Lecture 5 : Proximate Analysis II (Protein + Carbs)

Question 1

Proximate analysis of crude protein

What are the 5 main steps in Kjeldahl method?

Answer 1

1. Sample prep
2. Digestion
3. Neutralisation
4. Distillation
5. Titration

Question 2

Proximate analysis of crude protein

What does the kjeldahl method measure?

Answer 2

Nitrogen content (%N)

Question 3

Proximate analysis of crude protein – Kjeldahl method

What reagents and conditions are used to digest the protein sample? State the equation of protein digestion.

Answer 3

Protein + H₂SO₄–> (NH₄)₂SO₄

• Conditions : CuSO₄ catalyst, heat

Question 4

Proximate analysis of crude protein – Kjeldahl method

State the equation of the first neutralisation step

Answer 4

(NH₄)₂SO₄ + 2NaOH –> 2NH₃ + Na₂SO₄ + 2H₂O

Question 5

Proximate analysis of crude protein – Kjeldahl method

After neutralising, the ammonia is is being distilled and it drips into a conical flask containing a reagent. State the equation for the chemical reaction happening during distillation.

Answer 5

Neutralisation occurs too (ammonia with boric acid)

NH₃ + H₃BO₃ (boric acid) –> NH₄⁺ + H₂BO₃^- (borate ion)

Question 6

Proximate analysis of crude protein – Kjeldahl method

After distillation, borate ion is obtained and the next step is back-titration. State the equation for titration.

Answer 6

H₂BO₃^- + H⁺ –>H₃BO₃

Question 7

Proximate analysis of crude protein – Kjeldahl method

Why is it necessary to do back titration?

Answer 7

From the distillation step, there is an aqueous mixture of NH₄⁺ + H₂BO₃^-, so you won’t know the exact volume of NH₄⁺ to caluclate the N content

Question 8

Proximate analysis of crude protein – Kjeldahl method

What is the conversion factor used for?

Answer 8

to convert %N (obtained from calculations in kjeldahl) to %protein in sample and vice versa

Question 9

Proximate analysis of crude protein – Kjeldahl method

What is the numerical value of the conversion factor? How to use the conversion factor to convert :

1. %N to %protein?
2. % protein to %N?

Answer 9

Conversion factor : in 100g of protein, there is 16g of N (16%)

• Conversion factor = 100/16 = 6.25

1. %N to %protein = % N x 6.25
2. %protein to %N = % protein / 6.25 (or multiply by 1/6.25 = 0.16)

Question 10

Proximate analysis of crude protein – official AOAC methods

What is the main drawback of Kjeldahl and Dumas method?

Answer 10

Since these methods only measure nitrogen content, it is not very specific to proteins, so adulteration can occur –> add N containing substances to food to artifically boost protein content

• e.g. Melamine adulteration in milk

Question 11

Proximate analysis of crude protein – Dumas method

What does Dumas method measure?

Answer 11

Nitrogen content (%N)

Question 12

Proximate analysis of crude protein – Dumas method

What are the 3 main steps in Dumas method?

Answer 12

1. Combustion
2. Reduction
3. Detection

Question 13

Proximate analysis of crude protein – Dumas method

Step 1 of Dumas method : combustion.

State the reagents and conditions, and the products produced from combustion.

Answer 13

Reagents and conditions:
- Pure O₂
- Heat : 700-1000℃

The nitrogen in proteins gets combusted into Nitrous oxides, NO_X, where X = 1/2/3
- products : NO, NO₂, NO₃

Question 14

Proximate analysis of crude protein – Dumas method

Step 2 of Dumas method : reduction.

State the reagents and conditions, and the products produced from reduction.

Answer 14

Reduction occurs when passing NO, NO₂, NO₃ through a Cu column during GC

Reagents : Cu GC column

NO, NO₂, NO₃ gets reduced into Nitrogen, N₂

Question 15

Proximate analysis of crude protein – Dumas method

Step 3 of Dumas method : Detection

How can you quanitfy the N content and thus, the protein content in a food sample from the detection step?

Answer 15

The detector in GC will give peak area of N₂

• Compare peak area of sample to peak area of a** known nitrogen standard**, calculate concentration of N₂ -> can get %N and thus %protein using conversion factor

Note : also carry out a blank to account for any inteference, then minus off the concentration of blank from concentration of N₂ obtained!

Question 16

Crude protein – Colourimetric methods – Dye-binding methods

Dye-binding method 1 : anionic dye-binding methods
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How do anionic dye-binding methods work, and how to find protein content from these methods?

Answer 16

Anionic dyes have a –SO₃^- group, which binds to amino terminal of amino acids (-NH₃⁺) or positively charged side chains of amino acids

• to quantify protein content:
  1. Create a standard curve with BSA (bovine serum albumin, a protein found in cow’s blood)
  2. Measure absorbance of mixture (sample + anionic dye)
  3. Measure absorbance of blank (DI water) and minus off abs_blank from abs_sample
  4. find concentration of proteins in sample with reference to the calibration / standard curve

Question 17

Crude protein – Colourimetric methods – Dye-binding methods

Dye-binding method 2 : Bradford dye-binding method

The Bradford dye binding method works in a similar way as anionic dye binding method, but it is more specfic than anionic dye binding methods.

What kinds of proteins is the Bradford dye binding method specific to?

Answer 17

Proteins that are** soluble in acidic conditions** (means need basic amino groups to react to acidic conditions)

• The dye binds especially to lysine / arginine / histidine (LAH) → basic amino acids

Question 18

Crude protein – Colourimetric methods – Dye-binding methods

Dye-binding method 2 : Bradford dye-binding method

How does the Bradford dye-binding method work?

Answer 18

Same as anionic dye binding, where the dye, Coomassie Brilliant Blue G-250, –SO₃^- group, which binds to positively charged side chains of amino acids

Question 19

Crude protein > Colourimetric methods > Copper-Ion based methods

What are the 3 components that makes up the Biuret reagent and state their functions if applicable.

Answer 19

Biuret reagent :
1. Cu₂₊ –> in the form of CuSO₄.5H₂O
2. NaOH : to provide alkaline conditions for reaction
3. Potassim tartrate : Chelates and stabilises Cu2+ so that there will not be any side reactions – before Cu2+ gets chelated by peptide bonds, it will exist in chelate form with potassium tartrate

Note : although Cu₂₊ is in the form of CuSO₄.5H₂O, it still exists freely as Cu₂₊ in aqueous solution and thus is highly reactive

Question 20

Crude protein > Colourimetric methods > Copper-Ion based methods

Method 1 : Biuret test / assay (direct assay)
State how to conduct the Biuret test
What qualitative and quantitative information can I get from the Biuret test?

Answer 20

Add Biuret reagent to sample in a cuvette and wait for it to react.

• Qualitative info : If biuret reagent turns from blue to violet, protein is present
• Quantitative info : put cuvette through UV-vis, get absorbance. calculate the protein concentration with reference to a standard / calibration curve (BSA)

NOTE : MUST CONDUCT A BLANK AND MINUS OFF BLANK ABSORBANCE

Question 21

Crude protein > Colourimetric methods > Copper-Ion based methods

Method 2 : Lowry assay (coupled assay)

What is the Folin-Ciocalteu (Lowry) reagent made of?

Answer 21

Phosphotungstic acid + Phosphomolybdic acid

• i think dont need to know in detail, just know its acids consisting of metals in D-block of periodic table (super reactive, will tend to get reduced)

Question 22

Crude protein > Colourimetric methods > Copper-Ion based methods

Method 2 : Lowry assay (coupled assay)

How can I use the Lowry assay to determine protein content in my food sample? (3 steps) State what compound is formed in order to detect absorbance

Answer 22

Step 1 : complexation with Biuret assay
- add Biuret agent to sample to form the Cu⁺ chelated complex with peptide bonds

Step 2 : Reduction of Lowry agent
- The “Cu⁺ - peptide” chelated complex (with peptide bonds) will reduce Lowry agent to form Molybdenum blue
- The “Cu⁺ - peptide” chelated complex gets oxidised into “Cu²⁺ - peptide” chelated complex

Step 3 : Quanitfy protein content
- measure absorbance of sample, and find concentration of protein with reference to BSA calibration curve

Question 23

Crude protein > Colourimetric methods > Copper-Ion based methods

Method 2 : Lowry assay (coupled assay)

Other than Cu</sup>+</sup>, which 3 specific amino acids can reduce the Folin-Ciocalteu (Lowry) reagent?

Answer 23

1. Cysteine
2. Trypthophan
3. Tyrosine

CTT

Question 24

Crude protein > Colourimetric methods > Copper-Ion based methods

Method 3 : Bicinchoninic acid (BCA) assay (coupled)

What are the steps in the BCA assay so that i can quantify protein content?

Answer 24

Step 1 : complexation with Biuret assay
- add Biuret agent to sample to form the Cu⁺ chelated complex with peptide bonds

**Step 2 : Reduction of BCA **
- Cu⁺ , cuprous ion, binds to BCA at a higher affinity compared to peptide bonds. The Cu⁺ chelated complex (with peptide bonds) will dissociate, and a new Cu⁺ chelated complex, Cu⁺ with BCA is formed.

Step 3 : Quanitfy protein content
- measure absorbance of sample, and find concentration of protein with reference to BSA calibration curve

Question 25

# **Crude protein > Colourimetric methods > Copper-Ion based methods** Copper-ion coupled assay : Does the Lowry assay or the BCA assay have a lower LOQ? Why?

Answer 25

BCA assay has a lower LOQ, meaning it is more sensitive **Reason** : - In the Lowry assay, the Cu⁺ in the Cu⁺-peptide chelate does not detach, the Cu+ is just oxidised into Cu2+ while still remaining in the chelate form w peptide - BUT in BCA assay, from the Cu+ - peptide chelate complex, Cu+ detaches from the peptide and forms a new chelate complex with the BCA → so more sensitive (check but — this leads to more obvious colour change)

Question 26

# **Carbohydrate quantification > sugars > Somogyi-Nelson Method** What is the general idea behind the Somogyi-Nelson Method for quantifying reducing sugars?

Answer 26

- react reducing sugars (reduce other species) with an oxidising agent - The oxidising agent will get reduced = product 1 - Using product 1, do a coupled assay with another compound to form coloured substance --> measure absorbance

Question 27

# **Carbohydrate quantification > sugars > Somogyi-Nelson Method** What are the 3 main reaction steps for the Somogyi-Nelson Method? State the reagents and products

Answer 27

1. amonium **molybdate** and sodium arsenate is used to generate **arsenomolybdate** 2. Reducing sugar (aldehyde) + Cu(OH)₂ --> carboxylic acid + Cu₂O * i.e. Cu²⁺ is oxidised into Cu⁺ 3. Arsenomolybdate reacts with Cu₂O (= Cu⁺) to form **arsenomolybdate blue**

Question 28

# **Carbohydrate quantification > sugars > Somogyi-Nelson Method** After all reaction steps to get **arsenomolybdate blue**, how to determine sugar concentration in sample? [3]

Answer 28

1. Measure absorbance of the sample with the arsenomolybdate blue 2. Measure absorbance of a **blank** (DI water) 3. substract the blank absorbance from sample absorbance, and **sugar concentration is expressed as glucose equivalents** (with reference to a glucose calibration / standard curve)

Question 29

# **Carbohydrate quantification > sugars > Dinitrosalicyclic acid method** What does the dinitrosalicyclic acid (DNS) method measure?

Answer 29

concentration of **reducing sugars** -- only aldehydes (doesnt work on ketones) - (simple sugars like mono and disaccharides)

Question 30

# **Carbohydrate quantification > sugars > Dinitrosalicyclic acid method** What is the principle behind the dinitrosalicyclic acid (DNS) method? State the colour change involved.

Answer 30

Aldehydes (mono and disaccharides) gets oxidised by DNS into carboxylic acid, while the --NO₂ group in DNS is reduced into --NH₂ becomes orange-red - colour change : yellow to orange-red

Question 31

# **Carbohydrate quantification > sugars > Dinitrosalicyclic acid method** How to quantify reducing sugar content by DNS method?

Answer 31

- Add DNS to sample in a cuvette - Measure absorbance at 540nm Measure absorbance of blank (DI WATER) and minus abs_blank from abs_sample - Calculate concentration based on **glucose** calibration curve ## Footnote **Remember to do a BLANK with DI water!!!**

Question 32

# **Carbohydrate quantification > sugars > enzymatic method for glucose** In the enzymatic method for glucose, what are the 2 main reaction steps to quantify glucose concentration? State all reagents and products.

Answer 32

1. β,D-glucose oxidised by glucose oxidase, producing α-gluconolactone and H₂O₂ 2. H₂O₂ undergoes a coupled reaction with **o-diansidine**, *catalysed by peroxidase*. this forms H₂O and a coloured oxidised dye, and absorbance can be measured.

Question 33

# **Carbohydrate quantification > sugars > enzymatic method for glucose** Peroxidase is an enzyme that oxidises H₂O₂ into O₂. True or False?

Answer 33

False, peroxidase helps to **oxidise another molecule** by transferring an oxygen atom from H₂O₂. - Thus, H₂O₂ is always being **reduced** into H₂O.

Question 34

# **Carbohydrate quantification > sugars > mono and oligosaccharides** What are the 2 methods that can be employed to analyse mono and oligosaccharides?

Answer 34

1. HPLC (high performance liquid chormatography) 2. GC (gas chromatography)