calibration curve also makes it possible to determine
ε
. As usual, the best line through a series of
independent points makes a better calibration curve than one made by one point and the origin, or
by several points obtained by dilution of a single sample. Your analyses cannot be better than your
calibration curve.
For an analysis, the unknown samples are simply run as usual, and from the
observed absorbance values the concentrations in the samples are read from the calibration curve.
Spectrophotometer
A spectrophotometer makes it possible to measure the fraction (
I/I
0
) of light of different
wavelengths transmitted by a sample;
I
0
is the intensity of the light beam incident on the sample
and
I
is the intensity of the beam which emerges from the sample.
To accomplish this, the
spectrophotometer must provide (a) a source of white light of steady light intensity, (b) a means
of selecting a beam of desired wavelengths from the white light, (c) a stable light-sensitive
detector, and (d) a convenient means for reproducibly placing samples in the light beam.
Most instruments use a simple tungsten filament lamp for the light source in the visible
region of the spectrum, with stability of light intensity provided by an electronically controlled
circuit.
Lamp stability is important in order to avoid oscillations of the meter, or drift in light
intensity from one sample to the next.
By alternating each sample with the blank it is possible to
check on the drift and to make a suitable corrective adjustment if necessary. A H
2
discharge lamp
must be used for a continuous spectrum in the ultraviolet.
The desired wavelength band is selected from the spectrum by rotating, with a calibrated
knob, a reflective diffraction grating
or a refracting prism
.
The grating consists of a glass plate,
containing about 15,000 parallel and uniformly grooved lines per inch, which has been rendered
reflecting by aluminizing. The prism is a 30° - 60° type which is aluminized on the back side so
as to reflect the beam back through the prism in the direction from which it came; this doubling-
back trick gives the same degree of refraction as a 60° - 60 ° prism and greatly cuts down on the
length of the instrument that would otherwise be required.
In either case, the reflected beam is
dispersed to produce a continuous spectrum.
The detector, which measures the amount of light transmitted, is usually a photoelectric
cell in which the electrons that are ejected by the photons falling on an alkali metal surface (usually
cesium) are collected by a positively charged wire and passed as an electric current through a
meter.
The higher the light intensity, the more p per unit time, the greater the number of electrons
ejected per unit time, and the higher the meter reading.
