WEBVTT

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A spectrophotometer is an instrument
used to measure the intensity

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of light at a particular wavelength
in the visible spectrum.

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This type of instrument can be used
in the clinical laboratory

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to determine the concentration
of a substance in solution, by

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measuring the amount of light
either transmitted or absorbed.

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When a beam of light is passed through a
sample reaction at a specific wavelength.

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For this video,
we will be using a single beam

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spectrophotometer with a digital display.

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This instrument has a wavelength
range of 340

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nanometers to 950 nanometers,
and a nominal spectral bandwidth

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of 20 nanometers.

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The main controls for routine operation
of this spectrophotometer

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include the power switch, zero control

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percentage T,
the transmittance absorbance control,

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100% T 0.0 A,

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the wavelength control,
and the mode selection button.

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The power

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Switch zero control knob is used to power
the instrument on and off,

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and to set the digital display
to 0% transmittance

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or T when the sample compartment is empty
and the cover is closed.

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The transmittance absorbance control
knob is used to set the digital display

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to 100% transmittance T or 0.0 absorbance

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A when a water blank or reagent
blank is inserted

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in the sample compartment
and the cover is closed.

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The wavelength control
knob is used to select

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the desired wavelength for the analysis.

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The mode selection button
is used to select between transmittance,

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absorbance, concentration, or factor mode.

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For most analysis, the absorbance
or transmittance mode will be used.

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Some spectrophotometer
may also have a filter level

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that must be adjusted to select
the appropriate filter,

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based on the wavelength to be used
to power on the spectrophotometer.

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Plug the power cord
into an appropriate outlet

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and rotate the power switch zero control

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percentage knob in a clockwise direction.

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When working
with instruments or equipment,

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always check the power cords for damage
before inserting into a power source.

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The instrument will click on
and the display will light up.

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Once the instrument has power.

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The instrument
should be allowed to warm up

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for at least 15 minutes prior to use.

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After the instrument is warmed up,

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set the desired wavelength
by turning the wavelength control knob.

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Set the filter lever to the appropriate
position for the selected wavelength.

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For example,

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if the desired wavelength for testing
is 450 nanometers,

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the lever should be positioned
to the left,

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so the 340 to 599 nanometer
filter is in place.

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If the required wavelength were instead
650 nanometers,

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the lever
should be positioned to the right,

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so the 600 to 950
nanometer filter is in place

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to account for any

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stray light in the sample compartment
that could affect measurements.

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The instrument is first blanked or zeroed

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with nothing in the sample compartment

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to zero the spectrophotometer.

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Ensure that the sample compartment is
empty and the cover is closed.

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Turn the power switch zero control
knob percentage T

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until the digital display shows
the transmittance as 0%.

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Please note that this may require several
turns of the knob.

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Some procedures require
that the transmittance

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is monitored
while others measure absorbance.

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Refer to your procedure
to identify the desired measurement,

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and use the mode selection button
to toggle to the appropriate setting.

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Most procedures also require
the instrument to be zeroed using a water

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blank or reagent blank, to account
for the effects of the solution itself.

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To prepare a water blank,
fill a clean cuvette

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with distilled water,
avoiding the formation of bubbles.

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If using a reagent blank, fill

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a clean cuvette
with the designated reagent.

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In general, corvettes should be filled
to at least half their volume.

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The minimum required volume for the
spectrophotometer is three milliliters.

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Using a lint free tissue, gently wipe
the outside of the cuvette

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to remove any surface interference,
such as fingerprints, dust, or liquid.

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Insert the cuvette
into the sample compartment and align

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the guide mark on the cuvette
with the guide mark on the compartment.

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Gently but firmly push the cuvette into the sample compartment and close the cover.

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Using the transmittance absorbance
control knob

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100% T 0.0 A.

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Adjust the display to 100% transmittance T

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or zero absorbance A

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depending on the mode previously selected.

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Remove the cuvette and appropriately
discard the water reagent.

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Do not discard the cuvette as it will be
reused.

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Rinse the cuvette three times
with distilled water and gently tap

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the rinsed cuvette
on a small stack of paper towels to remove

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the remaining water.

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Use a lint free tissue to wipe
the outside of the cuvette.

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To measure the transmittance
or absorbance of a sample reaction.

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Fill a clean cuvette
with at least three milliliters

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of the solution,
avoiding formation of any bubbles.

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Use a lint free tissue to wipe
the outside of the cuvette and insert

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the cuvette into the sample compartment,
making sure to align the guide marks.

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Close the cover

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and record the percentage transmittance
or absorbance from the digital display.

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Remove the cuvette and appropriately
discard the sample reaction.

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Rinse the cuvette three times

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with distilled water, and gently tap
the rinsed cuvette

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on a small stack of paper towels
to remove the remaining water.

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Use a lint free tissue to wipe
the outside of the cuvette.

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Repeat this process
for all remaining samples to be measured.

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Once all measurements are complete,
the spectrophotometer

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should be powered off
by turning the power switch

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zero control
knob percentage counterclockwise

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until the instrument clicks
and the display is blank.

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Carefully
unplug the power cord from the outlet.

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Clean up any spills on the instrument
using a lint free tissue

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dampened with water or ethanol,
followed by a paper towel dampened

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with plain water or around the instrument
with a bench cleaning solution.

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Completely dry the exterior
of the instrument with a lint free wipe.

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The spectrophotometer should be covered
and placed in a secure area for storage.