Glucose Measurement - UV Vis

Spectroscopy
How to Conduct Wine Glucose Measurement Using UV
Vis Spectrophotometry
This application note describes how to determine the glucose content in wine using a sample and reagent kit along
with a UV/VIS Excellence Spectrophotometer. To find out more, please proceed to download the application note
below.

Why does the amount of glucose in wine need to be measured?

Glucose measurement is an important part of the wine production process as the amount of sugar in wine is
indicative of the final alcohol content. Glucose, one of the primary sugars found in wine grapes, is a six-carbon sugar
molecule derived from the breakdown of sucrose. These sugars are broken down during the fermentation process to
form ethanol and carbon dioxide.

How is glucose measurement conducted?

In this experiment, a kit for the quantitative, enzymatic determination of glucose in wine and other materials is used in
conjunction with the UV7 Excellence Spectrophotometer from METTLER TOLEDO. A spectrophotometer is a popular
glucose measurement device due to its speed and ease of use. The absorbance of standard and sample
solutions are both measured at 340nm. The difference in absorbance is directly proportional to the concentration of
glucose in wine.

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What is…

UV-Vis Spectroscopy (or Spectrophotometry) is a quantitative technique used to measure how

much a chemical substance absorbs light. This is done by measuring the intensity of light
that passes through a sample with respect to the intensity of light through a reference sample or
blank.

Provided the molar absorptivity is high enough, UV/VIS absorption is a highly sensitive
detection method and is a useful tool for quantitative analysis. Since many substances absorb
 broad regions of the spectrum, it is prone to possible interferences from other components of
the matrix.

Spectroscopy is the branch of chemistry concerned with the investigative

measurements of spectrum. UV-VIS (ultraviolet-visible) spectroscopy or
spectrophotometry is the study of the interaction of light with matter at
electronic levels. It ranges from the vacuum level ultraviolet region i.e.
180nm to visible region i.e. 780nm. UV spectrum extends from 180nm to
400nm whereas the visible region ranges from 400nm to 780nm.

UV-VIS spectroscopy is an instrumental technique used for chemical analysis.

It is used for qualitative as well as quantitative analysis. This technique has
several applications for a large number of compounds. It is used to observe
the optical behavior of chemical compounds, identification of various
species, and quantification of specific analytes.

UV-Visible spectroscopy
In UV-VIS spectroscopy, the transition of electrons at various levels by
absorption of radiation from ultraviolet to visible region is plotted in a graph.
This line graph of various absorptivities on specific levels of radiations is
because of the absorption capacities of compounds at certain levels. These
levels are called regions of absorption and the compounds are termed as
chromophores.

The chromophores are present in almost every compound. This can be

deduced by the fact that almost all compounds and especially organic
compounds can be identified and quantified by the uv-vis spectroscopy.

Working principle of UV-visible

spectroscopy
When a chemical compound absorbs light, some excitation and de-excitation
processes of electrons occur in atoms which result in the production of the
distinct spectrum.
What is a UV-Vis spectrophotometer?

Uv-vis spectrophotometer is an instrument in which the radiation from

ultraviolet and visible regions interacts with the sample solution. The
electrons get excited when absorbing some of the incident radiation and
transmitting the other. This transmitted radiation is detected, amplified,
recorded, and displayed on a readout device in the form of percent
transmittance.

What information can be obtained from UV-Vis spectra?

UV-visible spectra explain the radiation absorbance region of a compound.

When UV-visible light strikes with the matter, some of the light is absorbed
within the molecules of the compound while the rest will be transmitted
through it. This transmitted light shows how much light is being absorbed by
the sample (absent from the refracted radiation).

What is the principle of UV visible spectroscopy?

The basic principle of UV-visible spectroscopy is based on the absorption of

light (ranges from -nm) by different chemical compounds. It is the interaction
of ultraviolet and visible light with matter. Every chemical compound has a
particular or distinct spectrum as it only absorbs a specific wavelength of
light (radiation).

How is beer Lambert law used in spectroscopy?

Lambert beer’s law is used to determine the transmittance ‘T’ and
absorbance ‘A’ of a solution in a transparent cell with path length ‘ℓ’.
According to this law, the concentration of the absorbing analyte is directly
proportional to the absorbance and path length of the cell.

A = – log T = log Po / P = ε . c . ℓ

What is UV visible spectroscopy used for?

UV-visible spectroscopy is used to identify functional groups, water analysis,

and measure an analyte’s concentration using Lambert Beer’s law. It is also
used for quantitative measurements of DNA, RNA, and proteins in the
sample solution. UV visible is widely used in forensic, chemicals,
environmental, and clinical laboratories. UV-visible spectrometer is used as a
detector for various analytical techniques i.e. chromatography.

What are the applications of UV-visible spectroscopy?

UV-visible spectroscopy has various applications in different fields, such as

clinical analysis, food industries, petrochemical industries, forensic
laboratories, research, educational laboratories, pharmaceutical industries,
and quality control purposes. It is also used to detect and identify different
organic and inorganic species, functional groups, isomeric studies, and the
presence of conjugation, etc.

Why is quartz cuvette used in UV-visible spectroscopy?

Fused silica and quartz cuvettes are most commonly used in ultraviolet
spectroscopy as they are transparent in the ultraviolet region i.e. quartz can
not absorb ultraviolet light so are used in ultraviolet spectrophotometers.
Plastic and glass materials absorb ultraviolet light which interferes with the
results.

Which detectors are used in UV visible spectroscopy?

The most commonly used detector in UV visible spectroscopy is a

photomultiplier tube. Repetition of the dynode is structured with a slight
potential difference at a particular angle. The incoming photon strikes the
cathode, after knocking out several electrons from the dynodes every time.

Which lamp is used in UV-vis spectroscopy?

Hydrogen & deuterium lamps (ranging 160nm-380nm) with a tungsten

halogen lamp (ranging 240nm-2500nm) are commonly used for UV-vis
spectroscopy.

What is the range of UV Visible Spectroscopy?

The range of UV-vis is from 180nm to 780nm. The ultraviolet region

comprises 180nm-400nm while the visible region extends from 400nm-
780nm.

How do you read the UV-VIS spectroscopy spectrum?

To read the UV-vis spectrum the graph is plotted between the wavelength
and the absorption. The wavelength at which maximum absorption occurs is
called the λmax.

What is maximum absorbance?

The characteristic wavelength at which a chemical species shows the

maximum absorption is called maximum absorption. It is represented by λ max.

What are chromophores?

Chromophores are atoms or groups of atoms that are responsible for the
absorption of incident radiation (UV-visible radiation mainly).

What is the application of UV visible spectroscopy in natural products?

Uv-vis spectroscopy is used in the analysis of many natural products. It is

used to identify organic and inorganic species present in a solution. It is also
used for quantitative measurements of different components in natural
compounds, for example, to find the concentration of vitamin C in orange
juice, etc.
What are the advantages and disadvantages of UV visible spectroscopy?

UV-Vis spectroscopy gives accurate results. The instrument is easy to use and
handle. Besides this, it is a time-consuming technique, its preparation is
difficult and effort is required because external light and small vibration can
cause interference with results.

Why is methanol a good solvent for IR and UV spectroscopy?

Methanol is transparent in both ultraviolet and visible regions. It has a cutoff

point below these two regions hence it cannot cause any interference in
results. So, it is a good solvent for UV and IR spectroscopies.

Why are most absorption bands in the visible UV spectra very broad?

Many electronic transitions occur by various molecules, each of which slightly

differs from one another. A large number of closely spaced lines are difficult
for the instrument to detect or resolve individually hence it gives a broad
spectrum.

What is the use of a UV-Vis spectrophotometer?

UV-Vis spectrophotometer is used for the quantitative determination and

measurement of different analytes i.e. organic, inorganic, biological
molecules, etc.

Why the UV spectrum is broader than the IR spectrum?

In the ultraviolet spectrum, changes in all the energy levels (i.e. rotational,
vibrational, and electronic energy levels) are observed while in IR only
vibrational energy levels are observed. So, a UV spectrum is broader than IR
one.

What is the difference between an absorbance, emission, and excitation

spectrum for UV visible spectroscopy?

The main difference between absorbance, excitation, and the emission

spectrum is the type of radiation understudy to analyze the respective
chemical compounds. In absorption spectra, the light being absorbed is
studied with transmitted light. That transmitted light shows the absorbed or
missing light as the difference in the blank and sample transmittance. While
in the emission spectrum, such as fluorescence spectrum the fluorescence
intensity is taken as a function of excitation wavelength. The energy that is
first absorbed by the electron of an atom is emitted back. As the energy is
quantized so electrons that excite, gain a certain amount of energy and
release the same when going back to their original state.

Why is ethanol a good solvent for UV measurement but not for IR?

Ethanol is good for UV measurement because its cutoff point is 210 nm

which is near the lower limits of UV (190 nm- 400nm) so it does not interfere
with the results of ultraviolet regions. Whereas, ethanol has a dipole
moment, which means it is IR active, so it interferes with the results.

What is the difference between colorimetry and spectrophotometry?

Colorimetry is only used for colored compounds while spectrophotometry is

used for various compounds either colored or colorless. Spectrophotometry
comprises a wide range of wavelengths i.e ultraviolet, visible, and IR.

Why UV absorption value goes negative?

The negative value of absorption indicates that the sample is having an

impurity in it, which causes interference with the result. The fluorescence
caused by the impurity can enhance the value of transmitted radiation as
compared to incident radiation. That is the reason it gives a negative
absorption value.

Why are solid forms of the sample not suitable for UV vis spectroscopy?

For UV-vis spectroscopy, the analyte must be in solution form because the
interaction of radiation is effective this way. The light interacts with all the
molecules of the analyte in solution form and there are very low chances for
losses as in solid form.

What is the difference between zero and baseline in UV Vis

spectroscopy?
The zero in UV spectroscopy indicates the total transmittance while baseline
is the amount of radiation absorbed by the cuvette and the sample solution.

Qualitative Analysis
In addition to the Quantitative Analysis, measuring the full UV/Vis absorption
spectra allows substance identification. More precisely, the position and, to some
extent, the profile of the absorption peaks allow specific compounds to be

identified. Figure 2. Example of

Chlorophyll UV/Vis spectraEach sample has a characteristic and unique UV/VIS
spectrum that can be used for its identification. In particular, this is achieved by
comparing the spectrum of the sample with spectra of known, pure compounds. As
an example of UV/VIS spectrum, the spectrum of chlorophyll a is shown above.