Spectra Collection Protocol Visible, Near Infrared, and Short
Wave Infrared Using the Cary 500 Spectrometer
Introduction
Spectral measurements of materials in the Visible (VIS), Near Infrared (NIR), and Short Wave
Infrared (SWIR) with a high quality laboratory instrument are made with relative ease. The Cary
500 has a very stable internal light source and a dual-beam optical design that results in a greatly
simplified measurement process, which results in high measurement accuracy and instrument
stability.
While the measurement methodology is simple, it is imperative that those using the Cary 500
understand the measurement phenomenology and the operational aspects of the instrument.
Much of this is covered in the accompanying Traceability protocol document. It is repeated
below in a form appropriate for making measurements with the Cary 500. For those with
spectral measurement experience in a chemical analytical laboratory, many of the methods
described here will be familiar.
This document will primarily focus on the Varian Cary 500 Spectrophotometer. However, the
principles covered in this document also apply to V/NIR/SWIR measurements in general. It is
assumed the operators of the Cary 500 (or other similar laboratory spectrometer) will have
sufficient training to properly operate the instrument according to the manufactures instructions.
This document is by no means meant to replace these instructions. Instead, it is intended to
provide guidance on laboratory usage to insure high quality and consistent data.
To that end, this document is a “living” work. One can never anticipate all the conditions
under which you will collect spectra. Since this is the case, this document needs to serve
as a basis of measurement methodology from which investigators will start when they
make VNIR/SWIR laboratory measurements. For a class of targets that has been
measured before, this document serves as a set of guidelines. For a new class of targets,
it serves to educate and remind the investigator of relevant phenomena that they need to
keep in mind. As new and/or improved methods are developed they will be reflected in
this document.
Background
The Cary 500 is typical of high performance laboratory spectrometers in that it has a
dual-beam design. The instrument alternately measures the spectral radiance from a
reference material and from the sample material of interest (or the unknown). The
instrument uses a common light source, common monochrometers, and common detector
for both beams. A chopper wheel controls which beam the detector is viewing during the
measurement cycle. The chopper wheel further facilitates the measurement of the
detector dark current during each measurement cycle. The spectral transmission or
reflectance of a material in the most basic form is determined by the ratio of the two
measured beams, Eq. 1.
   Lsample ( ) / Lreference ( )
(1)
Unfortunately, the influence of an integrating sphere, reference standards, optics, etc. in a
spectrometer must be accounted for. As a result, Eq. 1 will not sufficiently describe the
measurement. To better understand the operation of a dual beam spectrometer, let us first
consider the components in each beam. The case of reflectance measurements with an
integrating sphere will be described first since that is the more complicated configuration.
A brief discussion will follow describing the simpler transmission measurement.
Reflectance Measurements
The reference beam as sensed by the detector is:
Lr (  )  E r (  )  r (  ) k S , r (  ) k O , r (  )
(2)
where:
Lr ( )
is the reference beam spectral radiance measured by the detector
Er ( )
is the spectral irradiance incident on the reference target
 r  
is the spectral reflectance of the reference target
k S , r ( )
is the spectral efficiency of the integrating sphere for the reference beam
k O , r ( )
is the optical spectral efficiency for the reference beam
The kO terms in the above and following equations account for the differences in the
optical paths the two beams follow in the instrument. These two paths (and associated
optical elements) can contribute to an imbalance between the two beams that must be
accounted for.
For the sample beam we have:
L s (  )  E s ( )  s (  ) k S , s ( ) k O , s ( )
where:
(3)
L s ( 
is the sample beam spectral radiance measured by the detector
Es()
is the spectral irradiance incident on the sample
s()
is the spectral reflectance of the sample
kS,s()
is the spectral efficiency of the integrating sphere for the sample beam
kO,s()
is the optical spectral efficiency of the sample beam
It would be convenient if Es() = Er(), kS,r() = kS,s(), and k O ,r ( ) = kO,s() in Eq. 2
and 3. A simple ratio of these equations would reduce easily reduce to Eq. 1. While
instrument designers strive for such perfection, it is seldom realized and cannot be
expected to be stable over the life of the instrument. For now, lets assume these terms are
not equal. To account for these inequalities, a baseline scan across the wavelength range
of interest is necessary to characterize the response of the instrument. For this document,
the methodology for 100% Baseline and 0% Baseline is discussed.
100% Baseline
For the 100% Baseline [B100()], a white reference target is placed at both the sample and
reference ports. While it is desirable for these two reference targets to be of equal
reflectance, it is not always possible, nor is it absolutely necessary. As will be shown, it
is necessary that a NIST-traceable white reference be placed in the sample port while
acquiring the baseline. From now on, let s() in Eq. 3 be the spectral reflectance of the
NIST-traceable white reference during the baseline scan. The instrument will scan these
targets and compute the ratio of the sample vs. reference beam radiance values. This
ratio will form the 100% Baseline response of the spectrometer.
B100 ( ) 
L s ( ) E s ( )  s ( ) k S , s ( ) k O , s ( )

Lr (  ) E r (  )  r (  ) k S , r (  ) k O , r (  )
(4)
After the 100% Baseline scan is performed, the target material of interest (the unknown)
is placed in the sample port of the integrating sphere for measurement. The sample beam
signal is now made up of:
Lu ( )  E s ( )  u ( )k S ,u ( )k O , s ( )
where:
Lu()
is the unknown sample spectral radiance measured by the detector
u()
is the spectral reflectance of the unknown sample
kS,u()
is the spectral efficiency of the sphere with the unknown material in the
sample port
The resulting ratio of the sample beam with the reference beam is:
D ( ) 
Lu ( ) E s ( )  S ,u ( )k S ,u ( )k O , s ( )

Lr (  )
E r ( )  r ( ) k S , r ( ) k O , r ( )
D() will contain within it the baseline response B100() of the instrument. To remove
B100(), D() is divided by B100(), resulting in a relative reflectance rel().

 rel ( ) 
E s ( )  u ( ) k S ,u (  ) k O , s (  ) E r (  )  r (  ) k S , r ( ) k O , r ( )
D ( )

B100 ( ) E r ( )  r ( )k S ,r ( )k O ,r ( ) Es ( )  s ( )k S , s ( )k O , s ( )
Reducing this equation results in Eq. 5.
 rel ( ) 
 u (  ) k S ,u (  )
 s ( ) k S , s ( )
(5)
For unknown materials with scattering properties similar to those of the white reference
in the sample port during the baseline scan, kS,u() = kS,s().
The above equation then further reduces to:
 rel ( ) 
 ( )
D ( )
 u
B100 ( )  s ( )
Rearranged into a practical form:
u() = rel()s()
(6)
This model illustrates the need to have a NIST-traceable standard in the sample port of
the spectrometer during the baseline scan. All subsequent reflectance measurements of
unknown samples are easily corrected for the spectral properties of the NIST-traceable
standard by multiplying by the absolute spectral reflectance of this standard, Eq. 6.
0% Baseline
While a 100% Baseline is simple and commonly used, it is not sufficient to fully
characterize a reflectance spectrometer. At very low spectral reflectance values, some
stray light from the sample beam may reflect off the integrating sphere, and be measured
by the detector. Furthermore, any variations in the detector response at these extremely
low light levels will also contribute to measurement error. The error is removed by
following up the 100% Baseline scan with a 0% Baseline scan. A 0% Baseline [B0()]
characterizes the instrument with a 0% reflectance target or light trap in the sample port.
The Cary documentation recommends using the black inside of the sample cover as the
light trap. However, experience has shown that a low level of reflectance (1 – 2%)
results from the closed sample cover. Until a better light trap is available, it has been
determined that the best practice is to completely darken the room, open the sample
cover, and leave the sample port empty. Black velvet fabric hanging across the room (~ 8
feet) from the Cary will greatly reduce any stray reflections.
Note: While tempting to merely block the incoming sample beam to the sphere (as
suggested in some of the Cary documentation), this will not properly characterize the 0%
response. Such a practice will only replicate the sample beam dark current measurement,
which is made during each measurement cycle. Using a light trap as directed will fully
account for stray light reflecting off the sphere.
When determining B0(), there is another equation involved with the reflectance
measurement.
L0() = Es()0()kS,0()kO,s()
where:
L0()
is the light trap spectral radiance measured by the detector
0()
is the spectral reflectance of the light trap
kS,0()
is the spectral efficiency of the integrating sphere with a sample port light
trap
The 0% Baseline spectra will be:
B0 (  ) 
L0 ( ) E s ( )  0 ( )k S , 0 ( k O s   

Lr (  ) E r (  )  r (  ) k S , r (  ) k O , r (  )
(7)
The instrument will perform the following computation when an unknown sample is
scanned when the 0% Baseline method is used.
 rel ( ) 
D( )  B0 ( )
B100 ( )  B0 ( )
Substituting in the appropriate expressions into Eq. 8 and rearranging yields:
(8)
 rel ( ) 
E s ( )  u ( ) k S ,u (  ) k O , s ( )  E s ( )  0 ( ) k S , 0 ( k O  s   
E s ( )  s ( ) k S , s ( ) k O , s ( )  E s ( )  0 ( ) k S , 0 ( k O  s   
(9)
As above, we will assume the scattering properties of the unknown and the white
reference sample are approximately equal as with the 100% Baseline case. If there is no
stray light in the sample beam, 0() will be assumed to be 0 since no light will be
reflected from the light trap. Eq. 9 will now reduce to:
u() = rel()s()
(10)
However, if there is stray light and detector/electronic variations, the error will appear in
the Bo() term, which is subtracted from both the sample and reference beams during the
measurement of the unknown. The stray light and detector/electronic error can be very
subtle; however, this subtle correction is particularly important when measuring low
reflectance materials.
Transmission Measurements
Transmission measurements typically are made of liquids in cuvettes or solid materials
that have transmissive properties. If the sample has scattering properties and the total
transmissive properties are of interest, the transmission measurements need to be made
with an integrating sphere. The sample would be placed at the sample beam entrance
port, and the resulting measurement will be the transmittance of the sample. Given the
need for the integrating sphere, much of the phenomenology and methodology described
for reflectance measurements will apply.
If the sample is non-scattering, or only the non-scattered transmissive properties are of
interest, the integrating sphere is removed from the Cary 500, and the instrument is used
in the transmission configuration.
The standard practice for transmission measurements of liquids requires the placement of
a blank solution in the reference beam, and the solution of interest in the sample beam.
The blank solution is often the solvent used to dissolve the material of interest. It may be
water, methanol, benzene, etc. In a remote sensing application, it is likely that the sample
will be the combination of the material of interest and the “solvent”. Examples include
sea water and algae in tidal pond water. In these situations, an empty cuvette would be
placed in the reference beam, and a cuvette filled with the combination liquid in the
sample beam. Since the cuvette is not part of the sample, having a cuvette in both beams
will ratio out the effects of the cuvette during the transmission measurement. For solid
transmissive materials, (such as glass, Plexiglas, window covering films, tree leaves),
there is no need to put anything in the reference beam.
The mathematics describing transmittance measurements are similar to those used for
reflectance measurements. Starting with Eq. 7, 8 and 9 for a 0% Baseline Correction, we
make the following adjustments:
- Since the sample beam will be attenuated by the sample, u() is added to Eq. 3.
-s() is substituted for u() and 0() since the Spectralon standard is replaced
in the sample port during transmittance measurements.
- kS,s is substituted for kS,0 in Eq. 7 since the Spectralon standard is replaced in
the sample port during transmittance meaurements.
A 0% Baseline Correction would be performed by placing an opaque material in front of
the sample beam entrance port to the integrating sphere requiring the addition of a t0 term
to Eq. 7.
Eq. 7 now appears as:
B0 (  ) 
L0 ( ) t 0 E s ( )  s ( )k S , s ( k O s   

Lr (  )
E r (  )  r ( ) k S , r ( ) k O , r ( )
Incorporating all these modifications into Eq. 9 results in Eq. 11.
 rel ( ) 
 u ( ) E s ( )  s ( ) k S , s ( ) k O , s ( )  t 0 E s ( )  s ( ) k S , s ( k O  s   
E s ( )  s ( ) k S , s ( ) k O , s ( )  t 0 E s ( )  s ( ) k S , s ( k O  s   
(11)
Since  = 0 for an opaque material, the B0() terms in Eq. 11 be 0.
Eq. 10 now easily reduces to:
 u ( )   rel ( )
(12)
For transmission measurements with the integrating sphere removed, the  terms are
removed since reflectance standards are not involved with these measurements. Since a
0% Baseline Correction is performed with an opaque material in the sample beam, Eq. 11
is simplified to Eq. 12 in the same fashion as the transmittance case.
Since the Bo() = 0 in the transmission and transmittance case, the need for a 0%
Baseline Correction is removed.
General Considerations
Spectral measurements between 0.35 and 2.50 m in the laboratory are relatively simple;
however, there are considerations that need to be taken into account that will insure the
measurements are of high quality. Even though this document is focused on the Cary 500
Spectrophotmeter, the follow considerations are common to any similar instrument.
Instrument Parameters
1.
Modern laboratory spectrometers are capable of amazing spectral resolutions and
signal to noise performance. Great care must be taken when selecting the measurement
parameters. It is important that realistic measurement parameters be selected that will
give the highest probability of producing high quality data. Generally, if wider spectral
bandwidths and/or longer integration times are used, the noise level will be decreased.
However, these two parameters must be balanced with the requirements of the data use
and the need for sample throughput.
Reflectance Measurements
1.
For reflectance measurements, an integrating sphere needs to be installed into
the spectrometer. Upon installation, it is crucial the alignment of the
integrating sphere and associated optics be checked and adjusted according to
the manufacturers instructions.
2.
Reflectance measurements rely on a NIST-traceable standard in the sample
port to return absolute reflectance measurements. A Spectralon standard is
suitable for this purpose, and is available with a NIST-traceable spectral
characterization. The accompanying Traceability Protocol document
describes how to maintain the calibration of these standards.
3.
The scattering properties of the unknown sample should be similar to those of
the Spectralon standard. If they are not, error will be introduced to the
measurement due to differences in sphere efficiency.
Transmittance Measurements
1.
For transmittance measurements, all the scattered light transmitted through the
sample will be collected by the integrating sphere and measured by the
detector. There may be a different optical layout for the optical elements of
the integrating sphere attachment. Therefore, it is necessary that the
manufacturer’s instructions be followed in setting up the sphere attachment.
Transmission Measurements
1.
Transmission measurements are probably the easiest measurements with a
dual beam spectrometer. However, care must be taken to insure the two
beams are balanced and properly baselined. For liquids, it is important the
cuvettes in the sample and reference beams are a matched set. Any
differences in the spectral transmission of the cuvettes will manifest itself as
error in the final spectral measurement. If a solution is used to dissolve or
suspend the unknown material, it is important that the solution also be present
in the reference cuvette. Otherwise, the resulting spectra will be that of both
the material and solution combined, which may not be the desired result.
Specific Considerations for the Cary 500
The Cary 500 Spectrophotometer is one of the premier instruments of its type. The
wavelength range is from 0.175 to 3.30 m in transmission mode. With a Spectralon
lined integrating sphere, the wavelength range is .25 to 2.50 m. It uses a dual-beam
optical system, and a dual-grating monochromator for increased spectral resolution. Like
many similar instruments, a photomultiplier tube (PMT) detector is used for the UV/VIS
and NIR wavelengths. For the transmission measurements in the SWIR wavelengths, the
Cary 500 uses a thermoelectrically cooled lead sulfide (PbS) detector. Reflective and
transmittance measurements at these wavelengths use an uncooled PbS detector. To
accommodate these different detectors and the broad wavelength range, different gratings
are used in the UV/VIS/NIR and the SWIR.
As good as the Cary 500 may be, there are specific considerations that must be taken into
account when using the instrument.
1.
The instrument must be allowed to warm up before use. Sufficient warm-up
time will allow the internal components and the thermoelectrically cooled PbS
detector to achieve thermal equilibrium. This will result in less drift from the
electronics, sources, and optics. Varian suggests a 1 - 2 hour warm up period.
Tests on the RIT instrument showed a linear signal drift over a two hour warm
up period of less than 0.10% reflectance. We conclude that a shorter warm up
period of approximately 30 minutes is sufficient.
2.
Dust on the optical elements or in the integrating sphere will degrade the
instrument performance. To reduce dust accumulation, it is helpful to have a
constant purge of filtered and dry air. The RIT instrument is continuously
purged with filtered air from a Balston dry air system that produces air with a
dew point of approximately –100oF. Purging with dry air has the added
benefit of reducing the interference of the water absorption bands in the SWIR
region. The repeated opening and closing of the sample compartment will
continue to allow dust to enter this part of the instrument. However, the purge
will maintain a positive air pressure and minimize further dust accumulation.
3.
As robust as the Cary 500 is, it is still sensitive to vibration. Therefore it is
important that the instrument be positioned on a firm and stable bench.
4.
The electronics of the Cary 500 are among the most sensitive for an
instrument of its type. While much design effort went into making the
electronics resistant to electrical noise and variable line voltage, it is prudent
to attach a high quality power conditioner to the instrument. Besides filtering
out electrical line noise and smoothing out line voltage fluctuations, a power
conditioner will also protect the instrument and attached computer from any
surges that may appear on the power line. Such inexpensive protection may
have significant cost savings in the future.
Making Measurements
Spectrophotometers are common instruments in analytical laboratories. As a result,
the measurement methods have become some what standardized. Spectral
measurements of remotely sensed materials are not a common application of a
spectrophotometer such as the Cary 500. However, the measurement protocols have
elements borrowed from the other common laboratory measurements.
Transmission Measurements:
Transmission measurements of liquids will utilize cuvettes typical of an analytical
laboratory. The measurement process is as follows:
1. Configure the Cary 500 for transmission measurements with the cuvette holders
mounted in the sample compartment.
2. Power up the instrument and allow to warm-up for at least ½ hour.
3. Select the “Baseline Correction” option for the baseline method.
4. Depending upon the samples to be measured, place cuvettes in the sample and
reference cuvette holders. If the unknown is to be dissolved or suspended in a
solvent, it is imperative to have the solvent in both of the cuvettes.
5. Once the solvent filled cuvettes are in place, a 100% transmission baseline scan is
performed to characterize the response of the instrument.
6. The unknown material is loaded into a cuvette and placed in the sample position
of the instrument.
7. Scanning of the sample is commenced.
Transmission measurements of solids are similar, except there will not be anything
placed in the reference beam. The measurement is made as follows:
1. Configure the Cary 500 for transmission measurements with the solid sample
holders mounted in the sample compartment.
2. Power up the instrument and allow to warm-up for at least ½ hour.
3. Select the “Baseline Correction” option for the baseline method.
4. A 100% transmission baseline scan is performed to characterize the response of
the instrument.
5. The unknown material is loaded into the solid sample holder in the sample
position of the instrument.
6. Scanning of the sample is commenced.
Transmittance Measurements:
Transmittance measurements are required when the total transmissive properties
(both scattered and non-scattered) of a sample are of interest. This includes both
solid and liquid samples.
1. The integrating sphere must be installed and aligned according to the
manufacturer’s instructions. For transmittance measurements, the large rear
mirror needs to be located in the “T” position.
2. Power up the instrument and allow to warm-up for a least ½ hour.
3. Select the “Baseline Correction” option for the baseline method.
4. A 100% transmittance baseline scan is performed to characterize the response of
the instrument.
5. For solid materials, there are clips to hold the material against the port. The
unknown material is placed in the sample holder on the input port of the sample
beam.
For liquids, a cuvette holder is positioned in the above clips. Note, since the
cuvette is not part of the sample, it will be necessary to do a scan with a blank
solution (or empty cuvette) to characterize the cuvette. The bland spectra is
subtracted from spectra of the sample during post processing.
6. Scanning of the sample is commenced.
Reflectance Measurements:
Reflectance measurements are required when the reflective properties of a material
are of interest. The measurement process is as follows:
1. The integrating must be installed and aligned according to the manufacturer’s
instructions. For reflectance measurements, the large rear mirror needs to be
located in the “R” position.
2. Power up the instrument and allow to warm-up for at least ½ hour.
3. Choose the baseline option that includes a 0% Baseline and Standard Reference
Correction. Be sure to select the current Standard Reference file.
4. With the NIST-traceable standard in the sample port, follow the software prompts
to perform a 100% Baseline scan.
5. Completely darken the room, remove the NIST-traceable standard, leave the
sample door open, and allow the sample beam to project onto black velvet
approximately 8 feet away. Follow the software prompts to perform a 0%
Baseline scan.
6. Insert the unknown material into the sample port and close the sample door. If the
sample is too large to close the sample door, wrap the integrating sphere with an
opaque fabric to block stray light from leaking into the sphere. (Note: the opaque
fabric must be opaque throughout the wavelength range being scanned.)
7. Scanning of the sample is commenced.