Kjeldahl Method

Introduction

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. These reactions can have either a favorable or detrimental effect on the overall properties of foods. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods.

Determination of Protein Concentration


1.Kjeldahl method
-a food digested with strong acid so that it releases nitrogen which can be determined by suitable titration techniques.
-does not measure the protein content easily
-Conversion factor is needed to convert the measured nitrogen concentration to a protein concentration


Principles:
A. Digestion
- sample is heated in the presence of sulfuric acid (oxidizing agent), anhydrous sodium sulfate, and catalyst.
- digestion converts any nitrogen in the food into ammonia and other organic matter to carbon dioxide and water.
- ammonia gas remains in acid solution because it is in the form of ammonium ion which binds to the sulfate ion to form ammonium sulfate.  


B. Neutralization
- the solution in the digestion flask is then made alkaline by addition of sodium hydroxide which converts  the ammonia sulfate into ammonia gas
- the ammonia gas moves out into receiving flask which contains an excess of boric acid
- in receiving flask. ammonium ion and boric acid to borate ion


C. Titration
- titrate with standard sulfuric or hydrochloric acid using a suitable indicator
- the concentration of hydrogen ions required to reach the end-point is equivalent to the concentration of nitrogen
- once nitrogen content has been determined, it is converted to a protein content using appropriate conversion factor

Advantages:
-universal
-high precision
-good reproducibility 


Disadvantages:
-does not give a measure of true protein
-different proteins need different concentration factors
-the use of concentrated sulfuric acid at high temperature
-time consuming


2. Enhanced Dumas method
-Principles: A sample of known mass is combusted in an high temperature to release carbon dioxide, water and nitrogen
- Nitrogen content is measured by separating nitrogen from carbon dioxide and water by using a column
-also used conversion factor to determine protein content


Advantages
-faster
-does not need toxic
-easy to use
-samples can be measured automatically


Disadvantages:
-high initial cost
-does not give a measure of true protein
-small sample size make its difficult to obtain a representative sample


3. Methods using UV-Visible spectroscopy
-use either the natural ability of proteins to absorb (or scatter) light or chemically or physically modify proteins to make them absorb (or scatter) light in the region
- calibration curve of absorbance versus protein concentration must be built first
-main difference: the chemical groups which are responsible for the absorption or scattering of radiation


A. Direct Measurement at 280nm
-Principle: tryptophan and tyrosine absorb ultraviolet  light strongly at 280 nm
-use the same wavelength to measure protein concentration
-Advantages: simple to carry out, non-destructive and no special reagents are required
-Disadvantages: nucleic acid absorb strongly at 280 nm
-To overcome: measure the absorbance at two different wavelengths.

Kjeldahl Method for Determining Nitrogen

There are three main steps in K jeldahl method namely digestion, distillation and titration.

Digestion- is usually done by boiling a homogenous sample in concentrated sulfuric acid and the end result is an ammonium sulfate solution. The general equation for this step that is shown below:  

Organic N + H2SO4    →

(NH4)SO4 + H2O + CO4 + other sample matrix byproducts

In distillation additional excess base to the digestion product in converting NH4 to NH3 as indicated in the follow up equation. NH3 is recovered by distilling the reaction product.

ammonium sulfate	heat	ammonia gas
(NH4)2SO4 + 2NaOH	→	2NH3 + Na2SO4 + 2H2O

Titration

Titration quantifies the amount of ammonia in the receiving solution. The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ion in the receiving solution.

There are two types of titration—back titration and direct titration. Both methods indicate the ammonia present in the distillate with a color change.In back titration (commonly used in macro Kjeldahl), the ammonia is captured by a carefully measured excess of a standardized acid solution in the receiving flask. The excess of acid in the receiving solution keeps the pH low, and the indicator does not change until the solution is "back titrated" with base.

ammonia	standard sulfuric acid acid		excess ammonium sulfate	sulfuric acid
2NH3 +	2H2SO4	→	(NH4)2SO4 +	H2SO4
(no color change)

ammonia sulfate	measured excess acid	measured sodium hydroxide		ammonium sulfate
(NH4)2SO4 +	H2SO4 +	2NaOH	→	Na2SO4 + (NH4)2SO4 + 2H2O
(color change occurs)

In direct titration, if boric acid is used as the receiving solution instead of a standardized mineral acid, the chemical reaction is:

ammonia gas	boric acid		ammonium- borate complex	excess boric acid
NH3 +	H3BO3	→	NH4 + H2BO-3 +	H3BO3
(color change occurs)

The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia collects, the color of the receiving solutions changes.

ammonium- borate complex		sulfuric acid		ammonium sulfate		boric acid
2NH4	+	H2BO-3	+	H2SO4 (NH4)2SO4	+	2H3BO3
(color change occurs in reverse)

The boric acid method has the advantages that only one standard solution is necessary for the determination and that the solution has a long shelf life.

Sources:   

Cole –Parmer Technical Library Cole (September 2, 2011). http://www.coleparmer.com/techinfo/techinfo.asp?htmlfile=KjeldahlBasics.htm&id=38