labmanual Physics

8/11/2019 labmanual Physics

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Laboratory Manual for PhysiologicalChemistrySpring 2009

Table of Contents

Handling Emergencies...............................................................................2

Safety Regulations.....................................................................................3

General Laboratory Procedures..................................................................3

ntroduction to !rganic Com"ounds...........................................................3

solation of Chloro"hyll and Carotenoid Pigments from S"inach.................3

Chemical Pro"erties of #li"hatic and #romatic #lcohols.............................3

!$idation and Structure of Carbonyl Com"ounds......................................3

!"tical somers..........................................................................................3

Carbohydrates...........................................................................................3

#cid%&ase Reactions 'ith Carbo$ylic #cids and Esters...............................3

Synthesis of #s"irin...................................................................................3

Synthesis ( "ro"erties of Soa"..................................................................3

solation and Characteri)ation of Casein from Mil*.....................................3

#mylase+ ,he #cti-ity of an En)yme..........................................................3

nteraction of / Light 'ith Matter............................................................3

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Laboratory Rules

and Guidelines

Handling Emergencies

While we will do everything possible to ensure a safe environment,accidents can occur. In case of the following emergencies, always informthe instructor and do the following+

Burns% lush 'ith cool ta" 'ater.

Chemicals in the eye% C#LL !R HELP. orce the eye o"en 4ush 'ith'ater for at least 20 minutes.

Chemicals on the skin% lush 'ith 'ater. Rinse acid s"ill 'ith sodiumhydrogen carbonate solution5 bases 'ith boric acid.

Chemicals on your clothes%REM!/E 6!R CL!,HES. 6ou do not 'antthe chemicals to reach your s*in.

Clothing on re% S,!P%7R!P%R!LL. se a 8re blan*et or sho'er only ifyou are standing 'ithin arm9s reach.

Cut in skin% Rinse immediately 'ith 'ater. ns"ect for glass. Getmedical attention.

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Safety Regulations

1: Acceptable eye protection must be worn at all times;earing contact lensesin the lab is strongly discouraged and may be forbidden by your instructor.

2: Bare feet and sandals are not allowed in the lab S"illed chemicals andbro*en glass on the 4oor can result in serious in nor shoulders may bee$"osed 'hether standing straight reaching or bending o-er.

?: Each student must know the location of the safety e!uipment" 8ree$tinguishers eye 'ashes safety sho'er and the e$it.

@: Horseplay and#or carelessness are prohibitedA: $o unauthori%ed e&periments are to be performedB: ;or* is "ermitted only at the assigned time unless other'ise authori)ed by the

instructor. n any case$E'ER work alone.: Chemicals and eDui"ment are not to be remo-ed from the laboratory.: (o not sit on bench tops.10:Eating) drinking) smoking or chewing anything is not permitted in the

laboratory11:$e*er pipet by mouth5 al'ays use a "i"et bulb.12:Be cautious when testing for odors. #l'ays 'a-e fumes to'ards your nose

'ith your hand. =E/ER smell a chemical directly.13:Always add acid to the water or base ne-er do the o""osite.1?:$e*er aim the opening of a test tube or +ask at yourself or anyone else1@:$e*er lea*e reactions unattendedif they in-ol-e heating or ra"id reactions.1A:!nly the lab manual and lab noteboo* should be at lab counter. Book bags)

outerwear) etc are to be placed out,of,the,wayin the location indication byyour instructor.

1B:Report any in-ury ho'e-er minor to the instructor at once.1:Always use tongs to handle hot ob-ects1:.oose clothingmust not be 'orn in the laboratory. !"en s'eatersFhoodies loose

slee-es e$cessi-ely 4o'y blouses.

20:Hair below chin must be tied back 'hile 'or*ing in the laboratory.21:Broken glass will be disposed of in the glass disposal bo&es not in the

regular trash.22:(ispose of waste properly) as directed by your instructor.23:Clean up all spills immediately2?:Always check glassware for chips and cracks

reali)e that these RLES ;LL &E E=!RCE7 for my safety and for the safety of my lab"artners and that failure to obser-e these rules in addition to resulting in unacce"tablesafety ha)ards and the loss of 'or*ing time 'ill result in e$"ulsion from the laboratory. ac*no'ledge that ha-e recei-ed a 'ritten co"y of these regulations and ha-e beengi-en the o""ortunity to discuss them 'ith the instructor.

=#ME IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIC!RSEIIIIIIISEC,!=IIIII

SG=#,REIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII7#,EIIIIIIIIIIIIIII

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/eneral .aboratory 0rocedures

,he follo'ing "rocedures are intended to "re-ent contamination of chemicals and to"romote safety smooth laboratory o"eration and laboratory eJciency.

Read labels

carefully

Some chemicals ha-e -ery similar names. n the case of acids and bases do

not assume that the reagent bottles are in the correct "laces.

Eyedroppers#0ipets

=e-er use an eye dro""er or "i"ettein a reagent bottle unless there is adro""er 'ith the bottle for use only 'ith that bottle. 6our seemingly cleaneyedro""er may actually be dirty and end u" contaminating an entire bottleof reagent. f you need to dis"ense a chemical 'ith an eyedro""er "our asmall amount into a bea*er and use the eyedro""er from there.

1nused ore&cessreagents

=e-er "ourthembac* into a reagent bottle. Contamination of the contentsmay result. 7is"ose of the e$tra "ro"erly. #long 'ith this do not ta*e largeamounts of reagents since e$cess amounts must be 'asted. 6ou can al'aysget more if you need it.

2btainingsolids

!btain solids using a s"atula s"eci8ed for the "articular reagent or by"ouring the solid out by rotating the bottle bac* and forth until the solid'or*s its 'ay out of the bottle or -ial.

CleaningE!uipment

&e sure your eDui"ment is clean before use and be sure to return borro'edeDui"ment either as clean or cleaner than you found it. ;ash glass'are 'ithsoa" and ta" 'ater. Rinse 'ith ta" 'ater. !ccasionally some chemicals andglass'are mar*ers must be remo-ed 'ith acetone. 7o a 8nal rinse 'ith asmall amount of distilled 'ater.

1singlitmus andother test

papers

#l'ays dolitmus "a"er or "H "a"er tests by "lacing the test "a"er on a clean'atch glass and transferring a dro" of the test solution to the litmus using astirring rod.

'ials and3ars

#l'ays reca" -ials)


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.aboratory$otebooks

# laboratory noteboo* is the most basic "iece of eDui"ment used in the laboratory. tis -ery necessary to de-elo" a "ro"er method of using a noteboo* no matter 'hat scienceone "ursues. t is one of the goals of this laboratory e$"erience to de-elo" good noteboo*techniDue. E-eryone has his or her o'n style of recording data and di>erent instructorsmay demand di>erent things. ,he guidelines described here are to gi-e you an idea of theminimum that is ty"ically acce"table.

6our laboratory noteboo* is for recording data and obser-ations 'hile "erforminge$"eriments. =otes from class discussion may a""ear in the noteboo* but they must beclearly distinguished from e$"erimental data. Ksee belo':

,he "ur"ose of a laboratory noteboo* is to "ro-ide 6! !R #=6!=E ELSE 'ith anaccurate record of 'hat 6! 77 in the laboratory not necessarily 'hat you 'ere su""osedto do. t should contain+1: ,itle of the "ro


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legitimate co"y. 6ou may 'rite in your noteboo* outside of lab as long as you "ro"erlydate the entries and 7! =!, CH#=GE #=6,H=G ,H#, ;#S PRE/!SL6 E=,ERE7.

2. 4rite what you did,his may be di>erent from 'hat you 'ere su""osed to do. f youha-e a record of 'hat you did you may ha-e a 'ay of 8guring out 'hat 'ent 'rong ifsomething does go 'rong.

3. The rst two pages of a notebook should be reser*ed for a Table of Contents)

'hich is continually u"dated. Sometimes noteboo*s already ha-e a table of contentssection in them.

?. The pages must be numberedstarting at 1 for the 8rst "age and continuing on each"age Keach side of the sheet: until the end of the noteboo*. =oteboo* "ages arenumbered


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:ntroduction to

2rganic Compounds/oals for the Student"

Learn to identify organic functional grou"s Learn to classify organic com"ounds based u"on their functional grou"s Learn to name organic com"ounds based u"on their functional grou"s Construct models of al*anes to -ie' three%dimensional structure of al*anes. n-estigate the relationshi" bet'een a structural formula and a three dimensional

molecule using molecular models

Construct models of isomers of al*anes 'ith the same molecular formula.

dentify isomers structural formula condensed structural formula and s*eletalformulas.

:ntroduction

n this e$ercise 'e 'ill be introduced to organic com"ounds. !rganic chemistry is thestudy of com"ounds that are "rimarily com"osed of carbon and hydrogen atoms. !ther"rominent elements in organic chemistry are o$ygen ! nitrogen = sulfur S and thehalogens K4uorine chlorine Cl bromine &r and iodine :. Since all organic com"oundscontain some amount of carbon and hydrogen atoms organic com"ounds are identi8edand classi8ed by the functional grou"s they "ossess. # functional groupis a grou" ofatoms that react in a "redictable 'ay. Com"ounds 'ith the same functional grou" areclassi8ed into a "articular class of organic com"ounds and the name is deri-ed from

belonging to that class.

;unctional /roups

Table 8" 2rganic ;unctional /roups;unctional /roup Class Characteristic E&ample

C C #l*ane !nly carbon%carbonsingle bonds

H3C CH3

C C #l*ene Carbon%Carbon double

bond

H2C CH2

C C #l*yne Carbon%carbon tri"lebond

HC CH

B


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#romatic Si$ atom carbon ring'ith alternating

double and single

bonds

H

H

H

H

H

H

X

X = F, Cl, Br, or I

Haloal*ane!ne or more halogen

atoms

H3C Cl

OH #lcohol Hydro$yl grou" K%!H: H3C OH

O Ether !$ygen atom bonded

to t'o carbon atoms

H3C O CH3

SH ,hiol # SH grou" bondedto a carbon atom

H3C SH

C

O

H

#ldehyde Carbonyl grou"Kcarbon%o$ygen

double bond: 'ith HH3C C

O

H

C

O

Qetone

Carbonyl grou"

bet'een t'o carbonatomsH3C C

O

CH3

C

O

O H

Carbo$ylic#cid

Carbo$yl grou"Kcarbon%o$ygen

double bond and !H:H3C C

O

O H

C

O

O

EsterCarbo$yl grou" 'ith

H re"laced by acarbon

H3C C

O

O CH3

N

#mine=itrogen atom 'ithone or more carbon

grou"sH3C NH2


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C

O

N #mide Carbonyl grou"bonded to a nitrogen

atom

H3C C

O

NH2

$omenclature

=omenclature of organic com"ound is go-erned by the nternational nion of Pure and#""lied Chemistry KP#C: system. ,he system is founded on t'o ma


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C

H3C

H3C

H iso"ro"yl

H3C CH2 CH2 CH2 butyl

C CH2

H3C

H3C

H isobutyl

C

CH3

CH3

H3C tert%butyl Ktbutyl:

X K Cl &r or : 4uoro chloro bromo iodo

H3C CH

Cl

CH2 CH

CH3

CH2 CH3

1 2 3 4 5 6

2%chloro%?%methylhe$ane

H3C C

Cl

CH2 CH

CH3

CH CH3

6 5 4 3 2 1

Cl

Br

2%bromo%@@%dichloro%3%methylhe$ane

= Cycloalkanesa. count the carbon atoms in the ring and add the "re8$ cyclo to straight chain

nameb. substituent rules from abo-e a""ly e$ce"t the 8rst substituent is al'ays

"laced on carbon 1c. al"habeti)e to determine substituent on carbon 1

CH2CH3H3C

1%ethyl%3%methylcyclo"entane

9 Alkenes and Alkynesa. name the longest continuous carbon chain that contains the double or tri"le

bondi. re"lace the aneending of the al*ane 'ith enefor an al*ene and

ynefor an al*yne

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b. number the longest continuous carbon chain from the end nearest thedouble or tri"le bond

c. gi-e the location and name of each substituent as a "re8$ to the al*ene oral*yne name

i. "lace a hy"hen bet'een the number and the substituent nameii. al"habeti)e the substituents

iii. use a "re8$ Kdi% tri% tetra% etc: if a substituent a""ears more thanonce and use commas to se"arate t'o or more numbers.

1 2 3 4 5

H2C CH CH CH

CH3

CH3

?%methyl%2%"entene

HC C CH

1 2 3 4 5

CH

Br

CH3

Cl

3%bromo%3%chloro%1%"entyne

> Aromaticsa. monosubstituted ben)ene rings are named as ben)ene deri-ati-es using the

substituent nameTable 9" Common ?onosubstited Aromatic Compuonds

Structure P#C =ame Common =ame

CH3 methylben)ene toluene

NH2 ben)eneamine aniline

OH hydro$yben)ene "henol

b. disubstituted ben)ene rings are numbered to gi-e the lo'est number to thesubstituents

i. common "re8$es are often used K12 substitution is ortho 13substitution is meta and 1? substitution is "ara:

c. if a ben)ene ring is a substituent Klongest chain is more than si$ carbons orcontains a double or tri"le bond: then it is named a "henyl grou"

so"ro"ylben)ene

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Cl

Cl

12%dichloroben)ene ororthochloroben)ene

H3C CH2 CH CH2 CH2 CH2 CH3

1 2 3 4 5 6 7

3%"henylhe"tane

A. Alcohols @ Thiolsa. name the longest continuous carbon chain containing the hydro$yl grou" K%

!H:i. re"lace the aneending of the al*ane 'ith olending

b. number the longest continuous carbon chain starting at the end closest tothe hydro$yl grou"

c. name and number other substituents relati-e to the hydro$yl grou"d. name a cyclic alcohol as a cycloal*anol 'ith all cycloal*ane rules a""lying forsubstituents

e. a""ly aromatic naming rules for ben)ene rings containing a hydro$yl grou".,he base name is then "henol

f. thiols are named by adding thiolto the al*ane name of the longestcontinuous carbon chain bonded to the SH grou"

i. the location of the SH grou" is indicated by numbering the mainchain from the closest end

1 2 3 4

H3C CH

OH

CH

CH3

CH3

3%methyl%2%butanol

OHI ?%iodo%3%cyclohe$anol

H3C CH

SH

CH2 CH

CH3

CH2 CH3

1 2 3 4 5 6

?%methyl%2%he$anethiol

Ethersa. 'rite the al*ane name of the larger al*yl grou" as the main chain

b. name the o$ygen and smaller al*yl grou" as a substituent called an alo!ygrou"

H2C O CH2H3C CH2 CH3 Etho$y"ro"ane

O CH3 metho$yben)ene

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Aldehydes @ etonesa. for an aldedyde name the longest continuous carbon chain containing the

carbonyl grou" by re"lacing the ein the al*ane name 'ith ali. name and number any substituents on the carbon chain by counting

the carbonyl carbon as carbon 1b. for a *etone name the longest continuous carbon chain containing the

carbonyl grou" by re"lacing the ein the al*ane name 'ith onei. number the main chain starting from the end nearest the carbonylgrou"

ii. name and number any substituents on the carbon chain

H3C CH2 CH2 C H

O

4 3 2 1

butanal

H2C CH CH2 C H

O

5 4 3 2 1

H3C

CH3

3%methyl"entanal

H2C C CH2 CH3

5 4 3 2 1

H3C

O

3%"entanone

H2C CH2

6 5 4 3 2 1

H3C C

O

CH

Cl

CH32%chloro%3%he$anone

D Carbo&ylic Acidsa. name the longest continuous carbon chain containing the carbonyl grou"

and re"lace eof the al*ane name 'ith oic acidb. number the carbon chain beginning 'ith the carbo$yl grou" as carbon 1c. gi-e the location and names of substituents on the main chaind. for the aromatic ben)oic acid number the ring from the carbo$yl grou" as

carbon 1

H2C CH2H3C C

O

OH

4 3 2 1

butanoic acid

C CH2H2C C

O

OHH3C

CH3

CH3

5 4 3 2 1

33%dimethyl"entanoic acid

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8Estersa. 'rite the name of the carbon chain from the alcohol as an alyl groupb. 'rite the name of the carbo$ylic grou" as carbo!ylate'ith an oateending

H3C CH2 O C

O

CH2 CH2 CH3

ethyl "ro"anoate

H3C O C

O

methyl ben)oate

88Amines @ Amidesa. for amines name the longest continuous carbon chain bonded to the

nitrogen atom and re"lace the ein the al*ane name 'ith aminei. number the carbon chain to sho' the "osition of the amine grou" and

any other substituentsii. in secondary and tertiary amines use the "re8$ "#to name smaller

al*yl grou" attached to the = atom.

b. amides are named by dro""ing the oic acidfrom the carbo$ylic acid nameand adding amide

H3C CH2 CH2 NH2 Pro"anamine

H3C CH2 CH2 NH

CH3=%methyl "ro"anamine

H2C CH2H3C C

O

NH2

butanamide

H2C CH2H3C C

O

N

CH3

CH3

==%dimethyl butanamide

#lso in this e$ercise 'e 'ill study the three dimensional structure of some al*anesusing a molecular model *it to reinforce the nomenclature for al*anes and some of theirTsderi-ati-es. n each ty"e of al*ane each carbon has four -alence electrons and mustal'ays ha-e four single bonds to other carbon hydrogen or halogen atoms. ,he bondarrangement of four single bonds used by carbon in al*ane is sho'n as belo'.

&onding Pattern ofCarbon

#rrangement of &onds aroundCarbon

S"atial Structure and &ond#ngles

C

,etrahedral

1?
http://www.windows.ucar.edu/tour/link=/teacher_resources/images/tetrahedron_big_jpg_image.html


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,o understand the three dimensional structure of organic com"ounds models can bebuild using a ball and stic* model *it. n this *it there are colored s"heres 'hich re"resentthe atoms drilled to recei-e connecting bonds. 7i>erent color s"heres blac* for carbonand red for o$ygen are used to re"resents di>erent *inds of atoms and a color code foratoms 'ill be included in the model *it. Each of the s"heres Katoms: has the correctnumber of holes for bonds K'ooden or "lastic stic*: that attach to other s"heres.

a: Kb: (c)

,he three dimensional structure of the al*ane models re"resents -ery closely resemblesthe a""ro$imate geometryKsha"e and angle: of the molecules they re"resent. ,'ostructures are identical if they are superimposable%that is if one structure can be "laceon to" of another so that all colored s"heres coincide. Methane is the 8rst member ofal*anes and three di>erent structure of methane is sho'n abo-e 'hich re"resents thestructural formula Ka: three dimensional structures Kb: and ball and stic* model Kc:.

Com"ounds ha-ing the same molecular formula can be re"resented by more than onestructure and each structure includes the same grou" of atoms but a di>erent s"atialarrangement of the atoms. ,hese com"ounds are called isomers. somers ha-e the samemolecular formula but di>erent three dimentional structures. !ne structure cannot becon-erted to the other 'ithout brea*ing and forming ne' bonds.

,he isomers ha-e di>erent "hysical and chemical "ro"erties. !ne of the reasons for the-ast array of organic com"ounds is the "henomenon of isomerism. Many biologicalreactions are -ery s"eci8c and in-ol-e only one isomer.

somers of C?H10

CH3 CH2 CH2 CH3 CH3 CH CH3

CH3

somers of C2HA!

H C C

H

H

H

H

O H

C O C

H H

HH

H H

n%butane 2%methyl"ro"ane

ethyl alcohol dimethyl ether

E&perimental

,his is a t'o 'ee* e$ercise. n the 8rst 'ee* you should com"lete and turn into yourinstructor the nomenclature re"ort sheet. 7uring the second 'ee* you 'ill "erform thestructure "ortion of the e$"eriment com"leting and turning in the second re"ort sheet.

1@


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,he model *it includes di>erent colored s"heres re"resenting di>erent atoms and greyconnectors for re"resenting bonds. Carbon atoms are blac* s"heres and ha-e four holesthat re"resent the four co-alent bonds that carbon atoms al'ays form. Hydrogen atomsare 'hite s"heres and only form one bond. ,he green s"here re"resents chlorine atom ando$ygen atoms are red s"here. Halogen atoms form one bond and o$ygen atoms form t'obonds5 s"heres for these atoms 'ill ha-e the a""ro"riate number of holes.

Single co-alent bonds are re"resented by grey connectors 'hich insert into holes ofthe atoms. ,o conser-e time and de"ending u"on the number of "ieces in your model *ityou may use only the stic* to re"resent the C% H bonding arrangement.

6ou 'ill be 'or*ing in grou"sKt'oFthree: to construct the models of di>erentcom"oundsKal*ane haloal*ane haloalcohol: using the model *its. Each model must bein-estigated for geometry Ksha"e ( angle:

1A


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$age intentionally blan.

1B


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RE02RT SHEET,:ntroduction to 2rganic Compounds 64eek87

$ameFFFFFFFFFFFFFFFFFFFFFFFFFFFF 0artnerGname FFFFFFFFFFFFFFFFFFFFFFFFF

SectionFFFFFFFFFFFFFF (ateFFFFFFFFF

$ame the following compounds

: Alkanes

Br

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

:: Alkenes # Alkynes

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

::: Aromatics


1


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:' Alcohols @ Thiols

OH

HS


' Ethers

O

O


': Aldehydes @ etones

H

O

Br

O


':: Carbo&ylic Acids

HO

O

HO

O I

I I


'::: Esters

1


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O

O O

O


: Amines @ Amides

NH2 N

O


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RE02RT SHEET I :ntroduction to 2rganic Compounds64eek


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a: ;hat is the geometry associated 'ith this structure N IIIIIIIIIIIIIIIIIIIIIIIIIIII

b: ;hat is the -alue of the HCH bond anglesN IIIIIIIIIIIIIIIIIIIIIIIIIIIII

:: Construct a model of chloromethane) CH=Cl

a: 7ra' a 'edge and hash mar* to re"resent the three dimensional sha"e of the molecule.

b: s the geometry the same as methaneN IIIIIIIIIIIIIIIIIII

c: #re the hydrogen atoms eDui-alent Ki.e. do they ha-e identical en-ironments 'ith res"ect to theother atoms ad


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b: #re the hydrogen atoms in CH3CH2Cl eDui-alentN IIIIIIIIIIIIIIIIIIIII

': Construct all possible models for dichloroethanes) C


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: Construct all possible models for C9H8 KHint+ straight -ersus branchedchain:

a: Ho' many structural isomers e$ist for C?H10N IIIIIIIIIIIIIIIIIIIIIIII

b: 7ra' condensed structural formulas for each structural isomers of C?H10. #lso usingsDuares triangles and For circles indicate the carbon atoms that are eDui-alent to eachother

2?


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1sing your C9H8 models from abo*e) remo*e one hydrogen atomand replace it with a chlorine atom to make diKerent structuralisomers of C9HDCl

a: Ho' many structural isomers e$ist for C?HCl.N IIIIIIIIIIIIIIIIIIIIIIIb: 7ra' condensed structural formulas for each structural isomer of C?HCl.

: (raw line,bond formulas for all possible structural isomers ofC9HCl


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:solation ofChlorophyll and

Carotenoid0igments from

Spinach

#da"ted from+ Pa-ia 7. L.5 Lam"man G. M.5 Qri) G. S.5 Engel R. G. Introduction to %rganic&aboratory 'echni(ues)# Microscale #""roach 3rdEdition Saunders College Publishing+ =e'6or* =6 1 and also uach H. ,.5 Stee"er R. L..5 GriJn G. ;.*. +hem. duc. 200? 13@%3B. ,oni &ell K200?:

/oals for the Student" Learn the techniDues of e$traction and "uri8cation of chemical com"ounds from

natural "roducts. Learn the techniDue of identi8cation of di>erent com"onents in chemical

com"ounds isolated fromnatural "roducts

:ntroduction

S"inach a green leafy -egetable usually can be gro'n as a s"ring and fall cro" inthe cooler =orth #merican climate. S"inach is a source of /itamin # and it is rich in ironand calcium. ,he lea-es contain a number of colored "igments generally falling into t'ocategories+ chlorophyllsand carotenoids.

Carotenoids are "art of a larger collection of "lant deri-ed com"ounds calledterpenes. ,hese naturally occurring com"ounds contain 10 1@ 20 2@ 30 and ?0 carbonatoms. :sopreneis the basic 8-e%carbon building bloc* of the ter"ene class of biologicalcom"ounds. #lso *no'n as 2%methyl%13% butadiene these units are lin*ed in a head to

tail fashion to build the structure of ter"enes. ,'o iso"renes are lin*ed together to ma*eone terpene unit. ,he branched end is the head and the unbranched end is the tail.Carotenoids are tetrater"enes Keight iso"rene units:.

S"inach lea-es contain chlorophyll a and band L,caroteneas 'ell smalleramounts of other "igments such as &anthophylls'hich are o$idi)ed -ersions of carotenesand pheophytins'hich loo* li*e chloro"hyll e$ce"t that the magnesium ion MgU2hasbeen re"laced by t'o hydrogen ions HU. Chloro"hylls a and b are the "igments that ma*e"lants loo* green. ,he double bonds are con-ugated meaning they occur bet'een e-ery

2A

Isoprene is the basic -ve#carbonbuilding bloc of the terpene classof biological compounds.head end

tail end


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other "air of carbons and allo' ca"ture the Knongreen: light energy used in"hotosynthesis.

V%Carotene is a carotenoidand it causes carrots and a"ricots to be orange. ;heningested V%carotene is clea-ed to form t'o molecules of /itamin #. /itamin # also calledretinol "lays an im"ortant role in -ision and ser-es as an anti,o&idant.

2B

+hlorophyll a left/ andchlorophyll b right/ arevery similar molecules. +an

you spot the dierences1'hese small changes areenough to change theircolor.

#+arotene top/, #

carotene middle/, and!anthophylls e!ampleon bottom/ have verysimilar structures. +an

you spot thedierences1 'hesesmall changes areenough to change theircolor. our body can

only use #carotene to

mae 3itamin 4. Why1


29/104

n this e$"eriment 'e 'ill isolate and use di>erences in "olarity of the "igments toe>ect a se"aration. Chloro"hylls and carotenoids are slightly di>erent in "olarity. 7ue totheir lo-ely color 'e 'ill easily follo' the se"aration -isually. V%Carotene is a hydrocarbonand it is -ery non"olar. &oth chloro"hylls contain C! and C= bonds 'hich are "olar

and also contain magnesium bonded to nitrogen 'hich is such "olar bond that it is almostionic. &oth chloro"hylls are much more "olar than V%Carotene. ,here is another structuraldi>erence in bet'een the chloro"hyll a and b Chloro"hyll a has a methyl grou" KCH3: in a"osition 'here chloro"hyll b has an aldehyde grou" KCH!:. ,his ma*es chloro"hyll bslightly more "olar than chloro"hyll a.

Since s"inach also contains cellulose iron and 'ater soluble -itamins in addition tochloro"hylls and carotenes 'e ha-e to ha-e a method of se"arating all these com"ounds.,he most common a""roach to isolating these bioacti-e natural "roducts is e&traction.Chloro"hylls and carotenes are relati-ely non,polarorganic substances com"ared to othercom"onents5 hence they are more soluble in organic sol-ents li*e dichloromethane oracetone. Since Wli*e dissol-es li*eT these sol-ents 'ill be suitable to selecti-ely e$tractthese com"ounds into organic sol-ent and lea-e the other com"ounds behind. ,o do thee$traction you 'ill 8rst grind u" the s"inach in a little bit of acetone. ,he green acetone'ith s"inach com"ounds is called e&tract. nfortunately acetone 'ill dissol-e almostanything including the stu> that you do not 'ant.

Chloro"hylls and carotenes do not dissol-e -ery 'ell in 'ater5 they dissol-e li*ecra)y in he$ane. ,he other com"ounds do not dissol-e 'ell in he$ane but dissol-e 'ell in'ater. Li*e 'ater and oil 'ater and he$ane are immiscible5 they Wdo not mi$T. ,hehe$ane 'ill form a layer on to" of the 'ater Kli*e oil does: because he$ane is less densethan 'ater. #fter -igorous sha*ing to mi$ the layers tem"orarily you 'ill allo' them tose"arate. ,he lo-ely green chloro"hylls and yello' carotenes 'ill lea-e the 'ater at thebottom to dissol-e in the he$ane layer at the to". #fter "i"etting%o> he$ane layer thedi>erent com"onents in the "igment mi$ture 'ill be analy)ed by using thin layerchromatogra"hy.

,he number of com"ounds in the he$ane e$tract can be Duic*ly determined by atechniDue called thin layer chromatography 'hich is abbre-iated T.C. 6ou 'ill "ut alittle s"ot of your e$tract on a "lastic "late coated 'ith silica gel. Silica gel is a -ery "olarsubstance. ,he "lates are "laced in a container 'ith a mi$ture of sol-ents. n this case thesol-ents 'ill be Duite non%"olar. ,he sol-ents 'ill begin to tra-el u" the "late li*e a 'ic*.Some of the com"ounds in the he$ane e$tract 'ill be more "olar and 'ill stic* to the s"oton the silica. !ther com"ounds in the he$ane e$tract 'ill be less%"olar to di>ering degreesand 'ill tra-el u" 'ith the sol-ent Since there are many le-els bet'een totally "olar andtotally non%"olar the com"ounds can be se"arated by "olarity. ,he more aJnity acom"ound has for the sol-ent the farther u" the "late it 'ill tra-el.

7i>erent com"ounds should rise to di>erent heights on your ,LC "late5 ho'e-er thee$act height a "articular com"ound rises de"ends on ho' high the sol-ent is allo'ed to

rise u" the "late. f the sol-ent tra-els higher then the s"ots all tra-el higher too. ,ocorrect for this di>erence and generate a number 'hich can be com"ared to re"orted-alues or to other indi-idualTs 'or* the retention fractionor Rf.-alue is calculated. ,heretention fraction is de8ned to be the fractional rise of the s"ot com"ared to the rise of thesol-ent. ,he Rf-alue for a com"ound 'ill change if a di>erent de-elo"ing sol-ent or adi>erent ty"e of "late is used. #fter you ha-e de-elo"ed your ,LC "late 'ith your he$anee$tract you ha-e to calculate Rf -alues for each s"ot on your "late. S"ots 'ith the same Rf-alues 'ithin e$"erimental error and the same a""earance should be the same com"ound.

2


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#n e$am"le if you had


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can you do this if you donTt *no'N:. Ma*e a labeled s*etch of the tube contentsand colors in your noteboo*.

11. Remo-e the 'ater layer 'ith a Pasteur "i"ette and transfer it to a small bea*erlabeled 'aste.

12. #dd another 2.0 mL of distilled 'ater to the he$ane layer in the centrifuge tube asa 'ash. Ca" and sha*e the mi$ture. &e sure to -ent the tube occasionally by

pointing away from you and othersand loosening the ca". #llo' the tube tostand for a fe' minutes so the layers may se"arate. Remo-e the 'ater layer 'itha Pasteur "i"ette and transfer it to a small bea*er labeled 'aste.

13. #lthough 'ater and he$ane Wdo not mi$TXin reality a little bit of 'ater 'ill stay inthe he$ane. 6ou can tell there is 'ater in the he$ane layer if it is a little cloudy.6ou must dryKremo-e 'ater from: the he$ane layer by adding a drying agentcalled anhydrous sodium sulfate K=a2S!?:. our instructor will show you how touse a drying agent. # cou"le of micros"atula scoo"s is usually suJcient.

1?. #llo' the drying agent to settle and then transfer the he$ane to a small -ial 'ith aca". Label the -ial and then "roceed 'ith thin layer chromatogra"hy.

,hin Layer Chromatogra"hy+

1.Pre"are the T.C chamberby "lacing one half of a 8lter "a"er into the X in 'hich case you 'illto get a ne' ,LC "late. sing a ca"illary tube ma*e a s"ot of your e$tract on the"encil line. 6ou may ha-e to let the s"ot to dry and then s"ot it again if it isnTt dar*enough carefully "lace the s"otted "late into the chamber and re"lace the lid.

MM $ote" if your spot goes under the sol*ent) it will not tra*el up the plate0repare a new plate if this happensMM

3. 6ou 'ill immediately see the sol-ent start to tra-el u" the "late. ,he line of sol-entmo-ing u" is called the Wsol-ent frontT. !nce the sol-ent front is roughly 1 cm of theto" of the "late remo-e the "late and Duic*ly mar* the sol-ent front 'ith a "encil.

?. #lthough most of the s"ots are easily -isuali)ed by the na*ed eye use the / lam"to insure that you are noting all "ossible s"ots. Lightly circle each s"ot in "encil.Lightly label each s"ot K# & C etc.:.

@. 7etermine Rf-alues for all of your s"ots. ,his 'ill gi-e you Duantitati-e -alues forcom"arison. Measure the distance from the starting line to the sol-ent front. ,henmeasure to the center of each s"ot. 7i-ide the center s"ot distance by the sol-entfront distance5 this is the Rf-alue. ,he higher the Rf-alue the less "olar thecom"ound

A. ,ry to match them to the com"ounds sho'n belo' Klisted in order of decreasing Rf-alues:+

30


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Carotenes K1%2 yello'%orange s"ots:Pheo"hytin # Kgray intense:Pheo"hytin & K gray may only be -isible under /:Chloro"hyll # Kblue%green intense:Chloro"hyll & Kgreen:

antho"hylls Kas many 3 yello' s"ots:


31


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RE02RT SHEET,:solation of 0igments from Spinach

$ameFFFFFFFFFFFFFFFFFFFFFFFFFFFF 0artnerGs name FFFFFFFFFFFFFFFFFFFFFFFFFF


1. Mass of s"inach IIIIIIIIIIIIIIIIII

2. ,o the right s*etch the layers in centrifuge tubeand clearly label the 'ater and he$ane layers.

3. ;hy must you occasionally -ent the tube during sha*ingN

? 7ra' a s*etch of your ,LC "late labeling the s"ots. ,hen 8ll in the table

32

S"ot Rf

Probable dentity

,LC "late


34/104

@. ;ere there any s"ots your that does not match 'ith the "robable com"oundsN

A. ;hich com"ound is the most "olarN ;hich one is least "olarN E$"lain the reason foryour ans'er.

B. ;hat do you thin* 'ould ha""en if you used in* to mar* the s"otting lineN

33


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Chemical0roperties of

Aliphatic andAromatic Alcohols

/oals for the Student" Learn to identify the -isible obser-ations in a chemical reaction Learn the di>erences in reacti-ity of 1o 2o 3oalcohols 'ith strong o$idi)ing reagent Learn the di>erences in reacti-ity of ali"hatic and aromatic alcohol Learn the chemical reactions in-ol-e con-erting one functional grou" into another

:ntroduction

Subclass General formula E$am"les

Primary

C OH

H

H

R

2-methylpropan-1-ol

Secondary

C OH

H

R'

R

butan-2-ol

,ertiary

C OH

R''

R'

R

2-methylpropan-2-ol

Phenol

C

C

C

CC

CH

H

H

H

H

OH

phenol

Alcohols aldehydes and ketones are three -ery im"ortant classes of o$ygencontaining organic com"ounds. #lcohols are classi8ed into primaryK1o: secondaryK2o:

3?


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and tertiaryK3o: according to the "resence of substituents in the carbon containing thehydro&yl grou". 0henolis a class of aromaticcom"ounds containing a hydro$yl grou"attached to a ben%enering. ,hree subclasses of alcohols and "henol are sho'n on the"receding "age.

n 8rst "art of this e$"eriment you 'ill learn the di>erence bet'een 1 o 2o and 3oalcohols and aromatic alcohols 'ith res"ect to their reacti-ity 'ith the strong o$idi)ing

reagent sodium dichromate. ,his can be easily demonstrated by noting a color change'hen the Cr in the UA o$idation state of the orange colored dichromate ion Cr2!B2% is

reduced to the green colored chromium K: Cr3U ion. Simultaneously an a""ro"riatealcohol is o$idi)ed to either an aldehydeKand subseDuently to a carbo&ylic acid: or aketone. !f course if there is a no redo$ reaction there 'ill be no obser-ed color change.

n general the follo'ing unbalanced reaction describes the redo$ reaction

#lcohol U Cr2!B2% Carbo$ylic acid Kor *etone:U Cr3U

Kcolorless: Korange: Kcolorless: Kgreen:

or the remaining "arts of the e$"eriment the chemical "ro"erties of ali"hatic alcohols 'illbe e$amined. Here 'e 'ill com"are the solubility acidFbase "ro"erties and the reacti-ity'ith iron K: chloride Kferric chloride: of a similar si)ed ali"hatic alcohol 'ith that of thearomatic alcohol K"henol:.

E&perimental

!$idation 'ith #cidic 7ichromate

1. !btain Y @ mL of acidic dichromate solution K"re"ared "re-iously by mi$ing 3 mL ofa @Z sodium dichromate and 1 mL of concentrated sulfuric acid: and "lace Y 1 mLKY20 dro"s: into four se"arate clean dry small test tubes and note the color.Caution" the dichromate solution can potentially burn your skin or make

holes in your clothes f you s"ill any of thisreagents re"ort the s"ill immediatelyto your instructor so that it may be cleaned u" in an a""ro"riate manner.

2. ,o these"arate test tubesadd Y 0.@ mL KY10 dro"s: of ethyl alcohol iso"ro"ylalcohol t%butyl alcohol or aDueous "henol. Mi$ by 8nger 4ic*ing the test tubes.

3. !bser-e and record the colors of the resultant solutions. int) 4 table of resultsmay help you -nd things (uicly during a lab (ui:.

?. 7is"ose of the solutions in the s"ecial 'aste container for dichromate 'aste.

Solubility

1. sing a s"atula or force"s "lace a "ea si)ed amount of solid "henol crystals into

t'o se"arate clean dry small test tubes.2. ,o one add 1 mL KY20 dro"s: of distilled 'ater and to the other add 1 mL KY20dro"s: of 3M =a!H Ksodium hydro$ide:. S'irl the test tubes eDually and note therelati-e s"eed 'ith 'hich the crystals dissol-e. Record your obser-ations.

3. Re"eat this "rocedure using 1 mL K20 dro"s: of cyclo"entanol instead of the "henol.n this case you are adding a liDuid to a liDuid thus solubility is noted by a singlehomogenous solution and insolubility by t'o layers.

?. Record your obser-ation and dis"ose of these solutions as indicated by yourinstructor.

3@


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38/104


3B


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RE02RT SHEET,Chemical 0roperties of Alcohols



!$idation 'ith #cidic 7ichromate

1. a. 7ra' condensed structural formulas of ethyl alcohol iso"ro"yl alcohol t%butylalcohol and "henol.

b. Classify each of the "receding alcohols as 1[ 2[ 3[ or aromatic.

2. ;hat is the function of the acid solution of sodium dichromateN

3. a. ;hat did you obser-e 'hen the sodium dichromate solution 'as added to ethylalcoholN

b. ;hat did you obser-e 'hen sodium dichromate solution 'as added to iso"ro"ylalcoholN

c. ;hat did you obser-e 'hen the sodium dichromate solution 'as added to t%butylalcoholN

3


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d. ;hat did you obser-e 'hen the sodium dichromate solution 'as added to the"henol solutionN

?. 7ra' condensed structural formulas for the organic "roducts of the abo-e reactionsthat occur. f no reaction occurs 'rite =R.

Solubility

1. a. 7id the "henol crystals dissol-e better in 'ater or in the =a!H solutionN

b. 7id the cyclo"entanol dissol-e better in 'ater or in the =a!H solutionN

c. &ased on the di>erent beha-iors of "henol and cyclo"entanol 'hatgenerali)ations can you ma*e about the solubility of similar si)ed ali"hatic andaromatic alcoholsN

d. ;rite a full chemical eDuation for the chemical reaction that occurred 'hen =a!H'as added to "henol.

e. ;rite a net ionic eDuation for chemical reaction that occurred 'hen =a!H 'asadded to "henol.

3


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#cidF&ase Pro"erties

1. &ased on the ni-ersal ndicator Color Chart 'hat is the a""ro$imate "H of+

a. ,he distilled 'aterN

b. ,he ethyl alcoholN

c. ,he aDueous "henolN

2. ;rite a full chemical eDuation for the chemical reaction that occurs 'hen "henol is"laced in 'ater that e$"lains the obser-ed "H. s "henol an acid or a baseN

Reacti-ity 'ith ron K: chloride KeCl3:

1. Can ferric chloride be used to distinguish aromatic alcohols from ali"hatic alcoholsNE$"lain.

?0


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?1


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2&idation andStructure of

CarbonylCompounds

/oals for the Student" Learn about the families of re"resentati-e carbonyl com"ounds Study the beha-ior of re"resentati-e carbonyl com"ounds to'ard o$idi)ing agents Learn about the di>erent functional grou"s in carbonyl com"ounds and in o$ygen

containing organic com"ounds

:ntroduction

n organic chemistry a carbonyl grou" is a functional grou" com"osed of a carbonatom double bonded to an o$ygen atom+ C!. ,here are se-eral ty"es of carbonylcom"ounds de"ending u"on 'hat is attached to the carbon atom in C !. ,he aldehyde

grou" is often 'ritten as CH! the *etone grou" is 'ritten asC O

and the carbo&ylicacidgrou" is 'ritten as C!!H estergrou" 'ritten asC!!R and the amidegrou"'ritten asC!=H2. # carbonyl grou" characteri)es the follo'ing ty"es of commoncom"ounds 'here C! denotes a C ! carbonyl grou".

Compound Aldehyde Ketoe C!r"o#yl$% !%$d &'ter A($de

Structure

General

formula)CHO )CO)* )COOH )COO)* )CONH)*

,he aldehyde grou" occurs in molecules of most sugars li*e glucose. ,he *etonegrou" is occurs also in one common sugar fructose. ,he amide grou" occurs in all aminoacids the building bloc* of "rotein.

?2
http://en.wikipedia.org/wiki/Aldehydehttp://en.wikipedia.org/wiki/Ketonehttp://en.wikipedia.org/wiki/Carboxylic_acidhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Amidehttp://en.wikipedia.org/wiki/Image:Amide-general.pnghttp://en.wikipedia.org/wiki/Image:Ester.pnghttp://en.wikipedia.org/wiki/Image:Carboxylic-acid.pnghttp://en.wikipedia.org/wiki/Image:Ketone-general.pnghttp://en.wikipedia.org/wiki/Image:Aldehyde2.pnghttp://en.wikipedia.org/wiki/Image:Carbonyl-general.pnghttp://en.wikipedia.org/wiki/Aldehydehttp://en.wikipedia.org/wiki/Ketonehttp://en.wikipedia.org/wiki/Carboxylic_acidhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Amide


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Carbonyl com"ounds are -ery reacti-e due to the di>erence in electronegati-itybet'een the carbon and the o$ygen atom. !$ygen is more electronegati-e than carbonand thus "ulls electron density a'ay from carbon to increase the bondTs "olarity. ,heo$ygen is said to carry a partial negati*e chargeor delta minus and 'ill be attractedto "ositi-e s"ecies in solution5 for e$am"le a "roton in an acidi8ed solution or the carbon of

another carbonyl. ,he o$ygen is a nucleophile. ,he carbon is said to carry a partialpositi*e chargeor delta "lus and 'ill be attracted to negati-e s"ecies in solution5 fore$am"le the o$ygen of an alcohol or 'ater. ,herefore the carbonyl carbon is anelectrophile.

n this e$"eriment 'e are going to loo* at both the structure and the o$idation ofsome carbonyl com"ounds. n the 8rst "art the action of mild o$idi)ing agent 'ill bee$amined. n the ,ollenTs ,est 'e 'ill use an o$idi)ing agent called the ,ollenTsReagent. ,ollenTs Reagent contains sil-er diamine ion \#gK=H3:2]U'hich can o$idi)esome categories of carbonyl%containg com"ounds to carbo$ylic acids. ,he #gUisreduced to metallic sil-er "roducing a sil-er mirror on the glass'are. ,he ,ollenTs testreaction is sho'n in the follo'ing generic e$am"le+

RCH!KaD:U 2#gK=H3:2]UKaD:U 3!H%KaD: RC!!%KaD:U ?=H3KaD:U 2#gKs:U 2H2!

n the second "art of the e$"eriment ball and stic* models of o$ygen containingcom"ounds including carbonyl com"ounds distributed in the laboratory 'ill bee$amined. rom the models you 'ill determine 'hat functional grou" family Kalcoholsethers aldehydes *etones hemiacetals or acetals: it belongs to and dra' its structure.

E&perimental

,ollenTs ,est

1. !btain four small test tubes and clean thoroughly 'ith detergent and a brush. Rinse'ell 'ith ta" 'ater and 8nally 'ith distilled 'ater5 sha*e out e$cess 'ater. ,o each add1 mL K20 dro"s: of @Z sil-er nitrate solution and 1 dro" of 3M =a!H mi$ 'ell.

2. ,o each tube add 2Z ammonium hydro$ide dro" by dro" until the grey sil-er o$ideK#g2!:


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?. Mi$ the contents of each tube and "lace them into a hot 'ater bath. #fter se-eralminutes remo-e the tubes and record your obser-ations.

@. Pour the contents of the test tubes into a 'aste container designated by your instructor.

Clean the tubes 'ith detergent and a brush. ,o remo-e any sil-er adhering to the testtubes add small of concentrated nitric acid Kcaution conc. H=!3can burn s*in and

clothes:. #dd contents to the 'aste container and clean the tubes.

Structures of Carbonyl Com"ounds

Some ball and stic* models of carbonyl com"ounds belonging to the families of aldehydes*etones carbo$ylic acids ester and other o$ygen containing com"ounds such as alcoholacetal hemiacetal and ether 'ill be distributed in this e$"eriment. or each model dra' aline%bond formula and gi-e its functional grou" name Ki.e. alcohol *etone hemiacetaletc.:. sing your results from the 8rst "art of the e$"eriment "redict if the com"ound'ould react in the ,ollenTs ,est.

,he im"ortant "ieces are+

,he white spherere"resents hydrogen atom ,he black sphere'ith four holes re"resents carbon atom ,he red spherere"resents o$ygen atom ,he gray sticksare for connecting carbon atoms to one another and to connect the

carbon atoms to hydrogen and o$ygen atoms # stic* Kbond: attached to a carbon atom or an o$ygen atom and not connected to

anything else 'ill re"resent the CH or !H bonding arrangement.

??


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?@


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RE02RT SHEET,2&idation and Structure of CarbonylCompounds



,ollenTs ,est

1. Record your obser-ations for the ,ollenTs test+

Glucose+

ormaldehyde+

#cetone+

so"ro"yl alcohol+

2. 7ra' the structures and gi-e the names of the com"ounds that ga-e a "ositi-e,ollenTs ,est.

3. Circle the functional grou" in the abo-e structures that is res"onsible for the"ositi-e ,ollenTs test.

?A


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?. ;hat is the name of this functional grou"N

Structur

e O

Line%bond ormula unctional

Grou"Ks:

Should React in

,ollenTs ,estN1

2

3

?

@

A

?B


49/104

B

10

?


50/104


?


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/oals for the Student" ,o in-estigate the use of three dimensional structures to identify di>erent ty"es of

isomers

,o learn about stereoisomers and o"tical isomers

:ntroduction

Stereoisomersare isomers that ha-e same molecular and structural formulas butdi>erent s"atial arrangement. !"tical isomerism is one form of stereoisomerism. 2pticalisomersare named li*e this because of their e>ect on "lane "olari)ed light.

#ll o"tical isomers contain a carbon atom erent grou"s. ,he carbonatom of this isomers are called asymmetric carbon atom or chiral center and themolecule is called chiral.!nly chiral molecules ha-e o"tical isomers. Some e$am"les of o"tical isomers are sho'nbelo'. ,he chiral center is mar*ed 'ith a star.

&utane%2%ol

2-Hydroxy propionic acid

Or Lactic acid

2-Aminopropionic acid

or Alanine

# carbon atom 'ith the four di>erent grou"s attached 'hich causes this lac* ofsymmetry is described as a chiral centeror as an asymmetric carbon atom. f youcannot 8nd a "lane of symmetry the molecule is chiral. Practice on the molecules on the

ne$t "age. 7%alanine in a 'edge%and%dash formula belo'%right does not ha*e a planeof symmetry and is chiral. Glycine on the left belo' has a plane of symmetry andis thus achiral.

@0

2ptical :somers


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Glycine%"lane ofsymmetry

#lanine%no "lane ofsymmetry

Chemist de-elo"ed methods to facilitate the -isuali)ation of 3%dimensional s"atialarrangements of atoms or grou"s of atoms in a 2%dimensional en-ironment i.e. the "laneof this "a"er. ,he most common method for "resenting 3%dimensional structures in a 2%dimensional "lane is the ;ischer pro-ection. ischer "ro


53/104

can be identi8ed by noting that itTs ischer "ro


54/104

::: &uild the structure of all stereoisomers for#s"aragine.

a: 7ra' ischer "ro


55/104

RE02RT SHEET,2ptical :somers



: 7ra' and label the ischer "ro


56/104

b: Ho' many total stereoisomers are thereN IIIIIIIIIIIIIIIIIIIIIIIII

c: ;hat 'ould be general formula for determining the ma$imum number ofstereoisomers 'hen n is the number of chiral center

:' 7ra' and label the ischer "ro


57/104

a: Ho' many chiral centers are thereN IIIIIIIIIIIIIIIIIIIIIIIIIII

b: Ho' many total stereoisomers are "ossible based on general formula for isomersNIIIIIIIIIIIII

c: Sho' your ischer "ro


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@B


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the diagram abo-e.

f at least one of the anomeric carbons has a hydro$yl grou" directly attached it canre-erse the cycli)ation "rocess and form the linear aldehyde or *etone again. n the linearaldehyde or *etone form the molecule can "artici"ate in any aldehyde or *etone reaction.


61/104

3. Place all the test tubes at the same time into the boiling 'ater bath.

?. =ote and record the ho' long it ta*es for the red Cu2! "reci"itate to form5 also note ifthe blue &enedictTs reagent color disa""ears.

@. #fter 10 minutes remo-e all the tubes and *ee" the boiling 'ater bath going for theremaining three e$"eriments. 7id any sugars not "roduce the red "reci"itateN ;hichare reducing sugarN ;hich are notN

&arfoedTs ,est+

1. se the boiling 'ater bath from before and label a ne' set of clean small test tubes.

2. n se"arate test tubes add 1 mL of the &arfoedTs reagent. ,o each test tube add 10dro"s of the test carbohydrate solution. Mi$ the sam"les.

3. Place all of the test tubes at the same time into the boiling 'ater bath.

?. =ote and record the long it ta*es for the red Cu2! "reci"itate to form.

@. #fter 10 minutes remo-e all the tubes. 7etermine 'hich are monosaccharides 'hichare disaccharides

Seli'ano>Ts ,est+

1. se the boiling 'ater bath from before and label a ne' set of clean small test tubes.

2. n se"arate test tubes add Seli'ano>TTs reagent. ,o each test tube add 3 dro"s of thetest carbohydrate solution. Mi$ the sam"les

3. Place all of the test tubes at the same time into the boiling 'ater bath. =ote and recordho' long it ta*es for the 8rst clear red colored solution to form.

?. Remo-e all of the test tubes as soon as the 8rst "ositi-e test is seen as "rolong heatingKin e$cess of @ minutes: may cause s"urious results. ;hich sugar solutionKs: contain a*etoseN

&ialTs ,est+

1. se the boiling 'ater bath from before and label ane' set of clean test tubes.

2. n se"arate test tubes add 1 mL of the &ialTs reagent. ,o each test tubes add 10 dro"sof the carbohydrate solution.

3. Place all the test tubes at the same time into the boiling 'ater bath.

?. =ote and record ho' long it ta*es for the 8rst clear blue%green solution to form

@. Remo-e all the test tubes as soon the 8rst "ositi-e test is seen as "rolonged heatingmay cause s"urious results. ;hich sugar solutionKs: contain "entoseN

odine test+

1. Place 3 dro"s of each test carbohydrate solution in se"arate 'ells of a clean s"ot "late.

A0


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2. #dd 1 dro" of the iodine solution to each test carbohydrate solution. =ote and recordthe color of each sam"le.

3. 7id any other solutions besides the starch solution gi-e a "ositi-e testN


A1


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RE02RT SHEET,Carbohydrates



1. Com"lete the table of results. &e sure to include color_+

odine,est

&ialTs,est

Seli'ano>Ts,est

&arfoedTs,es

t

&e

nedictTs,est

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Carbohydrate

Glucose

Galactose

2ructose

#rabinose

Maltose

Lactose

Sucrose

Starch

2. Su""ose you sa' no sign of a color change in &enedictTs test no sign of a red"reci"itate after 10 minutes 'ith &arfoedTs test and a dar* red colored solution'ith the Seli'ano>Ts test. ;hat sugarKs: could you ha-eN

3. Su""ose you sa' a red "reci"itate 'ith the &enedictTs test a red "reci"itate after2 minutes 'ith the &arfoedTs test and stra'%colored solution after more than @minutes 'ith the Seli'ano>Ts test. ;hat sugarKs: could you ha-eN

?. Su""ose you sa' a red "reci"itate 'ith &enedictTs test no sign of red "reci"itateafter 10 minutes 'ith &arfoedTs test and a stra'%colored solution after more than@ minutes 'ith Seli'ano>Ts test. ;hat sugarKs: could you ha-eN

A3


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66/104


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3. Cool the mi$ture to room tem"erature by running cold ta" 'ater along the outsideof the test tube.

?. #dd 1@ dro"s of 3M HCl mi$ing by 8nger 4ic*ing the test tube after each addition.Record your obser-ations and 'rite the o-erall eDuation for this reaction.

AA


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RE02RT SHEET,Acid,Base R&ns with Carbo&ylic Acids andEsters



Solubility and #cid%&ase Reactions of Salicylic #cid+

1. 7oes salicylic acid dissol-e in the cold 'aterN

2. 7oes salicylic acid dissol-e in hot 'aterN

3. 7oes salicylic acid dissol-e in aDueous sodium hydro$ideN

?. ;rite an o-erall eDuation for the reaction of salicylic acid 'ith aDueous sodiumhydro$ide.

@. ;hat did you obser-e 'hen hydrochloric acid 'as added to the test tube during ste" AN

A. ;rite an o-erall eDuation for the chemical reaction that occurs in ste" A.

#cid%&ase Reactions of Methyl &en)oate+

1. ;rite an o-erall chemical eDuation for the sa"oni8cation of methyl ben)oate.

AB


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70/104

Synthesis ofAspirin

/oals for the Student" Learn to do the organic synthesis of an ester from an alcohol and an anhydride Learn ho' to "urify the "roduct in the organic synthesis Learn ho' to calculate the Z yield of a "roduct Learn di>erent techniDues to com"are the "urity of a synthesi)ed "roduct 'ith

commercial "roduct.

:ntroduction

#cetylsalicylic acid K#S#: commonly called as"irin is 'idely used as medicine toreduce fe-er Kan anti"yretic: to reduce "ain Kan analgesic: to reduce s'elling Kanti%in4ammatory: and to "re-ent "latelet aggregation that initiali)es thrombosis or hemostasisKanti%clotting:. #s"irin inhibits the en)ymes necessary for the formation of "rostaglandinsand thrombo$anes Khormones: that are associated 'ith "ain fe-er in4ammation andblood%clotting in the human body. t has been also suggested that as"irin small amount as0%100mg for daily ingestion can lo'er the ris* of stro*e and heart attac* in at%ris* adults.

#s"irin is an ester of acetic acid and salicylic acid. Salicylic acid is acting as thealcohol because this also has a hydro$yl grou" attached to the ben)ene ring besides a

carbo$ylic acid. ,hough esters can be "roduced from the direct esteri8cation of an alcoholand a carbo$ylic acid in the "resence of an acid catalyst ty"ically sulfuric acid the "resentmethod to "re"are as"irin uses acetic anhydride a deri-ati-e of acetic acid to form moreDuic*ly an acetate ester 'ith salicylic acid. #cetic anhydride as a substitute acetylatingagent reacts 'ith salicylic acid as follo's+

n the 8rst 'ee* of a t'o%'ee* e$"eriment you 'ill synthesis as"irin Kacetyl salicylicacid:. ,he follo'ing 'ee* you 'ill determine the "ercent yield of your synthesi)ed as"irin.

A


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,hen you 'ill analy)e the "urity of your "roduct by thin layer chromatogra"hy and by amelting "oint determination.

CH#R#C,ER`#,!= ! S6=,HES`E7 #CE,6LS#LC6LC #C7 K#S# as"irin:+

,he second 'ee* you 'ill determine the "ercent yield of the synthesi)ed "roduct

as"irin and "erform di>erent techniDues to determine the "urity of the "roduct. !f coursethis as"irin is not suitable for oral admission or any "hysiological test because reagentsused for this synthesis are not of suJcient "urity for ingestion.

7etermination of Percent 6ield+

Percent yield is de8ned as follo's+

Z yield mass of acetylsalicylic acidKactual yield of #S# in e$"eriment:$ 100

,heoretical yield of #S# Kcalculated from stoichiometricrelationshi":

,he actual yield is the number of grams of acetyl salicylic acid that you actuallymade in the laboratory. ,he theoretical yield is the number of grams of acetylsalicylic acidyou should get based on the stoichiometry of the chemical eDuation. ,he theoretical yieldis based on the number of grams of salicylic acid you used. irst con-ert the number gramsof salicylic acid to moles by di-iding the grams of salicylic acid by the molecular 'eight ofsalicylic acid K13 gFmol:. !ne mole of acetylsalicylic acid is formed for each mole ofsalicylic acid used. ,herefore the number of moles of acetylsalicylic acid is the same asthe number of moles of salicylic acid. ,o con-ert to grams of acetyl salicylic acid multi"lythe number of moles you calculated by the molecular 'eight of acetylsalicylic acid K10gFmol:

,hin Layer Chromatogra"hy+

!ne 'ay of determining the "urity of your "roduct is to do thin layerchromatogra"hy using your "roduct one of the reactant Ksalicylic acid: and an authenticas"irin sam"le. ,his is the same "rocedure you com"leted 'hen you analy)ed chloro"hylland carotenoids from s"inach. Re-ie' that e$"eriment if you ha-e forgotten.

7etermination of the melting "oint+

6ou should use the Meltem" a""aratus to determine the melting "oint of youracetylsalicylic acid. Carefully "lace a thermometer in the slot on the Meltem" de-ice.,hese are mercury thermometers and are Duite fragile so handle the thermometer 'ithcare. f you brea* the thermometer notify the instructor immediately so the mercury s"illcan be cleaned u". &efore measuring the melting "oint of your "roduct loo* u" the truemelting "oint of acetylsalicylic acid in ,he Handboo* of Chemistry and "hysics. ,his 'ill

gi-e you an idea of about 'here your "roduct should melt.

erric chloride test+

7issol-e a small amount of your "roduct in 'ater and test it 'ith ferric chloride. #"ur"le color indicates the "resence of a "henol Kunreacted salicylic acid: in your "roduct.Com"are your results 'ith those obtained from a sam"le of authentic as"irin.

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#cid hydrolysis of acetylsalicylic acid+

Esters can be hydroly)ed by heating them in the "resence of an acid. ,he originalalcohol and carbo$ylic are generated from the acid hydrolysis.

E&perimentalSynthesis+

1. Place Y @0 mL of distilled 'ater into a 2@0 mL bea*er add a cou"le of boiling chi"sand heat to boiling. #lso "ut Y20 mL of distilled 'ater in a @0 mL Erlenmeyer 4as*into an ice 'ater bath using another 2@0 mL bea*er Kig. # =ote+ use of hot "late'ill be more safe than using burner:.

2. ;rite your grou" number in "encil on a "iece of 8lter "a"er. ,are the balance and'eigh the 8lter "a"er to the nearest 0.001 g5 record this -alue in your noteboo*.!btain Y 1 g of salicylic acid and "lace it into a tarred "lastic 'eigh boat. ;eigh thesalicylic acid to the nearest 0.001 g and record this -alue in your noteboo*.

3. Place the salicylic acid into a clean dry medium test tube. #dd 2 mL of aceticanhydrideand 2 dro"s of concentrated sulfuric acid Kcaution+ acetic anhydride "roducesirritation and necrosis of tissues in liDuid or in -a"or state:. #-oid contact 'ith s*inand eyes. 7o this addition in the hood.

?. Place the test tube containing the reaction mi$ture into the boiling 'ater bath. Stirthe mi$ture -igorously 'ith a clean glass rod 'hile in the boiling 'ater bath. &ecareful not to brea* the test tube.

@. #fter the entire solid has dissol-ed remo-e the test tube into the ice 'ater bath. fthe crystals do not form induce crystalli)ation by scratching the inside of the test

tube 'ith a glass rod. ;hen crystalli)ation is com"lete add 10 mL of the ice cold'ater.

A. old your 'eighed initialed 8lter "a"er so that it is 4uted Kyour instructor 'illdemonstrate this: and "lace it into a short stem funnel.

B. Collect the solid on the 8lter "a"er in the funnel. Rinse the solid 'ith 2 or 3 small K@mL: "ortions of ice cold 'ater. &e sure to let the 'ater drain through the 8lterbet'een additions of the rinses.

. Carefully remo-e the 8lter "a"er from the funnel and s"read it out on a "iece of"a"er to'el. Set the "a"er to'el 'ith the 8lter "a"er in a dra'er and allo' it to dryuntil the ne$t laboratory "eriod.

. 7etermine the gram formula 'eight for salicylic acid. sing the mass of yoursalicylic acid determine the number of moles in the reaction. 7etermine the gramformula 'eight for the acetyl salicylic acid "roduct. inally calculate the mass of"roduct e$"ected if all of the salicylic acid is con-erted to acetyl salicylic acid.

10. 6our instructor 'ill demonstrate ho' to "re"are a sam"le for a melting "ointa""aratus. Practice ta*ing melting "oints using the salicylic acid.

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11. ,he follo'ing 'ee* carefully remo-e the 8lter "a"er containing the as"irin from thedra'er and 'eigh it to the nearest 0.001 g. Record this -alue in your noteboo*.

12. 7etermine the "ercent of the of yield of the "roduct as"irin. Sa-e as"irin for furtheranalysis.

,hin layer chromatogra"hy+

1. !btain Y 1 mL of methanol to three se"arate clean and dry test tubes. ntothese se"arate test tubes add a small amount of your synthesi)ed as"irincommercial as"irin and salicylic acid 'hich you ha-e used for the synthesis. &esure label each test tube so that you *no' 'hich is contained in each test tube.,o aid in dissol-ing the crush the solid gently 'ith a clean dry glass rod. &e sureto clean the glass rod "rior to using for the ne$t sam"le.

2. !btain B cm 1? cm 4uorescent silica gel ,LC "late for your grou". ollo' the"rocedure in "ages 2? 2@ and 2A to s"ot K? L: your "roduct authentic as"irinand salicylic acid. &e sure to mar* care fully the s"ot line at the bottom andlabel for the s"ots at the to". &e sure to *ee" at least K10 mm: s"ace bet'eenthe s"ots. 6our "roductTs s"ot should be on the left most "osition then the s"otof authentic as"irin on the middle "osition and salicylic acid s"ot on the rightmost "osition. ,ry to *ee" the diameter of the s"ot less than 2 mm. Se-eralDuic* small a""lications 'ill be better than one large a""lication.

3. Pre"are a ,LC de-elo"ing tan* similar to 'hat you used "re-iously. Pleace onehalf of a 8lter "a"er into the erence bet'een the t'orecorded tem"eratures: 'ill be small%"erha"s a degree or t'o. f your "roduct isnot "ure you 'ill ha-e a 'ider melting "oint range. f your "roduct is "ure the

B2


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melting "oint should be similar to the true -alue for acetylsalicylic acid. f it isim"ure your 'ill ha-e melting "oint lo'er than the true -alue.

erric chloride test+

7issol-e a small amount of your "roduct in Y 1 mL of 'ater. #dd t'o dro"s of ferricchloride solution and note the color. Re"eat 'ith an authentic as"irin sam"le ofas"irin and 'ith the salicylic acid that you ha-e used. Record your obser-ations inyour lab noteboo*.

#cid hydrolysis of acetylsalicylic acid+

7issol-e a small amount of your "roduct in Y 1 mL of 'ater. #dd 8-e dro"s of conc.Sulfuric acid and heat the test tube in a boiling 'ater bath for 10%1@ minutes.Remo-e the test tube cool to room tem"erature and test for the "resence of a"henol by adding t'o dro"s of ferric chloride.

B3


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B?


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RE02RT SHEET,Synthesis of Aspirin



Synthesis+

1. Mass of 8lter "a"erIIIIIIIIIIIIIIIIIIIIIIIIIIII

2. Mass of 8lter "a"er "lus "roductIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

3. Mass of acetylsalicylic acid Kas"irin:IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

?. Mass of salicylic acid usedIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

@. Moles of salicylic acid usedIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

A. Moles of acetylsalicylic acid e$"ected Ksho' theoretical yield:Krefer to the eDuation:

B. Z yield of acetylsalicylic acid Ksho' calculation:

. ;rite full chemical eDuation Kline%bond formulas for both reactants and "roducts: forthe synthesis of as"irin.

. #fter 8ltering your reaction mi$ture 'hat 'as the "ur"ose of rinsing the "roduct'ith 'aterN

10. ;hy it is im"ortant for the 'ater to be ice coldN

B@


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Characteri)ation+

1. ;rite the literature -alue for the melting "oint of acetylsalicylicacid.IIIIIIIIIIIIIIIoC

2. ;rite the melting "oint range of your "roductN IIIIIIIIIIIIoC to IIIIIIIIIIIoC

3. 7ra' a re"roduction of your de-elo"ed chromatogram. Sho' the "osition of alls"ots the sol-ent front and s"otting line. ;hat conclusions can be dra'n about the"urity of your "roduct from this chromatogramN

?. ;hat conclusion can be dra'n about the "urity of your "roduct from thischromatogramN

@. ;hat did you obser-e 'hen eCl3'as added to your "roductN

A. ;hat did you obser-e 'hen eCl3'as added to authentic as"irinN

B. ;hat did you obser-e 'hen eCl3'as added to salicylic acidN

. ;rite the full chemical eDuation Kline%bond formulas for both reactants and"roducts: for the hydrolysis of your acetylsalicylic acid.

. ;hat did you obser-e 'hen eCl3'as added to the as"irin hydrolysis solutionN

10. sing all e$"erimental data com"are the "urity of your as"irin 'ith that of theauthentic as"irin sam"le.

BA


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Synthesis @properties of Soap

/oals for the Student" Learn about the "rocess of soa" ma*ing Learn the "rocedure for "urifying and testing the of "ro"erties of a soa"

:ntroduction

# natural soa" is the sodium or "otassium salt of long chain fatty acids "roducedby the base cataly)ed hydrolysis of triacylglycerol Kthe fat storage molecule in "lants andanimals *no'n clinically as triglycerides:. n the 8rst "art of this e$"eriment you 'ill"re"are soa" by a sa"oni8cation reaction of a small sam"le of oil or fat. # generali)edsa"oni8cation reaction is sho'n belo'+

n the second "art of this e$"eriment some of the "ro"erties K"H and solubility: insolutions of your soa" 'ill be e$amined.

E&perimental

Synthesis+

1. Caution must be obser-ed as the concentrated sodium hydro$ide Klye: is corrosi-eand can cause burns to s*in destruction of clothing and irre-ersible cornea damage

to the eye. #t no time are your safety glassesFgoggles to be remo-ed during thise$"eriment.

2. Pre"are boiling 'ater bath in a A00 mL bea*er5 be sure to add a cou"le of boilingchi"s. Place 12 mL of oil or 10 g of fat into a 2@0 Erlenmeyer 4as*. #lso "re"are anice 'ater bath using another A00 mL bea*er.

3. #dd 10 mL of ethanol Kethylalcohol: and 12 ml of AM sodium hydro$ide to the-egetable oil. ^^6ou may add a small "iece of 'a$ crayon no' if you 'ant yoursoa" to ha-e a color. Many dyes used in crayons 'ill be altered due to the change

BB


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in "H. ,he "ro"er color should return 'hen you rinse your soa" later. Someone inyour grou" needs to lea-e theirs 'hite for the tests

?. Stir the mi$ture 'ith a glass stirring rod. sing a ring stand and a clam" secure the4as* and heat it in the boiling 'ater bath. Continuously stir the mi$ture during theheating "rocess to "re-enting the mi$ture from foaming u" the sides of the 4as*.

@. Heat the mi$ture in the 'ater bath 'ith stirring until the odor of ethyl alcohol is nolonger detected. ,his may ta*e 1@ to 30 minutes. Remo-e the 4as* from bath.

A. Place the 4as* into the ice bath. Cool the soa" solution for 10 minutes.

B. ,o the contents of the 4as* add 20 mL of a concentrated sodium chloride solution.sing a s"atula brea* u" the lum"s of soa" as com"letely as "ossible to "ermitcontact bet'een the solid and the sodium chloride 'ash solution. Carefully decantthe solution to remo-e the 'ash solution 'hile retaining the solid in the 4as*.

. Re"eat the 'ashing and decanting ste" t'o more times. #fter the 'ashing remo-ethe last traces of liDuid by dum"ing the solid into the "a"er to'els and carefullyblotting the soa" 'ith additional "a"er to'els. #-oid touching the soa" 'ith yours*in.

#nalysis+

1. 7issol-e a small "ea si)ed of your soa" in a small test tube containing @ mL of distilled'ater. #dd 3 to ? dro"s of ni-ersal indicator. =ote the color a""eared. sing theni-ersal indicator reference card determine the a""ro$imate "H of your soa"solution. Re"eat this e$"eriment using a "uri8ed commercial soa" Ke.g. -ory:. Recordcolor and "H -alue in your noteboo*.

2. n three se"arate clean test tubes "lace @ mL each of distilled 'ater ta" 'ater and10Z CaCl2Kcalcium chloride : . #dd a small "ea si)ed "ieces of your soa" to eachse"arate test solution. Ma*e sure that you use eDual si)ed amounts. Sto""er each tubeand sha*e them -igorously. 7escribe the relati-e amounts of lather and foam thata""ear in each tube. Record your obser-ations in your noteboo*.

3. 7issol-e a small amount of your soa" in a minimum amount of distilled 'ater5 estimatethe -olume of 'ater you used. ,o the dissol-ed soa" solution add an eDual -olume ofconcentrated sodium chloride solution. 7escribe and record in your note boo* 'hatha""ens.

?. /isit other grou"s and com"are the te$ture of soa"s made from di>erent fat sources.

B


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RE02RT SHEET,Synthesis and 0roperties of Soap



1. ;rite a chemical eDuation for the sa"oni8cation of a triglycerol that contains"almitic acid oleic acid and linoleic acid as the three fatty acid moieties.

2. ;hat 'as the "ur"ose of adding the concentrated salt solution to your soa""re"arationN

3. a:. ;hat 'as the "H of your soa" solutionN

b:. ;as your soa" solution acidic basic or neutralN

c: ;hat 'as the "H of the commercial soa" solutionN

d: ;as the commercial soa" solution acidic basic or neutralN

e: &ased on your result to "arts 3a%3d abo-e is it "ossible to ha-e a neutral solutionof a "ure soa" in distilled 'aterN

?. 7escribed the obser-ed beha-ior 'hen your soa" 'as added to each of thefollo'ing and sha*en u"+

a: 7istilled 'ater+

b: ,a" 'ater+

B


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c: Calcium chloride solution+

@. ;rite a net eDuation for the reaction of calcium ions 'ith the anion of "almitic acid.

A. ;hat did you obser-ed 'hen concentrated sodium chloride solution 'as added todissol-ed soa"N

B. ;hat does the obser-ation in A suggest about the e>ecti-eness of ordinary soa" insea'aterN

. ;ere there any di>erences in te$ture in soa" from di>erent fat sourcesN 7oes thisagree 'ith your *no'ledge about saturated -ersus unsaturated fatsN

0


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:solation andCharacteri%ationof Casein from

?ilk

#do"ted from+ Isolation of $rotein, +arbohydrate and =at from ;il Mohr. S. C. GriJn S.. and Gensler ;.. in &aboratory ;anual for =undamentals of %rganic and >iological+hemistryohn McMurry and Mary E. Castellion Engle'ood Cli>s Prentice%Hall 1?

;ayne P. #nderson K?F2002:

/oals for the Student" Learn the about the "rotein "resent in mil* and cheese Learn the "rocedure to isolate the "rotein from the mil* Learn the techniDues used to characteri)e the "rotein

:ntroduction

6ou may recall the Mother Goose nursery rhyme Little Miss Mu>et sat on a tu>eteating her curds and 'heyX.. ;hen mil* is acidi8ed it is transformed into a solidcom"onent called curd and a liDuid com"onent called 'hey. ,his method is used to

ma*e cottage cheese. ,he curds contain butterfat and a "rotein called casein 'hichcontain all of the common amino acids and is "articularly rich in the essential ones.Casein e$ist in the mil* as a soluble calcium salt that "reci"itates at "H -alues belo'?.A. So mil* can be curdled by acids such as lactic acid that forms during naturalsouring of mil*. ,he carbohydrate lactose is "resent in the 'hey. n this e$"erimentyou 'ill also isolate casein from mil* and carry out some Dualitati-e tests for "rotein.

,he &iuret test is generally used for "rotein. ;hen the "ale blue Cu2Uion forms acom"le$ 'ith ad


83/104

,he anto"eroteic acid test is on the other is a general test for the "resence of thearomatic amino acids try"to"han "henylalanine and tyrosine in "roteins. #romaticgrou"s that ha-e an amino grou" Ktry"to"han: or a hydro$yl grou" Ktyrosine: are easilynitrated by concentrated nitric acid to form yello' K$antho Gree* for yello': coloredaromatic nitro com"ounds.

E&perimental1. 7etermine the mass of a 12@ mL Erlenmeyer 4as*. #dd @0 mL of mil* to the 4as*

and re%'eigh the 4as* to determine the mass of the mil*. Chec* the label on themil* container and record the amount of "rotein "er ser-ing in your noteboo*.

2. Pre"are a 'ater bath by "lacing 200mL of 'ater in a A00 mL bea*er. Heat the 'aterbath to ?0oC5 as the tem"erature is critical for this e$"eriment monitor thetem"erature 'ith a thermometer. Place the 4as* containing the mil* into the 'aterbath.

3. Slo'ly add 10 dro"s of glacial acetic acid to the mil* 'hile stirring 'ith a glass rod.Continue to add dro"s of glacial acetic acid dro" 'ise until no more "reci"itate is

formed 'hen a dro" of acid is added. #llo' the mi$ture to cool.

?. ilter the mi$ture into a 2@0 mL bea*er by "ouring it through cheese cloth that hasbeen fastened to the bea*er 'ith a rubber band. SDuee)e out as much liDuid as"ossible from the solid. ,hen scra"e the solid into a 100 mL bea*er.

@. ,o remo-e any fat from the curd Kdo you e$"ect any for s*im mil*N: add 2@ mL ofethanol to the solid in the 100 mL bea*er. Stir the mi$ture for about @ minutes5 thenlet the solid settle. ,he fat 'ill dissol-e in the alcohol. 7ecant the liDuid into anotherbea*er.

A. nder a hood add 2@ ml of a 1+1 K-F-: mi$ture of diethyl ether and ethanol toresidue. &e sure that that there is no 4ames or s"ar*s "resent as the diethyl ether is

e$tremely 4ammable. Stir the mi$ture for about @ minutes. Let the "rotein solid dryin your dra'er for a 'ee* then 'eigh your solid and determine the Z yield duringthe ne$t class "eriod.

&RE, ,ES,+

1. #dd a "ea si)ed amount of your casein to a small test tube and dissol-e it in ? mL ofdistilled 'ater. 7i-ide this "rotein solution into t'o 2 mL "ortions. Sa-e one "ortionfor the ne$t test.

2. ,horoughly mi$ 2mL of the "rotein solution 'ith 2 mL of 3M sodium hydro$idesolution. #dd 1 dro" of 1Z of co""er sulfate solution. =ote the color and recordyour obser-ations in your noteboo*.

3. Continue to add the co""er sulfate solution one dro" at a time note and record yourobser-ations. Sto" after adding 10 dro"s of the co""er sulfate solution.

?. Re"eat this test 'ith a 1Z casein solution.

#=,H!PR!,EC ,ES,+

2


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1. ,o the second 2 mL "rotein solution carefully add 1 mL of concentrated nitric acid.Mi$ and note the a""earance of any hea-y 'hite "reci"itate.

2. ;arm the mi$ture carefully in a hot 'ater bath noting any change to a yello'colored solution.

3. Cool the mi$ture in a stream of cold ta" 'ater and carefully add a fe' dro"s of 3Msodium hydro$ide. # "ositi-e test is indicated by the yello' color changing intoorange color. ,he entire tube does not ha-e to turn to orange. Loo* for the color asthe sodium hydro$ide is added to the solution or on a "iece of "reci"itate on the'all of the test tube.

?. Re"eat this test 'ith 1Z casein solution.

3


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?


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RE02RT SHEET,:solation and Characteri%ation of Caseinfrom ?ilk



solation+

1. Mass of 12@ mL Erlenmeyer 4as* IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

2. Mass of 12@ mL Erlenmeyer 4as* Umil* IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

3. Mass of mil* IIIIIIIIIIIIIIIIIIIIIIIIIIIIII

?. Mass of crude casein Kmay not be totally dry: IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

@. Calculate Z yield Ksho' your calculation:+

A. 7etermine the amount of "rotein in a ser-ing of mil* Kuse the mass of crude casein:Ksho' your calculation:

&iuret test+

B. Color of 0.1Z of co""er sulfate solution+

. Color of your casein U sodium hydro$ide solution+

. Color of your casein solution after addition of one dro" of co""er sulfate+

10. Color changes obser-ed after adding additional dro"s of co""er sulfate+

11. Color of 1Z casein U sodium hydro$ide solution+

@


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12. Color of your 1Z "ro-ided casein solution after addition of one dro" of co""ersulfate+

13. Color changes obser-ed after adding additional dro"s of co""er sulfate+

antho"roteic acid test+

1?. !bser-ation for your casein mi$ed 'ith concentrated nitric acid+

1@. !bser-ation for your casein mi$ed 'ith concentrated nitric acid after heating+

1A. !bser-ation for your casein after adding 3M sodium hydro$ide+

1B. !bser-ation for 1Z casein mi$ed 'ith concentrated nitric acid+

1. !bser-ation for 1Z casein 'ith concentrated nitric acid after heating+

1. !bser-ation for 1Z casein after adding 3M sodium hydro$ide+

Mil* com"arison+

7id one of the -arieties of mil* ha-e a higher "ercentage by 'eight of "roteinN

A


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Amylase" TheActi*ity of an

En%yme

#da"ted from+ actorTs #>ecting En)ymatic #cti-ity in ohn R. Holum and Sandra L.!lmstead &aboratorty manual for =undamentals of ?eneral, %rganic and >iological+hemistry @thEd. =e' 6or*+ ;iley 1?. Michael E. Pugh and ;ayne P. #nderson KRe-.3F200?:

/oals for the Student" Learn about en)ymes that cataly)e biological reactions Learn about the factors that in4uence the acti-ity of en)ymes

:ntroduction

#mylase an en)yme that is found in sali-a cataly)es the hydrolysis of starchKamylase:. Since en)ymes are "roteins their secondary and tertiary structures are

e>ected by tem"erature "H and the "resence of hea-y metal ions. En)yme acti-ity isclosely associated 'ith the structure of an en)yme. So any change in the secondary ortertiary structure leads to a change in en)ymatic acti-ity.

n this e$"eriment you 'ill e$amine the e>ect of tem"erature and "H on the acti-ityof amylase. Molecular iodine forms a com"le$ 'ith starch that has a characteristic dee"blue color. #s starch undergoes hydrolysis to form oligosaccharides and glucose thecharacteristics color of the starch iodine com"le$ disa""ears. ,herefore loss of thedee" blue color can be used to measure of the e$tent of hydrolysis of starch. # secondtest for hydrolysis is the occurrence of a "ositi-e &enedictTs test for the solution. Starchis not a reducing sugar but glucose formed u"on hydrolysis is a reducing sugar.

E&perimental

P#R, . E>ect of ,em"erature on En)yme #cti-ity

n order to ma*e sure that the concentration s of en)yme and starch remainreasonably constant in di>erent "arts of the e$"eriment use an eye dro""er to measureDuantities of solutions. #ssume that 20 dro"s re"resent 1.0 mL.

B


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1. Label three medium test tubes as 0 rt and 100. nto each test tube "lace bu>eredsolution and 2.@ mL of distilled 'ater. Place the test tube mar*ed 0 into the ice baththe one mar*ed rt into room tem"erature and 100 into a bea*er of boiling 'ater.

2. Place 1 mL of freshly "re"ared amylase solution K100 mgF100 mL: into each of threesmall test tubes. Put one of these into the ice bath another into the thermostated'ater bath and the third into the bea*er of boiling 'ater.

3. #llo' the solutions to remain in the tem"erature baths for about @%10 minutes toeDuilibrate.

+arry out the following steps in se(uence for the solutions in the 0o, rt, and @00otemperature baths.

?. Remo-e the test tube containing the starch and the test tube containing the en)ymefrom the bath and "our the en)yme solution into the starch solution. Record the time tothe nearest second and call this starting time 0.

@. uic*ly "lace a "iece of "ara8lm o-er the end of the test tube mi$ thoroughly remo-ethe "ara8lm and remo-e a small amount of the solution from the test tube using a

dis"osable "i"ette. Place the remaining solution bac* into the tem"erature bath. Place? dro"s of the solution onto a s"ot "late that contain 1 dro" of the iodine solutionrecord the color. Put any e$cess starchFen)yme solution in the "i"ette bac* into thetest tube in the tem"erature bath.

A. ,a*e a sam"le from the solution using the dis"osable "i"ette e$actly 1 minute follo'ingtime 0. Place ? dro"s onto a second s"ot of the s"ot "late containing 1 dro" of iodinesolution record the color. Return any e$cess starchFen)yme solution in the "i"ette totest tube in the tem"erature bath.

B. Re"eat the "rocedure in at the follo'ing time inter-als until the color of the solutionfollo'ing addition of the iodine is yello'+ 2 min ? min A min min and 10 min. f thecolor of the solution in the s"ot "late remains yello' for successi-e trials. t is not

necessary to continue the run.

Part . E>ect of "H on En)yme #cti-ity

1. Label three medium test tubes as @ B and .

2. nto the test tube mar*ed @ "lace 2.@ mL of unbu>ered starch solution and 2.@mL of "H @ bu>er solution. nto the test tube mar*ed B "lace 2.@ mL of unbu>eredstarch solution and 2.@ mL of "H B bu>er solution. nto the test tube mar*ed "lace 2.@ mL of unbu>ered starch solution and 2.@ mL of "H bu>er solution. Placethese into either the room tem"erature bath or the ice bath de"ending on 'hichtem"erature ga-e the better results in "art .

3. Place 1 mL of the amylase solution into each of three small test tubes. Put theseinto the tem"erature bath containing the starch solutions from ste" 2.

?. #llo' the solutions to remain in the tem"erature bath for about @%10 minutes toeDuilibrate.

@. Carry out the ste"s ?%B in P#R, in seDuence for the solutions that bu>ered at "H @B .


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P#R, . E>ect of Metal ons on En)yme #cti-ity

n this "art you 'ill test the e>ect of a metal ion on en)yme acti-ity Cu 2U e3U `n2Uorother transition metal ions or hea-y metal ions may be tested.

1. Label a test tube 'ith the identity of the metal ion in the salt solution you 'ill use forthis "art.

2. nto each test tube "lace 2.@ mL of bu>ered starch solution and 2.@ mL of the metal ionsolution. Place these into room tem"erature bath or the ice bath de"ending on 'hichone ga-e better results in "art .

3. Place 1mL of the amylase solution into each of t'o small test tubes. Put them into thetem"erature bath containing the metal ion solution.

?. #llo' the solutions to remain in the tem"erature bath for about @%10 minute

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