Virtual Lab Manual

Kjeldahl Method

Synopsis
In the Kjeldahl Method simulation, you will learn how to use the Kjeldahl method to
determine the protein content of a food sample, and how to use LC-MS/MS to investigate if
the sample has been adulterated to make the protein content seem higher than it is.

Analyze a milk powder sample

Your lab has received batch samples of milk powder from 3 different producers. Your mission
is to analyze a sample from one of the batches using the Kjeldahl method, in order to
determine the exact protein content, and to make sure this is consistent with the reported
value.

Tracing the steps of the Kjeldahl method

An animation will introduce you to each step of the analysis, and show what goes on at the
molecular level.You will then perform the acid digestion, steam distillation and finally a
colorimetric titration, before being challenged with the calculations needed to transfer the
lab result to a protein content in the sample.

Is everything what it appears to be?

How can you be certain that your findings are correct? There might be ways to fool the
Kjeldahl method! In the second half of the simulation, you will look into how this can happen,
and explore the powerful technique LC-MS/MS, which can be used to detect if a known
adulterant has been added to any of the received milk powder batches.

Will you be able to safely release the milk powder batches for consumers around the world?

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Learning Objectives
At the
end of this simulation, you will be able to…
●Demonstrate a detailed understanding of the nucleophilic addition reaction
●Provide an overview and examples of nucleophilic addition to a carbonyl group
●Draw correctly the mechanism for common nucleophilic addition reactions
●Demonstrate a detailed understanding of the Grignard reaction
●Describe the role of each reagent in the Grignard reaction
●Explain the sensitivity of Grignard reaction conditions and be able to make procedural
adjustments
● Gain understanding and practical experience of essential laboratory techniques:

Techniques in Lab
● Reflux technique
● Air- and moisture-sensitive synthesis
● Synthesis and use of Grignard reagents in-situ

Theory
Quantitative analysis
A quantitative analysis in chemistry is the determination of the absolute amount or content
of a substance. This amount would often be expressed as a concentration. In a quantitative
analysis, it's important to minimize loss of substance e.g. when transferring it between
containers.

Water in Acid
As a general rule, you never add water directly to concentrated acid, as it might result in a
powerful, exothermic reaction leading to uncontrolled splashes of concentrated acid.
However, in some lab work, it’s necessary to do this, and in such cases, careful safety
measures must be taken:
1. Wear thick acid proof gloves to protect your hands.
2. Wear safety goggles.
3. Make sure the concentrated acid is cooled in an ice bath.
4. Don’t add the water in a single instance, but add it slowly over several seconds.

Points 3 and 4 reduce the risk of splashing, whereas point 1 and 2 protects the one
performing the lab work.

Acid digestion
Acid digestion is a central step in the Kjeldahl method, where a food sample is digested in
concentrated sulphuric acid. The nitrogen from proteins in the food sample is turned into

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ammonium ions, whereas the carbon and oxygen end up as the gas carbon dioxide and
water. The acid digestion frees up the nitrogen from the other elements in the food sample,
making it possible to detect it quantitatively in the later steps of the Kjeldahl method.

Alkaline steam distillation

In the distillation step of the Kjeldahl method, the pH of the digestion mixture is first raised
to change the ammonium ions into ammonia. In contrast to ammonium ions, ammonia is
volatile, and can thus be distilled from the rest of the mixture and be caught in an acid
trap.

Units and symbols in calculations

Units
L = liter
mL = milliliter
g = gram
mol = moles
M = mol/L

Symbols
M = molar mass [g/mol]
m = mass [g]
n = amount [mol]
C = Concentration [g/L] or [M]
V = volume [L] or [mL]

Direct titration
In the Kjeldahl method, the ammonia is absorbed in the acid trap where it reacts with boric
acid to form ammonium ions:
NH3 + H2O + B(OH)3 → NH4+ + B(OH)4-

It’s important to note here that n(NH 3) = n(B(OH)4-),

where n is the amount in moles.

A color change from pink to green is observed, as this reaction leads to an increase in pH.
When titrating with 0.10 M HCl, where the strong acid HCl has reacted with water to form
H3O+, the following reaction takes place:
H3O+ + B(OH)4- → B(OH)3 + H2O

This lowers the pH further, and the color of the mixture changes back to pink from green
when:
n(HCl) = n(B(OH)4-)

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In titration, the measurement gained is the volume of titrant, here mL of 0.10 M HCl. To
determine the amount of acid used, the following relationship can be used:
n(Acid) = C(Acid) * V(Acid),
where C is the concentration and V is the volume. Remember that the unit M (molar
concentration) is the same as mol/L.

A final very useful equation is the relationship between amount, mass and molar mass:
n = m/M ⇔ m = n * M,
where m is the mass and M is the molar mass.

Chromatography basics
Chromatography is a laboratory technique for the separation of compounds. In liquid
chromatography, a solution is dissolved in a fluid called the mobile phase, which carries it
through a column holding another material called the stationary phase. The point where a
compound is eluted from the column is called the retention time, which is one of several
factors taken into consideration when identifying compounds. If the retention time for an
unknown compound matches that of a known standard, this indicates that the compound
might be the same as the standard, though further qualification is needed.

Several methods of detecting the eluted compounds exist, but in common techniques, a
chromatogram showing the response over time is produced. An increased response over a
short time interval is called a peak. The area under such a peak is proportional to the
concentration of the compound in the injected sample, and can be used for quantification.

Fragmentation in Tandem Mass Spectrometry

In tandem mass spectrometry, fragmentation is used to break up the ions of interest. This
means that you can first filter for specific ions, break up these into fragments specific for
the ions, and then filter for the fragments. Though this results in a lower amount of ions
reaching the detector, as the fragmentation is never 1:1 from precursor to product ion, it
increases both sensitivity and selectivity as the noise is reduced considerably and the
so-called transition between precursor and product can be designed to be very specific.

Calibration standards
Calibration standards are used to establish the relationship between the measured
response and the concentration of an analyte. For this, a set of standards with known
analyte concentration are used. The number of standards used, and the concentration
range they cover, depends on what the calibration is to be used for. For quantification, a
7-point calibration is common, spread out evenly over the concentration range covered and
always covering the expected concentration of the samples that will be measured.

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Recovery control
What is a recovery control
A recovery control in analytical chemistry is a sample spiked with a known amount of
analyte. It is used to assess the recovery of the method, which is done by comparing how
much of the analyte is detected by the method to the known concentration at the start.
This way the amount of analyte lost during the sample preparation can be determined. It is
similar to a standard in that the analyte is added in a known concentration, but in contrast
to a standard, the recovery control goes through the whole sample preparation protocol. It
is also in the same matrix as actual samples, e.g. milk, which is not always the case with
standards.

How to use recovery controls

Several recovery controls would often be included, so you’re also able to estimate the
variation of the method from start to finish. To estimate the recovery, you would first
calculate the concentration based on the measured response, which in LC-MS/MS would be
the peak area. The mean of the found concentrations divided by the known spiked amount
will yield the recovery.

In some methods, you need to take dilution or concentration factors in the sample
preparation into account as well.

Blank
In analytical chemistry, a blank is a way of determining the background level detected by a
method. Commonly two kinds of blanks can be used: solvent blanks and matrix blanks. A
solvent blank only contains the solvents used, e.g. water, methanol, acetonitrile, often in a
composition similar to that which the sample is dissolved in when analyzed.

A matrix blank will be prepared in a matrix similar to an actual sample, e.g. milk, but where
it’s certain that no analyte is present. This way, it’s possible to determine if any other
compounds will be detected as analyte, or if samples are becoming contaminated with
analyte at any point.

Relative Standard Deviation

The relative standard deviation, commonly abbreviated RSD, is used in analytical chemistry
as a tool to estimate the precision of a method. It’s the standard deviation of a set of
measured concentrations, divided by the mean of the measured concentration and normally
given in percent.

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Sensitivity and selectivity
Sensitivity
The strength of the signal of an analyte is proportional to its concentration. The lower the
concentration, the lower the signal. Sensitivity means how high a response a certain
amount of analyte gives. So an increased sensitivity means a higher response from the
same amount or concentration of an analyte. It is important to know the lowest
concentration of the analyte needed to get a signal that can be distinguished from the
baseline noise. If the concentration of an analyte in a sample is lower than this limit, we
cannot detect it even though it may be present in the sample.

Selectivity
Selectivity in analytical chemistry is the ability of a method to correctly determine an
analyte without interference from other compounds. A method's selectivity is increased if
it's very specific for the chosen analyte(s), e.g. via chromatographic separation or
fragmentation patterns in mass spectrometry.

Mass-to-charge ratio
The common unit of detection in mass spectrometry. Due to the way the magnetic fields
work in the mass spectrometer, it's not an ion's mass that's being selected for, but the
mass-to-charge ratio which is noted m/z. For many ions, the number of charges carried is
simply 1, and the m/z value is effectively the same as the mass of the ion. But for some
ions, this is an important distinction that users should be aware of.

Melamine
The organic compound Melamine is a white powder at room temperature. It contains 67 %
nitrogen by mass, which includes three amine moieties, making it a weak base.

Industry uses melamine to produce different kinds of heat-resistant plastics, for example
in the production of dinnerware, flooring and insulation.

When exposed to concentrated acid and high temperatures, the structure of melamine
degrades and the nitrogen is released as ammonium ions.

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