Spectrophotometer UV Vis

UV Vis Spectroscopy is a simple and commonly used technique based on the absorption of light in a given sample. Depending on the amount of light and wavelength absorbed by the sample, valuable information can be obtained, such as the concentration or purity of a sample, for example in the caffeine spectrum below. In addition, because each substance absorbs light in a different way, a unique and specific relationship exists between the substance and its UV Vis spectrum. Spectrophotometry can then be used to identify a substance.

A UV Vis Spectrophotometer measures the intensity of light passing through a sample solution in a cuvette and compares it to the intensity of light before it passes through the sample. The main components of a UV Vis spectrophotometer are a light source, sample holder, dispersive device to separate the different wavelengths of the light, and a suitable detector, for example a photo diode detector.

There are five main reasons UV Vis spectra are measured:

• UV Vis spectra identify components present in a sample solution. More precisely, the peak position, and to some extent the spectrum profile, allows for the identification of specific compounds.
  
• Absorption peaks can be used to quantify the investigated sample. For example, the sample concentration can be calculated from the absorbance value of the peak.
  
• Based on the relationship between absorbance and sample concentration, a UV Vis Spectrophotometer is used for quantitative analytical techniques in market segments such as Biotech, Pharma, Cosmetics, Food and Beverages, Chemical and Water Testing.
  
• The spectrum may reveal specific physical properties of the sample molecules and makes it possible to calculate the extinction coefficient of the sample, calculate the melting point of proteins and nucleic acids by measuring the UV Vis spectra at different temperatures, and determine the rate of a reaction by monitoring the absorption spectra as a function of time.
  
• The position and profile of the peaks in the spectrum can give information about the microscopic environment of the sample molecules.

• The fixed wavelength is the simplest application of a spectrophotometer. It is a single or multiple wavelength measurement and the result can be reported in absorbance or transmittance.
  
• The quantification or concentration determination of a substance by a UV Vis Spectrophotometer is based on the Lambert-Beer Law, which states that the absorbance of a solution is directly proportional to the concentration of the absorbing substance in the solution and the path length of the cuvette. Thus, a UV Vis Spectrophotometer can be used to determine the concentration of a sample.
  
• Spectral scanning measurements determine the absorbance or transmittance of a sample over a specified wavelength range or over the full spectrum range, typically from 190 to 1100 nm. The most common analysis afterwards is the detection of peaks and valleys.
  
• A UV Vis Spectrophotometer is often used in order to monitor the change of the concentration of either the reactant or the product by measuring the absorbance at a specific wavelength over time. These kinetic methods are often used to study enzymatic reaction rates or to determine the concentration of specific carbohydrates such as glucose, fructose or lactose.

• A scanning spectrophotometer measures each wavelength separately, and therefore takes a significantly longer time. An array spectrophotometer measures the full spectrum in a short amount of time, as all wavelengths are measured by the array detector.
  
• A scanning spectrophotometer scans the spectrum with mechanically rotating elements. This can lead to errors in the wavelength reproducibility. A scanning spectroscopy instrument therefore requires a regular re-calibration to guarantee wavelength accuracy. An array spectrophotometer does not include any moving optical parts, which means performance deviations over time due to mechanical imprecisions do not occur.
  
• An array spectrophotometer does not require a closed sample compartment because it is immune to ambient light due to its optical design.

Our spectrophotometry portfolio has been designed to fit in several industry segments:

• The UV5Bio and UV5Nano Spectrophotometers are typically used in Pharmaceutical, Life Science, Biotech, and Academia.
  
• The UV7 and UV5 Spectrophotometers are typically used in Pharma, Food&Beverage, Chemical, Cosmetic, Petro, and Academia.

A micro-volume spectrophotometer performs measurements using small volumes of sample (as low as 1 microliter). Highly concentrated samples can also be measured without further dilution due to LockPath technology on the METTLER TOLEDO UV5Nano, which can set the path length at either 0.1 or 1 mm.

• The METTLER TOLEDO UV5Nano and UV5Bio Spectrophotometers offer a large collection of Life Science applications, such as:
  
  • Qualitative analysis of DNA, RNA and proteins
    
  • Quantitative analysis of DNA, RNA and proteins
    
  • BCA, Bradford, Lowry and other colorimetric protein assays
    
  • Pre-configured dyes and option to add custom dyes
    
  • Oligo calculator for the concentration determination of DNA and RNA oligos
    
  • OD600 for live cells
    
• Additionally, the UV5Bio and UV7 Spectrophotometers offer kinetics methods for carrying out enzyme kinetics
  
• For more information on Life Science applications with METTLER TOLEDO spectrophotometers download the Life Science toolbox.

METTLER TOLEDO offer a solution to performance verification in the form of the CertiRef™ unit, which is the most efficient way to check whether your spectrophotometer is working according to pharmacopeia regulation. This accessory automatically performs tests for wavelength accuracy and repeatability, photometric accuracy and repeatability, resolution, stray light, photometric noise and drift, as well as baseline flatness.

METTLER TOLEDO LabX software enables users to have higher flexibility by creating custom workflows exactly for the user's needs. Calculation and transcription errors are completely avoided. It also ensures data integrity with all information safely stored in a secure database, including all performance verifications and services. With security features such as electronic signatures and user management, it assists to comply with 21 CFR part 11 regulations from the FDA.