Prof. dr. Kees Jalink
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“A main aim in basic cancer research is to unravel the differences between normal cells and cancer cells. These differences might then be exploited in the hunt for specific cancer vulnerabilities: any trade that is exclusive for cancer cells might give us leads on how to attack cancer cells while leaving healthy cells untouched”, explains Prof. dr. Kees Jalink of The Netherlands Cancer Institute in Amsterdam (NKI).

Today Prof. dr. Kees Jalink shares his story about the research the ‘Biophysics and Advanced Imaging Group’ the NKI (The Netherlands Cancer Institute) is working on and the role of flow meters and controllers in their research.

Biophysics and Advanced Imaging Group

Unraveling these differences between normal cells and cancer cells has proven a difficult task because most cancer cells are for 99.9% like healthy cells. In the Biophysics and Advanced Imaging Group, we zoom in on cells using advanced microscopy techniques, including live-cell imaging, fluorescence microscopy and “functional imaging” techniques. In the latter, tricks like Fluorescence Resonance Energy Transfer (FRET), Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy are used to extract information about proteins (biomolecules) and their interactions in single living cancer cells.

Image description Photo Jalink Group | Composite living cells NKI

Living cells yield much more information

Not too long ago, for visualization by high-resolution microscopy, cells were typically killed, fixed, stained for specific components and embedded in resin. However, imaging living cells yields much more information:

  • living cells may go through division
  • migrate
  • interact to form tight monolayers just like living (cancer) cells in our body Only with living cells, we can get a grasp of the dynamics of the internal biochemical processes.

A whole range of colored Fluorescent Proteins (picture 1) are available that enable us to label a single protein species and learn what we want to know about that protein. The trick is, to keep those cells alive and healthy on the microscope.

A two-color photomicrograph of a few cells through the microscope

A two-color photomicrograph of a few cells through the microscope

On the microscope, cells are kept in a glass bottom dish filled with DMEM medium: a bloodplasma-like salt solution with vitamins and nutrients. In the early days, we just kept them at room temperature in dishes in free air (~20 % O2, 80 % N2, 0.05% CO2). However, that does not mimic the atmosphere in our bodies at all, and consequently, results were not as expected.

For example, cells typically refused to divide and most cells died after 1-2 days. Also, control of pH in the medium appeared next-to-impossible. Therefore we needed to set up a dedicated incubator that houses the cells and most of the microscope. In this incubator, the air needs to be warmed to 37°C, moisturized and the atmosphere must be different from air:

  • it must contain at least 5% CO2
  • and the % of oxygen must be adjusted between ~2 % and 20%

This is to resemble the various oxygen tensions encountered in the body. For example, solid tumours are well known to be hypoxic (contain less than a few % of O2) and this completely alters the physiology of the cells, as well as their response to anti-cancer drugs. Read the process solution in our customers application note.

But how to achieve precise atmospheric control?

At a exhibition we learnt about Bronkhorst and their mass flow meters and controllers. With assistance of Bronkhorst Nederland, we have chosen three thermal mass flow controllers of the EL-FLOW Select series and hooked them up to the outlets of compressed CO2, N2 and air present in our lab.

The rest was simple: by adjusting the relative gas flows with the mass flow controller, we can now set CO2, N2 and O2 levels to all the relevant values. These ranges are 2-19 % for oxygen, 0-20 % for CO2 and 80 – 100% for nitrogen.

Ever since, we have carried out all our experiments under such controlled conditions and the results have been much more consistent -and also much more relevant- due to this incubator. We have used it to investigate how cancer cells migrate during metastasis and how they can penetrate layers of other cells and survive in this ‘niche’. We also used it to explore how cells use chemical signals to communicate with each other, and how these signals are received and subsequently processed within the cells, in detail.

Bronkhorst instruments in experimental setup

Bronkhorst instruments in experimental setup

The µ-Flow liquid mass flow controller comes to the rescue

As it goes in science, solving one problem led to the identification of another. We noted that at 37°C, the medium evaporated more rapidly, leaving the cells dry after a few days unless we tightly closed the imaging dish. But that restricts access to the cells, it makes it impossible to add growth factors, hormones or cancer drugs for our studies during the experiment.

Moisturizing the air only partly solved that, because at high humidity, condensation formed that might damage the sensitive electronics in our setup, and we therefore had to remain below 60 % relative humidity. Again, mass flow controllers provided a simple and reliable solution. We selected a µ-FLOW mass flow controller for liquids to supply a very stable flow of deionised water. Using a local BRIGHT controller with PiPS (Plug-in Power Supply) allowed us to control water influx between 0.5 and 9.6 microliter per minute.

Empirically we found that with the valve adjusted to 1.3 ul/min, we completely compensated for evaporation, and we are now capable of keeping cells alive for weeks on the microscope. The system has been very-low maintenance: simply install, and forget about it, so that we can focus on our core business. The mass flow controllers have been pivotal in constructing the microscope incubator, and the cells, they divide and develop happily.

Read and learn more about this application ‘Accurate flow control for cancer research’ from our customers experience. • Check out our instrumentsused in this application: µ-FLOW mass flow controller, EL-FLOW Select mass flow controller and BRIGHT controller

• Read more about this research on the website of The Netherlands Cancer Institute, NKI.

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Lynn Woerts
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2020 is a few days old and the plans and goals of this new year are starting to take shape. Let’s look back on last year. What did we achieve? Which blog post was the most fun, useful, gripping or interesting to you? Oh and by the way, we’ll assure you that we’re going to share all our knowledge about low flow, mass flow and flow meters even more often this year. From the overview of the 2019 statistics, we’ve come up with a top 5 of most popular blog posts.

  1. How to deal with vibrations using Coriolis mass flow meters
  2. Do you know why Mass Flow reference conditions matter?
  3. Real-time pressure and temperature compensation to optimize flow control
  4. Flow Meter Accuracy & Repeatability
  5. Flow control valves; the most used accessory in flow control

Top 5 most popular blog posts in 2019

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1. How to deal with vibrations using Coriolis mass flow meters A Coriolis mass flow meter is known as a very accurate instrument and it has many benefits. To be quite frank we were quite surprised that this blog post came in first place. In industrial applications, all kinds of vibrations with different amplitudes are very common. However, the question is whether these vibrations influence the measuring accuracy of a Coriolis mass flow meter. Ferdinand Luimes, Liquid Flow Technologies product manager, shares the advantages as well as the disadvantages of these flow meters and provides some handy hints in using these instruments.

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2. Do you know why Mass Flow reference conditions matter? The world of flow measurement applies reference conditions, which can be further divided into standard reference and normal reference. Another distinction is between European and American style. Chris King, Bronkhorst USA General Manager, sheds light on this apparently complicated construction in his blog post, detailing exactly what the differences are and explaining why these reference conditions matter.

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3. Real-time pressure and temperature compensation to optimize flow control This blog post topped the charts in 2018 and is still in the top 5 today, once again proving the relevance of this topic. As it turns out, various external factors can have an influence on the measurement accuracy and control stability of mass flow controllers. Vincent Hengeveld, Gas Flow product manager, explains the theory behind real-time pressure and temperature compensation.

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4. Flow Meter Accuracy & Repeatability Choosing which flow meter is right for your application is a pivotal element in its success. Generally speaking, the two important statistics are flow meter accuracy and repeatability. In his blog post, Chris King explains what these two parameters mean and why they are crucially important.

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5. Flow control valves; the most used accessory in flow control Finishing the list is a blog post about control valves, perhaps the most often used accessory in flow meters. A control valve is used to control a flow by varying the size of the flow passage. Do you know which valve is best for your flow meter? Stefan von Kann, senior engineer in applied physics, provides a number of tips and tricks for the most pressing areas of attention.

2019 guest bloggers

We wish to thank our guest bloggers very much for their fascinating studies and compelling stories. It fills us with pride that you contributed content to our website in 2019.

• Roland Snijder, medical physicist resident at Haaglanden Medisch Centrum (NL), worked as a researcher on the multi-infusion project at the department of Medical Technology & Clinical Physics of University Medical Center Utrecht. In his guest blog, he focuses on investigating physical causes of dosing errors in multi-infusion systems. • Jean-François Lamonier (University of Lille) is an expert in the catalytic treatment of volatile organic compounds. In this blog post, he explains how his team uses flow meters for this purpose. • Jornt Spit, researcher at the Radius research group at Thomas More University of Applied Sciences in Belgium, has a background in biochemistry and biotechnology. He is working on renewable biomass. Read his blog post on controlled CO2 supply for algae cultivation and its valuable contribution as an alternative source of carbon. • Prof. Michaela Aufderheide (Cultex Technology GmbH) has been working in the field of cell-based alternative methods with a focus on inhalation toxicology for more than 30 years. Increasing pollution of the environment and workplaces demands new testing methods. Read her blog post: ‘The e-cigarette – A blessing or a curse?’

Would you like to become even more inspired? All blog posts can be read on our website.

On behalf of the entire Bronkhorst team, I wish you a healthy, wonderful and innovative 2020!

Which topic would you like to be the focus of a blog post in 2020?

Sandra Wassink
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This weekend it’s the Easter Weekend! It’s the top selling season if you look at chocolate gifts. If you have a look in the supermarket now, you will find chocolate eggs, easter bunnies and bonbons in many variations. In our office we also have a large bowl filled with colourful chocolate eggs already, delicious!

And if we talk about various flavours of chocolate, that’s part were flow instruments come into the picture.

Chocolate confectionery industry

I would like to share my findings within the growing chocolate confectionery industry and the trends in using flavours. Who else can do this better than a woman you should think, as 75% of the women report that they indulge in chocolate, against 68% of the men.

Chocolate… a growing worldwide market of $100 billion once started with a simple choice between Milk, Dark or White chocolate. Nowadays the choice in variations is huge due to flavourings.

Chocolate as a seasonal gift is still very popular. Around the holidays we tend to buy more chocolate. The top selling season for chocolate is not Valentine’s Day, as you might think, but Easter. Besides treating yourself with chocolate, there is an emotional aspect. Chocolate can have a positive effect on your mood, especially with young adults. A popular reason for the increasing sales. The majority of the chocolate buyers are looking for options with mix-ins as opposed to the conventional unflavoured varieties.

Chocolate production

Flavour and textures

The global chocolate market has seen considerable innovation in flavour and texture. New product development continues to be imaginative, with more exploration of flavours and textures in addition to the traditional sweetness. However, the consumer base tends to be rather traditional as the most popular flavours still are Hazelnut, Caramel and Almond.

Older consumers tend to have a lower engagement with chocolate. The lack of interest reflects their desire to eat healthy. To regain this group of adult customers, companies have turned to tactics such as using alcohol flavours, organic ingredients, and premium positioning such as dark chocolate with Limoncello or chocolates filled with sweet liqueur.

Coriolis flow meter dosing block

Healthy lifestyle

It may come as a surprise, but a healthy lifestyle, which is one of the major trends worldwide, is also responsible for a substantial growth of the chocolate market and that’s not without reason. Chocolate, specifically dark chocolate with more than 85% cocoa, can offer beneficial health benefits. This results in labels mentioning:

  • Rich in fiber, iron, magnesium, copper, manganese and other minerals
  • Powerful source of antioxidants
  • Protective against cardiovascular disease

The growing awareness of the health benefits of dark chocolate is one of the reasons why consumption of chocolate is increasing. With the rising popularity of dark chocolate, the sales for other variations are also going up. People are seeking other ‘healthy’ variations, such as sugar-free, gluten-free, kosher or fair trade chocolate. Due to these ethical claims, the industry has seen an enormous growth in variations.

In order to enhance a healthy image for chocolate, functional ingredients such as fibers, proteins, micronutrients, quick energy (guarana extracts), green tea extract, or chia seeds are more and more often added to the chocolate.

Cocoa

The increasing demand for chocolate also has its downside. About 3 million tons of cocoa beans are consumed annually of which more than 70% are produced by four West African countries: Ivory Coast, Ghana, Nigeria and Cameroon. Cocoa is a delicate crop and trees planted a quarter century ago have hit their production peak and the land they grown on are not as fertile as it once was. A large rehabilitation of land and trees is necessary to prevent the loss of crop production. Also climate changes are taking their toll.

This results in high costs for raw materials and unstable economic conditions in cocoa-producing nations. To prevent a supply shortage, a number of well-known chocolate producing companies have decided to invest in rehabilitation of the land and trees to make sure that cocoa will be available in the future. This happens in a time that developing countries such as China, India, and Russia expect to increase their chocolate sales volume by 30%.

Mass Flow Meters in your production process

Due to the enormous growth of chocolate variations, using flavours and functional ingredients, mass flow meters and controllers find their way into the confectionery industry. Coriolis flow meters in combination with a pump are an ideal solution for dosing flavours and functional ingredients. Using the Coriolis instruments for additive dosing means less downtime between batches, traceability of ingredients, and higher product consistency and quality.

Watch our video about an additive dosing solution for the confectionery market.

Download our brochure (Ultra) low flow Coriolis competence for the confectionery industry.

Rob ten Haaft
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If we want to live healthy lives, we need to know the nature and content of undesirable chemical elements in our environment. If a municipal council wants to clean up a piece of land to develop a new suburb, it needs to know whether heavy metals or toxic substances such as arsenic remain in the soil from the previous use of the land. Likewise, the managers of drinking water sources, surface water bodies and fishing areas need to know about the quality of their water, to determine whether it contains excessive levels of undesirable substances that will have to be removed. And in order for air quality to be considered good, the trace element content in the solid particles floating in the air must not be too high.

Outside of the environmental field, there are other places where it is helpful to be able to identify and quantify the elements that are present – such as establishing the concentration of metal in lubricating oil to determine how quickly an engine will wear out, or the concentration of fertilisers in agricultural soil to determine whether additional fertiliser is required. Flow meters and regulators also play a major role here. As an industry specialist in the analytical market, allow me to explain how it all works.

Inductively Coupled Plasma – Atomic Emission Spectrometry, ICP-AES

As you can see, there are many applications in which it is useful to know what chemical elements are present and in what quantities. ICP-AES is a good analytical technique for measuring the nature and concentration of elements in solids, liquids and gases. This acronym stands for Inductively Coupled Plasma – Atomic Emission Spectrometry. Due to its high accuracy – up to the ppb (parts per billion) range – ICP-AES is particularly well suited to analysing trace elements, i.e. very low concentrations. This technique is excellent for detecting metals (such as mercury) and metalloids (such as arsenic), and dozens of elements can be analysed simultaneously. But what is behind this technique – and how does the careful delivery of gases play a role?

Controlled supply of argon gas through a flow controller

The short version: The ICP-AES method of elemental analysis uses an inductively coupled plasma to produce excited atoms and ions of the elements in the sample to be measured, whose characteristic spectrum is measured using atomic emission spectrometry (AES) as they return to their ground state. The intensity of the lines in the spectrum is directly proportional to the concentration of the elements in the sample.

The ICP-AES equipment can only analyse samples in liquid form. That’s not really a problem for water, but things get a bit tricky with soil samples and other solid substances. To unlock the chemical elements, you have to dissolve the sample in a strong acid: aqua regia, a mixture of hydrochloric acid and nitric acid. A peristaltic pump sucks the sample liquid out of a storage vessel and transports it to the nebuliser, which turns the liquid into an aerosol form or mist. To accurately regulate the concentration of the mist – and to dilute it if necessary – a flow of argon gas is supplied to the nebuliser, with the assistance of a flow controller. The mist then enters the reactor chamber, where it collides with the plasma that is already in the chamber.

If you supply a gas with sufficient energy – by passing a high electrical voltage through the gas using a coil – then some of the gas atoms release electrons. In addition to the original gas particles, you now have a mixture of negative electrons and positively-charged ions. This ‘ionised gas mixture’ of charged particles is called a plasma; plasma is considered to be the fourth state in which matter can exist, in addition to the solid, liquid and gaseous states. With ICP, argon gas forms the basis for the plasma, and this gas must be supplied with great precision, using flow controllers. The plasma has a very high temperature of around 7000 degrees Celsius. Because the plasma must have the correct composition at all times, a precise and continuous flow of argon gas is important. And to protect the outside world from this high temperature, a cooling gas (often but not always argon) is channelled around the outside of the reactor.

Regulating the mist

When the mist with the chemical elements to be measured collides with the plasma, these elements are also converted into plasma. The elements absorb so much energy that they enter an excited state. Elements don’t like to be in an excited state, so they try to return to their ground state at a lower energy level. During this transition, the elements emit radiation that is characteristic of each element. This radiation is measured by a spectrometer, and the intensity of the measured radiation is directly proportional to the amount of the element in question in the sample. Since each element has its own characteristic set of wavelengths of the emitted radiation, you can use this technique to identify multiple elements at the same time. And if you have a calibration curve for the elements concerned, or if you entered an internal standard into the nebuliser earlier in the process, then you can also quantify these amounts.

Spectrometer, ICP-AES or ICP-OES

The spectrometer within the AES part is a combination of mirrors, prisms, bars, monochromators/polychromators and detectors, which guide and ultimately measure the emitted radiation. To prevent any disruption to this process – such as the absorption of radiation by gases containing oxygen – the area where these optical objects are located is continuously flushed with nitrogen. This gas flow does not have to be very precise, but it does have to be highly reproducible. The use of flow controllers is essential to ensure this reproducibility. Incidentally, you may come across the term ICP-OES (optical emission spectrometry), which is an alternate name for ICP-AES (atomic emission spectrometry). These are two different names for the same technology.

ICP-MS

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ICP-MS is a similar technique for elemental analysis; the biggest difference is that the method of detection is not optical. The charged particles from the plasma enter a mass spectrometer (MS); here, they are separated on the basis of their mass-to-charge ratio, and the relative ratio of each of these charged particles is recorded. ICP-AES is performed at atmospheric pressure, but ICP-MS requires a vacuum. The detection limit for ICP-MS is lower than for ICP-AES.

In an environmental analysis, you can look not only at the total quantity of an element in a sample, but also at whether the element occurs in its free form or as a component of a chemical compound. By way of illustration: inorganic arsenic compounds are often more toxic than their counterparts in organic compounds. You can use ICP-AES and ICP-MS to distinguish between different forms of elements, a process known as ‘speciation’. However, this requires the different forms to be separated from each other before the ICP process, for example through ion exchange chromatography (IC). For this reason, the IC/ICP combination is very common.

Mass flow meters and flow controllers for ICP-AES

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When ICP was first invented, the gases were added manually. When ICP became automated gas regulation was automated too, and mass flow meters were introduced. Mass flow meters and flow controllers are devices used in ICP-AES to supply inert gases. If you have good gas regulation, the entire system is more accurate and more stable, enabling lower detection limits. Which is helpful, given the increasingly strict quality and environmental standards.

Bronkhorst supplies flow meters for the analytical market; our customers include a number of large suppliers of analytical equipment. These customers are often supplied with specific ‘manifold’ solutions. In these solutions multiple functionalities are integrated into a single body, custom built for the customer. Compact instruments with small footprints are becoming more and more important in laboratories where space is increasingly restricted.

Read the application story “Controlled supply of gases in Inductively Coupled Plasma (ICP-AES) for environmental analysis”.

Jos Abbing
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Industrial low flow applications have to cope with a wide variety of environmental and process conditions, but what does this mean when we talk about ‘industrial’? Knowledge about the specific application and low flow fluidics will help a lot to prevent slipping.

We often refer to ‘uncontrolled macro-environments’ for equipment, when we talk about ‘outdoors’. However, it can also be a room or factory without (local) climate control in which equipment is experiencing comparable temperature and humidity variations as outdoors.

What is important in low flow applications and what kind of challenges do you encounter? Let me share my ideas in this blog.

What is IP-rating?

I experienced that IP-rating is not always interpreted correctly. Having the highest possible IP-rating is often mistaken with having an ‘industrial-device’. But what does the IP-rating actually indicate? The first digit of an IP-rating only refers to dust ingress protection and the second digit refers to the liquid ingress protection.

Therefore, a higher IP-rating does not always mean that the instrument is better and more suitable for your application. Hence, it can even make things much worse in practice. A reason for this is that even the tightest IP-rated constructions may breathe in and out, caused by internal and external temperature variations. This can lead to internal condensation, especially in high humidity environments, if no further precautions are taken.

The importance of dedicated low flow equipment

Not surprisingly, things are often a lot smaller in low flow applications. The other side of this coin is that common process and environmental disturbances have a proportional larger impact on these low flow applications compared to traditional ‘normal’-flow applications.

In general, an industrial flow instrument, like a flow meter, needs to be suitable to a lot of external influences, such as resistance to corrosion and impact or pressure ratings. These requirements often lead to selecting more standard industrial flow meters instead of specialised low flow instruments. This is not always the best solution for the required low flow ranges and can lead to unsatisfactory results.

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What we want to achieve is to have rigid flow measurement and control, suitable for the application during the economic lifetime of the installation. Therefore, it would be best to select the best flow instrument fit for purpose. In case of low flow applications I therefore recommend to use dedicated low flow equipment. These flow meters are designed and tested for these kinds of applications. Our industrial low flow mass flow meters and controllers can be equipped with integrated control valves or dedicated pumps, especially designed for low flow purpose. Stable control characteristics are combined with signal-to-noise ratio plus being proportionally less sensitive for disturbances. Bronkhorst industrial low flow instruments We gladly support you in process and environmental equipment selections including system design aspects, starting with selecting the most suitable measuring and control principles. Our flow meter product portfolio contains laboratory-style and light-industrial flow meters to heavy-duty IEC-Ex/ATEX-rated industrial versions (…having a “high” IP-rating as well).

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Drive your low flow control reliable and safely!

Our product manager for liquid technologies, Ferdinand Luimes, explains how to deal with vibrations using Coriolis mass flow meters

Visit us at the Hannover Messe (April 1-5, Hall 11, booth A50)) and have a sneak preview at our new industrial Coriolis flow meter.

Kevin van Dijk
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For most people the classic summer treat is ice cream. Around 7 billion gallons of ice cream and other related frozen desserts are produced every year worldwide, with production peaking (as you might expect) in the summer months, according to the International Dairy Foods Association. Yet, the moment you consume an ice cream, you will probably not wonder how this delicacy is being made. To get that perfect ice cream, a mass flow controller is often used.

What does ice cream have to do with mass flow meters?

Ice cream contains many different ingredients, such as fat, sugar, milk solids, an emulsifying agent, flavouring and sometimes colouring agents. But there is one main ingredient that you may not have thought about, probably because you can’t see it—air. Ice cream is made by freezing and simultaneously blending air into the ingredients. So why is air so important?

If you have ever had a bowl of ice cream melt, and then refroze it and tried to eat it later, it probably did not taste very good. Moreover, if you leave a carton of ice cream out in the hot sun and let it melt, the volume of the ice cream would simply go down. Air makes up anywhere from 30% to 50% of the total volume of ice cream, therefore, aeration in the production process is crucial.

The amount of air in ice cream (often called overrun) affects the taste, texture and appearance of the finished product. Higher aeration will produce a tastier and smoother ice cream. A side effect of adding air to ice cream is that it tends to melt more quickly . Thus, for attaining an optimal structure of the ice cream, it is important to have a stable inlet air flow in the production process with a constant cream/air ratio. This can be achieved by using a mass flow controller.

The process of whipping ice cream into shape

To guarantee the right consistency and structure which ensures a full flavoured ice cream, the cream must contain the correct proportion and composition of air bubbles. Hence, aeration mixer manufacturers use a mass flow controller to dose an exact amount of air into the cooled mixer. Such a mass flow controller will ensure a continuous air delivery, proportional to the cream flow . The mass flow controller must be capable of maintaining its performance regardless of any possible back pressure variation. Occasionally, a check valve is mounted downstream of the mass flow controller. If inlet pressure drops, such valve will avoid ice back stream into the instrument. A pressure meter is also used with the purpose of monitoring the inlet pressure.

flowschemeofwhippingicecreamprocess

The SEM (Scanning Electron Microscope) picture below shows the ice cream microstructure. Air bubbles are a critical ingredient. Experts claim its optimal size, distribution and quantity are one of the secrets for having a creamy texture recipe. Hence, according to meet such demands, Bronkhorst has provided efficient solutions for enhancing continuous aeration processes.

Ice cream structure

So, the next time you head to the ice cream parlor with your friends, be sure to keep in mind the importance of Bronkhorst when it comes to that delicious refreshment.

EL-FLOW select

  • Watch the video about the EL-FLOW Select to learn more about the thermal mass flow instrument which can help you create ice cream.

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