us the name
of t h i s instrument and where it can be obtained because it seems
t o be most u s e f u l . I t h i n k we could a l s o ask him i n t h i s regard,
does it work i n combined sulphur-containing compounds; t h a t i s ,
could it be used t o determine the l e v e l s of sulphur compounds j u s t
merely a s an elemental sulphur a n a l y s i s ? I would l i k e t o t u r n some
time over t o you f o r questions although f i r s t I would l i k e t o ask
Dr. L i s k if he would speak t o us very b r i e f l y concerning the quest i o n s which I have asked.
DR. LISK: A s f o r t h e source of t h i s device, t h e o r i g i n a l
paper by Cook was published i n the Analytical Chemistry i n 1965.
I d o n ' t have t h e page number, b u t can g e t it f o r anyone who wants
it. I am r i g h t next door about 100 f e e t away from t h i s building so
I would be glad t o show you t h e device and w e can give you f a c t s
and f i g u r e s about i t . Now your second question had t o do w i t h
determining elemental sulphur i n combined compounds. If the compound contains sulphur and if it w i l l go through a gas chromatography
column you w i l l see t h i s sulphur band emission and you can do compounds t h i s way. In f a c t , I had a s l i d e (I d i d not show it today
because of l a c k o f time) i n which we followed t h e sulphur l i n e instead o f the phosphorus. The reason we chose t h e phosphorus l i n e
i s because the phosphorus gives us t h e s e n s i t i v i t y t h a t corresponds
t o about one times t e n t o t h e minus 13 grams p e r second. In o t h e r
words, i f you i n j e c t a t e n t h of an anagram of phosphorus you can
see it. The sulphur l i n e i s about t e n t o a hundred times l e s s
s e n s i t i v e . So therefore, t h e a b i l i t y t o look a t e i t h e r sulphur o r
phosphorus i n one end of a molecule w i l l depend on t h e concentrat i o n . You could s u r e l y do it on a formulation a n a l y s i s . But f o r
residue a n a l y s i s , you might have t o make two separate i n j e c t i o n s ,
105.
one i n which t h e phosphorus peak would go o f f s c a l e , b u t you could
see the sulphur peak. So you make your choice f o r a n a l y s i s j u s t
l i k e we a r e based on the s t r o n g e s t emission you g e t . This i s one
of t h e unfortunate p a r t s of emission work. It i s an o l d technique
and many of you a r e probably f a m i l i a r w i t h it o r heard o f it, b u t
the emission t h a t you g e t off w i l l vary i n s t r e n g t h depending upon
t h e p r o b a b i l i t y of t r a n s i t i o n taking place. In o t h e r words, when
you e x c i t e an atom, i f a c e r t a i n e l e c t r o n i s being kicked up i n t o
a higher o r b i t and dropping back and it i s a very probable occurrence, then you w i l l have a l a r g e number of these atoms undergoing
t h i s t r a n s i t i o n . Therefore, you w i l l g e t a strong s i g n a l and
t h e r e f o r e have a s e n s i t i v e response f o r it. If it i s less probable,
then your s e n s i t i v i t y w i l l vary as your p r o b a b i l i t y v a r i e s . With
phosphorus, as I say, w e can do p a r t s p e r b i l l i o n of a compound
of organo-phosphorus and i n s e c t i c i d e i n a crop. This i s p l e n t y
s e n s i t i v e f o r residue a n a l y s i s . For sulphur you probably would
have t o be satisfied w i t h 10 p a r t s p e r b i l l i o n . Iodine i s about
t e n times l e s s s e n s i t i v e than phosphorus. You could probably do
t e n p a r t s p e r b i l l i o n of an iodinated compound. B u t t o answer
your question, if the compound contains sulphur and if it w i l l go
through t h e chromatographic column you can s i t on t h e sulphur
l i n e and do it. By using a s e r i e s of standards you can t e l l how
much i s p r e s e n t .
DR. PEARSON: What i s the name o f t h e instrument o r
doesn't it have one y e t ?
DR. LISK: Well, I guess it depends on how
want t o t a l k . Cook has named it micro wave emission
some people j u s t c a l l it t h e micro wave d e t e c t o r . I
micro wave d e t e c t o r emission i s t h e most d e s c r i p t i v e
i s a c t u a l l y what you a r e doing. You a r e using micro
t o cause e x c i t a t i o n and then looking a t t h a t emitted
DR. PEARSON:
long you
d e t e c t o r and
think the
because t h i s
wave energy
energy.
Is it commercially a v a i l a b l e ?
DR. LISK: There are some companies t h a t have v i s i t e d
Cook's l a b and have a l s o seen o u r lab but have not committed
themselves a s y e t as t o whether they a r e working on it o r a r e
going t o come o u t w i t h it o r n o t . We suspect t h a t it w i l l come
o u t s h o r t l y though.
DR. PEARSON: Thank you. Are there o t h e r questions?
I would l i k e t o a s k D r . Kuksis very b r i e f l y t o comment on t h e
o r d e r i n which t h e compounds come o f f . I noticed, if you give
these peak numbers, do the same numbers always correspond t o t h e
same compound ?
DR. KUKSIS: Yes and no. I should say today t h a t i t ' s
very simple. We j u s t count t h e carbons i n t h e f a t t y a c i d s and
add a l l of them up. When c o r r e l a t e d t o a number of o t h e r compounds one could c e r t a i n l y c o r r e l a t e t o any kind of glycerides
one has and t o a number of o t h e r compounds. B u t when one goes t o
stearyl e s t e r s and what a r e you going t o name t h e r e ? Are you
106.
going t o count t h e number of s t e a r y l s , t h e carbons i n the stearyls
o r a r e you going t o count t h e carbons i n t h e f a t t y a c i d s ? Because they a r e eluted ahead of t r i g l y c e r i d e s and i n some cases
overlap t h e t r i g l y c e r i d e s over approximately t h e same carbon numb e r . For instance, c h o l e s t e r o l arachadonate 47 (27 carbons i n
c h o l e s t e r o l , 20 i n arachadonic a c i d ) . That overlaps t h e C46 t h a t ' s
w i t h the t r i g l y c e r i d e t h a t w i l l contain any combination of f a t t y
a c i d s t h a t w i l l give a t o t a l number of 46 carbons. So we counted
a l l of t h e carbons t h e r e . We d i d t h e same thing when we came t o
s t e a r y l . Again, one can say f o r instance, c h o l e s t e r o l 27 carbons
combestol 28, b e r o s t e r o l 29, they run a l l ahead of tridecanoin
which i s C30. But when one g e t s down t o f a t t y a c i d s t h a t we can
run i n t h e f r o n t of the chromatogram, we w i l l have t o count whatever we had i n the event of monoglycerides. We ended up using t h e
same numbers f o r t h r e e f a t t y a c i d s as f o r monoglycerides.
Diglycerides we counted as s t r a g g l e r s as t h r e e d i g l y c e r i d e s . B u t
t h e problems r e a l l y a r i s e when you use monoglyceride a c e t a t e s o r
when you use diglyceride a c e t a t e s . Does t h i s s a t i s f y t h i s ?
DR. PEARSON: Yes, thank you very much. I think t h i s
answers t h e question. Are there o t h e r questions? I wonder i f D r .
Elston would comment on the method t h a t they have used f o r t h e
preparation of methyl e s t e r s .
DR. ELSTON, Harvard University: Currently I wish I
could give you a good method of preparation of methyl e s t e r s . We
have extended the range of o u r d e t e c t i o n t o t h e point where we a r e
g e t t i n g a l o t of trouble. The b e s t I can describe i s oxidation
products. We a r e using t h e o l d procedure developed by D r . Ahrens
a t t h e Rockefeller I n s t i t u t e . So t h i s i s simply reflexing t h e
l i p i d mixture with methyl alcohol-sulphuric a c i d o r hydrochloric
a c i d , n e u t r a l i z i n g with sodium bicarbona tc aud e x t r a c t i n g i n p e t
e t h e r . I t h i n k Dr. Dugan from Michigan S t a t e r e c e n t l y developed
a procedure t h a t s e e m t o be & x d f o r a l a r g e q u a n t i t y . Is t h i s
what you were r e f e r r i n g t o , D r . Pearson?
DR. PEuRSON:
Yes.
DR. ELSTON: There i s a low tercperature o f methylation
i n t h a t procedure (I am not e x a c t l y sure o f i t s temperature) b u t
i t ' s u s u a l l y on a dry i c e ethanol bath. It involves dissolving
the glyceride o r t h e l i p i d mixture i n e i t h e r d i e t h y l ether,heptane,
hexane o r some such solvent, adding concentrates t o sulphuric
a c i d a t about 23 mls o f concentrated sulphuric a c i d t o 20 m l s
o f t h i s p e t e t h e r . I should not say p e t ether you have some
sulphur there; d i e t h y l e t h e r o r heptane. He s t i r s t h i s under
nitrogen f o r 10 o r 15 minutes and then n e u t r a l i z e s with sodium
hydroxide. A t t h i s p o i n t he allows it t o warm a t room temperat u r e s o t h a t t h e s a l t form w i l l s t a y i n s o l u t i o n . It i s e a s i l y
separated i n a separatory funnel and I believe he washes t h e
mixture t h r e e times. I am n o t sure how good a micro procedure
t h i s i s . It i s a very rapid macro procedure and I have used i t
with q u i t e good success. It takes very l i t t l e equipment.
107.
DR. PEARSON: Thank you, Charlie.
A comment from D r . Kuksis .
Any o t h e r questions.
DR. KUKSIS: I would l i k e t o amplify my comments here
about f a t t y a c i d chromatography. I n respect t o the preparation of
f a t t y a c i d methyl e s t e r s , I would l i k e t o c a l l your a t t e n t i o n t o
t h e decision of the American O i l Chemists' Society because they
have not proposed t h a t two percent s u l f u r i c acid-methyl alcohol
should be used f o r t h e methylation of f r e e f a t t y acids o r f o r t h e
transmethylation of f a t t y a c i d e s t e r s . This one i s described i n t h e
January, February o r Wrch i s s u e of 1966 of t h e Journal of t h e
American O i l Chemists' Society. They also give some procedures.
I t ' s s o r t o f a combined e f f o r t of a committee and f i v e o r s i x cooperating l a b o r a t o r i e s t o decide how t h i s should be done. I would
l i k e t o say t h a t i n o u r experience t h e s u l f u r i c a c i d methyl system
has proved t o be f a r superior t o t h e HCL methyl system. You can
over methylate with sulf'uric a c i d q u i t e r e a d i l y without any noticea b l e d e s t r u c t i o n o f f a t t y a c i d s . When one works with very small
q u a n t i t i e s of f a t t y a c i d s o r f a t t y a c i d methyl e s t e r s , it i s of
g r e a t i n t e r e s t w i t h work i n t h i n - l a y e r chromatography, t o take
these bands o f f t h e t h i n l a y e r p l a t e s , one c a n ' t possibly be
swabbing t h e p l a t e with a d d i t i o n a l solvent and then complete the
methylation o r transmethylation i n the presence of the s i l i c a g e l .
This s o r t o f transformation w i l l take place very e f f e c t i v e l y i n t h e
same methylating agent. Say we have been using 10 percent by
weight of s u l f u r i c a c i d methanol, one has t o increase t h e r e a c t i o n
time t o complete t h e methylation maybe two hours i n the absence of
s i l i c a g e l . I n t h e presence of s i l i c a g e l , you may have t o run
overnight and i f you have some possible l i p i d s present l i k e
sphingomyelin you would not g e t a complete y i e l d of t h e f a t t y a c i d
e s t e r unless you increased t h e temperature from 85 degrees t o n;aybe
110 degrees a s Feld and Reiser reported l a s t f a l l .
DR. PEARSON: I would l i k e t o ask M r . S t a l l i n g a question
ooncerning the r e l a t i v e time i n making a n a l y s i s of t h e amino acids
f o r the e s t e r s of t h e amino acids as compared t o t h a t when you u s e
your ion-exchange column.
DR. STALLING: In reference t o the t i m e required f o r
a n a l y s i s o f the amino acids i n t h e procedure we a r e p r e s e n t l y employing, t h e r e i s a half hour involved i n e s t e r i f i c a t i o n and then
two and a half hours f o r i n t e r e s t e r i f i c a t i o n which gives a t o t a l
amount of about t h r e e hours. The a c y l a t i o n can be done i n about
5 o r 15 minutes depending upon the conditions you a r e working w i t h .
The operator o r technician who i s doing the a n a l y s i s can be doing
six o r twelve amino a c i d samples. If he s t a r t s i n t h e morning he
can, on one chromatograph, run e i g h t p r o t e i n hydrolysates i n an
e i g h t hour working day and we f e e l t h i s i s representative. If you
a r e i n t e r e s t e d i n t h e l y s i n e content of a feed o r i f you are int e r e s t e d i n methionine o r c e r t a i n of the amino a c i d s , you can g e t
these analyses w i t h i n 5 minutes estimation t i m e on t h e chromatograph,
which means you can run twenty samples an hour.
108.
DR. PEARSON: Thank you very much. Now I w i l l t u r n t h e
program back t o our chairman. I t h i n k we have used a l l t h e time he
has a l l o t t e d us and I am sure we a l l want t o eat our lunch.
DR. CARPENTER: Thank you, D r . Pearson. For our lunch we w i l l be
a t the Dairy Ear in Stocking Hall. O.K. we a r e adjourned. Thank you.
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