Chemistry Laboratory 204: Organic Chemistry and Biochemistry Laboratory Manual

Laboratory Procedure

Analysis of Saliva

Activity

12

INTRODUCTION Saliva is a viscous fluid ith a !" of around #$%$ &t contains mucins' amylase' and other !roteins$ &norganic constituents include sodium' !otassium' calcium' chloride' and !hos!hate ions$ (aily out!ut of saliva is in the range of )$0 * 2$0 liters$

APPARATUS/MATERIALS Bunsen burner iron ring iron stand funnel s!ot !late test tubes test tube rac/ test tube holder bea/er stirring rod graduated cylinder dro!!ers atch glass s!ot !late ire gau7e filter !a!er PROCEDURE Coll"#tion of Saliva

C EMICALS/REA!ENTS + mL starch solution ) mL 0$), !hos!hate buffer ) mL 0$), sodium chloride' -aCl + mL )- acetic acid' C"+COO" . mL acetone ) mL +, hydrochloric acid' "Cl . mL +, sodium hydro0ide' -aO" + mL Benedict1s solution ) mL 2$., sodium hydro0ide' -aO" ) mL 0$0.2 co!!er3&&4 sulfate' CuSO4 ) mL )2 acetic acid' C"+COO" . mL 22 !otassium o0alate' 52C2O4"2O ) mL ammonium molybdate' 3-"44#,o6O24 ) mL nitric acid' "-O+ ) mL silver nitrate' Ag-O+ distilled ater

8inse out the mouth to get rid of food !articles$ 9a/e about )0 ml of arm ater into mouth and move it about by the tongue for nearly a minute$ Collect the fluid in a clean bea/er$ Perform the tests ith this diluted saliva$

)$ 9est for amylase

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Chemistry Laboratory 204: Organic Chemistry and Biochemistry Laboratory Manual

9o 2$. ml of starch solution in a test tube' add one ml of 0$), !hos!hate buffer' !" 6$0' follo ed by ) ml of 0$), -aCl solution$ Add ) ml of salivary fluid$ ,i0$ At intervals of one minute' transfer one dro! of the mi0ture to de!ressions in a s!ot !late' each containing a dro! of iodine solution$ Blue colour fades and disa!!ears ith sam!les collected ith increasing time intervals$ Amylase hydrolyses )$4 glycosidic bonds in starch at random$ 9he brea/<do n !roducts do not have the ca!acity to bind iodine$ 2$ &solation of ,ucin from Saliva 8inse the mouth ith ater and collect ).ml ofsaliva in a 20 ml test tube$ Add + ml of )- acetic acid dro! ise' s irling the mi0ture after each addition$ Observe the formation of a thread<li/e !reci!itate$ 9his is the mucin$ Let stand for at least +0 minutes$ (ecant' filter and ash the !reci!itate using t o 2<ml !ortions of acetone$ Allo the !reci!itate to drain com!letely$ 8emove the filter !a!er fro the funnel' s!read it out on a atch glass and allo the !reci!itate to dry$ ;se the !reci!itated mucin to test for the !ossible com!onents of saliva$ a$ Carbohydrate Place about one<half of the isolated mucin in a test tube$Add ) ml of +, "Cl and immerse in a boiling ater bath for 20<+0 minutes$ Cool and ma/e faintly basic ith +, -aO"' then add + ml of Benedict1s solution$ "eat again in boiling ater bath until a visible change occurs$ b$ Protein (issolve the remaining !ortion of mucin in ) mlof 2$., -aO"' then add + dro!s of 0$0.2 CuSO4 solution$ ,i0 +$ 9est for Calcium 9o 2 ml of salivary fluid' add . dro!s of )2 acetic acid and . ml of 22 !otassium o0alate solution$ A faint hite !reci!itate is formed$ Calcium ions are !reci!itated as calcium o0alate under neutral or slightly acidic conditions$ 4$ 9est for &norganic Phos!hate ,i0 )ml of saliva and ) ml of ammonium molybdate solution$ "eat and observe$

.$ 9est for Chloride ,i0 ) ml of saliva solution$ ith +<. dro!s of nitric acid follo ed by . dro!s of Ag-O +

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Chemistry Laboratory 204: Organic Chemistry and Biochemistry Laboratory Manual

PROPER DISPOSAL : (is!ose of solutions in the !ro!er acid or basic astes' and organic or inorganic astes4$

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$UESTIONS )$ 2$ +$ 4$ .$ =hat is the !ur!ose of each of the com!onents of saliva> "o does each hel! in the digestion of food> (oes digestion already ha!!en in the mouth> &f so' hat ty!e of digestion> =hat im!airment ill a !erson e0!erience ithout salivary glands> Cite three salivary gland disorders and the corres!onding medical treatment$

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(ate Performed Aacilitator1s &nitials

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Chemistry Laboratory 204: Organic Chemistry and Biochemistry Laboratory Manual

(ata Sheet

Analysis of Saliva

Activity

12

Data Substan#"s T"st"% for Amylase Carbohydrate Proteins Calcium &norganic Phos!hate Chloride T"st R"a&"nts 0$), !hos!hate buffer' 0$), -aCl -aO"' Benedict1s solution' heat -aO"' CuSO4 ). acetic acid' 22 !otassium o0alate Ammonium molybdate "-O+' Ag-O+ R"sults

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