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FST3111 : Food Analysis & Lab > Lecture 5 : Proximate Analysis II (Protein + Carbs) > Flashcards

Lecture 5 : Proximate Analysis II (Protein + Carbs) Flashcards

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1
Q

Proximate analysis of crude protein

What are the 5 main steps in Kjeldahl method?

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

Proximate analysis of crude protein

What does the kjeldahl method measure?

A

Nitrogen content (%N)

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3
Q

Proximate analysis of crude protein – Kjeldahl method

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

A

Protein + H2SO4–> (NH4)2SO4

  • Conditions : CuSO4 catalyst, heat
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4
Q

Proximate analysis of crude protein – Kjeldahl method

State the equation of the first neutralisation step

A

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

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5
Q

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.

A

Neutralisation occurs too (ammonia with boric acid)

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

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6
Q

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.

A

H2BO3- + H+ –>H3BO3

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7
Q

Proximate analysis of crude protein – Kjeldahl method

Why is it necessary to do back titration?

A

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

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8
Q

Proximate analysis of crude protein – Kjeldahl method

What is the conversion factor used for?

A

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

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9
Q

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?
A

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)
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10
Q

Proximate analysis of crude protein – official AOAC methods

What is the main drawback of Kjeldahl and Dumas method?

A

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
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11
Q

Proximate analysis of crude protein – Dumas method

What does Dumas method measure?

A

Nitrogen content (%N)

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12
Q

Proximate analysis of crude protein – Dumas method

What are the 3 main steps in Dumas method?

A
  1. Combustion
  2. Reduction
  3. Detection
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13
Q

Proximate analysis of crude protein – Dumas method

Step 1 of Dumas method : combustion.

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

A

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

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

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14
Q

Proximate analysis of crude protein – Dumas method

Step 2 of Dumas method : reduction.

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

A

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

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15
Q

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?

A

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!

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16
Q

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?

A

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
17
Q

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?

A

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
18
Q

Crude protein – Colourimetric methods – Dye-binding methods

Dye-binding method 2 : Bradford dye-binding method

How does the Bradford dye-binding method work?

A

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

19
Q

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.

A

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

20
Q

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?

A

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

21
Q

Crude protein > Colourimetric methods > Copper-Ion based methods

Method 2 : Lowry assay (coupled assay)

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

A

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)
22
Q

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

A

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

23
Q

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?

A
  1. Cysteine
  2. Trypthophan
  3. Tyrosine

CTT

24
Q

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?

A

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

25
Q

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?

A

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)
26
Q

Carbohydrate quantification > sugars > Somogyi-Nelson Method

What is the general idea behind the Somogyi-Nelson Method for quantifying reducing sugars?

A
  • 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
27
Q

Carbohydrate quantification > sugars > Somogyi-Nelson Method

What are the 3 main reaction steps for the Somogyi-Nelson Method? State the reagents and products

A
  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
28
Q

Carbohydrate quantification > sugars > Somogyi-Nelson Method

After all reaction steps to get arsenomolybdate blue, how to determine sugar concentration in sample? [3]

A
  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)
29
Q

Carbohydrate quantification > sugars > Dinitrosalicyclic acid method

What does the dinitrosalicyclic acid (DNS) method measure?

A

concentration of reducing sugars – only aldehydes (doesnt work on ketones)

  • (simple sugars like mono and disaccharides)
30
Q

Carbohydrate quantification > sugars > Dinitrosalicyclic acid method

What is the principle behind the dinitrosalicyclic acid (DNS) method?
State the colour change involved.

A

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
31
Q

Carbohydrate quantification > sugars > Dinitrosalicyclic acid method

How to quantify reducing sugar content by DNS method?

A
  • 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

Remember to do a BLANK with DI water!!!

32
Q

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.

A
  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.
33
Q

Carbohydrate quantification > sugars > enzymatic method for glucose

Peroxidase is an enzyme that oxidises H2O2 into O2. True or False?

A

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

34
Q

Carbohydrate quantification > sugars > mono and oligosaccharides

What are the 2 methods that can be employed to analyse mono and oligosaccharides?

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

FST3111 : Food Analysis & Lab (3 decks)

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