Lecture 5 : Proximate Analysis II (Protein + Carbs) Flashcards
Proximate analysis of crude protein
What are the 5 main steps in Kjeldahl method?
- Sample prep
- Digestion
- Neutralisation
- Distillation
- Titration
Proximate analysis of crude protein
What does the kjeldahl method measure?
Nitrogen content (%N)
Proximate analysis of crude protein – Kjeldahl method
What reagents and conditions are used to digest the protein sample? State the equation of protein digestion.
Protein + H2SO4–> (NH4)2SO4
- Conditions : CuSO4 catalyst, heat
Proximate analysis of crude protein – Kjeldahl method
State the equation of the first neutralisation step
(NH4)2SO4 + 2NaOH –> 2NH3 + Na2SO4 + 2H2O
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.
Neutralisation occurs too (ammonia with boric acid)
NH3 + H3BO3 (boric acid) –> NH4+ + H2BO3- (borate ion)
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.
H2BO3- + H+ –>H3BO3
Proximate analysis of crude protein – Kjeldahl method
Why is it necessary to do back titration?
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
Proximate analysis of crude protein – Kjeldahl method
What is the conversion factor used for?
to convert %N (obtained from calculations in kjeldahl) to %protein in sample and vice versa
Proximate analysis of crude protein – Kjeldahl method
What is the numerical value of the conversion factor? How to use the conversion factor to convert :
- %N to %protein?
- % protein to %N?
Conversion factor : in 100g of protein, there is 16g of N (16%)
- Conversion factor = 100/16 = 6.25
- %N to %protein = % N x 6.25
- %protein to %N = % protein / 6.25 (or multiply by 1/6.25 = 0.16)
Proximate analysis of crude protein – official AOAC methods
What is the main drawback of Kjeldahl and Dumas method?
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
Proximate analysis of crude protein – Dumas method
What does Dumas method measure?
Nitrogen content (%N)
Proximate analysis of crude protein – Dumas method
What are the 3 main steps in Dumas method?
- Combustion
- Reduction
- Detection
Proximate analysis of crude protein – Dumas method
Step 1 of Dumas method : combustion.
State the reagents and conditions, and the products produced from combustion.
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
Proximate analysis of crude protein – Dumas method
Step 2 of Dumas method : reduction.
State the reagents and conditions, and the products produced from reduction.
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
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?
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!
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?
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
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?
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
Crude protein – Colourimetric methods – Dye-binding methods
Dye-binding method 2 : Bradford dye-binding method
How does the Bradford dye-binding method work?
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
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.
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
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?
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
Crude protein > Colourimetric methods > Copper-Ion based methods
Method 2 : Lowry assay (coupled assay)
What is the Folin-Ciocalteu (Lowry) reagent made of?
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)
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
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
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?
- Cysteine
- Trypthophan
- Tyrosine
CTT
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?
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
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?
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)
Carbohydrate quantification > sugars > Somogyi-Nelson Method
What is the general idea behind the Somogyi-Nelson Method for quantifying reducing sugars?
- 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
Carbohydrate quantification > sugars > Somogyi-Nelson Method
What are the 3 main reaction steps for the Somogyi-Nelson Method? State the reagents and products
- amonium molybdate and sodium arsenate is used to generate arsenomolybdate
- Reducing sugar (aldehyde) + Cu(OH)2 –> carboxylic acid + Cu2O
* i.e. Cu2+ is oxidised into Cu+ - Arsenomolybdate reacts with Cu2O (= Cu+) to form arsenomolybdate blue
Carbohydrate quantification > sugars > Somogyi-Nelson Method
After all reaction steps to get arsenomolybdate blue, how to determine sugar concentration in sample? [3]
- Measure absorbance of the sample with the arsenomolybdate blue
- Measure absorbance of a blank (DI water)
- substract the blank absorbance from sample absorbance, and sugar concentration is expressed as glucose equivalents (with reference to a glucose calibration / standard curve)
Carbohydrate quantification > sugars > Dinitrosalicyclic acid method
What does the dinitrosalicyclic acid (DNS) method measure?
concentration of reducing sugars – only aldehydes (doesnt work on ketones)
- (simple sugars like mono and disaccharides)
Carbohydrate quantification > sugars > Dinitrosalicyclic acid method
What is the principle behind the dinitrosalicyclic acid (DNS) method?
State the colour change involved.
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
Carbohydrate quantification > sugars > Dinitrosalicyclic acid method
How to quantify reducing sugar content by DNS method?
- 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!!!
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.
- β,D-glucose oxidised by glucose oxidase, producing α-gluconolactone and H2O2
- H2O2 undergoes a coupled reaction with o-diansidine, catalysed by peroxidase. this forms H2O and a coloured oxidised dye, and absorbance can be measured.
Carbohydrate quantification > sugars > enzymatic method for glucose
Peroxidase is an enzyme that oxidises H2O2 into O2. True or False?
False, peroxidase helps to oxidise another molecule by transferring an oxygen atom from H2O2.
- Thus, H2O2 is always being reduced into H2O.
Carbohydrate quantification > sugars > mono and oligosaccharides
What are the 2 methods that can be employed to analyse mono and oligosaccharides?
- HPLC (high performance liquid chormatography)
- GC (gas chromatography)
