1	. SPECTROPHOTOMETER The spectrophotometer is an analytical instrument that makes possible a quantitative measurement of the light passing though a solution. The spectrophotometer is also known as a colorimeter or a spec-20. This instrument has a diffraction grating monochromator, an electronic detection, amplification and measuring system. It operates in a wavelength range of 390 – 800 mu.
2	HOW A SPEC WORKS White light emanating from the tungsten lamp of the Spec-20 passes through an entrance slit and is dispersed by a diffraction grating. From the dispersed beam, a narrow band of a selected wavelength is passed through a second slit and into the sample solution being measured.
3	HOW A SPEC WORKS Any of this light which is not absorbed by the solution, but which passes through it, falls upon the phototube of the instrument where the intensity of the transmitted light is measured electronically.
4	THE DIFFRACTION GRATING The diffraction grating is a precision grating having 600 grooves to the millimeter and they are accurately spaced. The white light falling upon the grating is dispersed into a horizontal fan of beams (violet and ultra-violet) at one end and the long wavelengths (red and infrared) at the other.
5	THE DIFFRACTION GRATING The whole spectrum of light falls on a dark screen with a slit cut in it. Only that portion of the spectrum (wavelength) which happens to fall on the slit goes through into the sample, and we can project any part of the spectrum onto the slit that we wish simply by turning the grating.
6	THE DIFFRACTION GRATING The grating is turned by the knob on the top of the instrument (the wave-length control knob). Attached to this knob is a dial calibrated in wavelength. This dial may be set to the wavelength wanted.
7	WAVELENGTHS The wavelengths are given in millimicrons [1mu = 10A = 10^-7 cm]. The slit of the instrument passes a band of wavelength 20 millimicrons wide.
8	OPERATING THE SPEC To turn on the Spec-20 rotate the amplifier control (left-hand knob) clockwise. This should be done at least 20 minutes before measurements are made. After the instrument has warmed up, the amplifier control knob may be adjusted so the meter needle will read “0” on the percentage transmission scale when no light is striking the phototube.
9	OPERATING THE SPEC The right-hand knob regulates the amount of light passing through the second slit of the phototube. After the colorimeter has been “zeroed” (by means of the amplifier control knob), a “blank” solution is placed in the light path and the light control knob is rotated until the dial reads 100 %T.
10	OPERATING THE SPEC The “blank” solution (the medium in which the substance being measured is located) may absorb light of certain wavelengths and interfere with the light being absorbed. We will use distill water as the blank because all of our solutions have water as the solvent.
11	OPERATING THE SPEC If a sample solution is now placed in the light path, any change in the %T reading is due to the particular light-absorbing species in the sample and the %T reading is a measure of the quantity of that species present.
12	OPERATING THE SPEC Of course, whenever a change in wavelength is made, however slight, the 0 %T and 100 %T must then be reset, since the amount of “compensation” needed varies with wavelength.
13	OPERATING THE SPEC The special tubes the “blank” and sample are placed in to be read by the spec-20 are called cuvettes. The handling of the cuvettes is extremely important. 1. Do not handle the lower portion of a cuvette (through which the light beam will pass).
14	OPERATING THE SPEC 2. Wipe off any liquid drops, or smudges on the lower half of the cuvette, with a Kimwipe before placing the cuvette in the instrument. NEVER wipe the cuvettes with paper towel or handkerchiefs. 3. Many precision instruments come with matched sets of cuvettes that have index lines on them that must be matched up when placing one in the sample holder.