Ultraviolet-Visible (UV-Vis) spectroscopy

Ultraviolet-Visible (UV-Vis) spectroscopy, is a widely used analytical technique in the field of chemistry and materials science. It provides valuable insights into the electronic structure of molecules and materials by measuring the absorption or reflection of light in the ultraviolet (UV) and visible (Vis) regions of the electromagnetic spectrum. This technique is an essential tool for researchers and scientists to analyze and characterize compounds, ranging from simple organic molecules to complex biological macromolecules and nanomaterials. The simplicity, speed, and non-destructive nature of UV-Vis spectroscopy make it a versatile technique for both routine laboratory analysis and advanced research.

The interaction of molecules with UV and visible light is based on the principle that electronic transitions occur when electrons within a molecule absorb energy from photons and move from one energy level to another. These transitions often involve the movement of electrons between different molecular orbitals, leading to the absorption of specific wavelengths of light. By measuring the absorbance or transmittance of light at different wavelengths, UV-Vis spectroscopy provides information about the energy levels and electronic transitions present in a sample.

 

Beer-Lambert Law

The relationship between absorption strength (absorbance) and optical path length is described by the Beer-Lambert Law in the equation below. Samples are placed in cells (cuvette) for measurements. Generally, the square cell with an optical path length of 10 mm is used. However, in cases where absorption is low, a cell with a longer optical path length is used, whereas when absorption is high, a cell with a shorter optical path length is used. The Beer-Lambert law equation can be used to determine the appropriate optical path length for effective measurement.

Absorbance: A=ϵlc

ϵ: Absorption coefficient
c: Concentration of sample
l: Optical path length

 

Fluorescence spectroscopy

Fluorescence spectroscopy, is a widely used analytical technique in the field of chemistry for studying structural changes in conjugated systems, aromatic molecules, and rigid or planar compounds that are caused by changes in factors such as temperature, pH, ionic strength, solvent, and ligands. Certain molecules or atoms absorb photons of a specific wavelength and subsequently emit them at longer, lower-energy wavelengths. This emission of light provides valuable information about the structure, composition, and behavior of the molecules or atoms under study. Compared to UV-Vis spectroscopy, fluorescence spectroscopy provides a much higher sensitivity as it is not limited by the instrument's ability to differtiate incident and detected intensities. This allows measurement to be done at lower concentrations.

Unlike UV-Vis spectroscopy, the emitted fluorescene light is detected perpendicular to the excitation beam. Therefore, fluorescence cell (cuvette) are designed with all four side polished to allow accurate detection of the emited light.

 

Choice of Cell

A square cell with a stopper is generally used to measure volatile liquid samples, and a micro-cell is used to measure a sample with a small volume.

Shimadzu offers 2 different types of sample cells. Quartz cell can be used for both UV and visible light range while glass cell can only be used in the visible light range .Below table shows the usable range and the range over which the transmission are guaranteed to be higher than 80%. 

Window Material	Path lengths	Wavelength Range	Tolerance Range	Usable Range From	T>80% From Nm (Guaranteed)	Matched Pair Tolerance range (Guaranteed)
Spectrosil® Quartz	0.5 to 30mm	200 nm – 2500 nm	± 0.01mm	190 nm	200 through 2500 nm	1.5% measured at 200nm
	40 to 100mm		± 0.02mm
Special Optical Glass	up to 20mm	320 nm – 2500 nm	± 0.01mm	320 nm	320 through 2500 nm	1.0% measured at 320nm
	30 to 100mm		± 0.02mm

 

Types of Cuvettes:

Standard cell with PTFE Lid	Semi-Micro cell with PTFE Lid	Semi-Micro cell with PTFE Lid, Single cell – Lower sample volume	Micro cells	Micro short cell
Ultra-micro lens cell	Cylindrical Cell	Standard Fluorescence Cell with PTFE Lid	Micro Fluorescence Cell with PTFE Lid	Flow cells

 

Standard cell with PTFE Lid

• Open top, comes with either non-sealing PTFE cover or a stopper providing a liquid-tight seal.
• Two polished windows.
• Walls polished internally, fine ground externally.
• Suitable for use with all standard cell holders.

 

Cell with PTFE Lid

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)	Quartz cell (Matched)	Glass cell (Single)
1	400	3.5 x 12.5 x 45	226-85101-01	226-85102-01	226-85111-01
2	700	4.5 x 12.5 x 45	226-85101-02	226-85102-02	226-85111-02
5	1700	7.5 x 12.5 x 45	226-85101-05	226-85102-05	226-85111-05
10	3500	12.5 x 12.5 x 45	226-85101-10	226-85102-10	226-85111-10
20	7000	22.5 x 12.5 x 45	226-85101-20	226-85102-20	226-85111-20
30	10500	32.5 x 12.5 x 45	226-85101-30	226-85102-30
40	14000	42.5 x 12.5 x 45	226-85101-40	226-85102-40	226-85111-40
50	17500	52.5 x 12.5 x 45	226-85101-50	226-85102-50	226-85111-50
100	35000	102.5 x 12.5 x 45	226-85101-00	226-85102-00	226-85111-00

 

Cell with stopper

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)	Glass cell (Single)
1	400	3.5 x 12.5 x 45	226-85201-01	226-85211-01
2	700	4.5 x 12.5 x 45	226-85201-02	226-85211-02
5	1700	7.5 x 12.5 x 45	226-85201-05	226-85211-05
10	3500	12.5 x 12.5 x 45	226-85201-10	226-85211-10
20	7000	22.5 x 12.5 x 45	226-85201-20	226-85211-20
40	14000	42.5 x 12.5 x 45	226-85201-40	226-85211-40
50	17500	52.5 x 12.5 x 45	226-85201-50	226-85211-50
100	35000	102.5 x 12.5 x 45	226-85201-00	226-85211-00

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Semi-Micro cell with PTFE Lid, Single cell

• Reduced nominal volume to <50% of Standard rectangular.
• Open top, comes with non-sealing PTFE cover.
• Two polished windows.
• Walls polished internally, fine ground externally.
• Suitable for use with all standard cell holders.
• Self-masking solid black walls enhance sensitivity and improve linearity at higher absorbances.

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)	Glass cell (Single)	Black Quartz cell (Single)
5	700	7.5 x 12.5 x 45	226-85301-05	226-85311-05	226-85401-05
10	1400	12.5 x 12.5 x 45	226-85301-10	226-85311-10	226-85401-10
20	2800	22.5 x 12.5 x 45	226-85301-20	226-85311-20	226-85401-20
40	5600	42.5 x 12.5 x 45	226-85301-40	226-85311-40	226-85401-40
50	7000	52.5 x 12.5 x 45	226-85301-50	226-85311-50	226-85401-50

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Semi-Micro cell with PTFE Lid  Single cell – Lower sample volume

*9mm thick base, not suitable for an instrument with an 8.5mm 'Z' dimension

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Black Quartz cell (Single)	Clear Quartz cell (Single)
10	1160	12.5 x 12.5 x 45	226-85401-99	226-85501-10

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Micro cells

• Reduced nominal volume to <20% of Standard rectangular.
• Open top, comes with non-sealing PTFE cover or a stopper providing a liquid-tight seal.
• Two polished windows.
• Walls polished internally, fine ground externally.
• Suitable for use with all standard cell holders.

 

Cell with PTFE Lid

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)	Glass cell (Single)
5	350	7.5 x 12.5 x 45	226-85601-05	226-85611-05
10	700	12.5 x 12.5 x 45	226-85601-10	226-85611-10
20	1400	22.5 x 12.5 x 45	226-85601-20	226-85611-20
40	2800	42.5 x 12.5 x 45	226-85601-40	226-85611-40
50	3500	52.5 x 12.5 x 45	226-85601-50	226-85611-50

Cell with stopper

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)	Glass cell (Single)	Black Quartz cell
5	350	7.5 x 12.5 x 45	226-86301-05	226-86311-05	226-85001-05
10	700	12.5 x 12.5 x 45	226-86301-10	226-86311-10	226-85001-10
20	1400	22.5 x 12.5 x 45	226-86301-20	226-86311-20	226-85001-20
40	2800	42.5 x 12.5 x 45	226-86301-40	226-86311-40	226-85001-40
50	3500	52.5 x 12.5 x 45	226-86301-50	226-86311-50	226-85001-50

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Micro short cell

• Two polished windows.
• Open top, comes with non-sealing PTFE cover.
• Walls polished internally, fine ground externally.

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Black Quartz cell (Single)	Clear Quartz cell (Single)
5	200	7.5 x 12.5 x 25	226-89029-05	226-86101-05
10	400	12.5 x 12.5 x 25	226-89002-10	226-86101-10

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Ultra-micro lens cell

• Ultra-micro volume ranges from 0.5μl to 10μl.
• Two polished windows.
• Sample inserted and retrieved with micro pipette tip.
• Two micro pipette tips provided with each cell.
• Z height = 15

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz flow cell (Single)
0.1	0.5	12.5 x 12.5 x 45	226-89031-01
1	5	12.5 x 12.5 x 45	226-86201-01
5	2.5	12.5 x 12.5 x 45	226-86201-05
5	5	12.5 x 12.5 x 45	226-89001-05 *
10	5	12.5 x 12.5 x 45	226-86201-10

*Has integral focusing lens which increases the energy entering the sample

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Cylindrical Cell, single cell

• Two polished windows.
• Closed by PTFE stopper, providing a liquid-tight seal.
• 10 & 20 mm cell closed by a single PTFE stopper
• 50 & 100 mm cell closed by two PTFE stoppers
• Internal diameter = 19mm

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)	Glass Cell (Single)
10	2800	22 x 12.5	226-85701-10	226-85711-10
20	5600	22 x 22.5	226-85701-20	226-85711-20
50	14100	22 x 52.5	226-85701-50	226-85711-50
100	28200	22 x 102.5	226-85701-00	226-85711-00

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Standard Fluorescence Cell with PTFE Lid, Single cell

• Open top, with non-sealing PTFE cover.
• Four windows and base polished.

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)	Glass Cell (Single)
5	1700	7.5 x 12.5 x 45	226-85801-05	226-85811-05
10	3500	12.5 x 12.5 x 45	226-85801-10	226-85811-10
20	7000	22.5 x 12.5 x 45	226-85801-20	226-85811-20
40	14000	42.5 x 12.5 x 45	226-85801-40	226-85811-40

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Micro Fluorescence Cell with PTFE Lid, Single cell

• Four windows and base polished.
• Comes with non-sealing PTFE cover.
• Base thickness - 3mm
• Suitable for use with all standard cell holders.

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)
10	700	12.5 x 12.5 x 45	226-85901-10

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Flow cells

• Two polished windows, long aperture.
• Path lengths of 0.5mm or less incorporate by-pass tubes to avoid back pressure and assist laminar flow through the sample compartment.
• M6 Screw-in connections.
• Profiled sample compartment to optimize flow characteristics, reduces carry over and bubble retention.
• Z height = 15

Light path /mm	Maximum Vol /μL	External Dimension (L x W x H) / mm	Quartz cell (Single)
0.1	41	12.5 x 12.5 x 45	226-89021-01
0.2	47	12.5 x 12.5 x 45	226-89022-02
0.5	95	12.5 x 12.5 x 45	226-89023-05
1	120	12.5 x 12.5 x 45	226-89024-10

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