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Spectrophotometers are prominent devices in many industries and offer value for many purposes. The biological concentration of a chemical can be determined using spectrophotometry data. Constructing a calibration curve is one of the ways users employ spectrophotometers. This technique, much like the device, works across many applications. At HunterLab, we specialize in spectrophotometers and support operations that create calibration curves. Calibration curves have applications in several domains, including analytical chemistry and biology.

What Is a Calibration Curve?

A calibration curve, also known as a standard curve, is a way to identify the concentration of an unknown substance. It calculates the concentration of the unknown material and the limits of detection and quantitation by comparing the sample to a set of known concentration reference samples. The data fits into a model that predicts unknown concentrations. The concentration of the material causes a change in the analytical signal or instrumental response, as demonstrated by a calibration curve.

Calibration curves are beneficial for compounds that indirectly cause absorbance. Typically, a dye reacts with the molecule of interest, and the dye is measured to determine what is going on with the molecule. Begin with a known chemical, measure its absorbance, and then graph it. Second, calculate the absorbance of the unknown sample, plot it on a graph, and apply a calibration curve to get the estimated concentration. 

These curves use data points of substances at varied concentrations, and researchers can use these curves to find where an unknown substance plots. An ultraviolet-visible (UV-VIS) spectrophotometer measures the absorbance of a chemical that absorbs light in the ultraviolet or visible parts of the electromagnetic spectrum. This system of identifying unknown substances is valuable to many industries. For example, you may find a calibration curve in coffee production for determining a roast. With a plot of caffeine content, roasters can discover where a bean falls. A calibration curve for the food industry may chart food dye concentrations for gelatin.

Other applications can include:

• Pharmaceuticals
• Paints
• Plastics
• Chemical production

Simply put, a calibration curve in spectrophotometry is helpful in any situation where a substance is unknown.

How to Make a Calibration Curve

To make a calibration curve, you need the following:

• A standard solution
• A solvent for dilution
• Pipette and tips
• Five microtubes
• UV-Vis spectrophotometer
• Computer

Key steps include:

• Standard preparation: Create a concentrated standard solution and dilute it to generate five multiple known concentrations.
• Sample measurement: Using a spectrophotometer, measure the absorbance of each standard solution and your unknown samples.
• Data analysis: Plot the absorbance (y-axis) vs. concentration (x-axis)to establish a linear calibration curve. Use statistical software to fit the data to a linear regression equation (y = mx + b) and determine the coefficient of determination (R²) to assess the goodness of fit.

How a Calibration Curve Is Used

Calibration curves are used in many scientific areas to calculate the quantity of unknown chemicals in a sample. Here are some popular applications:

• Environmental monitoring: Calibration curves are essential for measuring environmental health. They may be used to determine the concentrations of contaminants in soil samples, such as heavy metals or pesticides.
• Neuroscience research: Measuring neurotransmitter levels is frequently used to understand the complex workings of the brain. Calibration curves allow researchers to calculate the concentration of these signaling chemicals in brain fluids.
• Pharmaceutical quality control: It is critical to ensure pharmaceuticals are effective and safe. Calibration curves are used to accurately measure the quantity of active drugs and other components in pharmaceutical samples.
• Food and beverage analysis: Calibration curves play an important role in guaranteeing the quality of food and drinks, from monitoring caffeine concentration in beverages to ensuring vitamin potency in fortified products.

What Is an Ultraviolet-Visible (UV-Vis) Spectrophotometer?

Coffee can be the best way to start your morning, but before it gets to your favorite mug, a roaster ensures it has the right color for its roast. Color can be complex and challenging to thoroughly examine with the human eye. With special measuring tools, like a spectrophotometer, roasters can meet color standards in the coffee industry.

Color Standards in Coffee Assessment

Coffee beans start green, and roasting darkens the bean and changes the flavor. This roasting process is why there are light, medium and dark roasts. Bean color is a significant indicator for the kind of roast and the flavor the coffee will have.

When it comes to deciding on the right color for a coffee bean, you need to consider how we see color. Color comes from the amount of light a substance absorbs or reflects, often called optical properties. The color of the light and where a person stands can affect how they perceive color. While the human eye can perceive a series of wavelengths to register color, it can’t see all of them, which may affect color quality.

Color consists of three properties — hue, saturation and brightness. To get accurate measurements of these quantities, coffee roasters can use color measuring instruments. These tools, like spectrophotometers, remove human subjectivity from color and make coffee roasts more definable.

Paper was invented in China, where people began writing on sheets of cloth to keep records. From there, a Chinese court official created a pulp using natural materials and let it dry in a thin mat shape. Paper production has changed over the years to become what we know today. At HunterLab, we are dedicated to helping the paper industry meet color standards and best practices for paper production.

Meeting Color Standards for Paper

White paper may seem like a simple color standard to meet, but getting the right white shade can be challenging when we consider surface properties. While there may be a specific white that all paper manufacturers want to achieve, they must factor how light reflects on different surfaces, from low-gloss matte to high-gloss.

Colors have reflected light and absorbed light, and these measurements affect our perception of the color. Reflected light, however, is not an indicator of the actual color, while absorbed light is. For mid-gloss and high-gloss paper, their shiny surfaces offer a lot of reflected light, and it will skew our perception of the color.

To find the right color, paper manufacturers rely on instruments that offer a quantified measurement for color, rather than counting on the human eye. Machines like spectrophotometers can register the light a surface reflects and absorbs to provide a definitive color measurement. With this method, manufacturers can achieve a consistent white paper in all glosses.

We may not think much about the color of our foods, but this characteristic is valuable to the industry. Color can communicate a food item’s safety, and it can create a safe environment for food production. Meeting color standards in the food industry is possible with the right tools, and at HunterLab, we have the right products for the job.

USDA Color Standards for Food

The United States Department of Agriculture (USDA) uses color to determine many foods’ safety and health, from produce to frozen french fries. The organization provides color charts for agricultural and food processing operations to compare their products to. A critical part of this process is having the proper equipment, like a spectrophotometer.

Food colors can communicate many characteristics of an item. They can show:

• Refrigeration
• Temperature variation
• Color content
• pH levels

Plastic is a widespread medium for various applications, from children’s toys to kitchen tools. Coloring plastics can take on many different forms, and it’s vital to meet industry standards for safety and performance. At HunterLab, our spectrophotometers help plastic manufacturers create ideal color concentrates for their products.

ASTM Color Standards for Plastic

The American Society for Testing and Materials (ASTM) provides a series of standards for plastic production. While plastic is easy to mold and ideal for many applications, it’s a synthetic material that may be poorly made or unsafe for use.

ASTM standards provide rules and guidance for plastic production to ensure products last and stay safe throughout their lifespan. The organization’s website offers extensive documentation for you to follow these standards in your operation. Criteria include descriptions like:

• Specifications for materials
• Testing methods
• Production procedures

Color is an essential feature of plastic standards because plastic colorants can affect the quality of the material. Plastic producers use color charts to compare their colors, and the best way to handle this comparison is with color measuring devices. Machines like spectrophotometers quantify color based on the light it absorbs and reflects, so manufacturers can create a match to a color on the chart.

What Are Color Concentrates for Plastics?

Color concentrates are plastic colorants in a pellet or grain that manufacturers form into plastic. Rather than adding a colored layer to the outside of a plastic product, concentrates make up the product.

To create a concentrate, a pigment is mixed with a polymer or carrier resin. For concentrates to work, the polymer must work with the base resin that the concentrate mixes with. The challenging aspect of concentrates is the lack of a universal polymer. Developers must identify the best polymer and base resin for production.

Many industries follow regulations to introduce safety and consistency into their practices. The American Society for Testing and Materials (ASTM) offers some of these standards for various sectors to verify their processes. Many companies choose to follow these standards to ensure the quality of their product, and with HunterLab spectrophotometers, meeting ASTM standards is within reach.

What Is the American Society for Testing and Materials?

The American Society for Testing and Materials, known as ASTM International, is an organization that publishes production and testing standards for various industries. These standards cover multiple materials, systems and products to encourage consistency and safety among manufacturers.

ASTM International offers training to keep up with their standards and ensure the best performance on the job. From industry-specific continued training to onsite certification programs, ASTM provides plenty of resources to companies who want or need to remain compliant.