BFST 2101 FOOD ANALYSIS

Discuss the application of chromatographic techniques in

quantitative analysis of macronutrients in a food product.

W.J.M.V.R. Arambepola
AG/16/FT/048
Nutrients present in food can be defined as the intake of food, considered in relation to
the body's dietary needs. Nutrients can be grouped into two major categories as macro
nutrients and micro nutrients. There are several major classes of nutrients available in
food material. carbohydrates, fats, fiber, minerals, protein and vitamins. Macro-
nutrients (needed in relatively large amounts) - The macro-nutrients are carbohydrates,
fats, fiber and proteins. Micro-nutrients (needed in smaller quantities) - The
micronutrients are minerals and vitamins. The macro-nutrients (excluding fiber and
water) provide energy. Quantitative analysis of macronutrients can be carried out with
the appropriate equipment because carbohydrates, fats, proteins and fiber material of
the food matrix are present in relatively larger quantities than vitamins, minerals and
contaminants and additives in food. One such method possible for quantitative analysis
of macronutrients is Chromatography.

Chromatography is a laboratory technique used for the separation of material. The

mixture containing the compound of concern is dissolved in a fluid called the mobile
phase which carries it through a structure holding another material called the stationary
phase. The various constituents of the mixture will then travel at different speeds due
to their specific chemical natures and thus cause them to separate. There are many
different methods of chromatography available that can be used based on the nature of
the food substance we are handling. However, the fundamental thing we have to
consider is that the food matrix is complex and the material of concern may be
interacting with the other food components. Therefore the method chosen must result
in minimum loss and damage of the material to be quantitatively measured depending
on the functioning principles, operating conditions and reagents used. Partition
chromatography is based on the distribution of a substance between two immiscible
phases based on their polarity. Paper chromatography which uses partition can be done
as ascending or descending paper chromatography. In absorption chromatography, the
substance of concern gets absorbed onto the stationary phase and gets washed out by
the mobile phase. There are many methods that use the principle of absorption which
are – column chromatography, Thin layer chromatography, High performance liquid
chromatography and gas-liquid chromatography. Gas-liquid chromatography can be
combined with mass spectrometry for more accurate quantitative measures. Ion
exchange chromatography is based to retain the substance based on its charge which
can be anionic or cationic. Substances can also be separated based on their respective
molecular sizes through size exclusion chromatography e.g. gel permeation
chromatography or based on the affinity of the constituent to the stationary phase.

Carbohydrates are structurally classified as monosaccharides, oligosaccharides, and

polysaccharides. Monosaccharides and some oligosaccharides have a sweet taste.
Polysaccharides, in combination with proteins, lipids, and nucleic acids, play an
important role in animal metabolic systems. In food systems, carbohydrates provide
flavor, structure, and texture. (Thin layer chromatography (TLC), Gas chromatography
(GC) and High Performance Liquid chromatography (HPLC) are commonly used to
separate and identify carbohydrates. Carbohydrates are separated on the basis of their
differential adsorption characteristics by passing the solution to be analyzed through a
column. Carbohydrates can be separated on the basis of their partition coefficients,
polarities or sizes, depending on the type of column used. Many monosaccharides and
oligosaccharides are polar non-charged molecules and can therefore be separated from
charged molecules by passing samples through ion-exchange columns. By using a
combination of a positively and a negatively charged column it is possible to remove
most charged contaminants. Non-polar molecules can be removed by passing a solution
through a column with a non-polar stationary phase. Thus proteins, amino acids, organic
acids, minerals and hydrophobic compounds can be separated from the carbohydrates
prior to analysis. HPLC is currently the most important chromatographic method for
analyzing carbohydrates because it is capable of rapid, specific, sensitive and precise
measurements. In addition, GC requires that the samples be volatile, which usually
requires that they be derivitized, whereas in HPLC samples can often be analyzed
directly. High Performance Liquid Chromatography and Gas Chromatography are
commonly used in combination with Nuclear Magnetic Resonance (NMR) or Mass
Spectrometry so that the chemical structure of the molecules that make up the peaks
can also be identified. Gas-liquid chromatography can also be used in the analysis of
fatty acids and sugars by converting them into methyl ethers and acetates.
Carbohydrates can be identified using retention times in GLC or the retention volumes
in HPLC. In either case a standard curve can be drawn using the known standards of
known purity and concentration and detect the concentration of the sample. According
to the chromatograph obtained, using standards can see the peak identity and peak
purity can be used to find how pure the sample is and the peak area can be integrated
to find the concentration of the carbohydrate. Other than this partition chromatography
is used to detect lactose disaccharide amount present in a food sample.
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 also the major structural components of many natural foods, often
determining their overall texture. Methods used to quantify protein from food samples
are often inaccurate with significant variability that needs care to reduce the errors. The
errors result from losses during protein preparation and purification and false detection
of interfering compounds or elements. Amino acid analysis (AAA) involves a series of
chromatographic techniques that can be used to measure protein levels, avoiding some
difficulties and providing specific compositional information. However, unstable
derivatives,that are toxic, incomplete reactions, inadequate chromatographic
separations, and the lack of a single hydrolysis method with sufficient recovery of all
amino acids hinder precise protein quantificaton using AAA. Amino acids with or without
derivatization are separated utilizing, (ultra) high performance liquid chromatography
(UHPLC/UPLC or HPLC) and gas chromatography (GC) prior to detection by absorbance,
fluorescence, or mass spectrometry (MS). but the derivatives have limited stability and
measurement can be additionally compromised by interfering signals from naturally
fluorescing compounds. MS linked to GC (i.e. GC–MS) can avoid the latter complication,
but routine derivatizations such as making silyl derivatives that enhances compound
volatility also reduces stability and may degrade amino acids or produce multiple
reaction products that complicate analysis. In addition, the derivatized standards must
be made fresh prior to each analysis. Liquid Chromatography does not require
derivatizations to enhance volatility and when coupled with mass spectrometry (LC–MS)
provides a larger set of connected techniques to complement polar compound analysis.
LC–MS has high selectivity and is sensitive to detection of both derivatized and
underivatized amino acids. however, the coupling of LC with MS is restricted to
compatible volatile solvents unless additional desalting processes are introduced. Gel
permeation chromatography can also be used to separate large molecules usually
polymeric substances such as carbohydrates and proteins on the porous silica gel. The
molecules are separated based on the size. The larger molecules that does not fit in the
pores of the gel will flow faster and the smaller ions will flow through the pores and
results in desalted carbohydrates and proteins. This method is ideal for the purification
of the carbohydrate or protein sample of interest from a larger sample. It is known that
carbohydrates cannot be separated from mixtures effectively using the size exclusion
chromatography rather than any other method.

Long-chain fatty acids (LC-FA) are organic compounds in which the hydrocarbon chain
length may vary from 10 to 30 carbons. The hydrocarbon chain can be saturated or
unsaturated (contains one or more double bonds). Based on the number of double
bonds, unsaturated fatty acids are classified into the following groups -
(i)monounsaturated fatty acids (monoenoic acids, MUFA), containing one double bond,
for example, oleic acid, (ii) polyunsaturated fatty acids (polyenoic acids, PUFA), having
two or more double bonds, for example, γ-linolenic acid, (iii) eicosanoids, which are
derived from polyenoic fatty acids, for example, prostaglandins. HPLC analysis of all fatty
acids can be based on nature of stationary phase (solid or liquid), the high performance
liquid chromatography is divided into liquid-liquid chromatography, adsorption
chromatography, and reversed-phase chromatography. Among abovementioned high
performance liquid chromatographic techniques Reversed Phase(RP)- HPLC plays a key
role in PUFA analysis . This technique allows separating those fatty acids which cannot
be separated by normal phase HPLC. The retention of fatty acids in RP-HPLC depends on
the polarity of the stationary phase, mobile phase, and chemical structure of examined
fatty acids. In general, the retention time is proportional to the chain length and the
number of double bonds present in examined fatty acids. Additionally, the influence of
geometric isomerization of fatty acids plays a very important role in reversed-phase
HPLC analysis. Another popular method for the analysis of these fatty acids is the use of
Gas chromatography. The main step in GC analysis of fatty acids is the process of
conversion of these compounds into suitable volatile derivatives . The most frequently
applied derivatives are alkyl derivatives (methyl-, ethyl-, and isopropyl-) obtained by
means of esterification of fatty acids. This procedure facilitates volatility of fatty acids
and also improves their separation and sensitivity to GC detectors.

Chromatographic determination of fatty acids composition by Thin layer

Chromatography (TLC) including Liquid Chromatography (LC) -MUFA and LC-PUFA
content is mandatory for lipids analysis in food, agricultural, and also in biological
samples. Also Absorption chromatography methods such as column chromatography
can also be used to separate fatty acids from food samples using non-polar solvents such
as petroleum ether in combination with other solvents if progressive separation is
required. Moreover, the mono- and polyunsaturated fatty acids can be
chromatographically determined by TLC. It is well known that thin-layer
chromatography is a classical method of separation, identification, and quantification of
fatty acids. there are many advantages which make TLC very competitive with HPLC (high
performance liquid chromatography) in the fatty acids field such as simplicity of use, less
expensive cost, small consumption of solvents in comparison with HPLC, availability of
analysis of several samples in parallel, and possibility of easy visualization of unsaturated
fatty acids after TLC fractionation by use of suitable dyes. One of the primarily used TLC
impregnating procedure to separate fatty acids in complex lipid samples was silver ion
TLC (Ag-TLC). Silver ion chromatography is based on the ability of Ag+ to form weak
reversible charge transfer complexes with electrons of the double bonds of unsaturated
fatty acids. The retention of long-chain unsaturated fatty acids depends on the strength
of complex with Ag (I), on the number of double bonds and their configuration, and also
on the distance between double bonds. Partition chromatography is used to determine
the fat content using the rose-gottlieb method.

In all the above, whenever HPLC or GLC methods are used, It is important that for
possible analysis Peak Identity, Peak purity, and Quantification is important and using
standards of known concentrations and purity, chromatographs with peak patterns can
be obtained and compared with the sample chromatograph and using peak integration
or the standard curve, their concentrations can be determined.

Conclusion is, There are many methods that are effective to quantitatively analyse
macronutrients present in the food matrixes. However the use of the relevant method
depends on the amount of food material available, the relative abundances of macro
nutrients in food, the resources and equipment and chemical availability, Time
constraints and the error factor of the method used and the accuracy expected from the
measurement obtained. The chemical structure of the analyte and the physico-chemical
properties of the matrix together with the desired selectivity and sensitivity can be used
to determine the most suitable isolation and quantitation system. Depending on the goal
of the analysis all the available options must be reviewed with respect to accuracy,
precision, cost, The amount of labour, time required and the degree of automation.