EXPERIMENT 7: Separation of Amino Acids by Thin Layer

CHROMATOGRAPHY

• SEPARATION TECHNIQUE

• 2 PHASES

  1. STATIONARY

  2. MOBILE (MOVING)

PRINCIPLES OF CHROMATOGRAPHY

• DIFFERENT COMPOUNDS, DIFFERENT SOLUBILITIES/ADSORPTION TO 2 PHASES BETWEEN WHICH THEY ARE TO BE PARTITIONED.

USES OF CHROMATOGRAPHY

• SEPARATION OF MIXTURES

• IDENTIFICATION OF CPDS

• DETERMINATION OF THE NUMBER OF COMPONENTS IN A MIXTURE

• DETERMINATION OF PURITY OF SAMPLE

STATIONARY PHASE

• FILTER PAPER

DEVELOPER(Mobile Phase)

• CLOSED VESSEL WITH SHALLOW POOL OF LIQUID

  • CAPILLARY ACTION OF ADSORBENT

• FUNCTION:

  • SELECTIVELY DESORB CPDS

    • TRANSPORT SOME CPDS FURTHER

• MAJOR CONSIDERATION: POLARITY

  • TOO POLAR: WILL CARRY ALL OF THE COMPONENTS BY THE SAME DISTANCE

    • POOR SEPARATION

  • TOO NON-POLAR: NONE OF THE SPOTTED MATERIALS WILL BE MOVED

    • POOR SEPARATION

  • HIGHER POLARITY, HIGHER DESORBING AND TRANSPORTING CAPABILITY

DEVELOPMENT

• STRENGTH OF BINDING OF COMPOUND TO FILTER PAPER:

  • ACID & BASES>AMIDES>CARBOXYLIC ACIDS>ALCOHOLS>KETONES~ALDEHYDES>HALIDES>ESTERS>ETHERS>UNSATURATED HYDROCARBONS>SATURATED HYDROCARBONS

  • STRONGER BINDING: SHORT DISTANCE TRAVELED

• FACTOR AFFECTING RATE IN MOVING UP THE FILTER PAPER

  • POLARITY OF:

    a. SP

    b. MP

    c. cpd

• INTERACTIONS AMONG SP,MP AND SPOTTED COMPOUNDS

1. CPD BOUND TO ADSORBENT

2. MP ATTRACTS AND DESORBS IT

3. CPD CONTINUALLY ADSORB AND DESORB (Equilibrium) AS SOLVENT MOVES UP THE PLATE

• IF CPD IS:

  • MORE SOLUBLE IN MP: EASILY DESORB

  • TIGHTLY BOUND TO CELLULOSE: SPEND MORE TIME IN SP

• GOOD SOLVENT IN CHROM: GIVES GOOD RESOLUTION(SEPARATION)

  • HAS DIFFERENT INTERACTIONS WITH DIFFERENT SAMPLE COMPONENTS DESPITE SIMILARITY OF COMPONENTS

  • USUALLY MIXTURE OF LIQUIDS

DEVELOPING THE CHROMATOGRAM

1. KEEP COVERED TO KEEP CHAMBER SATURATED WITH SOLVENT VAPOR

   • MAINTAIN IN EQUILIBRIUM AS SOLVENT MOVES UPWARD

   • ADSORBENT WETTED WITH SOLVENT WILL LOOK LIKE WET SNOW

2. WHEN ABOUT 5mm FROM THE OPPOSITE UNSOAKED EDGE, REMOVE PLATE

   • FORGETTING TO REMOVE THE PLATE WHEN SOLVENT REACH THE TOP OF THE EDGE WILL ENABLE THE SPOT TO CONTINUALLY ASCEND

   • Rf VALUES WILL THEN BE HIGHER

3. MARK SOLVENT FRONT WHEN STILL VISIBLE

4. ALLOW SOLVENT TO EVAPORATE

5. IF SPOT ALREADY VISIBLE, OUTLINE THE SPOTS

NINHYDRIN

• VISUALIZING AGENT

• TEST FOR: FREE AA(ALPHA-AMINO GROUP)& FREE AMINO GROUPS

• (+) RESULT: PURPLE COLOR

RETENTION FACTOR

• MEASURED DISTANCE TRAVELED FROM ORIGIN BY:

  • EACH MIGRATED SPOT

  • AND SOLVENT FRONT

• FOR TAILING(SPOT ELONGATED): MEASURE TO CENTER OF SPOT

Rf= dSOLUTE/dSOLVENT

• Rf IS VERY DEPENDENT ON:

1. MOISTURE CONTENT(HUMIDITY)

2. THICKNESS OF ADSORBENT

3. PURITY OF SOLVENT

4. OTHER FACTORS

• SAME Rf VALUES POSSIBLY SAME COMPOUNDS

• IDENTITIES OF COMPOUNDS ESTABLISHED BY SPOTTING KNOWN COMPOUNDS TOGETHER WITH UNKNOWN COMPOUNDS

• PRESENT AMINO ACIDS

  1. GLYCINE (non polar)

  2. TRYPTOPHAN (non polar)

  3. TYROSINE (polar)

EXPERIMENT 8: NUCLEIC ACIDS

NUCLEIC ACIDS

• MACRO-BIOPOLYMERS OF HIGH MW

• REPEATING UNIT MONONUCLEOTIDE

• 2 STRUCTURAL KINDS OF NUCLEIC ACIDS

  1. DNA

  2. RNA

NITROGENOUS BASES

• PYRAMIDINE

  • CYTOSINE (C)- DNA AND SOME RNA

  • THYMINE (T)- DNA ONLY

  • URACIL (U)- IN RNA ONLY

• PURINE

  • ADENINE(A)- DNA AND RNA

  • GUANINE (G)- DNA AND RNA

DEOXYRIBONUCLEIC ACID

• CARRIES GENETIC INFORMATION RESPONSIBLE FOR DEVT AND FXN OF AN ORGANISM

• LOCATION: NUCLEUS

  

DNA IN FRUITS

• 0.1-1% DNA BY WEIGHT

  1. STRAWBERRY- OCTOPLOID (8 CHROMOSOME SET)

  2. KIWI- HEXAPLOID

  3. BANANA- TRIPLOID

DNA EXTRACTION

1. LYSIS

   • BREAK OPEN:

     • CELL TO RELEASE NUCLEUS

     • NUCLEUS TO RELEASE DNA

EXTRACTION BUFFER

• H2O

• DETERGENT

  • CONTAINS SURFACTANTS

  • DISSOLVED CELL MEMBRANE (LIPID BILAYER)

  • SALT

    • REMOVES PROTEINS BOUND TO DNA

      • KEEPS PROTEINS DISSOLVED IN AQUEOUS LAYER

    • Na+IONS NEUTRALIZE(-) CHARGES IN DNA MOLECULES

      • MAKE DNA LESS WATER-SOLUBLE AND MORE STABLE

2. PRECIPITATION

   • SEPARATES DNA FROM INTERFERRING MATTER

   • COLD ETHANOL

     • PRECIPITATE DNA OUT

       • DNA: LOW SOLUBILITY IN EtOH

         • LEAST WHEN COLD

       • LOW Ts PROTECT DNA

         • SLOW DOWN ACTIVITY OF ENZYMES (NUCLEASES) THAT COULD BREAK IT APART AND DEGRADE IT

3. PURIFICATION

   • REMOVES ALL REMAINING CELLULAR DEBRIS AND UNWANTED MATERIAL

   • RINSING WITH ALCOHOL

HYDROLYSIS OF NUCLEIC ACIDS

2 METHODS:

1. CHEMICAL

2. ENZYMATIC

HYDROLYSIS PRODUCTS

• PURINE BASES

  • A&G

• PYRIMIDINE BASES

  • C,T,&U

• OLIGONUCLEOTIDES

  • <20 RESIDUES

• NUCLEOTIDES

  • BASE + SUGAR

• DEOXYRIBOSE

• PHOSPHATES

INTERACTION RESPONSIBLE FOR RIGID MOLECULAR CONFIGURATIONS OF NUCLEIC ACIDS

• PHOSPHODIESTER BONDS

  • LINK THE NUCLEOTIES TOGETHER IN EACH CHAIN

• HYDROGEN BONDS

  • BETWEEN COMPLIMENTARY BASES (eg. A-T)

• VAN DER WAALS FORCES

  • BETWEEN STACKED BASES

QUALITATIVE TEST FOR NUCLEIC ACIDS

1. BENEDICTS TEST

   • FOR REDUCING SUGARS

   • (+) RESULT: BRICK RED PRECIPITATE

   • REACTION EQ.

     • RCHO-[O]-RCOO-

     • CU2+-[R]-CU++CuO

2. ORCINOL TEST

   • FOR PENTOSES

   • (+) RESULT: BLUE-GREEN

   • PRINCIPLE

     • PENTOSES+HCL+6-COMPOSITION+BLUE GREEN

     • FURFURAL+ORCINOL-CONDENSATION+BLUE GREEN PPT

       • ORCINOL=3,5-DIHYDROTOLUENE

3. TEST FOR PURINE BASES

   • HYDROLYSATE+NH4+ +AgNO3= WHITISH PPT

   • UNHYDROLYZED+ NH4+ + AgNO3= GRAYISH-WHITE PPT

4. TEST FOR INORGANIC PHOSPHATE

TEST FOR NUTRIENTS IN FOODS

NUTRIENTS

• SUBSTANCE/CPDS THAT SUPPLY BODY WITH ENERGY

• BUILDING BLOCKS OF MACRO-MOLECULES

• IMPORTANT TO BODY

CARBOHYDRATES

• Eg. SUGARS AND STARCHES

  • SUPPLY BODY WITH ENERGY

  • SOME STARCHES: PROVIDE INDIGESTIBLE FIBER (OR ROUGHAGE) WHICH AIDS DIGESTION

PROTEINS

• FOR GROWTH AND REPAIR

• FACTORS AFFECTING AMOUNT NEEDED IN DIET

  • WEIGHT

  • GENDER

  • AGE

  • HEALTH

• NUTRITIONAL VALUE: MEASURED BY QUANTITY OF ESSENTIAL AMINO ACIDS

COMPLETE PROTEINS (HAS ALL 9 TYPES OF AA)

• FISH

• POULTRY (CHICKEN,DUCK, OR TURKEY)

• EGGS

• DAIRY PRODUCTS (MILK, YOGURT, OR CHEESE)

• BEEF OR PORK

• SOY PRODUCTS (TOFU AND EDAMAME)

LIPIDS

• ORGANIC COMPOUNDS

• SUPPLY 4X AMOUNT OF ENERGY FROM CARBOHYDRATES AND PROTEINS

  • CARBS: 4kcal/g

  • LIPIDS: 9kcal/g

  • WHEN ENERGIES AROUND C ATOM IN FA ARE TRANSFERRED TO 0: MORE ENERGY FREED THAN WITH CARBS

QUALITATIVE TEST FOR NUTRIENTS IN FOODS

CARBOHYDRATES

• PRESENT IN ALL FOOD SAMPLES

MOLISCH TEST

• FORMATION OF PURPLE/VIOLET RING

• 1-Napthanol REACTS WITH THE CYCLIC ALDEHYDES TO FORM PURPLE COLORED CONDENSATION PRODUCTS (furfuryl-diphenyl-methane-dyes)

  

OSAZONE TEST

• GLUCOSAZONE & FRUCTOSAZONE

• 

TEST FOR FATS

• TRANSLUCENT PAPER REGION

• PRESENT IN ALL FOOD SAMPLES

• OCCURRENCE:

  • TRACE (0.1-0.5%) IN FRUIT JUICE

  • TRACE (0.03%) IN EGG WHITE

  • TRACE (0.8%) IN COOKED RICE

TEST FOR VITAMIN A - CARR-PRICE REACTION

• PRESENT IN MARGARINE & EGG YOLK

MINERALS

• Ca

• Cu

• I

• Fe

• Mg

• Mn

• P

• K

• Se

• Na

• Zn

MINERAL MATTER

• PRESENT IN ALL SAMPLES

• INDICATED BY THE FORMATION OF WHITE POWDERY RESIDUE

• ONLY 1-2% IN COOKED RICE

PRACTICE QUESTIONS WITH ANSWERS

SET 1:

1. In chromatography, what is used as the stationary phase?

- a) Solvent

- b) Developer

- c) Filter Paper

- d) Chromatogram

- Answer: c) Filter Paper

2. Which phase in chromatography is mobile?

- a) Stationary Phase

- b) Developer

- c) Adsorbent

- d) Amino acid

- Answer: b) Developer

3. What indicates a positive result for the presence of free amino groups with ninhydrin?

- a) Red color

- b) Green color

- c) Purple color

- d) Yellow color

- Answer: c) Purple color

4. The Retention Factor (Rf) in chromatography is calculated by dividing the distance traveled by:

- a) Solvent only

- b) Solute by solvent

- c) Solvent by solute

- d) Sample size

- Answer: b) Solute by solvent

5. Which of these solvents is the least polar, providing the lowest desorbing capability?

- a) Ethanol

- b) Chloroform

- c) Water

- d) Hexane

- Answer: d) Hexane

6. Which factor does NOT influence the Rf value in chromatography?

- a) Humidity

- b) Thickness of adsorbent

- c) Color of the spots

- d) Solvent purity

- Answer: c) Color of the spots

7. What type of bond links the nucleotides in a DNA chain?

- a) Hydrogen bond

- b) Van der Waals forces

- c) Phosphodiester bond

- d) Ionic bond

- Answer: c) Phosphodiester bond

8. Which nitrogenous base is present only in RNA?

- a) Thymine

- b) Uracil

- c) Adenine

- d) Guanine

- Answer: b) Uracil

9. What is the purpose of using cold ethanol in DNA extraction?

- a) To dissolve the DNA

- b) To neutralize charges on DNA

- c) To precipitate DNA

- d) To keep the DNA warm

- Answer: c) To precipitate DNA

10. The Carr-Price reaction is used to test for:

- a) Reducing sugars

- b) Vitamin A

- c) Fats

- d) Minerals

- Answer: b) Vitamin A

11. Complete proteins contain which of the following?

- a) Only essential amino acids

- b) All nine essential amino acids

- c) Only non-essential amino acids

- d) Primarily carbohydrates

- Answer: b) All nine essential amino acids

12. Which carbohydrate test produces a purple/violet ring as a positive result?

- a) Orcinol Test

- b) Benedict’s Test

- c) Molisch Test

- d) Ninhydrin Test

- Answer: c) Molisch Test

13. A positive Benedict’s test for reducing sugars is indicated by the formation of:

- a) Purple precipitate

- b) Green color

- c) Brick-red precipitate

- d) Blue color

- Answer: c) Brick-red precipitate

14. What is the major factor affecting the rate of movement of compounds in thin-layer chromatography?

- a) Molecular weight

- b) Solubility

- c) Polarity

- d) Viscosity

- Answer: c) Polarity

15. An amino acid with a non-polar side chain from those used in the provided chromatography example is:

- a) Glycine

- b) Tyrosine

- c) Glutamine

- d) Serine

- Answer: a) Glycine

16. A blue-green color in the orcinol test is a positive result for the presence of:

- a) Hexoses

- b) Pentoses

- c) Amino acids

- d) Lipids

- Answer: b) Pentoses

17. What type of chromatography phase is involved when the mobile phase moves by capillary action?

- a) Gas Chromatography

- b) Liquid Chromatography

- c) Thin-Layer Chromatography

- d) Paper Chromatography

- Answer: d) Paper Chromatography

18. In DNA, guanine pairs with:

- a) Thymine

- b) Cytosine

- c) Adenine

- d) Uracil

- Answer: b) Cytosine

19. An amino acid's retention factor (Rf) is low if:

- a) The stationary phase is too polar

- b) The mobile phase is non-polar

- c) It binds tightly to the stationary phase

- d) All of the above

- Answer: d) All of the above

20. In DNA extraction, what is the purpose of detergent?

- a) Neutralizing DNA charges

- b) Precipitating DNA

- c) Dissolving cell membrane

- d) Visualizing amino acids

- Answer: c) Dissolving cell membrane

21. The Benedict’s test detects:

- a) Non-reducing sugars

- b) Reducing sugars

- c) Proteins

- d) Fats

- Answer: b) Reducing sugars

22. What type of force exists between stacked bases in a DNA molecule?

- a) Ionic bonds

- b) Covalent bonds

- c) Van der Waals forces

- d) Hydrogen bonds

- Answer: c) Van der Waals forces

23. Amino acids like tyrosine, that contain a polar side chain, travel

- a) Farther in a non-polar mobile phase

- b) Not as far as non-polar amino acids in a polar stationary phase

- c) The same distance regardless of the mobile phase

- d) Only in non-polar stationary phases

- Answer: b) Not as far as non-polar amino acids in a polar stationary phase

24. When a spot appears elongated on a chromatogram, measurement should be taken from:

- a) The top of the spot

- b) The bottom of the spot

- c) The center of the spot

- d) The origin point

- Answer: c) The center of the spot

25. What does the positive test for purine bases with AgNO3 look like?

- a) Grayish-white precipitate

- b) Brick-red color

- c) Blue-green color

- d) White powdery residue

- Answer: a) Grayish-white precipitate

26. In chromatography, which substance serves as the mobile phase?

- a) Stationary phase

- b) Developer solvent

- c) Filter paper

- d) Adsorbent

- Answer: b) Developer solvent

27. In paper chromatography, high polarity solvents:

- a) Lead to poor separation

- b) Transport all components at the same distance

- c) Prevent separation by holding compounds together

- d) Both a and b

- Answer: d) Both a and b

28. A substance with a lower Rf value generally:

- a) Is highly polar

- b) Is non-polar

- c) Moves quickly

- d) Dissolves entirely in the solvent

- Answer: a) Is highly polar

29. Which amino acid does not interact well with polar mobile phases?

- a) Tyrosine

- b) Tryptophan

- c) Glycine

- d) Serine

- Answer: b) Tryptophan

30. Why is polarity important in chromatography?

- a) It affects how compounds dissolve in the solvent

- b) It determines the movement rate of compounds

- c) It only impacts the stationary phase

- d) It has no effect on separation

- Answer: b) It determines the movement rate of compounds

31. In the context of DNA extraction, what is the primary role of a lysis buffer?

- a) To precipitate DNA

- b) To dissolve nucleotides

- c) To break open cells and release DNA

- d) To increase the temperature for DNA extraction

- Answer: c) To break open cells and release DNA

32. What principle explains the movement of substances between the stationary and mobile phases in chromatography?

- a) Molecular diffusion

- b) Adsorption and desorption equilibrium

- c) Ionic bonding

- d) Solute crystallization

- Answer: b) Adsorption and desorption equilibrium

33. What type of visualization might be necessary to observe amino acids on a chromatogram if they are not naturally visible?

- a) Heat application

- b) Use of a color-developing agent like ninhydrin

- c) Applying a polar solvent

- d) Measuring solubility

- Answer: b) Use of a color-developing agent like ninhydrin

34. When performing a separation of amino acids using thin-layer chromatography, why might the plate need to be removed before the solvent reaches the top?

- a) To maintain equilibrium

- b) To prevent over-elongation of spots

- c) To avoid incorrect Rf values

- d) All of the above

- Answer: d) All of the above

35. Amino acids differ in their Rf values primarily because of differences in their:

- a) Solubility in non-polar solvents

- b) Adsorption to the stationary phase due to polarity

- c) Chemical composition

- d) Interaction with other amino acids

- Answer: b) Adsorption to the stationary phase due to polarity

36. What is a major purpose of using chromatography in biochemical analysis?

- a) To destroy mixtures

- b) To combine all components of a sample

- c) To separate and identify components in mixtures

- d) To change the color of the substances

- Answer: c) To separate and identify components in mixtures

37. In the orcinol test, a blue-green color is an indication of:

- a) Pentose sugars

- b) Hexose sugars

- c) Amino acids

- d) Fatty acids

- Answer: a) Pentose sugars

38. Which of the following best describes the function of a mobile phase in chromatography?

- a) It remains fixed and binds to solutes

- b) It helps in moving the compounds along the stationary phase

- c) It only supports non-polar molecules

- d) It absorbs light to reveal spots

- Answer: b) It helps in moving the compounds along the stationary phase

39. During DNA extraction, cold ethanol helps in DNA precipitation because DNA is:

- a) Very soluble in ethanol

- b) Less soluble in cold ethanol

- c) More soluble in the lysis buffer

- d) Warmed by the ethanol

- Answer: b) Less soluble in cold ethanol

40. What does the term "retention factor" (Rf) represent in chromatography?

- a) The solubility of compounds in a liquid

- b) The fraction of solvent absorbed

- c) The ratio of distance traveled by the solute to distance traveled by the solvent

- d) The color intensity of the sample

- Answer: c) The ratio of distance traveled by the solute to distance traveled by the solvent

41. In an experiment, a spot representing an amino acid travels 3 cm from the origin, while the solvent front travels 6 cm. What is the Rf value for this amino acid?

- a) 0.2

- b) 0.5

- c) 1.0

- d) 1.5

- Answer: b) 0.5

- Solution: Rf = distance traveled by solute / distance traveled by solvent = 3 cm / 6 cm = 0.5

42. A chromatography experiment shows a dye spot moving 4.5 cm from the starting point, and the solvent front reaches 9 cm. Calculate the Rf value of the dye.

- a) 0.25

- b) 0.5

- c) 0.75

- d) 1.0

- Answer: b) 0.5

- Solution: Rf = 4.5 cm / 9 cm = 0.5

43. In an experiment, the distance from the origin to a compound's spot is measured as 2 cm, and the solvent front traveled 8 cm. What is the Rf value of the compound?

- a) 0.25

- b) 0.5

- c) 0.75

- d) 1.0

- Answer: a) 0.25

- Solution: Rf = 2 cm / 8 cm = 0.25

44. A scientist observes that a compound travels 5 cm on a chromatography plate, while the solvent moves 10 cm. What is the Rf value of the compound?

- a) 0.25

- b) 0.5

- c) 0.75

- d) 1.25

- Answer: b) 0.5

- Solution: Rf = 5 cm / 10 cm = 0.5

45. If a pigment travels 6.5 cm on the chromatography plate and the solvent front is 13 cm from the origin, what is the Rf value of the pigment?

- a) 0.25

- b) 0.5

- c) 0.75

- d) 1.0

- Answer: b) 0.5

- Solution: Rf = 6.5 cm / 13 cm = 0.5

SET 2

Separation of Amino Acids by Thin-Layer Chromatography (Questions 1-20)

1. In thin-layer chromatography, which material typically serves as the stationary phase?

- a) Ethanol

- b) Filter Paper

- c) Water

- d) Amino acids

- Answer: b) Filter Paper

2. What is the purpose of the mobile phase in thin-layer chromatography?

- a) To keep compounds in place

- b) To move compounds along the stationary phase

- c) To dissolve the stationary phase

- d) To stop the reaction

- Answer: b) To move compounds along the stationary phase

3. Which factor primarily affects the movement of amino acids on a chromatography plate?

- a) pH

- b) Temperature

- c) Polarity

- d) Volume of amino acid

- Answer: c) Polarity

4. What is the Retention Factor (Rf) in chromatography?

- a) The total distance the solvent moves

- b) The distance traveled by solute relative to solvent

- c) The distance from the top of the plate

- d) The time taken for separation

- Answer: b) The distance traveled by solute relative to solvent

5. An Rf value close to 1 indicates that the compound:

- a) Barely moved

- b) Traveled very far along with the solvent

- c) Remained at the origin

- d) Has high molecular weight

- Answer: b) Traveled very far along with the solvent

6. Which amino acid would likely have the highest Rf value?

- a) Tyrosine (polar)

- b) Glycine (nonpolar)

- c) Lysine (charged)

- d) Glutamate (charged)

- Answer: b) Glycine (nonpolar)

7. If a solvent is highly polar, it may result in:

- a) Poor separation due to equal movement

- b) Strong binding of compounds at the origin

- c) Faster movement of nonpolar compounds

- d) Separation of only charged compounds

- Answer: a) Poor separation due to equal movement

8. Why is it important to cover the chromatography chamber during development?

- a) To maintain solvent vapor saturation

- b) To prevent spills

- c) To increase temperature

- d) To decrease the Rf values

- Answer: a) To maintain solvent vapor saturation

9. In TLC, ninhydrin is used to visualize:

- a) Carbohydrates

- b) Lipids

- c) Amino acids

- d) Nucleic acids

- Answer: c) Amino acids

10. A spot that appears elongated on the chromatogram should be measured:

- a) From the bottom

- b) From the top

- c) From the center

- d) From the origin

- Answer: c) From the center

11. What result would you expect for an amino acid tightly bound to the stationary phase?

- a) High Rf value

- b) Low Rf value

- c) Moves as far as the solvent

- d) No movement

- Answer: b) Low Rf value

12. In chromatography, a substance that barely moves from the origin is likely:

- a) Very polar

- b) Nonpolar

- c) Neutral

- d) Highly volatile

- Answer: a) Very polar

13. Thin-layer chromatography can be used to:

- a) Separate amino acids

- b) Destroy amino acids

- c) Measure protein content

- d) Decrease amino acid reactivity

- Answer: a) Separate amino acids

14. The Rf value for an amino acid is influenced by:

- a) Humidity

- b) Temperature

- c) Solvent purity

- d) All of the above

- Answer: d) All of the above

15. In TLC, which result indicates that two compounds are likely identical?

- a) Different Rf values

- b) Same Rf values

- c) Same color but different distances

- d) No movement

- Answer: b) Same Rf values

16. If an amino acid spot is very faint, you should:

- a) Apply more amino acid

- b) Change the solvent

- c) Use a stronger developing agent

- d) Adjust the stationary phase

- Answer: c) Use a stronger developing agent

17. In TLC, a mobile phase that is too nonpolar will likely result in:

- a) No movement of solutes

- b) High Rf values

- c) Movement of polar compounds

- d) Uneven separation

- Answer: a) No movement of solutes

18. Which of the following is critical in chromatography experiments?

- a) Room ventilation

- b) Solvent evaporation rate

- c) Solvent polarity

- d) Plate temperature

- Answer: c) Solvent polarity

19. The stationary phase in paper chromatography works by:

- a) Adsorbing the compounds

- b) Evaporating compounds

- c) Providing a smooth surface

- d) Reacting chemically with solutes

- Answer: a) Adsorbing the compounds

20. If a compound has an Rf of 0.5, and the solvent moved 10 cm, the compound traveled:

- a) 5 cm

- b) 10 cm

- c) 15 cm

- d) 2.5 cm

- Answer: a) 5 cm

Experiment for Nucleic Acids (Questions 21-40)

21. What are the two main types of nucleic acids?

- a) DNA and RNA

- b) DNA and ATP

- c) RNA and ATP

- d) DNA and proteins

- Answer: a) DNA and RNA

22. In DNA extraction, cold ethanol is used to:

- a) Increase enzyme activity

- b) Precipitate DNA

- c) Dissolve proteins

- d) Break cell walls

- Answer: b) Precipitate DNA

23. DNA contains which unique nitrogenous base?

- a) Uracil

- b) Thymine

- c) Adenine

- d) Cytosine

- Answer: b) Thymine

24. Which nitrogenous base is found only in RNA?

- a) Thymine

- b) Uracil

- c) Adenine

- d) Guanine

- Answer: b) Uracil

25. The bonds that hold together complementary bases in DNA are called:

- a) Ionic bonds

- b) Covalent bonds

- c) Hydrogen bonds

- d) Peptide bonds

- Answer: c) Hydrogen bonds

26. Which enzyme might be deactivated during DNA extraction to protect DNA?

- a) Polymerase

- b) Nuclease

- c) Helicase

- d) Ligase

- Answer: b) Nuclease

27. In a DNA extraction buffer, salt is used to:

- a) Increase DNA solubility

- b) Neutralize DNA charges

- c) Dissolve cell walls

- d) Precipitate DNA immediately

- Answer: b) Neutralize DNA charges

28. Which base pairing is correct in DNA?

- a) A-T

- b) G-U

- c) C-A

- d) T-G

- Answer: a) A-T

29. What method of nucleic acid hydrolysis yields free bases and oligonucleotides?

- a) Chromatography

- b) Chemical hydrolysis

- c) Enzymatic hydrolysis

- d) Sublimation

- Answer: c) Enzymatic hydrolysis

30. In a cell, DNA is primarily located in the:

- a) Cytoplasm

- b) Nucleus

- c) Mitochondria

- d) Ribosomes

- Answer: b) Nucleus

31. What test produces a blue-green color for pentoses in nucleic acids?

- a) Benedict’s test

- b) Orcinol test

- c) Ninhydrin test

- d) Iodine test

- Answer: b) Orcinol test

32. **Which DNA extraction step involves breaking

open the cell to release the nucleus?**

- a) Precipitation

- b) Lysis

- c) Purification

- d) Hydrolysis

- Answer: b) Lysis

33. The phosphodiester bond in DNA links:

- a) Two sugar molecules

- b) A base to a sugar

- c) Nucleotides in each DNA strand

- d) DNA and RNA

- Answer: c) Nucleotides in each DNA strand

34. Van der Waals forces are crucial in DNA for:

- a) Forming base pairs

- b) Linking nucleotides

- c) Stabilizing base stacking

- d) Hydrogen bonding

- Answer: c) Stabilizing base stacking

35. Which nitrogenous bases are purines?

- a) Adenine and Cytosine

- b) Thymine and Guanine

- c) Adenine and Guanine

- d) Cytosine and Uracil

- Answer: c) Adenine and Guanine

36. The Benedict’s test in nucleic acids indicates:

- a) Proteins

- b) Reducing sugars

- c) Phosphates

- d) Carbohydrates

- Answer: b) Reducing sugars

37. The role of detergent in DNA extraction is to:

- a) Dissolve proteins

- b) Dissolve lipids and cell membranes

- c) Enhance DNA precipitation

- d) Denature DNA

- Answer: b) Dissolve lipids and cell membranes

38. A nucleotide is composed of:

- a) Sugar, base, and phosphate

- b) Sugar and lipid

- c) Amino acid and phosphate

- d) Only a base

- Answer: a) Sugar, base, and phosphate

39. To protect DNA during extraction, cold conditions are used to:

- a) Denature enzymes

- b) Slow down nuclease activity

- c) Increase DNA solubility

- d) Dissolve cell walls

- Answer: b) Slow down nuclease activity

40. A strawberry is often used in DNA extraction due to its:

- a) High acidity

- b) Large number of chromosomes

- c) Low water content

- d) Small genome

- Answer: b) Large number of chromosomes

Test for Nutrients in Food Samples (Questions 41-60)

41. Which nutrient supplies the most energy per gram?

- a) Carbohydrates

- b) Lipids

- c) Proteins

- d) Vitamins

- Answer: b) Lipids

42. Which test produces a purple ring for carbohydrates?

- a) Benedict’s test

- b) Sazone test

- c) Molisch test

- d) Iodine test

- Answer: c) Molisch test

43. A brick-red precipitate in the Benedict’s test indicates the presence of:

- a) Proteins

- b) Reducing sugars

- c) Starch

- d) Lipids

- Answer: b) Reducing sugars

44. The test used to detect pentose sugars in food is:

- a) Benedict’s test

- b) Orcinol test

- c) Ninhydrin test

- d) Sazone test

- Answer: b) Orcinol test

45. A translucent spot on paper indicates the presence of:

- a) Carbohydrates

- b) Fats

- c) Proteins

- d) Minerals

- Answer: b) Fats

46. Complete proteins are foods that:

- a) Contain all nine essential amino acids

- b) Are entirely composed of lipids

- c) Contain only carbohydrates

- d) Lack essential amino acids

- Answer: a) Contain all nine essential amino acids

47. Which type of carbohydrate is tested with the Iodine test?

- a) Glucose

- b) Sucrose

- c) Starch

- d) Fructose

- Answer: c) Starch

48. What result is expected if a food sample contains Vitamin A?

- a) Positive Benedict’s test

- b) Positive Carr-Price reaction

- c) Positive Ninhydrin test

- d) Positive Iodine test

- Answer: b) Positive Carr-Price reaction

49. Carbohydrates are essential because they:

- a) Provide amino acids

- b) Provide the body with energy

- c) Store genetic information

- d) Synthesize enzymes

- Answer: b) Provide the body with energy

50. The Molisch test detects:

- a) Lipids

- b) Proteins

- c) Carbohydrates

- d) Vitamins

- Answer: c) Carbohydrates

51. For growth and repair, the body needs:

- a) Vitamins

- b) Lipids

- c) Proteins

- d) Carbohydrates

- Answer: c) Proteins

52. What is indicated by a positive Osazone test?

- a) Lipids

- b) Carbohydrates

- c) Fats

- d) Proteins

- Answer: b) Carbohydrates

53. In biochemistry, lipids are important because they:

- a) Provide 4 kcal/g

- b) Provide 9 kcal/g

- c) Do not provide energy

- d) Are made of amino acids

- Answer: b) Provide 9 kcal/g

54. Which compound would test positive in the translucent paper test?

- a) Lipids

- b) Proteins

- c) Starch

- d) Minerals

- Answer: a) Lipids

55. For detection of amino acids, which test would you use?

- a) Benedict’s test

- b) Orcinol test

- c) Ninhydrin test

- d) Iodine test

- Answer: c) Ninhydrin test

56. Amino acids required in the diet are classified as:

- a) Non-essential amino acids

- b) Essential amino acids

- c) Carbohydrates

- d) Fatty acids

- Answer: b) Essential amino acids

57. Which mineral is commonly detected as a white powdery residue in food tests?

- a) Iron

- b) Potassium

- c) Calcium

- d) Iodine

- Answer: c) Calcium

58. What type of test would reveal the presence of starch in food samples?

- a) Ninhydrin test

- b) Iodine test

- c) Molisch test

- d) Carr-Price test

- Answer: b) Iodine test

59. Which macronutrient is primarily responsible for building and repairing tissues?

- a) Carbohydrates

- b) Vitamins

- c) Proteins

- d) Minerals

- Answer: c) Proteins

60. Nutrients that do not provide energy but are necessary for cellular functions are called:

- a) Carbohydrates

- b) Minerals

- c) Lipids

- d) Amino acids

- Answer: b) Minerals

Identification (10 Items)

1. Test that identifies the presence of reducing sugars, with a positive result indicated by a brick-red precipitate.

- Answer: Benedict’s Test

2. Test used to detect pentose sugars, showing a blue-green color as a positive result.

- Answer: Orcinol Test

3. Test that detects carbohydrates, where a purple/violet ring formation indicates a positive result.

- Answer: Molisch Test

4. A test for the presence of amino acids (specifically free amino groups), with a positive result shown by a purple color.

- Answer: Ninhydrin Test

5. This test identifies Vitamin A in a sample, with a blue-green color indicating a positive result.

- Answer: Carr-Price Reaction

6. A test for carbohydrates in food samples, specifically identifying starch with a positive result shown by a blue-black color.

- Answer: Iodine Test

7. Test that reveals fats in samples, where a positive result is indicated by a translucent spot on paper.

- Answer: Translucent Spot Test

8. Test for the presence of fats or lipids, which results in a translucent oily mark as a positive indication.

- Answer: Grease Spot Test / Test for Fats

9. A qualitative test for protein presence, resulting in a purple or violet color as a positive outcome.

- Answer: Biuret Test

10. A test used to detect glucose and fructose by forming glucosazone or fructosazone crystals.

- Answer: Osazone Test