CHAPTER 1

INTRODUCTION

1.1 Background of work

Proteins are polymers of amino acids. Proteins differ from each other according to the
type, number and sequence of amino acids that make up the polypeptide backbone. 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 (Nelson & Cox, 2011).
Kjedahl method is an analytical chemistry of quantitative in determination of nitrogen
contained in organic substances with the nitrogen in inorganic ammonia and ammonium.
The Kjeldhal procedure measures the nitrogen content of a sample. The protein content
then, can be calculated assuming a ratio of protein to nitrogen for the specific food being
analysed (Nielson, 2010). This Kjedahl method was developed by John Kjedahl in 1883.
This method was firstly introduced by heating of substance with sulphuric acid that
decompose the organic substances by oxidation. Next, potassium sulphate are added to
increase the boiling point of the sample. The chemical decomposition are complete when the
dark colour sample become colourless and clear. The amount of nitrogen present in sample
are determined by titration of the sample with sodium carbonate solution using methyl
orange pH indicator (Kjeldahl Method for Determination of Nitrogen, n.d.).
This laboratory method for nitrogen and protein analysis is still universally used for this
analysis. Even though, other methods are faster and more efficient but none can adapt with
the variety of sizes or conditions of samples than Kjeldahl's original method (Blamire, 2003).
Lastly, The Kjedahl method is highly used all around the world and still the standard to
compare with other method. This method has higher precision, good reproducibility and it is
universality. It has become the major method for estimation of protein in food. Although the
method has its own advantage but this method does not give a measure of true protein since
all nitrogen in food is not in the form of protein. Protein has different amino acid sequence,
so different protein need different correction factor. This method using concentrated
sulphuric acid at high temperature that can cause hazard. The Kjedahl method is also time
consuming to carry out (McClements, 2007).
1.2 Objectives
The objectives of determination of protein using Kjedahl mehod was to determine
percentage of nitrogen by titration the light green solution with hydrochloric acid (HCl) and
calculate the amount of protein in bicuits.
1.3 Scope of work
The Kjedahl method was introduced to determine the amount of protein in sample. It
consist of three steps. The first step is digestion where 2g of the sample and filter paper are
weighed, filled in digestion tube. Then, 2 kjeltabs Cu 3.5 and 12ml of H2SO4 were added
into the tube, and heated 420C. The second step is distillation where 80ml of deionised
water were added into the digestion tube. Then, the digestion tube and a 250ml conical flask
containing 25ml of 4% (excess) boric solution were put together in distillation unit. The
receiver solution which is the boric acid then changed colour to light green. The third step is
titration where the light green solution was titrated with hydrochloric acid (HCl) and the light
green solution changed colour to light grey. During titration, the light green solution that were
titrated with the hydrochloric acid (HCl) standard can determine amount of nitrogen
percentage by calculating the amount of hydrochloric acid (HCl) used to reach end-point.