What is a Spectrophotometer? 

A spectrophotometer is a popular optical instrument that measures light intensity in relation to color (or wavelength). They’re used in the lab to see how much light a coloured chemical dissolved in the solution absorbs. This enables us to quantify chemical concentrations and purity, evaluate particular chemical properties, and monitor and measure chemical reactions in real-time.

What are Calibration Curves? 

Calibration curves are used to calculate the concentration of unknown substances based on measurements of known concentration solutions. The calibration curve determines the precision and accuracy of the measurements. The more accurate the answer, the better the curve; the less accurate the answer, the worse is the curve. Calibration curves are used for a wide range of measurements on a wide range of devices. 

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Why is Calibration Important? 

Calibration increases the accuracy of precise measurements needed in R&D, innovation, and the final output of millions of goods and services around the world. Take a moment to look around your room right now; the majority of what you see has been manufactured to strict measurement specifications that were ensured by calibration.

Calibration is secretly behind the scenes assuring precise measurements vital to the convenience, efficiency, and protection of goods and innovation we enjoy and rely on a daily basis. 

For example – 

  • All modes of transportation and associated services, including automobiles, trains, planes, and gas stations
  • Phones, mobile phone service, computers, appliances, and televisions are all examples of electronics.
  • All modes of transportation and associated services, including automobiles, trains, planes, and gas stations
  • Phones, mobile phone coverage, computers, appliances, and test and measurement instruments.
  •  Prescriptions and pharmacy products
  •  Medical instruments, such as pacemakers, and sterilization procedures
  • Rockets, spacecraft, and the International Space Station associated structures.
  • Producing, transporting, and storing food.
  • Commodities such as oil, grains, and gold.

In an ever-expanding universe of millions of other utilities and manufactured products around the world, the list goes on and on.

How to determine Concentration using a Spectrophotometer? 

For the classification and analysis of substances, the ultraviolet and visible light spectrum (UV/VIS) is commonly used in research, manufacturing, and quality control. The absorption of light by a sample is the basis for UV/VIS spectroscopy. Valuable details, such as the purity of the sample, can be obtained based on the amount of light and its wavelength absorbed by the sample. Furthermore, since the amount of absorbed light is proportional to the amount of sample, optical spectroscopy allows for a quantitative study.

Now, let us specifically explores techniques when using a spectrophotometer to determine the concentration of an analyte. (a substance whose chemical constituents are being identified and measured)

A UV/VIS spectrophotometer compares the strength of light before and after it travels through a sample solution in a cuvette (straight-sided clear container for holding liquid samples in a spectrophotometer). A light source, a sample holder, a dispersive apparatus to isolate the various wavelengths of light, and an appropriate detector are the key components of a UV/VIS spectrophotometer. The ratio of the transmitted intensity I to the initial intensity of light is measured by this instrument, which is known as transmittance. The Absorbance, which is described as A = log, is another significant derived (calculated) variable stated by the instrument (Transmittance).

A UV/VIS spectrum is usually interpreted graphically as absorbance as a function of wavelength. The benefit of this representation is clear: the height of the absorption peaks is proportional to the species concentration. The Lambert-Beer Law governs the measurement of concentration. To determine the concentration, use the following formula: C = (epsilon) x d = A.

Where C is the sample concentration in mol/L or g/mL, D is the length of the Cuvette direction in cm, and E is the sample unique constant – specific to the instrument and in epsilon. (describing how much the sample absorbs at a given wavelength)

Calibration Curve

Each instrument would have its own constant E that is unique to the wavelength and sample being measured, as described above. The value of constant varies depending on the instrument’s age and its components. As a result, it must be determined prior to each experiment. This is accomplished by preparing different known quantities of the sample and calculating the absorbance. Then you should make a graph of absorbance (y-axis) vs. concentration (x-axis). This graph will produce a straight-line plot with E as the slope, according to the equation above.

Then, one will use the calibration curve to calculate the concentration of an unknown sample by calculating its absorbance after it has been formed. After the graph shows the calculated absorbance, a horizontal line is drawn, from the absorbance to the calibration curve, followed by a vertical line down from the curve to concentration. This will indicate the concentration of the unknown sample.

Advantages of Spectrophotometer 

A spectrophotometer has the advantage due to its high-tech spectral analysis possibilities along with its easy and fast techniques. Users will gather data in a matter of seconds for the correct spectrophotometer model, making it suitable for a variety of applications. Developers can make more progress in less time with quicker calculations.

In the food industry, for example, food quality and safety are two of the most critical factors for consumers. If these two conditions are not met, the company may lose earnings or even its prestige, ultimately leading to bankruptcy. Thus, in addition to the general sense of food quality variables such as colour, appearance, scent, and taste, a spectrophotometer may also assist the food provider by providing instrumentation for chemistry and biology research. Furthermore, the spectrophotometer also helps maintain high-quality management, and manufacturing techniques are used to extend the shelf life of foods while ensuring their protection.

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