Transmission Sampling Techniques

• KBr Pellets
• Liquid Cells
• Thin Films
 Transmission Sampling

• Transmission analysis of samples is the most common

and most universal method of analyzing solids, liquids
and gases.
• Transmission spectra provide the highest sensitivity and
best detection levels of all infrared sampling techniques.
• Numerous documented analytical methods rely on
transmission spectra of samples and in many cases, it is
the only recognized analysis method.
• Transmission methods include KBr pellets, sample
mulls, the use of liquid (salt) cells and thin films of
liquid or polymer samples.
 The KBr Pellet
• KBr (potassium bromide) pellets are the most recognized
method for obtaining infrared spectra of powder or ground
solid samples.
• Numerous USP and forensic analysis methods utilize KBr
pellets for collection of infrared spectra.
• Other ionic salts may be used for the pellet matrix such as
NaCl, KCl or CsI, but KBr is preferred.
• These materials are very moisture-sensitive and must be
handled carefully as they will quickly absorb atmospheric
water vapor.
• It can be a challenge to make a pellet properly if the procedure
is not followed carefully.
 The KBr Pellet
• KBr pellets are made by grinding the sample to a fine powder and mixing
it with KBr powder. The powder mixture is placed into a KBr pellet press
and pressure is applied to make a clear disk, or window, of KBr which
contains the sample. The pellet is fixed into a holder and placed into the
sample compartment to collect the sample spectrum.
 The KBr Pellet - Pellet Presses

• KBr pellet presses are made of stainless steel and come in 3 main
varieties:
– The standard 13 mm pellet die is placed in a hydraulic press to create
the pellet. Approximately 8,000 pounds pressure is applied to the die
to press the pellet.
– The hand press uses two anvils (bolts without threads, basically) and a
smooth barrel to contain the KBr/sample mixture. The two polished
anvils hold the sample mixture between them in the barrel and the
whole assembly is placed into a hand press which applies pressure to
the die.
– The mini-press is simply a threaded barrel with two bolts and the
KBr/sample mixture is placed between the bolts. The ends of the bolts
are highly polished and they are screwed into the barrel to create a
pellet.
 The Standard 13 mm KBr Pellet Die
The die is assembled with the sample in the barrel between the plunger
and lower anvil. The die is placed into a hydraulic press and the pellet
created by fusion of the KBr and sample matrix. The pellet is removed
from the die and placed in the instrument. A vacuum can be applied to
the die to remove adsorbed moisture.
 Alternative KBr Pellet Dies

For the hand press and the mini-press, the die assembly is put
together with the KBr mix in the barrel between the two anvils, or
bolts. Pressure is applied with either the hand press or by screwing
the bolts into the barrel. After the pellet is created, the anvils are
removed and the entire barrel, containing the pellet, is placed into
the sample compartment such that the beam transmits through the
pellet. The pellet cannot be saved when using these KBr dies.
 KBr Pellets - Procedure

1.) Grind the sample using an agate mortar and pestle until the sample
is glossy or shiny in appearance (particle size <50 ). Generally,
the sample should be approximately 1-3% of the total weight. For
organic samples use 5-10 mg, for inorganics 2 - 5 mg. A final
KBr/sample weight of 300 mg will make a pellet that is 1 mm x 13
mm in diameter.
2.) Add 1/3 the KBr to the ground sample and mix. Add the
remaining KBr and mix well.
3.) Pour the KBr/sample mixture into the pellet die. Place the plunger
on top of the sample and rotate a few times to level the mixture
between the two anvils.
4.) Apply pressure for 2 - 5 minutes. Disassemble the die, place the
pellet into the instrument and collect the spectrum.
 KBr Pellets - Precautions

• ONLY use spectral grade KBr powder. It is ground to a particle

size of ~50 microns and is dried before packaging.
• ALWAYS keep KBr (KCl, CsI, etc,) powder in a desiccator until
ready to use. KBr powder can be dried in an oven set to 110o C.
• ALWAYS use clean, dry KBr powder. Collect a spectrum of a
blank KBr pellet to verify the quality.
• ALWAYS add the KBr to the ground sample. NEVER grind the
KBr with the sample, simply mix with a spatula.
• When a new bottle of KBr is opened, aliquots of the powder should
be stored separately for laboratory use to prevent contamination of
the primary source.
 KBr Pellets - Precautions

• Use only an agate or alumina mortar and pestle for grinding of the
sample. Glass or ceramic mortars may be softer than some samples
and will chip during grinding, contaminating the sample.
• Clean the mortar, pestle and pellet dies AFTER EACH USE! Use
hot water and dry the hardware thoroughly. Acetone may be used
to rinse the die, mortar and pestle after washing. Alternatively, the
pellet dies, mortar and pestle may be dried in a warm oven.
• Always read the instructions for the KBr pellet die and hydraulic
press before using it for the first time.
• Always use a safety shield around a hydraulic press.
 What can go wrong?

• Sloping baselines are often the result of improper grinding of the

sample. Check the pellet; if it is cloudy or there are visible
particles, the sample needs to be ground to a finer particle size.
• The strongest sample peaks should be greater than 20 %T but no
stronger than 5 %T.
• The appearance of moisture bands at 3300 and 1640 cm-1 indicates
wet KBr. Collect a spectrum of a 'blank' KBr pellet to verify and
dry the KBr thoroughly if needed.
• A pellet that is too thin (smaller than 1 mm) can exhibit a fringe
pattern (wavy baseline) in the spectrum.
• Even though the pellet may not look really good, run the spectrum
anyway because you can get a good spectrum from a bad pellet.
What can go wrong?
 What can go wrong?
• A pellet that is smaller than the instrument beam diameter can vignette
(restrict) the beam. This can cause wavenumber shifts and incomplete
compensation for atmospheric water bands. If in doubt, collect the
background through the empty pellet holder or die to compensate for the
beam restriction.
 What can go wrong?
Incomplete grinding or improper pressing of the pellet can
produce distorted spectra with severely sloping baselines.
8 0

6 0

% T

4 0

2 0

1 0

4 0 0 0 3 0 0 0 2 0 0 0 1 0 0 0 4 0 0

W a v e n u m b e r [ c m - 1 ]
 What can go wrong?
Too much sample in the pellet can also severely distort the
spectrum.
5 0

4 0

3 0

% T

2 0

1 0

4 0 0 0 3 0 0 0 2 0 0 0 1 0 0 0 3 5 0

W a v e n u m b e r [ c m - 1 ]
 A Good KBr Pellet Spectrum
1 0 0

8 0

6 0

% T

4 0

2 0

1 0

4 0 0 0 3 0 0 0 2 0 0 0 1 0 0 0 3 5 0

W a v e n u m b e r [ c m - 1 ]
 Caffeine Calibration – KBr Pellet

1 .7 1 5 1 3

1 .5

R a ti o

0 .5

0
0 20 40 5 0 .9 5 1 9
C a ff %
 Ibuprofen Calibration – KBr Pellets

1 .7 9 3 1 7

1 .5

1
R a ti o

0 .5

0
0 20 40 6 2 .7 9 0 7
% Ib u p r o fe n
 Mull Samples

• A 'mull' is a mixture of either Nujol (mineral oil) or

Fluorolube and a powdered sample. The powder is mixed
with mulling oil and the mixture placed between two salt
windows to collect the transmission spectrum.
• Mulls are generally used for samples that may exchange
ionic components with the KBr salt. It is also a simple and
inexpensive method for sample analysis.
• Nujol and Fluorolube oils also have an infrared spectrum and
must be subtracted from the mull spectrum. A spectrum of
the oil is collected without a sample and used as a reference
during subtraction.
 Mull Samples - Procedure

1.) Grind the sample to a fine powder using a mortar and

pestle. (Use the same procedure as for a KBr pellet without
adding the KBr.)
2.) Add a few drops of the Nujol or Fluorolube and thoroughly
mix with the sample powder. The final mixture should look
something like toothpaste.
3.) Use a rubber spatula to transfer the mull to a KBr (or other)
window. Place another window on top of the mull and
squeeze gently to obtain a thin film of the sample mixture.
4.) Place the sample into the instrument and collect the
spectrum.
 Mull Samples

• Always subtract a reference spectrum of the mulling oil from the

mull sample spectrum to obtain the spectrum of the powder only.
 Liquid Sampling Techniques

• The majority of procedures for liquid transmission

sampling involve the use of liquid cells made with
either water soluble or water insoluble windows.
• Water soluble windows of KBr, NaCl or CsI are
hygroscopic, or moisture-sensitive. Materials such as
these must be handled carefully as they will quickly
absorb water and either fog, or start to dissolve.
• Insoluble windows such as BaF2 or CaF2 may not
immediately react with aqueous solutions, but repeated
use will eventually fog the windows and reduce
throughput through the cell.
 Liquid Sample Cells

• There are several types of cells used for liquid samples

with a variety of windows:
– Dispersive infrared instruments use cells with rectangular
apertures while FT-IR instruments require cells with circular
apertures.
– Sealed liquid cells have a fixed pathlength and the cell assembly is
glued together. The fixed path length makes them ideal for
quantitative analysis.
– Demountable liquid cells are used for a wide variety of samples
and are excellent for qualitative analysis. These cells can be taken
apart and are usually supplied with a variety of 'spacers' that can
be used to change the cell path length.
 Liquid Sample Cells - Windows

• There are numerous window materials that can be used for

a liquid cell. The window selected should be based on the
type of sample to be examined.
• Water soluble windows should be used only with organic
solvents and samples. ANY contact with water will fog
the windows and significantly decrease spectral quality.
• Water insoluble windows are useful for almost any liquid
sample but may not have the required spectral range (see
following tables) or may be more expensive than other
windows.
 Water Soluble Windows

Material Useful Spectral Range (cm-1)

KBr 40,000 - 400

NaCl 40,000 - 650

KCl 33,000 - 500

CsI 33,000 - 200

 Water Insoluble Windows

Material Useful Spectral Range (cm-1)

SiO2 (Quartz) 50,000 - 2500
CaF2 66,000 - 1300
BaF2 50,000 - 1000
KRS-5 16,000 - 200
AgCl 23,000 - 400
AgBr 20,000 - 300
ZnSe 20,000 - 500
ZnS 33,000 - 200
33,000 - 2500;
Diamond (C)
1600 - 50
 Cells and Windows - Care and Feeding

• CLEAN the cell or window(s) immediately after use.

• NEVER use a solvent containing water to clean or rinse liquid
cells or windows composed of ionic salts. Use only a pure,
volatile solvent such as acetone or other small ketone; dry
alcohol; carbon disulfide or chloroform. DO NOT use
methanol for cleaning as it will fog a salt window or cell.
• Windows or cells that are water insoluble may be washed with
water, but volatile solvents are preferred. Read manufacturer
recommendations for the cell or window before use.
• ALWAYS keep liquid cells and windows in a desiccant
chamber.
 Cells and Windows - Care and Feeding

• Windows and cells can be dried in an oven set to 110 C.

• Never force sample into a liquid cell because it may rupture
the seals or gaskets.
• Use care when handling cells and windows as they scratch
easily and will crack or break when dropped.
• When taking apart or assembling a demountable cell, apply
pressure evenly to the windows during the procedure or they
may crack.
• Always work in a hood when using volatile solvents and do
avoid inhaling the vapors.
 Cells and Windows - Care and Feeding

• Only salt windows (KBr, NaCl, etc.) should be polished.

DO NOT polish water insoluble windows as the dust
from many of these windows is toxic (i.e. ZnSe, KRS-5
(thallium bromide iodide), ZnS, etc.).
• Salt windows may be polished by rubbing the window in
a figure 8 pattern using a leather chamois dampened with
100% ethanol. The polishing procedure will gradually
remove minor scratches and fogging of the window by
moisture. Commercial polishing kits are also available.
• Always use pure, dry solvents for washing and cleaning
of all cells and windows.
 Liquid Cells - Pathlength
• Selection of cell path length is crucial for useful spectra. Generally, a path
length of .015 to 0.1 mm is adequate for a polar sample (alcohols, ketones,
esters, amines, etc.) while non-polar liquids (hydrocarbon chains) require a cell
with a path length > 0.05 mm. For qualitative analysis, a path length of 0.01 to
0.25 mm is sufficient while quantitative analyses usually require a path length
of 0.1 mm or greater, dependent upon concentrations of the solutes.
Cell Pathlength for Optically Flat and Parallel Windows

1. Consider an empty cell:

2.Infrared spectrum of empty cell: (obtained with constant λ interval scale):

Transmission Cell Pathlength Determination
Transmission Cell Pathlength

n=68

5(68)
b= = 0.1122mm
(3633.5-603.6)
 Isooctane Calibration for Liquid Cells

Each peak can provide a calibration curve

for a cell in an optimum thickness range.
1206 cm-1 1282 cm-1

826 cm-1

1950 cm-1
 Sealed Liquid Cells

• Cells can be obtained with either salt or water insoluble windows.

• Sealed liquid cells are provided with specified pathlengths
ranging from .015 to 1.0 mm. Other pathlengths may be
available.
• Sealed liquid cells are ideal for quantitative analysis of solutions,
either aqueous or non-aqueous.
• The top cell window of a liquid cell is drilled and the cell fitted
with sample ports for filling of the cell using a Luer-lock syringe.
• A sealed cell is assembled at the factory to prevent leaking and
should never be disassembled to maintain proper sealing of the
gasket and spacers.
 Filling Liquid Cells

• Two syringes are used to fill a sealed

(or fixed path demountable) cell.
One syringe contains the sample, the
other is used to create a vacuum
within the cell volume and draw the
sample into the cell. Slight pressure
on the sample syringe may be used to
fill the cell. The same method can be
used to clean the cell with a volatile
solvent, flushing the sample from the
cell cavity.
• Never force sample into a liquid cell
because it may rupture the seals or
gaskets.
 What can go Wrong?

• A spectrum with a sloping baseline is an indication of

either a fogged or scratched window.
• If the window is clear but the spectral absorption bands
are distorted near a sloping baseline, the cell has air
bubbles and must be re-filled to remove them.
• Sealed cells are susceptible to cross-contamination and
must be thoroughly cleaned immediately after each
sample.
• Quantitative results obtained from a demountable cell
should be used carefully as the path length determined by
the spacer may not be repeatable.
• Always compare spectral data for unknowns with standard
spectra collected in the same manner as the unknown.
 Liquid Sampling - Solutions

• Solvents used for solutions can be subtracted to give the spectrum of

the solute. The strongest bands of the solvent may not subtract cleanly
from the solution spectrum.
 Demountable Liquid Cells

• Demountable cells:
– can be taken apart for cleaning or to sample
viscous liquids, greases, waxes or mulls.
– are used for capillary films of liquids and mulls.
– can create thin films of pastes, oils and greases.
– are used to analyze cast films, created by
dissolving a solid in a suitable solvent and
evaporating the solvent from the solution placed
on the window surface.
– can be assembled with a specific spacer and the
cell filled with a liquid using the sample filling
ports.
Demountable Liquid Cells - Capillary Films

• Capillary films are a thin layer of sample between two windows. The
windows of a demountable cell can be removed from the cell, used to
create the sample film and then reassembled for data collection.
• For qualitative analysis of a liquid sample, a few drops are placed
between the two windows. Capillary action and the weight of the top
window will create a thin film of the liquid sample.
• Viscous liquids, mull samples, oils and greases are spread onto a
window and the other window used to squeeze the sample into a thin
film.
• A thin spacer (.010 to .025 mm) can be used to control pathlength for
a capillary film sample or to enhance sample or contaminant bands.
• For optimum data collection, make sure the windows are clean and
there are no bubbles in the sample volume inside the cell.
 Capillary Film Spectrum
1 0 0

8 0

6 0

% T

4 0

2 0
Benzonitrile
1 0

4 0 0 0 3 0 0 0 2 0 0 0 1 0 0 0 3 5 0

W a v e n u m b e r [ c m - 1 ]
 Capillary Film Spectrum
1 0 0

8 0

6 0
% T

4 0

Isopropanol
2 0

4 0 0 0 3 0 0 0 2 0 0 0 1 0 0 0 4 0 0

W a v e n u m b e r [ c m - 1 ]
Protein Deuterium Exchange

Red- initial Blue-Exchanged

 Cast Films - Procedure

• Cast films are made by dissolving a solid sample in a volatile

solvent, placing the solution onto a demountable cell window and
evaporating the solvent, leaving a thin film of the solid behind.
• Solvent evaporation is performed in a hood by placing the
window with the sample solution and allowing the solvent to dry.
• Alternatively, the window can be gently warmed with a hot-plate
set on very low heat to promote evaporation. Place a paper towel
under the window to prevent rapid heating. Cover the crystal
with a beaker to prevent air drafts and to obtain a uniform film.
• A sloping baseline or band distortion may indicate large particles
in the casting or a sample that does not completely cover the
sample beam.
 Cast Films - Precautions

• Always collect a spectrum of the solvent to identify residual

solvent bands.
 Thin Films
• Thin film samples can be a simple method for qualitative or
quantitative analysis of polymers.
 Thin Films

• Similar to capillary and cast films, many polymer samples can be

heated and pressed to produce a thin 'sheet' or film of polymer.
– Polymer pellets or powder is placed between two aluminum plates and
the assembly warmed on a hot plate until the polymer melts.
– The plates are pressed together to create the thin film.
– After the plates are cooled, the polymer is removed and placed in the
instrument.
– Commercially available film making systems include a set of platens and
'shims' or spacers to control the film thickness for quantitative analyses.
– Polymers should not be heated too rapidly as they may burn.
– Some polymers may change crystalline structure or react chemically
during heating and may not be suitable for analysis by thin films.
– Wherever possible, always compare spectral data for unknowns with
standard spectra collected in the same manner as the unknown.
 Infrared Transmission Analysis

• KBr Pellets, mull solutions, liquid cells

and cast films are traditional methods of
collecting infrared spectra.
• Transmission spectra have greatest
sensitivity for infrared analysis.
• Most accepted analysis method.