SOURCE

MONO-

CHROMATOR

DETECTOR

SAMPLE

37.7

SLIT

The visible-UV spectrometer we use permits the quantitative determination of the absorption and

transmission of light in the wavelength range 200 nm* - 900 nm by a clear liquid sample. The device consists of simple components.

* We actually encounter a lower wavelength limit of ~350 nm due

to the use of glass sample containers. Glass absorbs

significant amounts of radiation below 350 nm.

sample compartmen

t

wavelength adjustmentzero

adjustment

wavelength switch

100% transmittance

adjustment

mode switch

The The ApparatusApparatus

display

Variations in SpectrometersVariations in SpectrometersSome models of spectrometers include other

switches and capabilities which do not play a do not play a rolerole in our exercises, e.g., mode switches labeled

– “concentration” or “factor”

• Some models of spectrometers do not include the wavelength switchwavelength switch shown in the preceding picture.

• A few of the spectrometers have an analoganalog instead of digitaldigital output.

While the details may differ slightly, the basic procedures

described herein are the same for all the models.

wavelength switch

Step 1 - Set Step 1 - Set WavelengthWavelength

If spectrometer has wavelength switch, be sure is it set properly

Adjust wavelength

using wavelength adjustment

knob

wavelength reading

Initializing the SpectrometerInitializing the Spectrometer

Once the desired wavelength is set, we adjust the electronics at the selected wavelength. We must set (or confirm) that spectrometer displays:

– 0% transmittance when no light reaches the detector

• When sample compartment cover is closed with no sample in chamber, the light beam is interrupted internally.

– 100% transmittance when the sample absorbs no light at the given wavelength

• This latter step requires the preparation of a BLANK,

BLANK: a solution containing all the substances in the solution except the one whose absorption we seek to determine.

Step 2 - Set/Check 0% Step 2 - Set/Check 0% TransmittanceTransmittance

EMPTY SAMPLE

CHAMBERSample

compartment cover

closedAdjust

transmittance to zero

Mode switch set to

transmittancetransmittanc

e reading

Having adjusted the electronics so the detector reads 0 (zero) percent 0 (zero) percent transmittancetransmittance when no lightno light reaches it, We next want to insure that the detector reports 100 percent 100 percent transmittancetransmittance (at the appropriate wavelength) when the blankblank, but no no samplesample, is in the beam.

The setting of 100% is a critical reference point.

It does not mean that the blank does not absorb light at this wavelength, but that we will measure only the absorption due to the samplesample.

Step 3 - Set/Check 100% Step 3 - Set/Check 100% TransmittanceTransmittance

Place blank in sample

compartment. Cover

closed

Adjust transmittanc

e to 100%

Mode switch set to

transmittancetransmittanc

e reading

DO NOT TOUCH THIS

SETTING

Initializing the Spectrometer

• Major changes in the 0% or 100% controls may require repeating both settings to be sure that adjusting one of them has no effect on the other

– I.e., confirm that transmittance is 0% with no sample after setting 100% with blank

• If 0% or 100% have changed, repeat the initialization steps

In principle, the 0% setting should need no adjustment after the spectrophotometer electronics have stabilized. In practice, small adjustments may be required.

Step 3a – Re-Check 0% Step 3a – Re-Check 0% TransmittanceTransmittance

Remove Blank Sample compartment

cover closed

Re-Adjust transmittanc

e to zeroONLY

if necessary

Mode switch set to

transmittancetransmittanc

e reading

After the Spectrometer is InitializedAfter the Spectrometer is InitializedOnce the 0% and 100% settings are stable,

the spectrometer is ready to measure samples at the setat the set wavelength wavelength

the subsequent steps depend on whether the spectrometer will be used:

to make a series of measurements at the same fixed fixed wavelengthwavelength (e.g., in a Beer’s Law

determination)

to change change wavelengthwavelength (e.g., to determine the

absorption spectrum of a sample)

Only periodic checks of the 0 and 100%

settings are necessary

The 100% setting must be readjusted

each time the wavelength is

changed

OROR

Summary1. Prepare Blank2. Turn on spectrophotometer3. Adjust wavelength setting4. Adjust zero setting with empty sample

compartment5. Adjust 100% setting with blank in sample

compartment6. If working at fixed wavelength, check both

zero and 100% settings periodically 7. If working at varying wavelengths, adjust

100% setting whenever wavelength is changed. Also check zero setting periodically.