| Analytical Instrumentation
II CHT 212 |
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PRINCIPLE
TYPES Of SPECTROPHOTOMETERS 
There are two principle types of uv-visible
instruments.
- Filter photometers
- Spectrophotometers
FILTER PHOTOMETERS
Filter photometers use filters to select the desired
wavelength and are either single beam or double beam. A single beam instrument uses
a single light path for both the reference and the sample. A double beam uses
separate light paths for the reference and sample.
SINGLE BEAM PHOTOMETERS
Single beam photometers use the same light path for
both the solvent (100 %T) and the solvent-sample. Typically, the light passes
through a collimating lens that is directed at the entrance slit where the light is then
passed through a filter. The unabsorbed light is passed through the sample holder,
passes a closeable shutter (0 %T), to a photovoltaic cell which is attached to a readout
device.
A stabilized power supply
is very important. Using a stabilized power supply avoids errors resulting from changes in the beam intensity during the
time required to measure 100 %T and the %T of the analyte.
Normally, the process that is followed for the analysis
of a sample at a predetermined wavelength (as per filter selection) is as follows:
| 1. |
The shutter is
closed to adjust the galvanometer to 0 %T. |
 |
| 2. |
The sample holder is filled with the
solvent (blank), the shutter is opened and 100 %T is adjusted. |
 |
| 3. |
The sample is
added to the sample holder and %T is read. |
 |
Advantages of single beam photometers
- low cost
- ease of operation
Disadvantages of single beam
photometers
- variation in light intensity — errors in %T
- light beam not monochromatic — deviation from Beer's
law
- %T and A are not "true" values
- not designed for spectral data
DOUBLE
BEAM PHOTOMETERS
Double beam photometers split the
light path into two segments. One segment is used as a reference and the other as
the working path. This allows the instrument to make automatic corrections for
variations in light intensity.
When the voltages between the sample resistor (CD) and reference resistor
(AB) are
equal, no current flows through the galvanometer (null detector) since there
is no voltage potential difference between the two resistors.
The 0 %T is set by moving contact A (between the meter and the reference
resistor) to no resistance (contact B). The shutter is closed which blocks all light
from hitting the phototubes. The null meter is mechanically set to read
0 %T since no current is produced by the phototubes.
100 %T is set by placing only the solvent in the sample container. The null
meter's contact A (between the meter and the reference resistor) is set for maximum
resistance (100 %T). The shutter is opened so that light does strike both
phototubes (sample and reference). The null meter's contact D (between the meter and the
sample resistor) is adjusted so that there is equal voltage difference between the
two resistors (points AB and points CD). At that point, no current flows through the null meter
and the meter reads 100 %T.
The analyte and solvent is now placed in the sample container. The
analyte will absorb some of the light passing through it which reduces the
intensity of the light striking the sample phototube. Since less light
is striking the sample phototube, the phototube produces less current.
Less current means less voltage across the sample resistor.
Now there is a voltage difference between the sample resistor (AB) and reference
resistor (CD). Current now flows through the null meter (AD).
To compensate, contact A (between the null meter and the contact reference
resistor) is moved downward until the voltages are once again equal and no
current flows (AD). The
%T is read directly from the meter.
Advantages of double beam photometers
- correction for changes in light intensity
- ease of operation
Disadvantages
of single beam photometers
- light beam is not monochromatic — deviation from Beer's law
- %T and A are not "true" values
- not designed for spectral data
SPECTROPHOTOMETERS
Spectrophotometers use
monochromatic dispersion elements to vary the wavelengths.
- continuously variable wavelength selection
- light source is UV or VIS
Optical Path of Single Beam
Spectrophotometers
Electronics of Single Beam Spectrophotometers
Steps for the use of a single beam spectrophotometer
| 1. |
Set the wavelength. |
| 2. |
Select readout display to the desired mode ( %T, A,
Conc). |
| 3. |
Set the readout display to 0 %T (no light) (shutter closed). |
| 4. |
Insert the reference cell which contains the solvent and set the readout display to
100 %T (shutter open). |
| 5. |
Insert the sample dissolved in the solvent and read %T from the display. |
Advantages of single beam spectrophotometer
- Wavelengths easily selected
- Low cost
Disadvantages of single beam spectrophotometer
- Cannot easily be used for absorption spectra (point by point).
- Changes in light intensity cause variations in readout.
- Changes in solvent causes variations.
- Changes in wavelengths cause variations and % T and has to be reset at 0 %T and 100 %T.
DOUBLE BEAM SPECTROPHOTOMETERS
Double beam spectrophotometers use a beam chopper to separate the reference beam from
the sample beam.
Beam Chopper
The chopper is divided into three equal segments. One segment is clear which will
pass light through the wheel, one segment is mirrored which will reflect light along a
different path, and the third segment is opaque so that no light is transmitted nor
reflected.
Optical Path of Double Beam
Spectrophotometers
Electronics of Double Beam
Spectrophotometers
Advantages of double beam
spectrophotometers
- speed of operation
- automatic compensation for
variation in lamp output
solvent absorption at various 8
changes in detector sensitivity
spectra scan
Disadvantages of double beam
spectrophotometers
OTHER SELECTIONS ON INSTRUMENT
Most instruments have various options and selections
which can be made during an analysis.
The mode switch allows the operator to choose what data the readout gives .
The three major choices are absorbance (a log amplifier converts transmittance to
absorbance), % T, or directly into concentration units.
The slit adjustment allows the operator to adjust the slit width of the instrument.
For instruments which have gratings which can be automatically turned the scan speed
(wavelength/min) can be adjusted. Higher scan speeds are used for surveys while
slower speeds are used to look at areas in more detail.
The chart recorder is used to record the data of scan instruments. The paper speed and
range (mv) can be adjusted to match the output of the spectrophotometer to give a spectra
scan.
SPECTRA SCAN
Spectra scans have two coordinated: the
X axis is normally wavelength, and the Y axis is either absorbance or transmittance.
The resulting graph is called a spectra scan. A spectra scan is similar to a
fingerprint of a compound.
PROBE TYPE
PHOTOMETERS
Dipping type photometers use fiber optics
to transport light.
The original light from a tungsten
lamp travels down a fiber optic cable which is dipped into the solutions of
interest.
The light then passes through the
solution and a mirror reflects the light back to a return fiber optics
cable.
The cell path length is 2 times the
distance between the ends of the optical cable and the mirror.
Interference filters are provided to
select wavelengths.
The reflected light is then passed
through a photodiode with an amplifier and an electronic chopper which is
synchronized with the lamp. This
results in the detector not responding to extraneous light.
