Refractive Index (RI) and Brix Standards – Theory and Application

Author: John Barron, Technical Director, Reagecon Diagnostics Ltd., Shannon Free Zone, Shannon,
County Clare, Ireland.

1.0 Summary
The measurement of Refractive Index (RI) is an important parameter in a whole range of industries.
There are several makes and models of refractometers available on the market, that vary in cost and
complexity. The instruments fulfil to a varying degree, fitness for purpose requirements for various
industries and various sample types. In some cases, RI measurement is heavily regulated, such as in
the sugar industry and there have been a wide array of ASTM written standards produced for many
applications. Irrespective of application, industry type, sample type, instrument used or target
measurement uncertainty, high quality calibration standards are an imperative. These can also be
used for quality control, method validation, analyst qualification or proefficiency testing. This paper
details a brief explanation of the science of RI, how a typical refractometer works and presents some
examples of instruments available in the marketplace. This is followed by narrative of the relevance
of RI measurement and various ASTM standards that have been published depending on application.

The final section of the paper explains the reasons why high-quality liquid standards are necessary
and then details specific RI standards available from Reagecon. It is important to point out, that there
are other high quality manufacturers of such standards, but the Reagecon standards are presented
here for example purposes, as these are most familiar to the author. In broad terms there are four
main classifications of RI standards that include ICUMSA compliant and non-compliant sucrose
standards expressed in Brix values, sucrose standards expressed as RI and solvent based standards
expressed as RI.

It is hoped that this publication will provide the reader with a sound theoretical knowledge of RI
measurement, and will provide a detailed knowledge of the metrology pertaining to RI and furnish the
analyst with the knowledge and tools to procure and use RI or Brix Standards optimally.

2.0 Theory of Refractive Index (RI)

The speed of light travelling through a vacuum is always the same. When light moves through another
medium, its speed slows down because the light is being constantly absorbed and re‐emitted by atoms
within the material through which the light is travelling. The ratio of the speed of light in a vacuum to
the speed of light in another substance is defined as the Refractive Index for that substance. This
relationship can be expressed as follows:

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 RI = speed of light in a vacuum
Fig. 1
speed of light in the substance measured

In terms of how a refractometer works, when light changes speed as it crosses the boundary from one
medium to another, as well as its speed changing, its direction also changes. In other words – it is
refractive. The relationship between the speeds of light through the two media, the angles of both
incident and refraction and refractive index of the two media is expressed in a simple formula.

Fig. 2

On the basis of this formula, it is not necessary to measure the speed of light to determine its refractive
index. Instead by measuring the angle of refraction and knowing the refractive index of the layer that
is in contact with the sample – it is possible to measure the refractive index of the sample. Nearly
every instrument on the market utilises this principle, although instruments may vary in optical design.

3.0 How a Refractometer Works

Samples with different refractive indices will produce different angles of refraction, bearing in mind
that the angle of incidence and the refractive index of the prism are constant (Diagram 1).

A different sample in each glass exhibiting different refractive indices.

Diagram 1

The different angles of refraction in different samples will be reflected in a change in the position of
the border line between the light and dark regions of the instruments.

By calibrating the instrument appropriately, the position of the border line can be used to determine
the refractive index of any sample. More specifically, the sample is placed on the prism of the
instrument, incident light is shone onto the prism, which is transmitted through the sample (See
Diagram 2).

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 Light being transmitted through a sample placed on a refractometer prism.

Diagram 2

This incident light is diffracted at the interface between the sample and the prism. The refractive index
value is then calculated based on the relationship between the angle of incidence and the angle of
diffraction for light hitting the boundary between the two media (See Diagram 3).

Comparison between angle of incidence and angle of refraction.

Diagram 3

However, accurate measurement is dependant, both on the temperature of the sample and the
wavelength of the light used. The importance of both parameters will now be briefly explained, but in
a lot of instruments the temperature is standardised to 20°C and the wavelength of the light is
standardised at 589.3nm.

First of all, to deal with wavelength. In most sample types the refractive index will vary with
wavelength. This variation is called dispersion and it is this phenomenon that causes white light
moving through a prism to be refracted into the component colours of the rainbow. (Red, Orange,
Yellow, Green, Blue, Indigo, Violet). For accurate measurement of refractive index, it is necessary to

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use monochromatic light and the commonest source of light used in refractometers is Sodium D Light
at 589 nm, although this does not apply to ABBE Refractometers.

In terms of the temperature coefficient of variation, the speed of light is slower in a sample, than in a
vacuum, due to absorption and emission of light by the atoms, as has been already stated. Since the
density of a sample normally decreases as temperature increases, the speed of light through the
sample will increase. Consequently, the refractive index will decrease. For water, this decrease in
refractive index will be approximately 0.0001/°C. For certain other types of sample including liquid
organics, the refractive index decrease will be approximately 0.0005/°C. Therefore, the management
of temperature is critical for accurate measurement of refractive index.

All high-quality bench, on line and handheld refractometers where accurate measurement is required
are equipped with temperature management systems. Refractive index results should always be
reported at the temperature of measurement, which is normally either 20 or 25°C and the
temperature of measurement is often denoted with the wavelength of the light used. Refractive Index
measurement can be performed manually or automatically. A selection of widely available types of
refractometer can be seen in Diagram 4.

A selection of widely available commercial refractometers.

Diagram 4

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4.0 Why Measure Refractive Index
Refractive index is measured as part of the characterisation of liquid samples and is analogous to the
way the measurement of melting point is used to characterise solid samples. Each substance has its
own refractive index and its measurement may be of value in the following situations:

Firstly, to either identify or confirm the identity of a sample by comparing its refractive index to known
values. Such known values can be derived experimentally, taken from the technical or scientific
literature, or obtained from the manufacturer of the liquid. Secondly, Refractive index may be used to
assess the purity of a sample by comparing the refractive index of the sample to the known value for
the pure substance. Thirdly, Refractive index can be expressed in BRIX values for the measurement of
the sugar content of sugar rich samples such as jams, jellies, syrups and fruit juices. The BRIX value
correlates to the sugar concentration in the sample measured and is expressed as the number of
grams of sucrose in 100 grams of sucrose/water solution. The relationship between the refractive
index and BRIX is determined by the International Commission for Uniform Methods of Sugar Analysis
(ICUMSA). Samples that have multiple ingredients such as salts and sugars (liquid sauces like soya
sauce) can be measured for total percentage concentration of these substances using refractive index.
The value can be expressed as BRIX or refractive index and the BRIX value can be read and interpreted
as the total concentration of dissolved materials in the sample.

In general, though, Refractive Index measurement is a non-selective technique, because different

solutions can have the same Refractive Index, is a fundamental property, and can be used to measure
purity and concentration. Finally, Refractometers can be used to determine the concentration of
solutions such as oil‐based solutions, pharmaceuticals and heavy chemicals such as machining oil,
cleaning solutions, glycols, Cesium Chloride, Sodium Hydroxide and others. Each solution has a
correlation value between BRIX or refractive index and the concentration of solute can be established
by comparing refractive index or BRIX to known concentrations of the solute using a standard curve.
A conversion table can be established from the standard curve and several manufacturers of
Refractometers input the conversion tables into the memory of their instruments and thus by
measuring their refractive index can display concentration directly. In the market place, up to 80
different instruments are available with dedicated, fit for purpose concentration scales dedicated to
specific end user needs and niches.

5.0 ASTM Standards

As well as several manufacturers offering quality control or calibration standards, dedicated for
specific applications, there are several dedicated ASTM written standards available for various
applications and industries. A selection of these are presented in Table 1.

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ASTM Standards Dedicated for Various Applications
D542 Index of Refraction of Transparent Organic Plastics
D1045 Sampling and Testing Plasticizers Used in Plastics
D1218 Refractive Index and Refractive Dispersion of Hydrocarbon Liquids
D1416 Rubber from Synthetic Sources – Chemical Analysis
D1747 Refractive Index of Viscous Materials
D1807 Refractive Index and Specific Optical Dispersion of Electric Insulating Liquids
D3321 Use of Refractometer for Field Test Determination of the Freezing Point of Aqueous
Engine Coolants
D4095 Use of the Refractometer for Determining Non-Volatile Matter (Total Solids) in
Floor Polishes
D5006 Measurement of Fuel System Icing Inhibitors (Ether Type) in Aviation Fuels
D5775 Rubber from Synthetic Sources – Bound Styrene in SBR (styrene-butadiene
copolymers)
Table 1

6.0 Why Use Refractive Index Standards

Understanding the theory of Refractive Index, the various instruments available and when and how
to use these instruments is important. However, neither the test method or the instrument can be
used in any meaningful way, without the availability of high-quality standards. From a metrological
point of view, most workers have focussed on the use of RI standards for the calibration or re‐
calibration of the Refractometer. Few have focussed on an equally important, but much more
widespread use of the standards as a control material for each test carried out in line with the
principles of metrology and good laboratory practice. The advent of the ‘science of chemical
metrology’ has given renewed emphasis to the use and necessity of control materials. The benefits
of such controls cannot be overstated. As with all analytical standards or reference materials, RI
standards should fulfil several criteria which are presented in Table 2.

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Criteria That Refractive Standards Must Fulfill1
• Provide traceability (needed to quote results in SI units)
• Demonstrate the accuracy of results
• Calibrate the equipment and methodology
• Monitor the user performance
• Validate the test
• Facilitate comparability, which is to ensure that when the correct procedures have been
followed, the same analysis of the same materials will produce results that agree with
each other whenever or wherever they are performed.
Table 2

Such materials must also be able to fulfil the criteria required for quality control, accreditation and
proficiency testing where appropriate. Standards and reference materials for RI should be produced
and characterised in a technically competent matter, should be homogenous, stable, certified and
have available a known uncertainty of measurement as a function of time. Production of primary
standards rarely focuses on measurement uncertainty as a function of time that is realistic in the
context of the production, transport and use of such standards under commercial or routine
conditions. Such standards are rarely suitable for use as secondary or working standards.

Secondary or working standards need to be affordable, widely available and certified. The
development, production, stability, assignation of uncertainty as a function of time and
commercialisation of an extensive range of fit for purpose standards that includes refractive index
standards has occupied the time and resources of the author’s laboratory for several years 2. In the
context of normal commercial or routine use, RI standards or any other standards need to be rugged,
have extensive shelf life, and be fit for purpose.

7.0 Applications of Refractive Index Measurement

In terms of markets and applications, these are wide and varied (see Table 3). They include the food
industry, the manufacture and testing of both alcoholic and non-alcoholic beverages, a wide range of
salts within the chemical industry, both in water based and petroleum-based chemicals. The
measurement of Refractive Index has particularly wide application and relevance in the petrochemical
industry, being relevant and useful for many types of products within that industry. Refractive Index
is also very relevant in the sugar industry, both in processing of sugar beet and sugar cane.

Refractive Index, expressed in Brix units is a widely used measure of sugar content. In fact, as described
earlier, ICUMSA, determines the relationship between Brix and sugar content and a whole host of
Refractive Index and Brix Standards are made from the disaccharide sugar sucrose. Finally, the

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measurement of Refractive Index has huge relevance in the pharmaceutical, flavours, fragrances and
perfume industries. For specific industries and applications within these industries, see Table 3.

Industrial Applications of RI or Brix

Industries Applications
Food and Beverages - Coffee Extract
- Soy Bean Oils
- Milk
- Wine
- Juice
- Jam
- Spirits
Sugar, Milling, Refining, Processing - Molasses
- Liquid Sugar
- Cane Sugar Milling and Refining
- Beetroot Sugar Milling and Refining
Water Based Chemicals - Gels
- Glycols
- Resins
- Polymers
Petroleum Based Chemicals - Plastics
- Solvents
- Oil Based Plants
- Resins
Petroleum Industry - Additives
- Lubricants
- Fuel Oils
- Paraffin, Waxes
Pharmaceutical Industry - Toxicology Testing
- Pharmacy Compounding
- Drug Testing
- EP and USP Test Methods
Flavour, Fragrance and Cosmetic - Perfumes
- Lemon Oil
- Waxes
- Creams
Table 3

8.0 Reagecon’s Refractive Index and Brix Standards

Reagecon’s standards fall into 4 different classifications (see Table’s 4, 5, 6 and 7). To start with, we
have Brix Standards which are comprised of ICUMSA compliant and ICUMSA non-compliant standards.
Both categories are available in many different values, but differ in that the guidelines from ICUMSA,
states that no preservatives can be added to Brix Standards, so as a result the ICUMSA compliant
products have a shorter shelf life. Our range of ICUMSA compliant Brix Standards are also ISO 17034
accredited. Although these standards have a shelf life of 12 weeks as opposed to 6 weeks from our

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competitors, the non ICUMSA compliant standards have a shelf life of 12 months. All of the standards
in both categories are expressed in Brix units.

Both of the other two categories of standards are expressed in Refractive Index Units. The first
category is also sucrose based, have a twelve-month shelf life and are accredited to ISO 17025. The
second category are solvent based Refractive Index standards, that have a higher upper range of up
to 1.65808 units at 20°C and an extended shelf life of 24 months.

These products are accredited up to a Refractive Index value of 1.44193. Generally, the products are
offered in 15ml dropper bottles that are offered singly or in a multipack of 6 bottles. The test result of
both ranges is verified using a high-performance refractometer and the test is controlled using
Certified Reference Materials. Customised pack options may be available upon request.

There are many good producers of RI standards, which are available on the market. Those available
from Reagecon are presented here, simply, because these are the products that are most familiar to
the author. We do believe, though, that the Reagecon list is comprehensive in terms of offering and
specification and serve as a valuable template, to enable an analyst to know what to look for, when
sourcing such standards in the marketplace.

Sucrose Standards (ICUMSA Compliant)

• Primary level refractive index standards
• Values and temperature: 0, 5, 7, 10, 11.2, 11.5, 12, 12.5, 15, 20, 25, 30, 35, 40, 45, 50, 55
and 60% Brix @ 20C. *14.9, 19.4 and 23.8% Brix @ 20C also available
• Manufactured acc. to ICUMSA (International Commission for Uniform Methods of Sugar
Analysis)
• Manufactured and certified in accordance with ISO 17034 requirements
• Extended shelf life of 12 weeks (competitors 6 weeks)
• Available in packs of 1 or 6 x 15ml dropper bottles
Table 4

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 Brix Standards (Non- ICUMSA Compliant with Preservative)
• The most extensive range available in the market place
• Values and temperature: 0, 5, 7, 10, 11.2, 11.5, 12, 12.5, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60 and 67.5% Brix @ 20C.
• Range with preservative and 12 months shelf life
• Tested and certified to ISO 17025 (values up to 60%)
• Available in packs of 1 or 6 x 15ml dropper bottles
Table 5

Refractive Index Standards – Sucrose Based

• Extensive range covers 1.34325 to 1.44193nD @ 20C
• Uncertainty of measurement up to ±0.00014nD
• Shelf life of 12 months
• Tested and certified ISO 17025 for the full range
• Available in packs of 1 or 6 x 15ml dropper bottles
Table 6

Refractive Index Standards – Solvent Based

• Extensive range covers 1.38779 to 1.65808nD @ 20C
• Uncertainty of measurement up to ±0.00014nD
• Shelf life of 24 months
• Tested and certified ISO 17025 up to 1.44193nD
• Available in packs of 1 or 6 x 15ml dropper bottles
Table 7

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9.0 Features and Benefits of Reagecon’s Standards
A summary of the features and benefits of Reagecon’s Standards can be seen in Table 8.

Features and Benefits of Reagecon’s Refractive Index Standards

Range Brix ICUMSA Brix Non-ICUMSA Refractive Index Refractive Index
Sugar Based Solvent Based
Accreditations ISO 17025 for the ISO 17025 up to ISO 17025 for the ISO 17025 up to
whole range. 60% Brix whole range 1.44193nD
ISO 17034 for
Sucrose in Water
(% Brix)
Shelf-Life 12 weeks (6 12 months 12 months 24 months
weeks from
competitors)
Traceability Traceable to International Standards
Applications Suitable for use on any brand and type of refractometer
Table 8

10.0 Conclusion
The measurement of refractive index is carried out in a wide range of industries as detailed in Section
3.0 of this publication. In such instances, critical decisions are made based upon these RI readings and
so it is essential that analysts not only achieve the correct RI test results, but also prove the validity of
these results.

Ultimately the provision of the correct result and proof of that correctness, is a direct function of the
choice, quality and fitness for purpose of the standards used. The overall objective of this paper is to
offer guidance on making the correct choice.

11.0 Bibliography
1. Barron, J., (1996) “Turbidity Standards and Reference Materials”, Self-published, Reagcon
Technical Paper, Available from the Reagecon Knowledge Site

2. Barron. J., Papacosta. K., (1997). Poster presentation, Pittcon Conference, New Orleans, USA

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