The Manual Book of Biochemistry Experiment

8/12/2019 The Manual Book of Biochemistry Experiment

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[THE MANUAL BOOK OF

BIOCHEMISTRYLABWORK] December 16, 2013

SEMARANG STATE UNIVERSITY |FMIPA

THE MANUAL BOOK OF

BIOCHEMISTRY

LABWORK

by

Biochemistry Lecturer Team

BIOORGANIC CHEMISTRY LABORATORY

FACULTY OF MATHEMATICS AND NATURAL SCIENCE

SEMARANG STATE UNIVERSITY

2013


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THE MANUAL BOOK OF

BIOCHEMISTRYLABWORK

by

Biochemistry Lecturer Team

BIOORGANIC CHEMISTRY LABORATORY

FACULTY OF MATHEMATICS AND NATURAL SCIENCE

SEMARANG STATE UNIVERSITY

2013


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PREFACE

Thanks to God for his blessings and mercies so that the lecturer team of

biochemistry successfully finished this manual book of biochemistry labwork.

This manual book is the final editors from the previous manual book of

biochemistry which published by lecturer team of biochemistry KBK bioorganic. Some

correction toward some parts is the result of evaluation based on the previous

experiment of biochemistry on last semester. Correction is done by creating the better

goal standard quality and relevance for the students, so that they have basic competence

to arrange research in biochemistry field.

In this manual book, also completed by the rules of experiment, the format of

report, basic theoretical of the experiment, working steps and assignments which must

be done and reported together with experiment book report.

Lecturer team already tried to give the best manual book in every edition, with

some correction annually related to up to date research so gives maximum benefits for

the students whom done the experiment.

Finally, arranged team want to say thank you for everyone who helps for

creating this biochemistry manual book. We are waiting for the critics and suggestion

from the reader especially the students to be the better.

Semarang, December 2013

Bioorganic laboratory

Chemistry department FMIPA UNNES

LECTURER TEAM OF BIOCHEMISTRY


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TABLE OF CONTENTS

PREFACE......................................................................................................................... ii

TABLE OF CONTENTS ................................................................................................iii

LECTURE PREFACE..................................................................................................... iv

THE LAB WORK REPORT ............................................................................................ v

EXPERIMENT I VITAMIN ............................................................................................ 1

EXPERIMENT II BLOOD .............................................................................................. 5

EXPERIMENT III URINE ............................................................................................. 11

EXPERIMENT IV DETERMINATION OF PROTEIN CONTENT

SPECTROPHOTOMETRICALLY ............................................................................... 16

EXPERIMENT V DETERMINING THE LEVELS OF GLUCOSE IN THE URINE

BY TITRIMETRIC ........................................................................................................ 19

EXPERIMENT VI ENZYME ........................................................................................ 23

EXPERIMENT VII IDENTIFICATION OF HUMAN CHORIONIC

GONADOTROPIN IN URINE ...................................................................................... 28

EXPERIMENT VIII BLOOD GROUP TEST ............................................................... 30

EXPERIMENT IX STRUCTURIZED JOB 1 (ENZYME) ........................................... 32

EXPERIMENT X STRUCTURED TASK II - CEP (CHEMOENTERPRENEURSHIP)

........................................................................................................................................ 33


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LECTURE PREFACE

LECTURES COURSE

a. Pre-testb. Practical workc. Students submit the provisional report after done the lab work and validated by

a lecturer or assistant that responsible

d. Students submit the lab work report result a week aftere. Presentation as the final project that scheduled by lecturer

THE RULES

The important things should be pay attention by students in the biochemistry lab work

are:

a. Prepare the laboratory coat before doing the lab work, napkin that easily canabsorb water, work book and the manual book of Biochemistry labwork.

b. The equipment that is used in the beginning and end must be in the cleancondition. If the equipment is broken, the group or class that is pertinent must

be responsible.

c. Prohibit to throw the rubbish/waste/substance in the washing vesseld. The result of lab work must be shown to assistant or lecturer along with

observation result to validate the propriety. The observation result will be

reputed done if on the each group provisional report book has been valued and

signed by assistant or lecturer

e. The lab work report must be submitted to the assistant/lecturer on the nextmeeting. The student that doesnt submit the report cant follow the pre-test and

lab work.


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THE LAB WORK REPORT

In order to make the last report of one kind, then the format made as :

Name :

NIM :

Major/Study Program :

Group :

Date of lab work :

The lab work name :

A. OBJECTIVES

Explain your objective of doing the lab work

B. BASIC THEORY

Described the theory that base the lab work you do in brief with references

C. EQUIPMENT AND MATERIALSMention the equipment used included glass tool, instrumentation tool and the other

tools. Mention materials used as lab work done

D. PROCEDURE

Show the procedure in the block diagram or table

E. OBSERVATIONAL DATA

Note the result of observation by filling the observation sheet or work book. Thenote is all the datas that can be observed during the lab work process included

qualitative and quantitative data.

F. DISCUSSION

Study the experimentation result refers to the basic theory. The things that need to

be explained are: explanation about the procedure, substance adding function,

theory suitability with practice, reaction equation, rendemen, result purity that is

LAB WORK REPORT


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shown by physics and chemistry characteristic. The other explanations addition

that is relevant with the experimental objectives.

G. CONCLUSION AND SUGGESTION

Make the conclusion from the experimentation and suggestion if there are

something needed to be repaired better

H. REFERENCES

Described the name book that refers to make the lab work report.

Name of writer. publication year. Book Tittle, Volume, edition.

publisher, publisher town.

The sum of book that can be referenced are (min) 3 reference books.

Semarang, date month year

Known,

Lab work Lecturer Student

. .

NIP NIM


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EXPERIMENT I

VITAMIN

A. OBJECTIVESAfter performing this experiment, students are expected to:

1. Identify vitamins A, D, E, B1, B2, B6, and C qualitatively with the colorreaction.

2. Describe the chemical reactions to be the basis for the identification of vitaminsin food.

B. BASIC THEORYVitamin is an organic compound that is needed to survival and function

normally. Body's need for vitamin is relatively small at around a few micrograms

to a few grams. However, vitamin must be present in the food, because the body

can not synthesize their own except the few vitamins such as vitamin K.

Deficiency or absence of vitamins in the body will cause to disruption of the

processes of metabolism and other vital processes. This is because most of the

vitamin plays a major role as a coenzyme of the enzyme that can catalyze chemical

reactions in the body. Vitamin based on solubility can be classified in two groups,

namely:

1. Vitamins are not soluble in waterVitamins are not soluble in water are: Vitamin A, D, E, and K

2. Water-soluble vitamins water soluble vitamins are: Vitamin B Complex(B1, B2, B6) and vitamin C.

C.

EQUIPMENT AND MATERIALS TOOLS:1. Test tube2. Beaker glass3. Universal Indicator4. Tripod5. Screen6. Spiritus Burner


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MATERIALS:

1. Fish Oil2. Capsules Nature3. Can-Price reagent4. Alcohol 95%5. Nitric acid 1 N6. H 2O27. 1N NaOH

8. Vitamin B1 Tablets9. Isobutanol10. Potassium Fericyanida11. Vitamin B complex tablets12. Aquades13. Vitamin C14. Fehling reagent A15. Fehling reagent B

D. PROCEDURES TEST VITAMIN A

1. Prepare fish oil samples and Can -Price reagents consisting of 10 ml CHCl3and 5 grams SbCl3.

2. Prepare samples of vitamin A to demolish gradually the tablets by takingvitamin A in the dark place then dissolved in fish oil samples.

3. Add 1 ml sample of vitamin A in fish oils into a clean test tube and dried.4.

Add 1 ml of Can-Price reagent.

5. Observe the color changes.

TEST VITAMIN D1. Prepare fish oil samples and reagents Can -Price consisting of 10 ml CHCl3

and 5 grams SbCl3.

2. Enter 1 ml sample of fish oil into a clean test tube and dried.3. Add 5 drops of H2O2 then heat until bubbles arise again but not boiling.


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4. Chilled with running tap water.5. After cool add 1 ml of Can-Price reagent.6. Observe the color changes.

TEST VITAMIN E1. Prepare samples of nature E or vitamin E samples provided.2. Cut vitamin E capsule or sample, insert it into the test tube.3. Add 0.5 ml of 95% alcohol and shake well.4. Add 1 ml of nitric acid, observe the color changes.

TEST VITAMIN B11. Enter vitamin B1 tablets into a clean test tube, add 5 ml distilled water and

shake until dissolved.

2. Take 1 ml of the solution to enter into another test tube, add a few drops of 1N NaOH until alkaline (pH-10)

3. Beat thoroughly, then add a solution of potassium fericyanida and shakeagain.

4. Observe the color changes, add the isobutanol to reinforce of color5. Check the color that occurs in the lining of isobutanol.

TEST VITAMIN B21. Dissolve tablets of vitamin B complex in 2 ml of distilled water.2. Take the solution and then add 1 ml alcohol 95% to almost tube (+, case

yellow)

3.

In another test tube, take 1 ml of vitamin B complex solutionthen add 1ml of2M AgNO3solution (+, if it occurs in red cherry).

4. Shake well and observe the color changes and observe this fluorescence.

TEST VITAMIN B61. Dissolve tablets of vitamin B complex into 2 ml of distilled water.2. Take a vitamin B complex tablet solution, enter into clean reaction tube.3. Add a few drops of a solution of FeCl3.


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4. Observe the color changes, check its fluorescence from the solution.

TEST VITAMIN C1. Dissolve sample of vitamin C into distilled water.2. Take 1 ml of vitamin C, add 3ml Fehling reagent Fehling (a mixture of

reagent Fehling A and Fehling reagent B) just as much.

3. Shake and then cooked in a water bath.4. Observe the color changes.

E. QUESTIONS AND TASKS1. There where and what happens when the body lacks vitamin D?2. Write the structure and the other name of vitamin D!3. Present in foodstuffs, and what happens if the body lacks vitamin C?4. Write the structure of vitamin E!5. Mention the function of vitamin E in living!6. Contained in any food, and what happens when the body lacks vitamin B1?7. Write the structure and another name for vitamin B1!8. There where and what happens when body lack of vitamin B6?9. Write the structure and other name of vitamin B6!10. What is the function of vitamin B6 in protein anabolism?11. Write the structure and another name for vitamin C!12. Write down the oxidation of vitamin C (reaction with Fehling reagent)!13. Why the vitamin C is used as an antioxidant?14. Why the Fehling reagent must be separated in the form of Fehling A and

Fehling


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EXPERIMENT II

BLOOD

A. OBJECTIVES1. Understanding of the main component that contained in blood.2. Skill of making blood plasma and blood serum.3. Skill in doing test to blood plasma, test of Fe in hemoglobin, test of Albumin

and globulin in blood serum and test of non protein substance in blood serum.

B. BASIC THEORYBlood is tissue that cycle in closed blood vessel system. Commonly, blood is

alkaline with pH is about 7.36, as oxygen transportation equipment and other

substances, organize constant reaction, regulation, and protector of infection. Blood

is divided into solid cells (contain of erythrocyte leucocytes) and plasma (consist of

fibrinogen and serum).

Blood components that have assignment to transport oxygen is pigment with

chromoproteida structure, contain of globins protein and hem color substance. Hem

is derivate of protophorpin with Fe2+ ion included prosthetic group. Hemoglobin (

Hb ) is blood color substance composed of group prosthetic protohem ( ferro-

protophorfirin ) and globins protein. Protohem in every animal is same, only its

protein. Blood is coagulated easily, to prevent it add anti-coagulated substance

such as oxalate compound, citrate or EDTA etc.

C. MATERIAL AND EQUIPMENTS

BLOOD

SOLID PLASMA

ERYTHROCYT

LEUCOCYT

FIBRINOGEN SERUM


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EQUIPMENTS

1. Microscope2. Tripod3. Gauze4. Cuvet Centrifuge5. Centrifuge6. Reaction tube7. Filter paper8. Porcelain cup9. Stirrer

MATERIALS

1. Blood2. Acetic Acid Glacial3. Dilute NaCl (NaCl 0.9%)4. Zn(OH)25. CaCl2 20%6. Dilute HCl7. K4Fe(CN)6 solution8. Dilute Potassium Rodanide9. Concentrated Nitric Acid10.Saturated of Ammonium Sulfate11.Solid of Ammonium Sulfate12.Glyserol13.

Solid of Sodium Carbonate

14.CH3COOH 2%15.Dilute HNO316.Dilute AgNO317.Dilute CuSO418.Dilute BaCl219.Dilute Ammonium oxalate 10%20.Red chlor phenol indicator


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D. PROCEDURE Making Blood Plasma and Blood Serum

1. Making Blood Plasmaa. Take 10 mL fresh blood, enter it in to cuvette centrifuge, add 1 mL

Zn(OH)25%.

b. Centrifuge it about 1015 minutes until precipitate occur.c. Filter the precipitate. Filtrate is plasma, the precipitate is packed cell.

2. Making Blood Seruma. Take 2 mL blood plasma and enter it in to reaction tube.

b. Dilute it with solution of NaCl 0.9% 30 mL.c. Add it 2 drops CaCl2 20% until gel precipitate occure.d. Filter it with filter paper.e. Filtrate is serum, the precipitate is fibrinogen.

Plasma Blood Test1. Blood Crystal Test ( Teichman)

a. Take a drop of blood, put it in to object glass.b. Drop it with a drop of a mixture of asetic acid glacial and dilute NaCl

solution.

c. Close it with closing glass, and then heated the object glass carefully withsmall fire until the solution boiled.

d. Observe the changes happened.b. Let the object glass cool.c. Paint the form of crystal that you observed carefully using microscope.

2.Fe in Hemoglobin Testa. Take 10 drops of blood, put it in the porcelain cup.

b. Heat it carefully until all fired up.c. Prepare a test tube contain a mixture of dilute hydrochloric acid and a

little concentrated nitric acid.

d. Pour that mixture into porcelain cup, heat while stir with stirrer glass untilall the ash dissolved.

e. Take the clean solution, put into 2 clean testtube.


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f. Test the first reaction tube with potassium ferrocyanide solution.g. Test the second reaction tube with potassium rodanide solution.h. Observe what happened and write the result.

Compound in Blood Serum Test1. Albumin and Globulin Test

1.1 Albumin Testa. Put 2 ml of blood serum into test tube, add 2 ml of saturated

ammonium sulfate.

b. Shake the test tube strongly, let the white precipitate of albuminformed.

c. Filter it with filter paper to obtain filtrate and precipitate.d. Wash the precipitate that obtained with half saturated ammonium

sulfate.

e. Take the precipitate that obtained, put it into a clean test tube.f. Add aquades, shake it carefully.g. Observe the precipitate, dissolved or not.a. Globulin Testa. Next, the filtrate that we obtained in step (c), put it into a clean test

tube.

b. Add excess solid ammonium sulfate, shake strongly until thesolution saturated with ammonium sulfate (until formed

precipitated).

c. Filter and move the precipitate that obtained into test tube and thendissolved with aquades.

d.

Observe the precipitate, dissolved or not.

2.Non Protein Substance Testa. Take 5 ml blood serum and put into a test tube.

b. Add 10 drops aquades, and then heated.b. Add CH3COOH 2% drop by drop until formed coarse sediment

suspension.

c. Filter and collect filtrate in clean test tube.


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d. Adjust pH filtrate become 5,4 (use red chlore phenol indicator, observeuntil light red color disappear).

e. Heat and filter if needed.

2.1 Ca2+Test (calcium ion)Take 1 ml filtrate in step e, put into test tube, add 1 ml ammonium

oxalate 10%. Observe and write what happened.

2.2 Cl-Test (chloride ion)Take 1 ml filtrate in step e, put into test tube, add 1 ml mixture of dilute

argentum nitrate and dilute nitric acid. Observe and write what

happened.

2.3 Glucose TestTake 1 ml filtrate in step e, put into test tube, add 2 drops of glycerol and

a little of solid sodium carbonate. Add 2 drops of dilute cuprisulfate ,

heat several minutes ( or use fehling reactant). Observe and write what

happened.

2.4 SO42-Test(sulfate ion)Take 1 ml filtrate in step f, put into test tube, add several drops solution

of dilute barium chloride, Observe and write what happened.

E. Question and task1. What purpose of heating with glacial acetate on experiment of blood crystal

test?

2. What function of dilute NaCl solution on experiment of blood crystal test?3. Write down reactions that may happen from Fe test in haemoglobin?4. What purpose of heating blood until burned perfectly on Fe test in

haemoglobin?

5. What function of mixing dilute HCl with concentrated HNO3 in experiment ofFe test in haemoglobin? What is the name of mixture?

6. Mention protein component that exists in blood serum!


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7. Explain in few words about uses of concentrated ammonium sulphate solutionand ammonium sulphate solid on test of albumin and globulin in blood serum!

8. What the meaning of salting out?9. Write down the reactions that may happen on experimental test of non protein

substance in blood serum!

10. Which part of blood serum that forms crude suspension?11. Why in blood serum contains glucose and amino acid?


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EXPERIMENT III

URINE

A. OBJECTIVESAfter did this experiment, observers are expected can :

1. Understand the components in urine2. Doing this experiment skillfully

B. BASIC THEORYNormal urine is called as excret substance which has odour and the color of

solution is yellowish. Most of it consists of water and the other components areammonium salts with chloride, phosphate, sulfate from sodium, potassium,

magnesium and organic substances form metabolism such as: uric acid, uric salts,

creatinine and ureum. Urine contains a little of pigmen from HB and amilase from

pancreas. Everyone can produces urine everyday (24 hours) about 1-1.5 litres.

C. EQUIPMENTS ANG MATERIALSEQUIPMENTS:

1. Test tube2. Water bath3. Glass stirer4. Filter Paper5. Beaker glass

MATERIALS:

1. Urine sample2. 2% of sodium carbonate solution3. Pp indicator4. Aquadest5. Dilute vinegar solution6. Soybean powder7. Picric acid8. 10% of NaOH solution


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9. Benedict reagents (are made of cupri sulphate solution and sodium citrate inalkaline Na2CO3anhydrate solution).

10. Concentrated nitric acid11. Diluted silver nitrate solution12. Concentrated ammonium hydroxide13. 2% of vinegar solution14. Potassium oxalate crystal15. Ammonium molibdic16. Potassium oxalate solution17. Diluted HCl solution

D. PROCEDURE Ureum test

1. Prepare two reaction tubes, one of them is filled with 2 ml of urine, and theother one is filled with 2 ml of aquadest.

2. Both of them are added with a drop of pp indicator and 2% of sodiumcarbonate solution until the color of the solution changes become pink.

3. Both of them are added with vinegar solution until acidic has reached (signedwith color changes of the solution, it becomes yellow solution), then heat it

until the temperature of the solution has reached 60C.

4. Soy powder is added slowly into it and shake it carefully.5. Observe the results, note the changes in each tube.

Amonium salts test1. Enter 2 ml of urine intotest tube.2. Add 1 drop of indicator PP and 2% sodium carbonatesolution till the mixture

color is red.

3. Boil it.4. Cover the test tube with filtering paper which moistened previously with

PP indicator.

5. Observe and note the result.

Creatinin Test


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1. Prepare 2 test tube.2. One of them filled by 1 ml of dilute picrate acid and 0.5 ml of 10%NaOH

solution, then shake it.

3. Divide mixture intotwo test tube.4. Add 3 mL of urine into a test tube and 3 mL of aquades into another test

tube.

5. Observe and note the result.

Test for non-nitrogen compounds1. Reducing Sugar Test

a. Enter 10drops ofurineintoa cleantest tubeb. Add 5ml ofbenedictsolutionc. Heatin a water bathfor 2 minutesd. Observe and note the result

2. Chloride Testa. Enter 1 ml of urine into a test tube

b. Add a few drops of concentrated nitric acid, then add a few drops of adilute solution of silver nitrate

c. Observe and note the result

3. Phosphate Testa. Enter 5 ml of urine into a test tube

b. Add 1 ml of rather concentrated ammonium hydroxide and heat itc.

Filter the precipitation formed and wash it with water

d. Then the precipitate dissolved into hot vinegar 2% in a test tubee. Add 1 drop of concentrated nitric acid and a few drops of a solution of

ammonium molybdate

f. Heat for a while, observe and note the result

4. Calcium Testa. Enter 2 ml of urine into a clean test tube


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b. Add 1 ml of concentrated ammonium hydroxide and heat itc. Filter the precipitation occur and wash it with waterd. Then the precipitate was dissolved in hot vinegar 2% into a test tubee. Add a few drops of a solution of potassium oxalatef. Observe and note the changes occur

5. Sulphate Testa. Enter 1 ml urine into a clean test tube

b. Acidify with a few drops of dilute hydrochloric acid solutionc. Add 1 ml of dilute barium chloride solutiond. Observe and note the changes occur

6. Ketone Object Testa. Enter 2 ml of urine into a test tube

b. Add with an excess of solid ammonium sulphate and shake it firmlyc. Add 2 drops of new dilute sodium nitro prussided. Add 1 ml of ammonium hydroxide, let it till 2 minutee. Observe and note the changes occur

E. QUESTION AND TASK

1. Write down the reaction that may occur from urea test trial2. Are the two test tubes in the urea test showed the same results?3. What is the function of soybean powder on experimental urea test?4. In the test ammonium salts, whether at the end of the rod raised red? If yes,

please explain why this happens?

5. Write the reactions that may occur in ammonium salts test6. Write theformulaofcreatinineandpicricacid7. Writereactions thatmayoccur increatininetesttrial8. Mentionedsugarsthat can reducesolutionbenedict9. write the reaction that occurs from reducing sugar test10. write the reaction that occurs fromchloridetesttrial


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11. in the experiment chloride test, predict the changes occurs if added to the testtube an excess ammonium hydroxide?

12. Write down thechemical reactionsthat mayoccur inthe testtrialsof calcium13. What happens if in calcium test, we use of dilute of sodium sulphate solution14. Write down thechemical reactionsthat mayoccur inthe sulphate test15. in the test sulfate, what The changes occurs when urine doesnt make

acidified first?

16. Writethe chemicalreactionsthat mayoccur intheketone object test17. Areketone object testcan distinguishbetweenaldehydesand ketones?18. Write thestructure ofketonebodiesandmention the name19. What willhappenif your bodyexcessof ketonebodies/ ketone object?


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EXPERIMENT IV

DETERMINATION OF PROTEIN CONTENT

SPECTROPHOTOMETRICALLY

A. OBJECTIVESAfter following this experiment, students are expected to:

1. Understand the using of spectrophotometer as a tool to analyze the proteincontent.

2. Explain the basic principles of the using of spectrophotometer in the analysis ofprotein content.

3. Use spectrophotometer skillfully to determine the protein content.

B. BASIC THEORYDetermination of protein content by biuret based on the measurement of light

absorption spectrophotometer by a complex bond with a purple color. It occurs

when proteins react with copper in alkaline environments.

C. EQUIPMENT AND MATERIALSEQUIPMENT:

1. Spectronic 202. Cuvette3. Pipette4. Test tube5. Erlenmeyer6. Beaker Glass7. Measuring flask8. Volume pipette

MATERIALS:

1. Protein samples2. CuSO4.5H2O3. Sodium potassium tartrate


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4. Aquadest5.NaOH 10%6. Pure albumin serum7. Casein8.NaOH 3%

D. PROCEDURE Making Biuret Reagent

Dissolve 1.5 grams of CuSO4and 6.0 grams of sodium potassium tartrate

(Na.KC4O6.4H2O) in approximately 500 ml of distilled water in a 1 liter

measuring flask. Then add 300ml of 10% NaOH and shake. Finally, adddistilled water up to the line. Blue solution can be stored for a long time. When

making unfavorable deposition can occur black or red. Such reagents should not

be used.

Preparation of Standard Solution ProteinMake a solution of pure albumin serum or casein in distilled water to yield

1.25 to 10 mg / ml. To facilitate solubility add a few drops of 3% NaOH.

Preparation of Calibration Curve1. Prepare a standard solution with a concentration of 1mg/ml protein, 2 mg /

ml, 3 mg / ml, 4mg/ml, 5 mg / ml.

2. Insert standard solutions into a test tube 5 pieces (each 1 ml). Add 4 ml ofbiuret reagent.

3. Shake and let it for 30 minutes at room temperature.4. Put the solution into the cuvette, measure the absorbance using spectronic 20

at a wavelength of 540 nm.

5. Use the blank solution that contains a mixture of 1 ml of distilled water and4 ml of biuret, that were reacted for 30 minutes at room temperature.

6. Make a calibration curve based on the absorption of standard proteinsolution.

Measurement of protein samples.


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1. Prepare the protein sample to be analyzed.2. Add 1 ml of the sample into a clean test tube, add 4 ml of biuret reagent.3. Shake and let it for 30 minutes at room temperature.4. Enter into the cuvette, measure the absorbance using spectronic 20 at a

wavelength of 540 nm.

5. Use the blank solution that was used in the calibration curve.6. Use the calibration curve obtained to calculate the protein content in the

sample.

E. QUESTIONS AND TASKS.7.

Explain the legal basis of the analysis using spectrophotometry tool.

8. Describe the work flow of spectrophotometer instrument. Describe the workflow of visible spectrophotometer instruments.

9. Explain the advantages and disadvantages of the using of the biuret methodwith analysis spectronic 20 for protein content.

10.Why the reaction in this experiment is called biuret reaction?


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EXPERIMENT V

DETERMINING THE LEVELS OF GLUCOSE IN THE URINE BY

TITRIMETRIC

A. OBJECTIVESAfter following this experiment students are expected to:

1. Understand the basic principles of analysis procedures and levels of glucose inthe urine

2. Detrmine glucose levels in the urine analysis using titrimetric method skillfully

B. BASIC THEORYNormal rates of glucose in the urine per 24 hours at 0.5 to 1 gram.

CuSO4alkalis in hot condition can be reduced by glucose to glucosal and

cuproxide, which precipitated the red brick. Blue color of the cupric salt solution

will dissappear when CuSO4 changed to Cu2O. These compounds can interfere

with the end point of the titration. To prevent the interference of potassium ferro-

cyanide is added to bind Cu2O into complex compounds soluble in water (brown).

Reaction occurs:

CuSO4(alkalis)

Cu2O

Cu2O + K4Fe(CN)6 Complex matter (brown)

C. EQUIPMENTS ANG MATERIALSEQUIPMENTS

1. Burner Methylated2. Tripod + Screens3. Pasteur Pipette4. Test tube5. Beaker Glass


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6. Buret7. Erlenmeyer

MATERIALS :

1. Glucose 0.5%2. Mixture of urine with 0.5% glucose (2:1)3. CuSO4solution (35 grams of CuSO4+ 5 ml concentrated H2SO4per liter)4. Potassium ferro cyanide 5%5. Signette solution (150 g K.Na tartrate + 90 grams of NaOH per liter) as the

catalyst.

D. PROCEDURE Titration using 0.5% glucose solution

1. Pipette 10 ml CuSO4enter into erlenmeyer2. Added 10 ml signette3. Added 5 ml of potassium ferro-cyanide4. Heat until boiling5. Titrated with 0.5% glucose solution above the fire6. Titrated until the color changes from blue to green and then to yellow.

Titrated dropwise until brown color

Titration using mixture of urin : 0.5% glucose solution (2:1)1. Do same steps no 1 to 42. Titrated with mixture of urin : 0.5% glucose solution (2:1) above the fire3. Titrated until the color changes from blue to green and then to yellow.


Titration using urine1. Do same steps no 1 to 42. Titrated with urine3. Titrated until the color changes from blue to green and then to yellow.


Example calculation:

Example:

Volume (ml)


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0,5 % glucose Mixture urin : glucose (2:1)

8.93 11.75

Titration results of weighting glucose = 0.4989 gramMethod 1:

Weight of glucose 0.4989 = 0.4989 gram/100ml x 8.93ml = 0.04455 gram Weight of glucose in urine glucose mixture (2 : 1)

Volume glucose 0.5% = 1/3 x 11.75 ml

So weight of glucose in urin glucose mixture is

= 1/3 x 11.75 x (0.4989 gram/100ml)

= 0.01954 grams

So urine volume is

= 11.75 ml - (1/3 x 11.75 ml)

= 2/3 x 11.75

= 7.833 ml

levels of glucose in the urine as penitir= (Weight of glucose Weight of glucose in urin glucose mixture ) x 100%) /

volume of urine

= (0.04455 - 0.01954) x 100%) / 7.837

= 0.3193%

Method 2:

Level of glucose in urine glucose mixture = level of 0.5% glucose = 0.4989grams

V1 x N1 = V2 x N2

11.75 x N1 = 8.93 x 0.4989

N1 = 0.3792%

Ratio of urine : glucose 0.4989 = 2 : 1Levels of glucose in urine glucose mixture = Glucose + glucose in the urine

within 0.4989%

0.3792% = 2/3x + (1/3 x 0.4989%)

0.3792 = (2x + 0.4989): 3

3 x 0,3792 = 2x + 0.4989

2x = 1.1376 - 0.4989


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x = 0.3194%

Note:

1. Titrating using pure urine is as a controller2. Do variation mixing of urine and glucose such as 4:3, 3:2; 3:1 etc.

E. QUESTIONS AND TASKS1. Draw the diagram for analysis of glucose in urine using titrimetric method.2. In this analysis, described the end point titration indicator so that the titration

can be done.

3. Why titration done during boiling? Explain!


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EXPERIMENT VI

ENZYME

A. OBJECTIVES1. Understanding enzyme function in the human body.2. Identify enzyme activities by the sympton and phenomenone which can be

observe.

3. be able to do critical experiment of enzyme activities.B. BASIC THEORY

Food which enter in digestive canal can not be used by the body if food can

not be absorbed through digestive canal wall and carried by blood to all over the

body. Food can be absorbed must micro molecules. Food digestion system from

macro molecules to micro molecules did by digestive canal which supported

digestive enzyme. Since food in the mouth, occured carbohydrate metabolism by

tooth and ptyalin enzyme become smaller saccharide molecule, such as

oligosaccharide moreover disaccharide and monosaccharide. whereas protein, fat

and other substance have mechanic metabolism, that are metabolism by tooth.Food which have metabolism in the mouth by physic or enzymatic, they will

entering to stomach.

In normal condition, food stay in the stomach for some hours while

hydrochloric acid (HCl) and pepsine scatter protein and carbohydrate become

oligopeptide and oligosaccharide. Then, digestive process occurs in small intestine

by enzymatic digestion. Digestive enzymes also secreted by pancreas, bile and

gland digestion and small intestine that finally it would become monosaccharide,

glycerol, fatty acid and amino acid. Furthermore result substance will be absorbed

through small intestine wall then enter to blood circulation and transfered to other

part body which is need together with vitamin and other mineral.

Based on the reaction, enzyme are grouped become 6 class, they are : (1)

oxide-reductase, (2) isomerase, (3) ligase, (4) liase, (5) Hidrolase, (6) transferase.

Enzyme is protein catalyst of a specific biochemical reaction. one enzyme product


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become another enzyme substrate. Enzyme catalytic activities is influenced by pH,

temperature, substrate level, enzyme level and inhibitor.

Enzyme mechanism reaction through forming complex of enzyme substrate

(ES). Therefor obstacle in reaction which use enzyme as catalyst can be occured

when substrate annexation(merge) in active side of enzyme have inhibition.

Molecule or ion which obstacle reaction is called inhibitor. Inhibition by inhibitor

as :

1. irreversible inhibition, generally caused by destruction process or modification afunctional group or more.

2. reversible inhibitiona. Competitive inhibition is inhibition caused there are molecule which similar

with substrate, so can able to make complexs, that is enzyme inhibitor

complex (EI).

b.Non competitive inhibition is inhibition which is not influenced by amountsubstrate and inhibitor consentration. example non competitive inhibitor is

heavy metals (Cu2+, Hg2+, Ag+).

C. EQUIPMENT AND MATERIAL :EQUIPMENT :

1. Test tube2. Filter paper3.balance4. Porcelain mortar5. Drop pipete6. Spititus burner7. Tripod8. Beaker glass

MATERIAL :

1. Saliva sample2. Amilum solution3. I2 solution in KI (lugol)4. Indicator universal


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5. gastric juices of animals6. aquades7. benedict reagent8. yeast bread9. clean sand10. toluene11. sodium carbonate solution12.buffer solution pH = 413. sucrose14. soybean filtrate15.pp indicator16. HgCl2 solution17. Urea 1%

D. PROCEDURE Activities Ptialin Test

1. Enter 3 ml of saliva into a clean and dry test tube2. Dilute with 6 ml distilled water3. Shake until homogeneous4. Filtered to obtain a clean filtrate5. Prepare 2 pieces of clean and dry test tubes6. Put 2 mL of starch solution into each test tube7. Add 2 ml of saliva on the tube 1, and add 2 ml of distilled water in tube 28. Shake each test tube9. Add 1-2 drops of Lugol's solution to both test tube, observe and record the

changes

Stomach Sap Test1. Take 2 pieces of tube, first tube with 2 ml of the liquid contents of the

stomach while the second tube filled with distilled water

2. Check the pH of each tube with universal idicator, record the pH obtained

Sucrase test


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1. Prepare enzyme with 1 gram of yeast bread, put in porcelain mortar2. Add 5 grams of clean and dry sand3. Add 10 ml of toluene, and then grinding the mixture until yeast bread smooth

and formed homogeneous mixture

4. Add 30 ml of distilled water form a homogeneous suspension5. Separate the liquid from filtrate6. Take the supernatant with a pipette7. Treated supernatant according to the following table:

No supernatant Buffer

acetate

Sucrose Amylum Aqua Na2CO3 Benedict Treatment

1 3 cc 1 cc - - 3 cc 5drops

5 cc heat

2 3 cc 1 cc 3 cc - - 5

drops

5 cc heat

3 3 cc 1 cc - 3 cc - 5

drops

5 cc heat

4 3 cc

boiling

1 cc 3 cc - - 5

drops

5 cc heat

5 3 cc

boiling

1 cc - 3 cc - 5

drops

5 cc heat

heated in a water bath 10 minutes.

8. Observe the color change.

Activity urease test in soybean.Ureum can analyzed by urease enzyme become CO2and NH3. Because of

NH3, so it can give red color in pp indicator. Enzyme work can be destroyed or

obstructed by hot temperature or by adding Hg. Reactant that used to urease test

is urea solution 1%. Soybean filtrate, pp indicator and HgCl2.

1. Prepare 3 clean reaction tube, then fill it with material as:

Tube Sample Adding HgCl2 Adding

ureum 5%

PP

I 1cc soybean - 5cc 1 drop


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filtrate

II 1cc soybean

filtrate

5 drop 5cc 1 drop

III 1cc soybean

filtrate that

boiled

- 5cc 1 drop

3. Put 3 reaction tube in water bath at 400C during 5 minute.4. Observe the color change!

E. Question and task1. Write the function of ptyalin in digestive process and what is enzyme included

in ptyalin?explain it.

2. how to make iodium solution? Explain it.3. why activity ptyalin test appear the color change?explain it.4. at gastric juice test , why do not use litmus paper?5. Why the color change at gastric juice test occurred in one of many reaction

tube? Explain it.

6. What the function of gastric juice in the body?7. What the enzyme that work in pH of gastric juice?8. In yeast bread there are enzyme. Mention and explain the function of it!9. What the function of buffer solution at sucrase test?10. At sucrase test, why the 3thtube do not happen the color change as in the 2nd

tube? Explain it.

11. At activity urease test, what the tube that happen the color change? Explain it.12. What the function of using HgCl2 in 2nd tube and mention the heavy metal

other?

13. Based on the reaction,what urease included in enzyme?14. Why enzyme can not work with the heavy metal?


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EXPERIMENT VII

IDENTIFICATION OF HUMAN CHORIONIC GONADOTROPIN IN URINE

A. OBJECTIVESAfter following this experiment, students are expected:

1. To understand the function of chorionic gonadotropin hormone in urine.2. To be competent to identify HCG in urine.

B. BASIC THEORYDuring pregnancy, there is HCG (Human Chorionic Gonadotropin) in the

urine of women. HCG is a hormone produced by the developing placenta tissueshortly after ovulation, which is 8 days after ovulation. HCG concentrations

continued to increase until reaching a peak at 60 days to 80 days of pregnancy or 8

weeks after the last menstrual period, and then drop in the next pregnancy.

After maternity, HCG levels will drop quickly and return to normal within a

few days. Determining the presence of HCG can be done by using immunologic.

By this way the pregnancy was detected on day 3-6 after a late period. In normal

pregnancy, the presence and increasing concentrations of HCG in a short time.

C. EQUIPMENT AND MATERIALEQUIPMENT:

1. HCG strip tester2. Beaker glassMATERIAL:

1. Urine sample of pregnant women2. Urine sample of not pregnant women

D. PROCEDURE1. Put the urine (first urine in the morning after waking up) in a clean container.2. Dip the HCG strip tester into the urine according to the boundary line for 3-6

seconds.

3. Lift the HCG strip tester, wait for 1-5 minutes.


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4. Reading results: when there are two pink lines, then the result is positive.When only one pink line appears, then the result is negative.

E. QUESTION1. Describe completely what is chorionic gonadotropin hormone?2. What media can be used to analyze HCG?3. Why there are difference in the results of HCG strip tester? Describe the

approach to chemical reaction!


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EXPERIMENT VIII

BLOOD GROUP TEST

A. OBJECTIVESAfter following this experiment, students are expected:

1. Understand the blood type of human2. Understand the agglutination process during blood test

B. BASIC THEORYBloods group test principle is agglutination process (clotting). The

agglutination condition requires the suitable between agglutinogen and agglutinin,

for example agglutinogen A with agglutinin alpha or there are titer / agglutinin's

rate that high enough to agglutinogen that will be clot. This is the basic that must

be done to avoid agglutination or clotting in blood transfusion.

Erythrocytes contain agglutinogen A and B also agglutinin alpha and beta.

From that concept, there are 4 kinds of blood group, blood group A, B, AB, and O.

This kinds of blood group based on the presence of agglutinogen A and or

agglutinogen B, called ABO system.

Table. Agglutinin and Agglutinogen

No Blood GroupAgglutinogen

(in erythrocyte)

Agglutinin

(in plasma)

1. A A Beta ()

2. B B Alpha ()

3. AB A and B -

4. O - Alpha and Beta

C. EQUIPMENTAND MATERIAL :1. Antiserum of person who already knows his/her blood group, which is

antiserum A (agglutinin ) test and antiserum B (agglutinin ) test.

Agglutinin beta was taken from a person who had blood group A, agglutinin

alpha was taken from a person that had blood group B.


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2. Lancet3. Object glass

D. PROCEDURE1. Take the blood from fingertip by using lancet.2. Drop blood on the two tips of object glass (right tip and left tip).3. Right drip given by antiserum A and left drip given by antiserum B.4. Flatten or swirled.5. See the sign of clotting (agglutination).

Agglutination Criteria Table

Blood Group Anti A test Anti B test Anti AB testA + - +

B - + +

AB + + +

O - - -

Information: + : Agglutination happened;

: Agglutination not happened.


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EXPERIMENT IX

STRUCTURIZED JOB 1 (ENZYME)

A. OBJECTIVESAfter do this experiment, students are expect to:

1. Found and understand the next procedure about the characteristic andapplication of enzyme.

2. Explain and presented about the procedure that was is founded (explain aboutthe technical procedure in laboratory)


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EXPERIMENT X

STRUCTURED TASK II - CEP (CHEMOENTERPRENEURSHIP)

A. OBJECTIVEAfter doing this task, students are expected:

1. To make PKM proposal with the benefit of natural source.2. To find the procedure of making CEP product from natural source.3. To explain by presentation or demonstration about the procedure of the product

making which technically found in the laboratory.

Documents

The Manual Book of Biochemistry Experiment