CHEM LAB FINAL EXAM NOTES Saccharomyces cerevisiae (yeast)

Experiment 10: Isolation & Hydrolysis of Yeast  unicellular fungus

RNA  contains 4% RNA by weight

Nucleic Acids Hydrolysis of Nucleic Acids

 macrobiopolymers of high molecular wt. Oil

with mononucleotide as the repeating RNA  2’, 3’ cyclic monophosphate  OH 
unit produces 2’ nucleotide and 3’ nucleotide,
 2 structural kinds: DNA & RNA –made respectively
up of nitrogenous bases, sugar, and
phosphate group Qualitative Hydrolysate Unhydrolyzed
 Pyrimidines: cytosine, thyamine, uracil Tests RNA
 Purines: adenine, guanine Benedict’s Brick-red ppt (+) Blue solution
Test (for (no visible
Isolation of yeast RNA Reducing reaction)
Sugars)
 It involves heating with alkali (NaOH)
Orcinol Blue green Blue green
which extracts nucleic acids & water-
Test (for solution (+) solution (+)
soluble proteins & inactivates nucleases
Pentoses)
which can degrade RNA.
 NA is then separated from the protein Test for Whitish ppt (+) Grayish-white
Purine ppt (+)
and other interfering subs. by acid
extraction at pH 4-5. Bases
 Treatment with alcohol and conc. HCl Test for Clear solution Yellow
can precipitate the RNA & repeated Inorganic Yellow ppt (+) solution with
washings with alcohol & ether/organic Phosphate yellow ppt (+)
solvents can remove lipids.
Negative result: Benedict’s Test in
Hydrolysis of Nucleic Acids unhydrolyzed RNA

Hydrolysis of NA by either chemical or Therefore, unhydrolyzed RNA has no reducing

enzymatic method, yields: sugars.

 Purine bases (A&G) Benedict’s Test

 Pyrimidine bass (C, T, U)
 Oligonucleotide containing up to 20  test for reducing sugars
residues  aldehydes & ketones are oxidized by
 Nucleosides (base + sugar) alkaline copper solution
 Ribose or D-ribose  cupric ions are reduced to cuprous and
 Phosphates brick-red Cu2O forms

Interactions (Responsible for the Rigid Mol. Orcinol Test

Configuration of NA)
 test for pentoses
 Phosphodiester bonds: join nucleotides  decomposition of pentoses when heated
in each chain with conc. HCl to form furfural which
 Hydrogen bonds: join the bases then condenses with orcinol (3,5 –
 van der Waal’s forces: between stacked dihydrotoluene) to form a blue or blue-
bases green compound

SANDIG, LYNETTE
Test for Purine Bases carboxy- proteins
peptidase proteins
Hydrolysate + NH4 + AgNO3  whitish ppt aminopeptidase
Intestinal maltase maltose
Unhydrolyzed + NH4 + AgNO3  grayish-white
Juice sucrose sucrose
ppt
lactase lactose
Test for Inorganic Phosphate dipeptidase dipeptides

Ammonium phosphomolybdate Saliva

[(NH4)3PO4(MoO3)12 + 12H2O] can be made by
heating ammonium orthomolybdate (3NH4+ +  watery secretion prod. by 3 pairs of
12MoO42- + 24H+) combined with phosphoric & salivary glands + numerous minute
nitric acid, yielding ammonium nitrate, water, glands, the buccal glands which are
and a yellow ppt of AP is obtained. located in the mucosal lining of the
mouth
Experiment 11: Salivary Digestion  speeds up chemical changes in the
carbs of ingested food materials
Alimentary Digestion
 it has no chemical effect on proteins
 process wherein ingested food materials and fats
are rendered assimilable or capable of
Components of Saliva
being transported thru the intestinal
mucosa & absorbed into the portal 1. Water: over 99% of saliva is water
blood system of the lymphatic system 2. Salivary amylase: hydrolyses starch to
 consists of a series of enzymatic maltose
hydrolyses reactions that convert 3. Mineral salts: (ex. sodium hydrogen
colloidal food into simple, non-colloidal carbonate) helps maintain a pH of 6.5-7.5
solutes which is optimum for the action of salivary
amylase
Breakdown of naturally occurring foodstuff into
4. Mucin: sticky material which helps bind food
assimilable forms in the work of hydrolytic
particles together & lubricate them to assist
enzymes (enzymes involved in digestion). They
swallowing
catalyze hydrolysis of proteins to amino acids,
starch to monosaccharides, and fats to acyl On average, we prod. 1 to 1.5L of saliva every
glycerols. 24hrs. The watery fluids produced by salivary
glands dissolve the food particles. Flavor can
Hydrolytic Enzymes
only be detected if food particles dissolved in
Digestive Enzymes Substrate the sol’n. penetrate the cells of the taste buds.
Juice
Paraffin Wax
Salivary a-amylase starch
Juice  waxy crystalline sub. that is white,
Gastric pepsin proteins transluscent, odorless, and tasteless
Juice when pure
Pancreatic lipase triglycerides  obtained from distillates of wood, coal,
Juice a-amylase starch or now usu; petroleum or shale oil
trypsin proteins  complex mixture of hydrocarbons
chymotrypsin proteins principally of the methane series
 resistant to water & water vapor
SANDIG, LYNETTE
  chemically inert Inorganic Matter
 stimulates prod. of saliva since saliva
is 99% water & paraffin wax is resistant Test for Chlorides: Chloride reacts with AgNO3
to it to yield a white ppt.

Approximate pH of Saliva with a pH paper (8.0) Color of ppt: White

Name of ppt: AgCl (silver chloride)
 saliva has a pH bet. 6.5-7.5 (salivary Equation: Ag+ + Cl-  AgCl
amylase functions best at this range)
 saliva is slightly alkaline owing to the Test for Inorganic Phosphates: Phosphate
presence of sodium bicarbonate residues of phospholipids will react with
ammonium molybdate, (NH4)2MoO4, in HNO3; a
Anywhere, enzyme actions depend on the pH yellow ppt is formed.
of its surroundings. For example, salivary
amylase funcs. best at pH 6.5-7.5, but the Color of ppt: Yellow
contents of the stomach are very acidic (1.0-
Name of ppt: Ammonium
3.5 pH). The acidic condition inactivates the
Phosphomolybdate
salivary amylase & no carb digestion takes
(NH4)3PO4(MoO3)12 + 12H2O
place in the stomach.
Equation: PO43- + 3NH4+(aq) + 12MoO2-
(aq) + 24H+ 
Test for Mucin
(NH4)3PO4(MoO3)12 + 12H2O
Mucin
Test for Sulfates: Sulfate reacts with BaCl2 to
 sticky material w/c helps bind food yield a white ppt.
particles together & lubricate them to
Color of ppt: White
assist swallowing
 heavily glycosylated protein found Name of ppt: Barium Sulfate (BaSO4)
extensively in salivary secretions Equation: Ba2+ + SO42-  BaSO4
 can be ppt-ed out of saliva by adding
dilute acetic acid Test for Calcium: Calcium reacts w/ (NH4)2C2O4
 ppt formed in the experiment: mucin to yield a white ppt.
 increases the viscosity of the fluids in
Color of ppt: White
saliva because of the highly extended &
hydrated structure Name of ppt: Calcium Oxalate (CaC2O4)
Equation: Ca2+ + C2O42-  CaC2O4
Test for Proteins (Biuret Test)
Calcium helps maintain or stabilize a compact
Proteins + CuSO4  violet-colored solution conformation of the molecule of α-amylase &
also helps maintain an enzymatically active
 Biuret complex: cpd. containing two conformation.
–CONH2 joined directly together or
through a N or C atom Test for Thiocyanate (FeCl3 Test): Thiocyanate
 if test sample is reacted with alkaline reacts with FeCl3 in acidic medium to yield a
CuSO4 and if it contains proteins in it, red-colored product.
then a violet color coordination complex
is produced Color of ppt: pale yellow solution
 the color intensity is dependent on the red ppt
# of peptide bonds present Equation: 2Fe3+ + 12CNS- 
2Fe(CNS)63-

SANDIG, LYNETTE
Thiocyanate is the metabolite of cyanide & the  maltose: colorless
end prod. of the detoxification of cpds.
containing cyanides thru a reaction catalyzed Digestion of Starch Paste
by the enzyme rhodanase.
Starch  Amylodextrin  Erythrodextrin 
For confirmation: Achroodextrin (colorless)  Maltose (colorless)

Addition of HgCl2: Fehling’s Test for Reducing Sugars

Hg2+ displaces Fe3+ to yield coloreless mercuric Color of ppt: blue solution
thiocyanate brick-red ppt
Equation: reducing sugar + CuSO4 
Fe(CNS)6 + Hg 2+
 Hg(CNS) 4
2-
+ Fe 3+
(yields) Cu2O(s) brick red ppt
colorless Reaction due to: Cu2+
Everyone has thiocyanates in their saliva. Its
sources are: smoking & cyanide-containing Phenylhydrazine Reaction
food.
 monosaccs prod. after salivary digestion
Digestion of Starch Paste of sarch will react with phenylhydrazine
reagent to prouce Osazone crystals
 Starch: forms a violet complex with I2
 possible bc of the helical form of a Benedict’s Test
mol. of amylose or amylopectin
 when starch is digested by saliva, it is  for reducing sugars
broken down into smaller saccharide  aldehydes & ketones are oxidized by
units and can no longer form the violet alkaline copper sol’n.
product when salivary digestion is  the cupric ions are reduced to cuprous
completed and brick-red ppt forms
 longer (minutes) = fainting of violet  Positive: all sugars, but not sucrose
complex (nonreducing sugar)

Achromatic Point Influence of Free Acid

 point where starch breakdown is  FA hinders the digestion of starch by

complete and starch has been the salivary amylase
converted to dextrin & smaller  higher conc. of dilute HCl = greater
monosaccs & disaccs intensity of blue color
 point at which the reaction mixture no  the more dilute the acid, the salivary
longer gives a color change with I2 digestion will proceed
solution
Experiment 12: Urinalysis
 activity of SA is enhanced by the Cl ion
which is present in saliva Gastric Digestion
Salivary Amylase Stomach
 cleaves α-1,4 linkages in starch  normal gastric juice is a light-colored,
 gives diff. colors in I2 thin fluid of high acidity, 0.25-0.50% HCl
 amylodextrin: purple  gastric juice is 97-99% water; remainder
 erythrodextrin: red consists of mucin and inorganic salts,
 chroodextrin: colorless

SANDIG, LYNETTE
 the digestive enzymes pepsin & rennin Transparency/Turbidity
and a lipase
 gastric pH (optimum) = 1.0-2.0 Cloudy urine: may be evidence of phosphates,
 Changes in pH of normal stomach may urates, WBs, bactria, epithelial cells, or fat
lead to conditions of hypoacidity (assoc.
Odor
with stomach/gastric carcinoma) and
hyperacidity (assoc. w/ ulcers)
Normal fresh urine has slight aromatic odor;
upon standing may have the odor of ammonia
Small Intestine
(drugs, food, and patho conditions change
 most important part: duodenum odor of urine).
 3 juices enter the intestine in the
Urine  NH3
duodenum: pancreatic juice, intestinal
(hydrolyze)
juice, and bile
Specific Gravity: 1.015-1.025
Most favorable pH for tryptic activity: 8.3
Less than 1.015 (24hr specimen): indicates
 TT with pancreatic extract & intestinal
extract showed fastest digestion of
excess fluid intake, diabetes insipidus, or
chronic renal failure
eggwhite (pancreatic extract w/c contain
pancreatic trypsinogen needs intestinal
More than 1.025 (24hr specimen): may indicate
extract w/c contain enterokinase to be
limited fluid intake, dehydration, fever, kidney
reactive)
inflammation
 optimum temp is 400C
Vol. excreted per day: 0.5-1.5L –can be
Urine
increased w/ ingestion of large amount of
lipids; decreased by loss of water by
Normal: Color varies from yellow to amber. It
perspiration
is due to pigment urochrome w/c is a cpd.
urobilin & a peptide. Upon standing: darkens
Organic Constituents of Urine
due to release of bilirubin
Urea/Uric acid: end prod. of protein
Abnormal: varies from nearly colorless
metabolism which take place in the liver
depending on the pathological condition
Protein is metabolized and produces urea
Milky: indicates presence of WBCs, bacteria, or
which goes into the blood and to the kidney
fat
where it is secreted by urine.
Reddish-amber: indicates urobilinogen which is
Urea Cycle: ornithine, citruline, arginine,
prod. in the intestine by action of bacteria on
ornithine
bile or porphyrin w/c may indicate evidence of
liver disease, Addison’s or other conditions The uric acid level increased in gout. The
quantity of uric acid in human urine is
Brownish-yellow/green: may indicate bile
generally 0.5 to 1.0 gm per hour.
pigments
Adenine (+H2O)  Hypoxanthine (with oxidase)
Red to smoky brown: may indicate blood &
 Xanthine (with oxidase)  Uric acid
blood pigments

SANDIG, LYNETTE
Murexide Test Indican

The uric acid is oxidized to dialuric acid &  potassium indoxyl sulfate which is easily
alloxan. These 2 subs. condense to form oxidized to indigo blue
“Alloxantin” which reacts with ammonium  comes from putrefactive decomposition
hydroxide to form purpuric acid. of tryptophan in the intestine

Uric acid  Dialuric acid + Alloxan (thru Obermeyer’s Test

condensation)  Alloxan (+NH4OH)  Alloxatin
purpuric acid/Ammonium purpurate/Murexide  colorimetric method used to test for
intestinal putrefication
The deposit turns bluish violet due to
formation of sodium purpurate. Indican (hydrolysis)  Indoxyl + H2SO4
(oxidation)  Indigo blue in the CHCl3 layer
Creatinine
Positive test: Chloroform layer becomes
 The quantity of creatinine excreted in greenish blue due to presence of Indican.
urine by normal healthy adults is from
1.2-1.7 gms per hour. The excretion Pathological Constituents in Urine
depends partly on the amount of
 present only in certain cases of disease,
creatinine in the diet—excreted in urine.
dysfunctioning of liver & kidney, or any
 The amount excreted is related to
other metabolic disorders in the body
muscular dvpt. to be higher in men than
in women & children.
Main Abnormal Constituents in Urine
 Creatine (-H2O)  Creatinine
 Protein (albumin)
Nitroprusside
 Sugar (glucose)
 Ketone bodies
 light-sensitive, breaks down in sunlight,
 Blood
producing cyanide
 Bile salts & pigments
contraindicated in patients with renal
failure
Pathological Organic Constituents
Nitroprusside Test
1. Albumin
A test for cystinuria in w/c sodium cyanide &
Albuminuria: presence of albumin in urine
then nitroprusside are added to urine.
Proteinuria: presence of protein in urine & may
Picric Acid
indicate a kidney damage or nephritis
 chem cpd. formally called 2,4,6-
2. Globulines
trinitrophenol (TNP)
 a yellow crystalline solid and is one of Sometimes found in urine; together with
the most acidic phenols albumin since they are larger molecules, their
presence would show more extensive kidney
Picric Acid Test
damage.
Positive result: deep red orange colored
Proteins are precipitated with HNO3 to form a
solution is obtained
fluffy ring at the zone of contact.

SANDIG, LYNETTE
3. Sugar

Glycosuria: presence of sugar in urine

Glucosuria: presence of glucose in urine

Occurrence of glucose may result from

excessive carb intake or diabetes mellitus.

4. Bile

When not properly excreted thru the intestinal

tracts (ex. obstructive jaundice) 
accumulation of bile pigments, bile acids, and
bile sals  excreted thru urine (yellowish
green to brown)

Boiling Test

Dvpt. of turbidity implies proteinuria. If ppt

disappears after addition of 3-4 drops of very
dilute acetic acid, the ppr is phosphate.

Nitric Acid Test

Positive Test: gives a white ring at the junction

of fluids

Benedict’s Test for Glucose

Cupric ion is reduced to Cu2O (cuprous oxide).

If only ≤ 0.1% or less glucose is present, the
ppt may not appear until cooling. Appearance
of blue to red color confirms presence of
glucose in urine.

SANDIG, LYNETTE