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5-1935

Chemical and Physical Considerations of Carbohydrates

Flora Cooke

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 C H E M I C A L AND PHYSICAL CONSIDERATIONS OF CARBOHYDRATES

SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR DEPiiRTMENT HONORS

BY

URSINUS COLLEGE

COLLEGEVILLE, PA.

APPROVED BY

DEPARTMENT OF CHBidlSTRY
 CHEinCAL

and:

PHYSICAL CONSIDERATIONS OF CARBOHYDRATES

 CARBOHYDRATES

GLUCOSE - Cgil;|_20g

The following tests v/ere iiiade on glucose as a typical c a r b o h y d r a t e ,

and a.re .not specific for this s u g a r .

I. SOLUBILITY -

Glucose is soluble in all ordinary solvents (HgO; lO/o LaC-1; 5)o W a g C O g ;

2% H C l ; concentrated K O H ; concentrated IIGl) and in a l c o h o l , >

2. HAPIITHOL R E A C T l O H (MOLISCH) -

vJhen 5 c . c . concentrated HpSO w e r e placed in a test tube and 5 c . c .

sugar solution to which 2 drops of ..olisch's reagent (15)1 alcoholic solution

of n a p h t h o l ) w a s added slowly dovm tne inclined t u b e , a reddish violet

zone was produced at the point of cont,.ct of tlie two s o l u t i o n s .

3. PHELYLHYDRAZIWE RBACTION -

5 c . c . of tlie sugar solution were added to a small amount of phenyl

hydrazine mizture (2 parts of phenyl hydrazine h y d r o c h l o r i d e ; 3 parts of sodium

aceta,te b y W6ig;ht) and shaken v/ell. lifter heating on a water bath for about

one-half h o u r , and allowing the tube t o c o o l , yellow crystals f o r m e d . These

uere prepared on a slide and examined m i c r o s c o p i c a l l y .

•3
4 . C I P O L L I M ' S TELT -

4 c . c . of dextrose s o l u t i o n , 5 drops of phenylhydrazine and c.c.

of glacial acetic acid were thoroughly mixed in a t e s t tube and heated

for about one rainute. A few drops of W a O H w e r e a d d e d , the solution r e -

maining a c i d , and it w a s again heated and t h e n c o o l e d . Yellow crystals

formed v e r y similar to the glucosazone v/ere foriaed and revealed w h e n

the crystals were mounted on a slide and examined m i c r o s c o p i c a l l y .

5 . PRECIPITATION BY ALCOHOL -

Dextrose solution added t o 9 5 % alcohol showed no r e a c t i o n . As c o m -

pared w i t h d e x t r i n , this was a distinction since 9 5 % alcohol throws

dextrin out of solution as a gummy w h i t e p r e c i p i t a t e .

6. IODINE TEST - '

Iodine added t o glucose showed n o r e a c t i o n . This w a s in contrast to

starch w h i c h yields a blue color w i t h i o d i n e .

7 . DXFFbSIBILITY OF GLUCOSE -

Glucose solution was diffusible through an animal membrane or parcliment

paper. A dialyzing bag'of collodion was n o t p r e p a r e d .

8 . kOORE'S TEST -

An equal volume of KaOH w a s added to 2/3 c . c . of glucose solution and

boiled. The solution darkened and finally assumed a brown c o l o r . The odor

of c a m e l was a p p a r e n t ,

9 . REDUCTION TESTS -

To their aldehyde or ketone structure raany sugars o re the property of

readily reducing alkaline solutions of oxides of m e t a l s . Upon this property

of reduction the most w i d e l y used tests for sugars are b a s e d , A S an example -

\
 a , TROMlaER'S TEST -

2 c . c . of NaOH v/sro added t o 5 c . c . of sugar solution and a

dilute solution of copper sulphate added u n u i l there v/as a slight per-

majient precipitate of cupric h y d r o x i d e . .,hen h e a t e d , the cupric hydroxide

v/as reduced to yellov/ish red cuprous o x i d e . Trornmer's test v/as not v e r y

satisfactory.

b . FEHLING'S TEST -

(Fehling's solution is a mixture of copuer sulphate, caustic

s o d a , and Rochelle salt v/hich holds the cupric hydroxide in solution.)

Good Fehling's solution v/as added to sugar solution a fev/ drops at a tirne

and the mixture heated after e a c h a d d i t i o n . The cupric hydroxide v/as r e -

duced to cuprous oxide v/hich varied in color from v/ellow through green to

r e d , due to the different sizes of the p a r t i c l e s .

3. ^ ^M.Mu^ c> ^ L^/ . ^/^o

c . B E N E D I C T ' S MODIFICATION OF FEHLING'S TEST -

Benedict's solution is modified in t h a t sodium carbonate replaces

soda and sodium citrate is used instead of Rochelle salt to hold t h e cupric

hydroxide in s o l u t i o n .

i/hen a few drops of dextrose solution was added t o Benedict's

s o l u t i o n , boiled v i g o r o u s l y , and c o o l e d , a precipitate formed which v/as

bluish green changing through green t o yellov/. The above modification will

serve to detect sugar w h e n present in as small quantity as .015 to .02 p e r c e n t .

d . B I S M D T H REDbCTIGN TEST ( N Y L A N D E R ' S TEST)

Nylander's iteagent is prepared by digesting 2 grams of bismuth

subnitrate and 4 grams of Rochelle salts in ICQ c . c . of 1 0 % K O H . When

Nylander's Reagent v/as added to su-gar solution and heated in a boiling

water b a t h , the solution d a r k e n e d , and u p o n standing for a fe?/ raoraents,

a black color due to the precipitation of bismuth f o r m e d .

C^J ^^^^^ ^ 734: ^ XM^'^

3

e. BARFOSD'D TEST - X

Barfoed solution is prepared b y dissolving 9 grams of neutral

crystallized copper acetate in 100 c . c , of HgO and adding 1.2 c . c . of

50% CHgCOOH.

When Barfoed's solution was added t o sugar solution and h e a t e d ,

reduction w a s indicated b y the f o r m t i o n of a red precipitate of cuprous

oxide. • ,

f. PICRIC ACID TEST -

Yiihen 2/3 c . c , of saturatf/d picric acid solution and 1 c . c . of

1 0 % K O H was added t o about 5 c . c . of sugar solution and w a r m e d , a mahogany

red color formed due to reduction of picric acid w i t h the formation of

picramic a c i d .

^ Z. k?

1 0 . ALCOHOuIC FERxEiHT.riTIOh -

A concentrated colution of .lucose to w h i c h a fragment of yeast w a s

added was placed in a test tube and inverted over H g O . COg collected at the

t o p of t h e tube and was identified b y addition of Ba(0H)2 w h i c h formed a

white pr ./cipitate of E a C O g .

Alcohol was revealed b y t h e iodoform test which consisted of rendering

the solution alkaline w i t h fiOH and adding a few drops of i o d i n e . Yellov/

crystals of iodoform v/ere formed and recognized by their characteristic

hosoital-like o d o r .
 ^ /20.C^J^ — ^ /20, ^ ^ x/^O
C /^^^ 7- ^ 7U.d7/^ ^ M > dW^ ^ // d€7zf^^
^ 7- ^ kv//%?

1 1 . FORMATION 0? CARkviEL -

i'ihen a small airiount of pulverized gkucose w a s melted and continued

t o be h e a t e d , a dark brown color formed w h i c h w a s soluble in H g O . This

coloring matter produced is Icnomm as c a r a m e l .

1 2 . The polariscope v/qs examined as t o mechanism and xaethod of o p e r a t i o n .

Trial readings were also t a k e n .

FRUCTOSE

1 . FRUCTOSE w a s identical w i t h glucose in its solubility and in its r e -

action t o the F e h l i n g ' s , P h e n y l h y d r a z i n e , B a r f o e d ' s , N y l a n d e r ' s , and

Fermentation t e s t s ,

2 . RESORCINOL - HYDROCHLORIC mCID REACTION (SSLB/ANOFF)

£eliw<anof f's reagent is prepared by d i s s o l v i n g .05 grarn. of resorcinol

in 100 c . c . of dilute H C l ,

When Seliwanoff's reagent was added to fructose solution a n d h e a t e d ,

a red color was produced and a brovm red precipitate separated. The p r e -

c i p i t a t e , when dissolved in a l c o h o l , imparted a striking red c o l o r .

3 . BORCILiRDT'S RE.,.CTION

iin equal volume of 2 5 % iiCl and a few crystals of resorcinol w e r e added

to a solution of f r u c t o s e . .dien heated t o b o i l i n g , a red color was p r o d u c e d .

After c o o l i n g , the mixture w a s made slightly alkaline w i t h solid K O H and

2/3 c . c . of acetic ether a d d e d . The acetic ether was colored y e l l o w .

 GALACTOSE - CsHigOs

1 . PHLOROGLUCIKOL - HYDROCnLORIG A C I D REACTION (TOLLSHS)

A littls phloroglucinol was added to equal volumes of galactose .

solution and lICl and the mixture heated on a boiling water b a t h , red

color y/as p r o d u c e d .

2 . IviUCIC A C I D TEST

when a solution of galactose v/as treated wit'ii concentre-ted H K O g , and

the mixture evaporated on a w a t e r - b a t h , a clear fluid containing a fine

w h i t e precipitate of inucic acid r e s u l t e d .

3 . PHEWLYhyDROZINE REACTION

This t e s t was carried out according t o t h e sanie directions as for

glucose, v.'hen the yellow crystals were raountsd on a slide and examined mdc

r o s c o p i c a l l y , it mas revealed t h a t they have a striking resemblance to

phenyl g l u c o s a z o n e .

DISACCILiRIDES - CqgH^pOll

MALTOSE

1 . Maltose v/as very soluble in the ''ordinary s o l v e n t s " . It reduced Fehling

N y l a n d e r ' s , and Barfoed's R e a g e n t s , H o w e v e r , its reducing power w a s only

about 2/3 that of g l u c o s e , i./altose gave a characteristic osazone with

phenylhydrazine.

LACTOSE

1 . Lactose was ;au.ch less soluble in w a t e r than g l u c o s e . It was insoluble-

in alcohol and soluble in dilute acid and a l k a l i . Lactose reduced F e h l i n g '

 B a r f o e d ' s , and Nylander's R e a g e n t s , . Its reducing power was a b o u t 7/10 that

of g l u c o s e .

This sugar formed a chara-cteristic osazone w i t h p h e n y l h y d r a z i n e .

2 . iSUGIC A C I D TkST / . % ''

Lactose solution, w h e n treatsd w i t h concentratod MO.^^ a n d e v a p o r a t e d ,

yielded a clear fluid and a fine white precipitate of inucic a c i d .

- SUCROSE - C^^H^^G^^

1 Sucrose w a s soluble in the "ordinary solv'ents" and did not r e d u c e F e h l i n g ' s ,

N.ylander's, or Barfoed's R e a g e n t s . It did n o t .yield a conipound with

phenylhydrazine.

2 . INVERSiax OF SUCRCSE.-

W h e n a few drops of IlgSO^were added to sucrose solution, b o i l e d , and

n e u t r a l i z e d , and the resulting solution tested, t h e f o l l o w i n g results were

obtained, lai o s a z o n e , p h e n y l g l u c o s a z o n e , was forined with phepylhydrazine,, arid

F e h l i n g ' s , N y l a n d e r ' s , and Barfoed's Reagents were r e d u c e d . This was due to

the inversion of the sucrose into g^lucose and fructose b y the a c i d .

c // o 7 /i' o 2c^^ ^ c /-/ o e M d)

STiiRGH (CgH^oOg)^

1 . A potato was p a r e d , g r a t e d , mixed w i t h w a t e r , and strained through cheese

cloth. After f i l t e r i n g ^ i t was dried in the a i r .

2 . SQLUBILITY - x 'r • '

Starch was insoluble in cold water and in a l o o h o l . It was soluble in

dilute alkali and a c i d .

3. MICROSCOPICAL EXAMIILHIOM -

Starch grains under the microscope appeared as r o u n d , concentric

granules, . P

4 . IODINE TEST - x

A Sow granules of starch were treated with a drop of a dilute solution

of iodine in potassium i o d i d e . The granules were colored blue due to the

formation of so-called iodide of s t a r c h . The cellulose of the granule was

n o t stained as shovm w h e n exarained m i c r o s c o p i c a l l y ,

5 . IODINE TEST O N STARCH PabTE -

Starch paste was treated with dilu-te iodine solution producing a blue

color, dhen h e a t e d , the color disappeared but it reappeared on c o o l i n g ,

iilcohol and alkali also removed the blue color w h i c h did n o t r e t u r n .

6 . T'EHLING'S TEST - '

. Starch paste reacts negatively to Fehling's s o l u t i o n . The blue color

of the reagent was not a l t e r e d .

7 . H Y D R O L Y S I S OF S T A R C I I -

10 drops of concentrated ilCl were added to about 25 c . c . of starch

paste and gently b o i l e d . At regular intervals a drop of the solution was

added to a test tablet containing iodine s o l u t i o n . As the testing p r o c e e d e d ,

t h e blue color gradually faded and finally d i s a p p e a r e d . After cooling and

neutralizing w i t h K O H , Fehling's test was positive y i e l d i n g a red precipitate

of cuprous o x i d e , edso, a portion of the solution yielded an osazone w i t h

phenylhydrazine. The osazone was phenyl glucosazone and this proved that

the final hydrolysis product of starch was g l u c o s e .

8 . iui excess of tannic acid solution added to a small amount of starch

paste yielded a yellowish white p r e c i p i t a t e . This is a test w h i c h distinguishes

starch frorri d e x t r i n .

9 . Starch paste was non-diffusible t h r o u g h parchment papej- and an aniraal

membrane.

DEXTRIH - (CeHioOs)^

1. Dextrin w a s insoluble in cold w a t e r . It formed a clear solution in hot

water. This test distinguishes dextrin from glycogen w h i c h gives an

opalescent s o l u t i o n .

2 . IODINE T E S T -

Iodine added to dextrin solution produced a brovmish red color due t o

the formation of red iodide of d e x t r i n . The color disappeared on h e a t i n g ,

and reappeared v e r y slowly u p o n c o o l i n g . Alkali and alcohol removed the

c o l o r , which did n o t r e a p p e a r .

3. FEELING'S SOLUTION -

Dextrin solution responded negatively to Fehling's solution.

4 . HYDRa^YSIS OF DLXTRIN -

5 drops of dilute HCl were added to about 25 c . c . of dextrin solution

in a small beaker and the solution b o i l e d . At regular intervals a drop of

the solution w a s added to a test tablet containing i o d i n e , the testing

proceeded the red color gradually faded, and finally d i s a p p e a r e d . When

neutralized and tested with Fehling's solution, there was a positive r e a c t i o n ,

proving the dextrin had been h y d r o l y z e d . By the phenylhydrazine t e s t , it

w a s proven that the dextrin was hydrolyzed t o g l u c o s e .

5. PRUCIFITATION Qi' A L C O H O L -

W h e n 9 5 % alcohol was added to d e x t r i n , dextrin was thrown out of

solution as a guuiuny white p r e c i p i t a t e . This test distinguished dextrin

from, g l u c o s e .
 '•- x
6 . II^FLUBNCE OF TANNIC A C I D -

An excess of tannic acid had n o effect on de:dsrin. T h i s result

differs from the result of a similar experiment u p o n s t a r c h ,

7 . DIFFUSIBILITY OF DE^iTRIN -

De>rbrin was non-diffusible t h r o u g h parchment paper and animal

membrane.

CELLULOSE

1 . Cellulose w a s insoluble in water and dilute acid and a l k a l i . It was

soluble in concentrated acid and a l k a l i .

2 . lODII-iE TEST - ' '

Dilute iodine added t o a few shreds of cotton in a t e s t tube produced

no color. Cellulose differs from starch an.d devrbrin in this r e s p e c t .

3 . FORlvATION OF AMYLOID -

when 10 c . c . of dilute and 5 c . c . of concentrated IHSO.was added to

d mt

some absorbent c o t t o n , the cotton dissolved w i t h o u t h e a t i n g . ..hen some

of this solution w a s cooled and diluted witn w a t e r , a gurany precipitate

of amyloid w a s f o r m e d . ^

Yihen a portion of t h i s same solution w a s neutralized ./ith K O H , and

tested w i t h Fehling's s o l u t i o n , a positive reaction was o b t a i n e d . Glucose

w a s formed from the cellulose b y the a c t i o n of the a c i d .

4 . AMMONIACAL CUPRIC HYDROXIDE SOLUBILITY T E S T - ( S C I L V E I T Z E R ) -

W h e n Schweitzer's reagent was added t o cellulose and stirred, the solid

dissolved. Upon acidification w i t h acetic a c i d , an amorphous precipitate

of cellulose w a s p r o d u c e d .
5 . HYDROCHLQRIC ACID - ZINC CHLORIDE LOLUBInlTY TEST (CROSS AKD BEVAN) -

W h e n a solution of two parts of hydrochloric acid and one p a r t of zinc

chlorice by w e i g h t (Cross and Sevan's reagent) was added t o cellulose, the

solid d i s s o l v e d . 95% alcohol reprecipitated the c e l l u l o s e .

GALACTAN'

1 . SOLUBILITY - '

The g a l a c t a n , a g a r - a g a r , was insoluble in cold w a t e r , soluble in hot

water and had a marked property of imbibing w a t e r .

2. IODINE TEST -

Iodine added to agar-agar yielded n o color r e a c t i o n .

3 . HYDROLYSIS OF ..GAR - AGX -

Agar-agar boiled with strong HCl and neutralized yielded a positive

reaction w i t h Fehling's s o l u t i o n , proving that the product of hydrolysis w a s

a reducing a g e n t . This reducing substance was g a l a c t o s e .

 I

BIBLIOGIUPHY

H a w k , Philip B . - "Practical Physiological Chemistry" - P . Blakiston's

Son and C o . , Sixth E d i t i o n , Philadelphia, 1 9 1 9 ; p p . 1 9 - 5 3 .