Using the Spectrophotometer for Analysis

The spectrometer, or spectrophotometer, is an analytical instrument used to identify or confirm the chemical species, chemical structure, or concentration of substances in a sample. The instrument emits an energy source to pass through a solution and measure light intensity at different wavelengths. If the solution is high in molecular concentration, then more light will be absorbed.

In most cases, a sample submitted for spectrometric analysis must be very pure in order to avoid poor or contaminated results. Spectrometers generally include a light or energy source (typically a lamp), a filter (or monochromator) to set desired wavelengths in which to read, a place for cuvettes or blanks, and a radiation detector (or phototube) to convert the energy received during the experiment into a measurable signal.

Important criteria to consider when selecting a spectrophotometer/spectrometer:

• Detection limits

• The density, shape, or size of the product you wish to measure

• Wavelength range

• Analytical working range

• Sample throughput (single sample vs. multi-sample)

• Data quality

• Cost of instrument and associated consumables

• Customizable and/or pre-configured method options

• Measurement time

• Footprint of instrument

Single Beam, Double Beam (Dual-Beam), and Split Beam Spectrophotometers

A single beam spectrophotometer has only one beam of light, while a double beam spectrophotometer has two beams of light, one passing through a reference solution and one passing through the sample. Single-beam instruments, because there is only one light path that passes through the sample, require manually switching a reference cuvette with the sample cuvette for calibration.

Double beam spectrophotometers operate faster and provide more reproducible results because they perform an automatic correction for the loss of light intensity as the beam passes through the sample and reference solution. Split-beam instruments are similar to dual-beam systems, but instead use a beam-splitter which rapidly alternates the light path between the reference and the sample while using one detector.

Guidelines for Evaluating Cuvettes

Windows: Cuvette windows should be uniformly clear, with no scratches, clouding, inclusions, bubbles, streaks, or “rainbowing”, as these issues will negatively affect the performance of the cuvettes. Scratches on non-optical sides are negligible.

Mold markings: Ideally, a single box of cuvettes should have identical mold markings. If they are not identical, then they may not have come from the same mold cavity and will be inherently more inconsistent in measurement.

Performance: Test your cuvette by running it several times in your spectrophotometer. It is always a good idea to rotate it 180 degrees and retest. Values should be very close. If results are inconsistent it could be due to poor molding or a poor fit in the cuvette holder. If values differ significantly, the cuvette windows are likely inconsistent from one side to another. Window inconsistencies, thickness, and quality of resins used make a big difference at critical wavelengths during testing and use.

See more spectrophotometers.