The principles behind preparing microscopic samples for infrared analysis are similar to those for preparing macroscopic samples, as discussed in Chapter 4. Since an infrared microscope can be used in transmittance and reflectance, both types of sample preparation come into play. Unfortunately, the preparation of microscopic samples for infrared analysis may be the hardest part of using an infrared microscope. Frequently, samples are too small to be seen by the naked eye, so the preparation of samples for infrared microscopy requires the use of a low-power stereo microscope, as shown in Figure 6.5.
The user looks at a magnified image of the sample through the binocular eye- piece. Preparative microscopes typically have a magnification of 5× to 25×. The sample is then placed on the working surface of the microscope and may be illu- minated by a lamp for better viewing. The focusing knob is adjusted to bring the sample into view. Special tools such as probes, scalpels, and tweezers are used to cut the sample, move it around, and mount it. It takes skill, patience, and practice
Binocular eyepiece Working surface Focusing knob
FIGURE 6.5 A photo of a preparative microscope: a low-power visible-light microscope
to manipulate small samples while looking through a microscope. The challenge is that in a magnified view of a sample, a small amount of movement can cause it to leave your field of view. Steady hands are a necessity, and one way to steady them is to plant your little fingers on either side of the working surface, and then manipulate the sampling tools with your other fingers.
Microscope transmission samples suffer from the same opacity problem as macroscopic samples. The solution to this problem is to prepare samples that are 1 to 20 microns thick. Fortunately, many microscopic samples can be turned into thin films by applying pressure and flattening them. This works because it does not take a lot of force to put a lot of pressure on a sample of small area, as shown in Equation 4.3. There are many ways to apply pressure to a microscopic sample to flatten it. In the author’s opinion the best tool for this purpose is a “roller knife,” as shown in Figure 6.6, so called because it has a roller at one end and a scalpel at the other.
Roller knives are available from companies that make infrared microscopes. To use one, the sample is typically placed on the working surface of a preparative microscope like the one shown in Figure 6.5. The roller is pressed and rolled over the sample several times while applying pressure, which serves to spread the sample
out and thin it. Many samples can be flattened in this manner including polymers, particles, and fibers. The flattened sample is then picked up with tweezers or a probe while still being viewed through the preparative microscope, and then placed on an infrared transparent window (typically KBr). The sample and window are then placed at the focal point of the infrared microscope. Using visible light the sample is found and brought into focus. The aperture(s) of the microscope are then adjusted so their image touches that of the sample, the microscope is switched to infrared transmission mode, and the sample spectrum is measured. The background spec- trum is obtained on a clean portion of the infrared transparent window using the same aperture as for the sample. The infrared spectrum of a cocaine hydrochloride crystal flattened with a roller knife, mounted on an infrared transparent window, and obtained in transmission mode is shown in Figure 6.7.
Since microscopic samples are much smaller than the infrared transparent win- dow upon which they sit, they are sometimes difficult to locate. A probe or tweezers can be used to place an identifying scratch on the window near the sample. Once the window is placed in the microscope the scratch can easily be found, and moving the field of view along the scratch will lead to the sample. Another problem with microscopic samples is that they can be easily blown away by a sneeze or a puff of wind, such as from a door opening, a ventilation system turning on, or by someone walking past. A way around this problem is to flatten the sample into the surface of the infrared transparent window, thereby embedding it. This works because KBr
Wavenumber (cm–1) 4000 3600 3200 2800 2400 2000 1600 1200 800 0 100 % Tr an sm itt an ce Cocaine 250 × 250 microns
FIGURE 6.7 The infrared spectrum of a cocaine hydrochloride crystal obtained by flat-
tening it with a roller, mounting it on an infrared transparent window, and measuring its spectrum using infrared transmission mode.
is soft. Embedding the sample makes it more secure and less prone to blowing away, and a number of samples can be embedded in the same window. However, if there are multiple samples embedded in a window one must be careful to take the spec- trum of the correct one. Samples embedded in windows can be removed by rubbing the KBr window briefly on a small piece of wet paper towel and then quickly drying it. This removes the first few atomic layers of the window along with any samples that are embedded.
Hard samples can be difficult to flatten with a roller knife, and rubbery samples such as elastomers will spring back to their original size and shape after pressure is removed. For these samples a device called a diamond anvil cell can be used to flatten them, a picture of which is shown in Figure 6.8. A diamond anvil cell consists of two metal halves that mate together, with each half containing a small gem-quality diamond. The two halves are separated and placed on the working sur- face of a preparative microscope like the one shown in Figure 6.5. The sample is placed on one of the diamond windows, then the two halves of the cell are brought together, sandwiching the sample in between. For softer samples finger pressure may be sufficient to flatten them; for harder samples there are screws on the device, as shown in Figure 6.8, that can be tightened. The cell is roughly 1 inch in diameter and three-quarters of an inch thick so it will fit on the sample stage of most microscopes.
FIGURE 6.8 A photo of a diamond anvil cell used to flatten samples for transmission analy-
There are holes in the top and bottom of the cell to let the infrared light through, as shown in Figure 6.8. The beam path for the diamond windows and the sample is illustrated in Figure 6.9.
For samples that will not pop back into shape it is sometimes convenient to remove the top window and analyze the flattened sample with it resting on the bottom win- dow. This makes the device smaller, allowing more room for focusing and moving the sample around. It also increases infrared throughput by reducing the pathlength of diamond through which the infrared beam must pass. The background spectrum should be obtained on a clean part of the diamond window using the same aperture as the sample. This can be tricky, because if the entire window is covered with sample it will be necessary to measure the sample spectrum, remove the diamond cell to clean it, place it back in the microscope, and then measure the background spectrum, all using the same aperture setting.
A beauty of the diamond anvil cell is it is hard enough to flatten almost anything including polymers, powders, particles, fibers, paint chips, and elastomers. The cell turns an infrared microscope into an almost universal sampling accessory for solids; in some labs they analyze all their macroscopic and microscopic solid samples this way. In labs without an IR microscope the diamond anvil cell can be used to analyze solids by placing it in a beam condenser mounted in the FTIR sample compartment.
The utility of the diamond anvil cell is great, but it comes with a price: the device costs several thousand dollars. This is no surprise since diamonds are expensive. Add to this the cost of an infrared microscope, which is typically tens of thousands of dollars, and you end up with a hefty price tag. Another disadvantage of the diamond anvil cell is that diamond absorbs in the mid-infrared between 2200 and 2200 cm–1. It will typically mask the peaks of samples that absorb in this region such as materials containing C≡C and C≡N bonds. If your sample has important peaks in this wave- number range, it may be necessary to analyze the sample using some other sample preparation technique.
Infrared spectra of microscopic samples can also be obtained in reflectance mode. Reflectance samples are placed on a gold or aluminum mirror, and then the infrared beam is bounced off them. The background spectrum is taken on a clean portion of the mirror because these materials are good infrared reflectors (which
Diamonds
Sample
IR beam
FIGURE 6.9 The path of the infrared beam through a sample mounted on a diamond
is also why they are used as mirror materials in FTIRs). The type of reflectance depends upon the sample as discussed in Chapter 4. So, for example, specular reflec- tance, diffuse reflectance, and reflection absorption spectra can be measured using an infrared microscope. Reflectance samples do not suffer from a thickness problem so they do not have to be flattened, and frequently they can be analyzed with little sample preparation, saving enormous amounts of sample preparation time. However, despite the best efforts of optical designers it is difficult to collect all the light that is reflected from the surface of a microscopic sample, particularly samples with rough surfaces. This means that in reflectance less light makes it to the infrared detector than in transmittance, so some microscope reflectance spectra are noisy. Increasing the number of scans can improve the SNR of such a spectrum. Reflectance spectra are also surface sensitive, and for samples where this information is important this mode should be used. For example, surface layers coated on a piece of metal can be analyzed in reflectance. Another example is when the sample must be analyzed in situ because it cannot be removed from its matrix for some reason. So, defects in polymer films or contaminants on circuit boards can be analyzed in this fashion. However, in many labs transmission is the sample preparation method of choice for most microscopic samples because of the better SNR obtained.