It demonstrates the repeatability of the Photopette and compares the Bradford Assay results with the Photopette to readouts done with a  benchtop spectrophotometer and microplate reader.

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Unless you are a trained microbiologist, or have additional experience working with bacteria and other organisms, the concept of OD600 might be unfamiliar to you.

In this article, we are going to give you an overview of what exactly OD600 is, how to measure it and some real-life applications.

What is OD600 and What Does it Mean?

OD600 is an abbreviation of two parts; ‘OD’ is short for ‘optical density’, whilst ‘600’ is in reference to the 600 nm wavelength used to measure said optical density.

The term ‘OD600’ is used in reference to a spectrophotometer method that is used to help estimate the concentration or “number of cells per volume” of bacteria or other cells within a liquid sample.

How is OD600 measured?

OD600 is typically measured using a bench-top spectrophotometer, but can also be measured using our Photopette® handheld spectrophotometer.

A sample is placed within a cell or cuvette, and a light source of 600 nm is directed towards the sample. The light is absorbed and scattered by cells and the transmitted light is picked up by a detector, which outputs a value.

The light scattering value that is generated by the sample is what is used to estimate the concentration of bacteria or other cells within a sample.

Light scattering is referred to as a factor of turbidity, rather than light absorption.

What is OD600 used for?

Determining the concentration of bacteria, or other cells within a sample has lots of real life applications, with a few outlined below:

1. Synthetic Biology

In synthetic biology, organisms are redesigned for additional purposes than what they originally are used for. To research and develop new cells, it is vital that cells can be grown and harvested at the optimum level of growth.

2. Microbial Production

The use of microbes is far-reaching across many fields. They play a big part in the food and beverage industry, as well as having the potential to provide fuel sources such as ethanol. Using OD600 to measure cell growth is of vital importance, particularly where microbes are grown for commercial purposes.

3. Antibiotic Resistance

Developing new antibiotics requires susceptibility testing, which determines the effectiveness of antibiotic treatment. To test the effectiveness of new antibiotics, measuring the optical density of bacteria, helps to understand if, and how bacteria is growing in response to new antibiotics.

Using OD600 to Determine Cell Growth Phase

When measuring OD600, the measured value that is given is an arbitrary number that needs further interpretation in regards to the overall cell growth cycle.

Using a standard or control growth curve, it is possible to plot measure OD600 values against a known cell growth chart and understand the cell growth phase. A typical growth chart for a bacterial cell or microorganism is shown below:

Spirulina is a microscopic cyanobacteria that can be consumed by both humans and animals, as either a whole food or food supplement.

The distinctive blue-green bacteria is often touted as a ‘superfood’, due to its high density of proteins, vitamins and antioxidants. The nutritional profile of the food means that it may help to lower cholesterol, improve diabetes management and prevent heart disease as well as all-round health. For animals, spirulina is often used as a fish feed or as a livestock supplement.

With plenty of applications for Spirulina, the commercialisation of spirulina is big business. But ensuring that maximum profits are generated, understanding how spirulina grows and when to harvest is an important aspect.

How Does Spirulina Grow?

Spirulina is a free-floating bacteria that typically grows on the water’s surface in tropical and sub-tropical climates. Because Spirulina are autotrophic, they produce their own energy source, but are most often found in conditions where water pH and temperatures of 9.5-10.0 and 30℃ respectively are present. A high alkalinity is often present by a high concentration of both carbonates and bicarbonates – both necessary for spirulina growth.

However, it is also possible for Spirulina to be farmed for commercial purposes. Replicating natural growth conditions requires monitoring and optimisation of the following properties:

1. Water Quality

Although spirulina is robust enough to grow in a broad of conditions, having a water source that is optimum pH and rich in carbonates and bicarbonates will maximise spirulina production.

2. Light

Growing spirulina at commercial scale will require the use of artificial light. The wavelength of the light, and the intensity will directly impact the colour and nutritional content of the spirulina.

3. Temperature

Spirulina growth is highest at 30℃, with temperatures either side risking production output.

4. Water Agitation

As spirulina grows, only the top surface will be exposed to sunlight. Water must be agitated to ensure that the bacteria is rotated to prevent any cells from dying.

Fluctuations outside of the typical growing conditions for spirulina can affect the growth rate, and properties such as the colour and protein content. This makes spirulina farming cost ineffective.

Growth Rate of Spirulina

Controlling the range of spirulina growth factors listed above will help to maximise spirulina output. But because spirulina is a bacteria, it follows the typical growth curve that we’ve previously outlined in our article about understanding OD600 and cell growth.

Therefore when it comes to harvesting spirulina on an industrial scale, understanding when maximum output has been achieved, but before cell death.

There are a few ways to determine spirulina biomass.

Biomass Determination Methods

When it comes to measuring spirulina, there are a few different properties of spirulina that can be analysed in the lab or directly at the spirulina tank.

Optical density (OD) is one of the most common parameters to monitor and typically involves the use of a benchtop spectrophotometer. The optical density correlates to the concentration of spirulina present and can help determine at which stage of the bacteria growth curve the spirulina is going through.

Alternative measurements to optical density include turbidity and light scattering. Both of these methods can determine the mass of spirulina present when compared to control data. The higher the concentration of spirulina, the higher the measured value of both turbidity and light scattering that will occur.

Improving Efficiency For Spirulina Farmers

Regardless of which methods (mentioned above) you currently use to measure your spirulina crop, all methods require analytical equipment that are typically found in the lab. Due to the size and complexity of these instruments, obtaining results typically means taking samples and transporting them to a lab. Not only is this time consuming, it can result in inaccurate results and be a costly method.

A better, more efficient method to measure your spirulina culture is using our Photopette® instrument. Our handheld spectrophotometer allows users to take measurements in seconds, with no training or specialist knowledge required.

Due to the flexibility of the instrument, we are able to build Photopette® Custom devices supporting the possible to take measurements at 565 nm (phycocyanin complex), 680 nm (chlorophyll) and 750 nm (turbidity caused by cells) to match the wavelength to optical density, turbidity or light scattering method respectively, of choice.

FAQs

• Where does spirulina grow?
  Spirulina typically grows in bodies of water that have a high pH level and are rich in minerals. Spirulina typically grows in tropical and subtropical climates.
• What are the benefits of spirulina?
  Spirulina is beneficial to both humans and animals. It can be consumed on its own as a ‘superfood’ or it can be added to foods or taken as a supplement for animals such as livestock and fish.
• How can you determine spirulina growth?
  To determine the mass of spirulina at any one point, you can typically use one – or a combination of – optical density, turbidity and light scattering. However, with our Photopette® option, it is possible to measure all three with one handheld instrument.
• What factors influence spirulina growth?
  Spirulina growth is impacted by factors including water quality, light, temperature and water agitation. Ensuring as many of these factors stay as close to the optimum as possible will maximise spirulina output.

An enzyme assay is the name given to any laboratory technique that measures enzyme activity within a sample.

Enzyme assays can be used for a variety of purposes, which include identifying the presence of an enzyme, investigation of specific enzyme kinetics or the activity of inhibition within a sample.

Measuring Enzyme Activity

When it comes to measuring enzyme activity, both qualitative and quantitative methodologies can be used.

Qualitative assays are used to identify the presence (or absence) of a particular enzyme. On the other hand, a quantitative assay can be performed to determine the amount of the target enzyme that is present within a sample.

For qualitative assays, the use of coloured compounds is typically enough to confirm or deny the presence of the target enzyme. However, to ensure reproducibility and avoid operator error, using an appropriate instrument such as a colorimeter or photometer may be more appropriate than visual confirmation alone.

Alternatively, quantitative assays are used in cases where instruments operating in the visual range are not suitable or determination of enzyme concentration of kinetic mechanisms are of interest.

Types of Enzyme Assay

There are two types of enzyme assay, which can be split into two; continuous and discontinuous assays.

1. Continuous Enzyme Assay

In continuous assays, the course of the reaction is continually followed until completion.

Sometimes referred to as ‘endpoint assays’, enzyme activity is measured via the quantity of substrate consumed, or the amount of product formed during the reaction over a fixed period of time. Both values are directly proportional to the concentration of enzymes present in the sample.

Examples of continuous assays include spectrophotometry, calorimetry, chemiluminescence and fluorimetry. In these methods, the progress of reactions are measured by light or heat.

2. Discontinuous Enzyme Assay

In contrast to continuous enzyme assays, discontinuous assays are performed when samples are taken at set intervals. This form of enzyme assay directly or indirectly measures changes in substrate or products over time, to understand how the reaction rate changes.

Examples of instrumentation used during discontinuous enzyme assays include radiometric assays as well as chromatographic assays such as HPLC or TLC.

Comparing the two methods, the continuous enzyme assay method is typically the easiest to perform and can give whilst discontinuous enzyme assays are used in cases where higher precision or complex sample matrices are present.

Factors That Affect Enzyme Assay

In order for an enzyme assay to remain accurate, controlling external factors so they do not influence the outcome of the assay is crucial.

1. pH

All enzymes have an optimum pH range where their rate of reaction is highest. Anything too far out of the optimum range will cause denaturation and a reduction in reaction rate.

2. Temperature

Generally speaking, as temperature increases, so does the reaction rate of an enzyme. However, once temperature exceeds a certain threshold, reaction rates can drastically drop. This is due to the effect that temperature has on the bonding of the reaction site.

3. Substrate Saturation

Increase in substrate concentration will increase the rate of reaction, but only up to a certain point. At the point of saturation, the reaction rate is at its upper limit and will not increase, regardless of how much substrate is added.

4. Salt Concentration

Enzymes typically work best in low salt concentration environments. Increase in salt concentration causes interference with bonds, which impact the structure of the bonding site and the resulting reaction rate.

Enzyme Assay Use Cases

Enzyme assays cover a wide range of real life use cases. A couple of examples of enzyme assays include the following:

1. Alcohol Concentration

The measurement of short chain alcohols (methanol, ethanol, propanol) can be useful to determine a range of properties, such as the level of alcohol in food and drink, or metabolism rates.

Alcohol can be metabolized via many pathways, and is broken down by two enzymes – alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) – so measuring the properties of these enzymes can give a wealth of information.

2. Lactate Enzymatic Assay

Lactate is a metabolite produced by the breakdown of glucose in humans and animals via anaerobic metabolism. It forms when NADH is oxidized to NAD+, due to the reduction of pyruvate to lactate, which is catalyzed by the enzyme lactate dehydrogenase (LDH).

Measuring the activity of LDH can help understand information about the process of the metabolism process.

Performing Enzyme Assays With Photopette® Cell

In traditional enzyme assays, performing measurements via a spectrophotometer typically means obtaining samples in a cuvette and repeating.

With Photopette® Cell, our handheld device has been designed for a sampling free workflow to increase efficiency and decrease cross-contamination. Dedicated to measurements at 340 nm, 570 nm and 600 nm, Photopette® Cell is the ideal instrument for enzyme assays.

FAQs

• Why do we need to measure enzyme activity?
  Performing enzyme assays provide a wealth of information related to enzyme activity. This includes the identification of an enzyme, the concentration of enzyme and the rate of reaction within a sample.
• What is an endpoint enzyme assay?
  An endpoint assay is an assay method that is run for a predetermined amount of time. The amount of substrate consumed and/or the quantity of product formed is measured over the course of the reaction.
• What is a kinetic enzyme assay?
  In the kinetic assay method, the progress of the reaction is continuously measured as substrates are converted into products. Changes in concentration of both substrate and product cause shifts in measurements.
• What is the difference between endpoint and kinetic?
  The main difference between the two methods is how measurements are taken. In the endpoint assay, a final measurement is taken to measure the total amount of substrate/products. In the kinetic assay method, multiple measurements are taken over the course of the reaction.
• How do you measure endpoint activity?
  Endpoint measurements can be performed qualitatively, via visual assessment, or quantitatively via instruments such as spectrophotometer or calorimeters. Photopette® Cell by Tip Biosystems is a handheld device which can be used to measure an endpoint.

Chlorine is a chemical element that is a yellow-green gas at room temperature. The element is part of group 17 in the periodic table – the halogens – which accounts for many of its chemical properties, including its high reactivity.

Because of its high reactivity, chlorine is naturally present as ionic compounds. Although it is commonly found within the earth’s crust, chloride is also found in sea water in the form of brine.

The majority of elemental chlorine that is commercially produced comes from brine, before it is used for a range of different purposes.

Uses of Chlorine

Chlorine has a wide range of industrial and commercial applications.

Approximately 20% of all chlorine produced is used to make PVC, a versatile plastic used in window frames, pipes and flooring.

Another major use of chlorine is within organic chemistry. It is used within most chemical reactions, with 85% of pharmaceuticals using it at some stage within the manufacturing process.

Additionally, chlorine is commonly used as a disinfectant. It is often associated with disinfectants of swimming pools, but has far wider use within the water industry.

Chlorine Treatment in Water

Most people are aware of the use of chlorine as a disinfectant in swimming pools, but chlorine is commonly used in the treatment of drinking water also.

Chlorine doesn’t work as a disinfectant in its gaseous form, instead it forms analogues such as hypochlorous acid, hypochlorite and hydrochloric acid to do so.

The Presence of Chlorine in Water

Due to the chemical properties that chlorine possesses as a halogen, it makes it a highly effective disinfectant. It is commonly added to water to kill diseases that can be caused by bacteria, parasites and viruses that grow within water supplies.

When chlorine is added to water, it forms hypochlorous acid (HOCl) in equilibrium with chlorine and hydrochloric acid (HCl), as shown below:

Cl₂ + H₂O HOCl + HCl

In aqueous solutions with set conditions, hypochlorous acid can dissociate into hypochlorite ions (ClO-):

HOCL ClO^– + H⁺

Depending on the pH will determine how much of the hypochlorous acid will dissociate into hypochlorite ion. At pH 4, hypochlorous acid is present at 100%, dropping down to 0% at pH 11, and vice versa for hypochlorite.

Although both molecules disinfect water, hypochlorous acid is the most effective of the two.

Hypochlorous acid is commonly used as a disinfectant due to its ability to penetrate the cell wall and destroying enzymes and proteins by its oxidative activity.

Free Chlorine, Combined Chlorine and Total Chlorine

When it comes to measuring the presence and concentration of chlorine, there are multiple forms to which chlorine is referred – free chlorine, combined and total chlorine.

1. Free Chlorine

Free chlorine is used in reference to all of the chlorine present in a water sample, as Cl2, HOCl and OCl.

As soon as free chlorine is added to water samples, it quickly reacts with contaminants. This will result in free chlorine becoming combined chlorine.

2. Combined Chlorine

Combined chlorine refers to free chlorine that forms with organic amines or ammonia that are present in water. Once chlorine becomes combined, it can no longer act to disinfect or sanitise.

This happens when chlorine is added to water, forming hypochlorous acid and hypochlorite ions, which are both reactive forms of chlorine.

Combined chlorine can be problematic, not only for the fact that it can no longer act as a disinfectant, but due to the pH imbalance it can cause. This can lead to a cause of corrosion for metals.

3. Total Chlorine

Total chlorine is the sum of both free chlorine and combined chlorine.

It is important to know the difference between all three forms of chlorine, as they can affect how you measure chlorine in water samples.

Impact of Chlorine in Water

Like with the use of chemicals in most industrial applications there are drawbacks to using it.

1. Taste and Odour

Chlorine has a distinctive odour and taste, which is noticeable in drinking water when it is added. Although it is considered safe up to levels of 4 ppm, many people can taste and smell its presence in treated water. Levels above 4 ppm can have more severe consequences.

2. Health Issues

There are a number of health issues that have been associated with chlorine-containing compounds used in water.

They start with mild health issues such as asthma or allergies and have been linked to more severe issues such as congenital abnormalities and cancers.

3. Aquatic Impact

Free chlorine is known to be toxic to aquatic organisms such as fish. Chlorine damages the gill structure of fish, which can make it harder for them to breathe, which is made worse as the pH level is decreased.

4. Safety Concerns

Pure chlorine is extremely volatile and hazardous, which means that it needs to be transported and processed properly. Due to the highly hazardous nature of chlorine, even the smallest accidents can have a large impact on human and environmental health.

5. Toxic By-Products

The reactivity of chlorine is one of its properties that makes it so useful as a disinfectant in water, however, it can easily form unwanted compounds that can be toxic in nature. It is crucial that these by-products undergo further processing to ensure they are removed and don’t find their way into the environment.

Testing For Free Chlorine

Testing free chlorine concentration in solution typically uses methods that prioritise ease of use over accuracy.

1. Test Strips

One of the simplest methods for measuring free chlorine is via the use of test strips. Dipping the strips in solution will change colour corresponding to a chlorine concentration. Test strips are easy to use, but are likely to be inaccurate and prone to error.

2. Colour Wheel

A method for measuring free chlorine is via the DPD method, typically at the 0.1-2.0 mg/L range. The DPD indicator and buffer solution are mixed with the chlorine-containing water samples, forming a pink solution.

Using a series of colours from a colour wheel, it is possible to approximate free chlorine concentration in a sample.

3. Colorimeter

In a process almost identical to the method listed above, digital colorimeters are used in place of colour wheels, to accurately measure the colour intensity by emitting a wavelength and determining the colour intensity.

This allows for greater accuracy with typically a larger measurement range.

Free Chlorine Testing with Photopette® Aqua

Typical free chlorine testing is simple and straightforward, where test methods have been created for ease-of-use. Unless using a colorimeter, testing is typically inaccurate or open to error.

As a new alternative to free chlorine testing we offer the Photopette® Aqua. The product is a handheld device that contains a digital colorimeter which can measure free chlorine, as well as analytes such as ammonia, nitrate, nitrite, phosphate and more.

Photopette® Aqua is suitable for water testing, both in the lab and the field. The device is waterproof, dustproof and very durable and because it is so easy to use, only minimum training is required. Photopette® Aqua allows for simple free chlorine testing, whilst providing better accuracy and precision of results.

Featuring five inbuilt calibrations for Merck Spectroquant® water test kits, it can measure a total of 18 analytes from the Spectroquant® family.

Removal of Chlorine

Once you have tested your water sample for the presence of free chlorine, it is likely that you are going to want to do something about it. Below are a few methods for removing free chlorine from your water.

1. Neutralisation

For neutralisation to happen, additional chemicals are added to water, typically in the form of a tablet. The chemicals react with free chlorine, neutralising it, before it evaporates from the water.

2. Evaporation

Chlorine is naturally removed from water via evaporation, but the rate of evaporation will depend on multiple factors.

Because of the volatility of chlorine, it will naturally evaporate from water. The rate of evaporation will be increased where temperature of water and air is increased, where there is a greater surface area of air to water or where water is agitated.

3. Filtration

Chlorine can be removed from water via reverse osmosis. Water is pressured to pass through a semi-permeable membrane which removes chlorine and other contaminants from the water.

• How Much Chlorine Is In Drinking Water?
  The WHO states that the level of chlorine present in disinfected drinking water is typically between 0.2 – 1.0 mg/L. The amount of chlorine present in your drinking water will depend on your geographical location as well as how your water is treated.
• Is It Safe To Drink Water With Chlorine?
  Drinking water containing chlorine is considered to be safe by many countries and governments, even in excess of the 1.0 mg/L stated by WHO.
• Are High Levels of Chlorine in Water Bad?
  What is considered a ‘high level’ of chlorine will vary on a case-by-case basis with different levels of free chlorine required for drinking water, or to disinfect a swimming pool for example.

As with all Photopette® models, the Aqua automatically attaches a geotag and timestamp to measurement results as well as optionally a photo or note, which dramatically streamlines data collection/documentation of samples in the lab and in the field.  This has proven beneficial in areas such as agriculture, aquaculture, environmental monitoring, wastewater analysis and other ecological/geological applications. The results are instantly available in digital form and can be exported as CSV file via e-mail or cloud.

The Photopette® Aqua was designed to utilize the Spectroquant® Reagent Test Kits from Merck for a variety of parameters, and with maximum user friendliness.

Please contact your local distributor for more information about Photopette Aqua, or feel free to contact us directly at info@tipbiosystems.com.

An Introduction to Turbidity

Turbidimetry is an analytical technique that is used to measure the cloudiness or haziness of liquid samples.

For example, consider the differences in water from a tap and water from a muddy river. The water from the tap is colourless and clear, with a very low turbidity. On the other hand, a muddy river is brown and opaque and therefore has a high turbidity.

When we consider these two samples and what makes the water appear so different, we have to consider the suspended solids that affect the clarity of the water. Turbidity is a measure of clarity of a sample, rather than the measure of the amount of dissolved or suspended solids within a sample, which can be caused by inorganic sediment, bacteria or precipitates.

Turbidity is a great indicator of water quality, whether that be regarding the quality of drinking water or the quality of aquatic environments, of which will be covered further in this article.

How is Turbidity Measured?

The way in which turbidity is measured has evolved over time.

The first known measure of turbidity was called the Jackson Candle Method, which used a vertical glass tube over a candle. A sample was poured into the tube until the user could no longer see the distinct image of the candle flame.

The final height of the sample which was added resulted in the corresponding turbidity measurement. Although crude in methodology, it was important to provide the basis of modern turbidity measurements.

Modern day methods for measuring turbidity consist of using turbidimeters, which consists of a fixed light beam, aperture, and detector.

In most modern turbidimeters, a sample is obtained, added to a vial and placed in the instrument. The fixed light beam is then shone in the direction of the sample to measure how much light is transmitted and how much is scattered, by photodetectors set 90-degrees to the sample.

Alternatively, in handheld instruments – such as our Photepette instrument – you take the instrument to the sample. The users put a CuveTip on the device, directly immersing the tip into the sample to take a reading – as demonstrated in the image below:

Modern instruments can determine turbidity in two ways, by the loss of light from the transmitted beam, and the measurement of light scattered onto surrounding detectors.

In the presence of suspended and dissolved solids, light will be scattered, resulting in a detection of light from photodetectors. The higher the concentration of particles or solids, the more light that is scattered, resulting in a higher turbidity measurement.

Measurements using the difference in light intensity from the transmitted beam is typically suitable for samples of high concentrations of solids, or turbidity, whilst light scattering is a suitable method for samples with a low concentration of solids.

The Units of Turbidity

When it comes to measuring and reporting turbidity measurements, you often see results reported in NTU or JTU.

 NTU – Nephelometric Turbidity Units – is the common unit used to report measurements from modern turbidity instruments, whereas JTU – Jackson Turbidity Units – are the original units used from the historic Jackson Candle Method described previously.

Today, NTU is the standard unit used to signify an instrument measuring scattered light at a 90-degree angle from the incident light beam.

Other than NTU and JTU, it is possible to come across alternative turbidity units as outlined below:

• FNU – Formazin Nephelometric Units – signifies the measurement of turbidity via light scattering and used when referencing ISO 7027
• FTU – Formazin Turbidity Units – Similar to NTU, but more commonly used when referring to formazin as the primary reference standard
• FAU – Formazin Attenuation Units – signifies the measurement of turbidity via light transmission, typically measured by colorimeters or spectrophotometers.

Because the optical design of the light source, aperture and photodetector are different in different instruments a calibration with a turbidity standard, or NTU standard, is always required.

Causes of Turbidity

In environmental water analysis, there are numerous sources that can contribute to high turbidity measurements, from both natural and human sources. This can include sediment which can be disturbed and suspended; a common event after rainfall.

But other causes of high turbidity include the growth of algae and bacteria, or the release of tannic acids from peats and bogs. These sources can be exacerbated by human activities, such as erosion or pollution.

But turbidity isn’t always a direct correlation with the number of dissolved or suspended solids in a sample. The size, shape and composition of the offending material all have a direct effect on the final turbidity, which can make diagnosing identifying causes of turbidity difficult.

Applications of Turbidity Measurements

Because turbidity is a measure of the clarity of water, it is a useful methodology to determine the ‘quality’ of water in the sample.

Typical drinking water is clear and colourless with a very low – almost zero – turbidity value. Turbidity measurements above the typical value may indicate the presence of bacteria, pathogens or particulates that may not be visible to the naked eye.

Other applications for turbidity measurements include the monitoring of the quality of water in rivers and streams and the impacts on aquatic life, or measuring the turbidity of wastewater effluent to monitor for the presence of harmful pathogens.

Turbidimetric measurements are also applied in the chemical industry, the food and beverage industry and for research purposes in various labs.

FAQs

How do you measure turbidity in water?

Measuring turbidity requires taking a sample from the source and using an instrument to analyze it. Ensure the turbidimeter has been recently calibrated using turbidity calibration standards and take a minimum of two readings

What is a good turbidity level in drinking water?

In Western countries, turbidity levels of drinking water should be below 1 NTU, ideally aiming for an average turbidity measurement of 0.2 NTU or less. In poorer or low resource countries, aiming to keep the turbidity of drinking water below 5 NTU should be the target.

What are the limitations of typical turbidity measurements?

Modern turbidity measurements require obtaining a sample, and then sending it to a lab to complete the measurement. The lab measurement is very accurate. However, the sample transport can cause large errors. During transport, solids may aggregate and sediment causing a decrease in turbidity or algae or bacteria continue to grow causing an increase in turbidity.

Using a solution such as our Photopette® Turbidity Handheld Turbidimeter provides a sampling free workflow to measure directly in the sample; no sample transport is required resulting in a much better accuracy.

It is an ideal solution for environmental monitoring in industries such as wastewater treatment plants or food and beverage. 

Colour is everywhere. Every chemical compound absorbs, transmits, or reflects light over an electromagnetic spectrum in wavelengths. When light passes through any solution a section of it is absorbed. Spectrophotometry allows both qualitative and quantitative analysis. As the concentration of a substance increases light absorption increases, and light transmission decreases.

Spectrophotometry is used in chemistry, biochemistry (for enzyme-catalysed reactions), physics, biology, and clinical studies (examining haematology or tissues). It allows scientists to analyse different samples without having any skin contact as the samples are contained in a small tube called a cuvette or in case of the Photopette, measurements are done directly in the sample container without having to transfer it.

How does a Spectrophotometer work?

Handheld Spectrophotometer – Photopette

Spectrophotometry is a standard and inexpensive technique to measure light absorption or the amount of chemicals in a solution. It uses a light beam which passes through the sample, and each compound in the solution absorbs or transmits light over a certain wavelength.

Spectrometry is measured by a spectrophotometer; an instrument that is made up of two instruments – a spectrometer and a photometer. The spectrometer produces the light of the wavelength and the photometer measures the intensity of light by measuring the amount of light that passes through the sample.

In addition to those two components, spectrophotometers consist of a light source, a monochromator, a sample chamber containing a cuvette, a detector (such as a photomultiplier tube or photodiode) to detect the transmitted light, a digital display and a data analysis software package.

Light Source

Spectrophotometers rely on light sources to operate. Because of the wide range of samples, light sources can vary in nature, and use a wide spectrum of wavelengths, including visible, UV and IR.

Monochromator

The monochromator (such as a prism or grating) inside the machine refracts the light into a single spectrum and disperses polychromatic light into the essential wavelengths. A grating divides the light available into different segments. Gratings are common in spectrophotometers that use UV, visible and infrared regions.

Sample Chamber

The sample chamber is where the operator inserts the sample for analysis. Samples are typically placed into a cuvette made of a material such as glass or quartz.

Detector

The detector is the light-receiving element that absorbs the energy of the incident light. Examples of typical spectrophotometer detectors include photomultiplier tubes and photodiodes. They convert the light energy into an electrical signal, which is converted into an absorption figure.

Digital Display

Modern day spectrophotometers typically have a digital display built into the instrument. This gives operators an accessible way to change instrument settings, set up method parameters and see results. It has no effect on the way the instrument works however.

Data Analysis

Alongside digital displays, most spectrophotometers have the ability to do any calculations and analysis. Once all the method parameters have been set up within the instrument, data and results are output once the method is complete.

Absorbance Wavelengths

In the spectrophotometer, the number of photons absorbed by a solution is called the absorbance readout. The longer the path-length that the light must travel through a solution prior to it reaching the detector, the greater the chance of a photon being absorbed.

Different compounds absorb best at different wavelengths. A UV-visible spectrophotometer uses light over the ultraviolet range (185 – 400 nm) and visible range (400 – 700 nm) of the electromagnetic radiation spectrum. Whereas an IR spectrophotometer uses light over the infrared range (700 – 15000 nm).

Ultraviolet (UV) and visible (VIS) spectroscopy show electronic transitions in atoms and molecules, to measure this a spectrophotometer is used. Compounds that absorb in the visible region are coloured, whereas ones that absorb only in the UV region are colourless.

UV-VIS spectrophotometer usually use two light sources. A deuterium lamp is used for the UV region and a tungsten lamp for the VIS region. These lights reach the monochromator via a mirror. The wavelength for red light is between 700 and 750 nm and blue between 400 and 450 nm. If the wavelength is shorter than 350 nm it is UV and has more energy. 

Single vs Double Beam Spectrophotometers

There are generally two types of spectrophotometers: a single beam, and double beam. Single beam spectrophotometers use a single beam of light – visible or UV – which passes through a sample in a cuvette. Light intensity is measured before and after the light passes through the sample, and using Beer-Lambert’s Law (see further below), the concentration of the analyte can be calculated.

Double beam spectrophotometers work in a similar way to single beam spectrophotometers but with a key difference. The initial light source is split into two; one beam passes through the sample, and the other through a reference solution or the solvent. The ratio of the two light beams then corresponds to the absorbance of the sample.

Single beam spectrophotometers are generally more compact and have a higher dynamic range but the optics in a double beam can permit higher levels of automation, better precision and can correct for background absorption of the solvent.

Transmittance and absorbance

Spectrophotometers measure absorbance (A) and transmittance (T). The intensity of light (I₀) measures photons per second. When light passes through a blank sample, it does not absorb light so is symbolised as (I). Scientists use blank samples without chemical compounds as a reference. They contain everything that is in the sample cuvette, except the one material which absorbance is being measured.

To calculate the transmittance the following equation is used:

Transmittance (T) = I_t/I­₀

I_t = Light intensity after passing the cuvette (transmitted light)

I₀ = Light intensity before passing the cuvette (incident light)

Absorbance (A) = – log₁₀ T = – log I_S/I_R

Using the Beer-Lambert Law (Beer’s Law)

The Beer-Lambert Law (sometimes just referred to as Beer’s Law) is the relationship between the attenuation of light, through a substance, and the properties of the substance.

The Beer-Lambert law indicates that the amount of light that is absorbed by a substance is proportional to the amount of the sample concentration. It is also determined by the amount of solute that is present. But to fully understand the Beer-Lambert Law, understanding the relationship between absorbance and transmittance is of importance.

Measuring Absorbance

The sample molecules or ions in a solution can be detected and quantified using a spectrophotometer and The Beer-Lambert Law with this equation: A = ƐCL

A = absorbance of light at a specific wavelength

Ɛ = molar extinction coefficient (the absorbance of 1 mole of a substance dissolved in 1 litre solvent)

C = the molar concentration of a sample

L = the optical path length of a sample

How to Measure Absorption With a Spectrophotometer

To measure absorption of a sample, you need to know the values of the three factors – molar extinction coefficient, molar concentration and optical path length.

Molar Extinction Coefficient – Ɛ

The molar extinction coefficient is a value at which a chemical species attenuates light at a given wavelength. The SI unit is m2/mol, but sometimes expressed as M-1 cm-1 or L mol-1 cm-1.

You can obtain the molar extinction coefficient for your target sample from literature sources.

Concentration

Concentration refers to the concentration of the sample. This is simply the molar concentration, and measured as mol/L.

Path Length

Path length refers to the distance that light travels through the sample as absorption is directly proportional to distance of light traveled. This is determined by the size of your cuvette.

Whilst determining the absorption from Beer-Lambert’s Law is relatively straightforward, make sure to use the correct units, or convert them correctly to avoid any errors in your end result.

Uses of a Spectrophotometer

Spectrophotometry is an incredibly robust technique that has been adopted by many areas of science, industry and manufacturing. Below, we look at some of the ways spectrophotometry gets used and why.

Pharmaceutical Production

Understandably, the process of manufacturing pharmaceuticals is carefully monitored to ensure the end product is exactly what it says it is.

Where possible, using spectrophotometry is a quick and effective method to perform QC testing on raw materials, intermediates and final products. By measuring samples and comparing them to samples, it is possible to understand whether the correct compound has been manufactured and whether any impurities are present.

Due to the low sample size typically required for spectrophotometry, it also makes it an incredibly cost effective method, where raw material costs are high.

Water Analysis

Water quality is incredibly important for industry, manufacturing and consumption, yet it can be hard to determine quality without some form of testing. Spectrophotometry provides a non-destructive method of analyzing water for quality, clarity and purity. Typically, water is measured on the APHA or Hazen scale, which was initially introduced to measure waste water, but can be applied to ‘purer’ samples also.

Measuring water quality has important applications, such as determining the presence of heavy metals in drinking water, determining the concentration of pollutants in wastewater and validating water purity for laboratory testing or manufacturing processes.

FAQs

What is the difference between a spectrometer and spectrophotometer?
Although similar words, both actually have different meanings. A spectrometer is just one part of a whole spectrophotometer, and is the part that is mostly responsible for measurement. A spectrophotometer is the word used to describe the whole instrument.

What is the difference between absorption and optical density?
Absorption and optical density may be used interchangeably, but they mean different things. Absorption measures the amount of light that hits the detector, whereas optical density measures the amount of attenuation, or the loss of light intensity. Therefore, both measurements are different, however, most spectrophotometers can produce both results.

What are the units of absorption?
Absorption has no units and should be reported as a figure only. However, it is common to see absorption reported with the units ‘a.u.’, which typically stand for ‘arbitrary units’, but sometimes in the case of absorption, are mistakenly referred to as ‘absorption units’.

What is photospectrometry?
Photospectrometry is simply another name for spectrophotometry, however, it is less commonly referred to. Spectrophotometry is the correct term to use to describe the method of measuring absorption.