Microvolume spectroscopy is a very new segment of the molecular spectroscopy market, but it already accounts for about 10% of the combined UV-vis and fluorescence spectroscopy markets. The market consists of both instruments and specialized aftermarket cuvettes and offers a greatly improved tool for bioanalysis application.

Microvolume spectroscopy demand by industry.			

Microvolume spectrophotometers, which include both UV-vis and fluorescence spectrofluorometers, can analyze sample sizes of under 5 μL, and in many cases can analyze samples as small as 0.5 μL. While some new systems are designed specifically to be able to analyze such small samples, many other new systems have been designed to analyze both microvolume and conventional volume cuvettes, generally by using specialized fittings. There is also a plethora of new microcuvettes on the market that work with conventional spectrophotometers.

Microvolume spectroscopy has been developed with bioanalysis in mind. Some of the most common applications include quantitation of DNA, RNA, and protein in biotechnology and academic laboratories. Such instruments also are becoming popular in clinical laboratories, where sample conservation is very important, such as in tissue matching for organ transplants.

The market for microvolume spectroscopy is estimated to have been close to $90 million in 2008, and will easily break the $100 million mark in 2009, despite the deep global recession. The fact that a significant amount of demand is from academia, which is seeing relatively consistent growth, and that these instruments are relatively inexpensive, are major factors in maintaining strong double-digit annual growth.

The foregoing data were based on SDi's market analysis and perspectives report entitled 

Global Assessment Report, 10th Edition: The Laboratory Life Science and Analytical Instrument Industry, September 2008. 

For more information, contact Stuart Press, Vice President – Strategic Analysis, Strategic Directions International, Inc., 6242 Westchester Parkway, Suite 100, Los Angeles, CA 90045, (310) 641-4982, fax: (310) 641-8851, 

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Microvolume spectroscopy is a very new segment of the molecular spectroscopy market, but it already accounts for about 10% of the combined UV-vis and fluorescence spectroscopy markets.

Market Profile: Microvolume Spectroscopy

In the previous installment of this column (1), I introduced the concept of base units and discussed several of them. Here, I will complete the discussion of the units themselves and present some associated issues.

In the first part of this column (1), I mentioned that the meter used to be based upon a line from the emission spectrum of xenon. I was wrong — it should have been krypton-86, not xenon. Figure 1 shows the correct spectrum, with the ~6057-Å line indicated by arrows. Apologies to my readers for the error, and thanks to Mike Mozil of Lubrizol Corporation for pointing this out to me. 

Base Units of Amount and Luminous Intensity

By the early 1800s, it became obvious to scientists that the modern atomic theory was a sound basis for understanding chemical transformations. Work early in that century was focused on establishing the relative weights (actually, masses) of the elements, especially since Lavoisier's 1789 

 established the necessity for the law of conservation of mass in chemical reactions. John Dalton himself proposed a scale of masses in which hydrogen was 1, while J.J. Berzelius, perhaps one of the finest chemical scientists of the first half of the 19th century, proposed a scale with oxygen at 100. 

In 1809, Guy-Lussac announced a law of combining volumes, which stated that the volumes of reacting gases and gaseous products had whole number ratios. In 1811, Italian scientist Amedeo Avogadro (Figure 2) became one of the first to differentiate between the terms 

 was used for both. In realizing that there was a fundamental chemical difference, Avogadro proposed his own gas law: equal volumes of gases at the same temperature and pressure have the same number of gas species. Ironically, given its central role in science today, Avogadro's proposal was not enthusiastically embraced by the scientific community. It wasn't until the Karlsruhe Congress in 1860, an international (though mostly European) conference of chemists, where fellow Italian chemist Stanislao Cannizzarro pushed Avogadro's ideas, that they finally took hold. The distinction between atoms and molecules became pervasive, and a mass scale with hydrogen as having mass 1 was adopted. 

The recognition of the difference between atoms and molecules allowed the modern concept of a balanced chemical reaction to be formulated. Issues such as the law of definite proportions, the law of multiple proportions, the law of conservation of mass, and the law of combining volumes are now obvious — chemical reactions count atoms and molecules, not grams or pounds. In understanding how substances behave and even change, we have to count atoms and molecules. 

Problem: Individual atoms and molecules are notoriously tiny. It would take about 360 million, million atoms to make a square with 1 cm to a side and only one atom thick. Real scientists don't deal with individual atoms; they deal with untold thousands and millions of atoms at a time. We would use the units "dozen" or "gross" or "ream" to describe the numbers of atoms or molecules that are present in any one sample, but even 12, 144, and 500 are still too small to be able to express such a quantity easily. We need to use a much, much larger number. 

Because of advances in the accuracy of analytical methods, by the late 1800s, oxygen had been chosen as the fundamental basis of relative masses, and it was defined as 16 units. However, with the discovery of isotopes in the 1920s, it was found that oxygen actually existed as a mixture of isotopes. Physicists, using physical rather than chemical methods, used a mass scale based on 

O, rather than the naturally existing isotopic blend of oxygen. However, it was also found that the exact isotopic blend of oxygen varied, depending upon the source of the oxygen — it varied too much to be used as a standard. Finally, in 1959–1960, international organizations of chemists and physicists agreed to use 

C as the standard for relative atomic masses, setting it equal to exactly 12 units. 

 was first used by Wilhelm Ostwald in 1893, the concept of "gram equivalent" amounts in chemical reactions was relatively well known. However, in 1971, the 14th Conférence générale des poids et mesures (CGPM, the General Conference on Weights and Measures) formalized it: the unit 

, abbreviated mol, is equal to the number of fundamental chemical entities that appear in exactly 0.012 kg of carbon-12. While similar in theory to a dozen, a mole of atoms or molecules actually has a mass that is easy to manipulate in the laboratory, kitchen, basement, garage, or wherever someone performs experiments. Careful experiments allow us to determine how many entities this is; the currently accepted value is that there are 6.022 136 7 x 10

  (602 213 670 000 000 000 000 000) things in a mole of things. That's a bit more than 12 in a dozen . . . 

 is the name given to the quantity of power given off by a light source per solid angle unit. The solid angle unit is the steradian; a unit sphere has 4π steradians of solid angle, or 129 600/π square degrees of solid angle. 

Luminous intensity was one of the last units to be standardized. In the early 1900s, the U.S., France, and the U.K. agreed on the international candle as the unit of luminous intensity. It was defined as the amount of light given off by a one-sixth pound spermaceti candle burning at a rate of 120 grains per hour (where 1 grain = 1/7000th of a pound), or about 7.78 g/h. In 1937, the international candle was defined in terms of a blackbody at the freezing point of platinum (1768 °C, or 2041 K). One square centimeter of blackbody at that temperature had a luminous intensity of 58.9 international candles. In 1946, the luminous intensity of the square centimeter was revalued to 60 "new candles," ever so slightly changing the size of the candle unit. In 1948, the word 

 (abbreviation "cd") was adopted as the new name for the unit. 

In 1979, the 16th CGPM adopted the current definition. One candela, they decided, would be equal to 1/683rd of a watt per steradian of light having a frequency of 5.40 x 10

 . Light of this frequency has a wavelength of about 555.2 nm and is the color that the human eye is most sensitive to. The fraction 1/683 seems unusual (indeed, 683 is a prime number). It was apparently used in order to make the new candela unit as close to the previous definition as possible. This definition of a candela represents about 4.092 x 10

  photons of green-colored light per second. 

To get an idea of how bright this is, a 100-W incandescent light bulb, a common bulb in pre–compact fluorescent days, has a brightness of about 120–135 cd. Bright sunlight is about 10,000 cd per square meter, while moonlight is only about 0.1 cd/m

The candela is unusual in that it is actually defined in terms of a derived unit (watts; see below), while all other base units are defined in terms of a specific number of things or phenomena. Nonetheless, a candela is a base unit of the SI system.  

Using the seven base units as the starting point, other units can be constructed; these are called derived units. Derived units are constructed by multiplying or dividing the base units. Occasionally, the derived unit itself is given a name, giving some people the misleading impression that they are more fundamental units than they actually are. For example, to describe a volume, we can describe it ideally as being a length times a width times a height; therefore, the basic unit for volume is m x m x m, or m

. This derived unit is actually too large to be useful for many purposes, so we use 1/1000 of this volume, which we rename the liter (abbreviation L). Units for speed include meters per second, which involves the division of one unit into another: m/s. Acceleration is m/s

  (which is renamed a newton, abbreviated N); energy is kg·m

  (which equals N·m and is renamed the joule); and power is kg·m

 , which is equal to a J/s and is renamed a watt (abbreviation W). 

Most units are derived units. The International Union of Pure and Applied Chemistry (IUPAC) publishes 

Quantities, Units, and Symbols in Physical Chemistry,

 which details many defined units (2). Chapter 2 of the book lists hundreds of types of quantities, all of whose units are either one of the seven base units or some combination thereof. 

In many cases, the base unit or a derived unit might not have the right size to succinctly describe a quantity — one example was given above, when using the cubic meter to describe volume. Although a system of units based upon decimalization was proposed as early as 1586 (by Flemish mathematician Simon Stevin), the first systematic proposal for a decimalization of units was made in 1875 by the Convention du Mètre, mentioned in the previous column. As part of the metric system (as it was called back then), a series of combinatorial prefixes and associated abbreviations was adopted to modify the fundamental unit (whether base or derived) to a more appropriate size. For example, consider the base unit kilogram. The kilogram is based upon the fundamental unit the gram, and has been modified by the prefix kilo-. The prefix kilo- is defined as "1000 times," meaning that it is 1000 times the size of the gram. Some prefixes represent multiples of the fundamental unit, while others represent fractions of the original unit — but all prefixes imply a certain power of 10, so all prefixes have the effect of moving a decimal point to the right or left of 1. 

Each prefix also has an associated abbreviation that can be combined with the abbreviation of the fundamental unit to give the abbreviation of the modified unit. The abbreviation for kilo- is k; when combined with the abbreviation for the gram, g, the abbreviation for kilogram is produced: kg. Table I gives the current list of approved units that are part of the SI system. 

Table: Unit prefixes, abbreviations, and multiplicative values.  

There are some conventions when using the prefixes and their abbreviations. Only one prefix is used at a time on a unit. If more than one multiplier is needed, use a different prefix. So, instead of saying "kilokilogram," say megagram. Note that the case of the abbreviation is important: if you use ps instead of Ps, you are implying picosecond instead of petasecond — both useful units, but hardly interchangeable! There is no space between the prefix and the fundamental unit, either when spelled out or when abbreviated. Hence, a femtosecond is not spelled "femto second," nor is it abbreviated "f s." Finally, note that some of the abbreviations are also abbreviations of units. The most obvious example is the use of the letter 

 to represent meter as well as the prefix milli-. People using these abbreviations need to understand what they represent based upon their usage — "mm" means "millimeter," while "ms" means "millisecond, not "meter second." (If, for any reason, a meter second is the appropriate unit, it would be written as "m s", with spaces between the unit, or "m x s" or "m·s," with the multiplication operation shown explicitly.) These conventions are neither capricious nor unnecessary — they are crucial to being able to communicate quantities and their units properly. 

The prefixes in Table I are getting more and more use as society progresses. Already we are measuring time periods on the order of attoseconds (10

 s) and computer memory in terms of terabytes and petabytes (10

  bytes, respectively). I predict we will be seeing more zepto-, zetta-, yocto-, and yotta-based units in the years to come.* 

*Although these last few prefixes sound like random syllables, they are actually derived from the Latin 

, respectively. This is because they represent 1000 raised to the seventh or eighth power, either as a multiplicative or as a divisive factor. They do remind this author of the Marx brothers, though — can a harposecond or grouchogram be far behind? And once we're finished with the Marx brothers, we can always use the Three Stooges as inspiration; curlycandela or moemeter, anyone? 

 is a professor of chemistry at Cleveland State University in Ohio. Many of his "Baseline" columns have been reprinted in book form by SPIE Press as 

 available through the SPIE Web Bookstore at 

 was published in May 2006 and is available from SPIE Press. He can be reached at 

 2nd ed. (Blackwell Science, Oxford, 1993). 

Columns: The Baseline

In the previous installment of this column, David Ball introduced the concept of base units and discussed several of them. Here, he completes the discussion of the units themselves and presents some associated issues.

The Seven Base Units: Part II

A two-dimensional gas-phase separation technique, ion mobility-mass spectrometry (IM-MS) is applied to the analysis of crude oils. In this approach, ions are generated by laser desorption/ionization and separated before MS analysis on the basis of collision cross-section (that is, apparent surface area of the gas-phase ion). The capacity to separate petroleum-derived molecules having the same nominal mass in the mobility dimension is demonstrated. Inspection of IM-MS spectra reveals a high degree of diversity in structure and chemical class for crude oil components.

Understanding the molecular composition of a crude oil is an essential component of the refining process (1) and a prerequisite for development of new and more efficient processes to convert the full mass balance of a crude into useful energy. Resolving petroleum samples to their molecular composition is a great challenge to analytical and petroleum chemists because of the complex nature of the samples (2). Moreover, petroleum samples are typically composed of thousands (~1000–100,000) of individual molecules (1–3) derived from highly diverse populations of compound types,  (such as porphyrins, paraffins, multi-ring aromatics, alkanes, and alkenes) (with varying degrees of branching), and other S-, N-, and O-containing hydrocarbons (2–10). Numerous approaches have been utilized to fully characterize crude oils, but no single technique is fully descriptive, and analyses by different techniques frequently generate conflicting data for the same sample. As a result, complete description of a crude oil is often difficult, if not impossible.  

Ultrahigh resolution mass spectrometry (MS) has shown potential to resolve components in a crude oil (1,11,12), but the practical limit is imposed by the rapid increase in the number of possible elemental formulas (with increasing mass), making it difficult to assign unique elemental formulas for ions greater than 300 

 (1). In the current work, we investigate the potential for ion mobility spectroscopy (IMS) to provide complementary information to MS for characterization of petroleum samples. IMS is a postionization separation technique that separates gas-phase ions based on their collision cross-section; we have shown that when coupled with mass (

) separation, ion mobility-mass spectrometry (IM-MS) has unique strength in resolving complex mixtures of peptides, carbon clusters, and various polymeric molecules that have significant overlap in their mass spectra (13–16). In particular, IM-MS provides rapid separation of isomers (17), conformers (18), and species of differing chemical class (19) (based on differences in functional groups, polarities, and atomic compositions), which would be advantageous for the rapid characterization of petroleum samples. 

The principles for IMS separation have been described in detail elsewhere (20,21). Briefly, ions enter a drift cell filled with an inert gas and migrate along the length of the drift cell under the influence of a weak electrostatic field. The ions experience collisions with the neutral drift gas establishing a constant drift velocity that is related to the electric field (

 is a mobility constant that is specific to the ion. The mobility of an ion (

) is inversely related to its ion-neutral collision cross-section (the apparent surface area of the ion) and forms the basis for typical IMS separations; that is, a separation based on size, composition, structure, and charge state.  

The IM-MS instrument used in these studies was constructed in collaboration with Ionwerks, Inc. (Houston, Texas). A schematic of the instrument is shown in Figure 1. A 355-nm, Nd:YAG laser was utilized to desorb and ionize samples from a stainless steel sample plate. After laser desorption–ionization (LDI), ions are separated in a 15 cm-long drift cell (periodic high-field, low-field regions) maintained at ~3 torr with He gas. Once IMS separation is complete, ions are focused into a narrow beam using a multielement einzel lens and mass analyzed with an orthogonal time-of-flight (o-TOF) mass spectrometer equipped with an electrostatic ion mirror (R ~2500). In all IMMS experiments, the LDI source was maintained at the same pressure and temperature (approximately 297 K) as the drift cell, and typical IMS field strengths were 10–20 V.cm

  The mass spectrometer was externally calibrated using C

 (Sigma). All spectra were acquired at a laser intensity equivalent to the observed signal threshold.  

To illustrate the potential of the IM-MS instrumentation, two samples were studied — carbon nanopowder (Sigma), and a Saudi Arabian crude oil (courtesy of Professor Dr. Daulat D. Mamora, Department of Petroleum Eng., TAMU). Samples were used as received without further purification before analysis. LDI-IM-MS analysis was performed by depositing a thin layer of the sample (~1 mL, either in solution or neat) directly onto a stainless steel plate, then inserted into the IM-MS instrument. 

IMS introduces an additional post-ionization separation before mass analysis, thus providing an inherent increase in the peak capacity of any mass spectrometer (22,23). At the same time, IMS will provide complementary information on the composition and structure of an ion as described earlier. To illustrate the effectiveness of IM-MS in the analysis of hydrocarbon-rich samples, carbon nanopowder is taken as a reference because (i) it provides a high ion yield, and (ii) it produces ions with chemical species ranging from small hydrocarbon clusters to polycyclic–branched aromatic hydrocarbons, to pure carbon clusters. An IM-MS spectrum of carbon nanopowder is shown in Figure 2, which plots the drift time of the ions (arrival time distribution) versus the ion mass (

). Notably in the figure, efficient IMS separation is achieved for the single and double charge states of fullerene clusters (inset, bottom right). On close inspection of the lower mass range of Figure 2 (inset, top left), it is evident that additional components that overlap in 

 are resolved in the IMS dimension. These components have been identified as various classes of hydrocarbon clusters that are either present in the sample or formed in the sample when irradiated with photons from the laser (24). These different classes of hydrocarbons are easily resolved in the experiment without incorporating complicated and lengthy prefractionation procedures. In fact, IM-MS separations occur on a timescale of milliseconds compared to a timescale of minutes to hours for traditional approaches such as liquid (4) or gas chromatography (9,25). 

The driving force behind the observed IMS separation in Figure 2 can be realized considering the inherent differences in the gas-phase structure: ions that are more compact, branched, or condensed in structure will have shorter arrival time distributions than the more extended and less compact ions. Similarly, ions with similar structure, or of the same molecular class from different samples, will occupy the same space in the 2D plot as the trendlines illustrated in Figure 2, thus enabling their identification based on the correlation between the apparent gas-phase surface area and 

Analysis of untreated crude oils generally provides complex MS spectra, and information-rich data has been restricted to very high resolution instruments (such as Fourier transform ion cyclotron resonance [FT-ICR]-MS) because of sample complexity in which there are multiple peaks at the same nominal mass. One way to overcome this limitation is by adding a complementary gas-phase separation. For example, Figure 3 contains IMMS spectra of a light crude oil that has not been subjected to pretreatment or prefractionation. The upper portion of the figure (Figure 3a) shows the mass spectra summed over all mobilities and is the MS spectrum that would be obtained if no IMS separation was included. As shown, the molecular weight distribution ranges from 200 to 1100 

, showing a peak at every nominal mass. When looking closely at the IM-MS data (Figure 3b), it is observed the that the ion signal is spread over a broad range of IMS drift times and includes overlap with both hydrocarbon trendlines obtained from the carbon nanopowder spectra (Figure 2). 

Figure 3d contains an expansion of the spectra in Figure 3b, and these data accurately illustrate the structural diversity of the petroleum sample while highlighting the utility of the IM-MS experiment. That is, for a series of ions that increase in mass but maintain similar molecular class, structural features, or conformation (as with the fullerene clusters in Figure 2), an increase in mass is typically accompanied by a near linear increase in arrival time distribution. When viewing the crude oil spectra in two dimensions, it is clear that this trend is not conserved, indicating a high degree of structural, and potentially compositional, diversity for the petroleum sample. It is likely that the relative differences in mobility observed in Figure 3 result directly from the differences in chemical class and type of molecules present in the sample. The utility of analyzing the spectra with two dimensions (mobility and mass) is further highlighted by the 3-D IM-MS spectra projected in Figure 4, in which numerous peaks are observed that coincide in mass (separated by less than a few milli-daltons) but are resolved in the mobility dimension. These data clearly illustrate the advantage of incorporating an IMS separation; that is, multiple, overlapping 

 can be resolved in the mobility dimension, thus greatly simplifying the challenge of petroleum characterization once the observed IMS separations have been related to the inherent nature of the various ions.

The separation strength offered by IMMS was shown to be highly applicable to petroleum research, where progress is traditionally hindered by sample complexity. Although careful characterization of the space occupied by petroleum samples in the IMMS plots is required at this point, the data herein show that accurate assessment of ion composition and structure from a single IMMS experiment is feasible. Currently, detailed molecular modeling and IMS analysis of model compounds are under way to better understand the behavior of petroleum molecules in the drift cell. Given the current mass resolution of our instrument (~2500), we estimate a three- to fourfold increase in peak capacity when the IMMS spectra are compared with MS-only spectra for analysis of petroleum samples.  

 are with the Department of Chemistry, Texas A&M University, College Station, Texas. 

(2) S.K. Panda, J.T. Andersson, and W. Schrader, 

(3) K. Qian, R.P. Rodgers, C.L. Hendrickson, C.L. Emmett, and A.G. Marshall, 

(9) K. Qian, G.J. Dechert, and K.E. Edwards, 

(11) C.A. Hughey, R.P. Rodgers, and A.G. Marshall, 

(12) K.E. Qian, J.H. Diehl,  and L.A. Green, 

(13) K.J. Gillig, B. Ruotolo, G.F. Verbeck, E. Stone, and D.H. Russell, 

(14) J.A. McLean, B.T. Ruotolo, K.J. Gillig, and D.H. Russell, 

(15) J.A. McLean, J.A. Schultz, M.V. Ugarov, T.F. Egan, and D.H. Russell, 

(17) A.B. Kanu, P. Dwivedi, M. Tam, L. Matz, and H.H. Hill, 

The Mobility and Diffusion of Ions in Gases

(22) B.T. Ruotolo, J.A. McLean, K.J. Gillig, and D.H. Russell, 

(23) B.T. Ruotolo, J.A. McLean, K.J. Gillig, and D.H. Russell, 

(24) C. Becker, K. Qian, and D.H. Russell, 

(25) F.C. Wang, K. Qian, and L.A. Green, 

The authors demonstrate the capacity to separate petroleum-derived molecules having the same nominal mass in the mobility dimension using IM-MS spectrometry.

Ion Mobility-Mass Spectrometry: A Tool for Characterizing the Petroleome

A demountable ICP torch from Glass Expansion includes standard quartz outer tubes that can be replaced when worn. According to the company, the ceramic outer tubes will withstand harsh conditions. The torch is available for specific PerkinElmer, Spectro, and Thermo Fisher Scientific ICP–OES and ICP–MS systems. 

Hellma's TrayCell cuvette requires a sample volume of 0.7 μL. According to the company, the surface tension of the sample forms a repeatable optical path of 0.2 mm or 1 mm. Applications include biotechnology, drug discovery, and forensic investigations. The lower part of the cuvette reportedly contains fiber optics and mirrors for the UV that interface to an upper part that contains collimating optics. The parts are optically connected through the sample. 

The model RA-3420 Gold+AFS mercury analyzer from Nippon Instruments North America is designed for analysis according to EPA Method 1631E. Samples are automatically digested and oxidized followed by automated analysis by cold vapor atomic fluorescence spectroscopy. According to the company, no user intervention is required. 

The Spectrolinker XL-1000 UV crosslinker from Spectronics Corporation is designed for biotechnology research. Applications include elimination of PCR contamination, crosslinking of DNA and RNA, nicking ethidium bromide–stained DNA, gene mapping, RecA mutation screening and testing, UV sterilization, UV-induced polymerization, UV intensity monitoring, and other UV-dosage applications. 

The Prodigy DC arc spectrometer from Teledyne Leeman Labs is designed for fast trace metals analyses of solid samples. The instrument reportedly can analyze samples including ceramics, refractory powders, graphite, geological materials, nuclear materials, and soils, sludges, and ashes. 

 automated mercury analyzer from Teledyne Leeman Labs is designed to provide 1 ppt detection limits and fast sample throughput. The instrument includes a 270-position autosampler. According to the company, the instrument's software automates the analytical process from method development to final report generation. The analyzer reportedly can be upgraded to perform direct solids analysis. 

Kaiser's RamanRxn2 general purpose Raman analyzer is designed for method development and reaction monitoring. The instrument features automatic calibration and is available in either four-channel or hybrid configuration. In the hybrid configuration, the analyzer can be used for small-spot Raman sampling and PhAT-based wide-area sampling. In four-channel configuration, the analyzer reportedly can be used to study mechanisms, refine reaction pathways, and optimize yields. 

The TRicorder handheld Raman system from BaySpec features no moving parts, volume phase gratings, a narrowband fiber laser, an ATR probe interface, battery operation, and a PC tablet interface. The system requires no periodic calibration. A Windows-based handheld PC is included with the system. The system reportedly can be used to identify chemicals through transparent and translucent materials, is capable of single-handed or wireless analysis, and can identify chemicals in mixtures with low concentrations. 

LECO's ChromaTOF 4.20 software integrates acquisition, processing, and reporting features and includes functions for signal deconvolution, peak identification, semiquantitation, and EPA reporting. The software is supplied with the company's Pegasus HT GC–MS and 4D GC×GC–MS systems, TruTOF HT GC–MS systems, GC×GC system, and Unique HT LC–MS instruments and will also be available as an upgrade for existing versions of the software. 

A mini gas cell with PTFE stopcocks from New Era Enterprises is designed for use with gas samples such as hydrochloric acid that will react with the lubricant used in all-glass stopcocks. According to the company, the cell has a 10-cm path and a 30-mL volume.The cell reportedly can be used to analyze pure gases or gas mixtures with component concentrations greater than 1% and it allows for experiments at slight variations from standard pressure. 

The TemPro lifetime system from HORIBA Scientific uses time-correlated single photon counting for fluorescence measurements. According to the company, the desktop system is capable of measuring lifetimes ranging from picoseconds to seconds. The instrument includes interchangeable pulsed laser diode and LED light sources that cover discrete emission wavelengths ranging from 255 nm to near-IR. Optional polarizers enable measurement of rotational correlation times. 

HORIBA Scientific's CLIP (collection of line intensity profiles) is designed to assist analysts in the development of methods for high-resolution sequential ICP–atomic emission spectroscopy. According to the company, the profile of each line is calculated according to the instrument's configuration, including focal length, slit combination, diffraction grating, and order used. The product is available as an upgrade module for the company's ULTIMA instrument. 

Thermo Fisher Scientific's OMNIC Specta software is designed for Raman applications. The software features multicomponent searching capability, a contaminant identification capability, and an interface that reportedly enables analysts to visualize the workflow on one screen, including data collection, processing, and final report. According to the company, the software automatically indexes all spectral information on a user's system. 

WITec's Project Plus software is designed for data evaluation and chemometric image processing. The software includes features for multivariate data analysis for confocal Raman imaging and scanning probe microscopy such as cluster analysis and principal component analysis. The software is also available as an add-on package for the company's Project data evaluation software. 

A look at the newest spectroscopy-related products.

Products

Tablet coatings are of great importance to both consumers and pharmaceutical manufacturers. By controlling the release of the active pharmaceutical ingredient, tablet coatings ensure bioavailability and minimize harmful effects and the waste of the drug by delivering it at the specific site and at the optimum level. These functions can be compromised if a coating is nonuniform or has defects.

The exceptional properties of terahertz radiation — including its low photon nonionizing energies and its ability to penetrate most nonpolar materials that can be opaque for visible light or low contrast for X-rays — make this radiation a very attractive tool for helping to understand pharmaceutical materials and products. 

Until recently, the analysis of coating thickness relied upon indirect techniques, and it usually was determined by the weight gain of the coated tablet compared to the uncoated tablet core. Near-infrared (NIR) and Raman spectroscopic and imaging techniques that have been developed in recent years to yield information about the quality and integrity of the coatings usually are restricted to the outer surface of the tablet. To test the coating for uniformity or to analyze buried structures or even multiple layers within the tablet, the tablet must be cut and spectroscopic images must be acquired for each layer. However, in contrast to the NIR and mid-IR regions of the electromagnetic spectrum, many of the excipients used for coating solid dosage forms are transparent or semitransparent to terahertz radiation. Moreover, terahertz radiation can easily penetrate commonly used coating polymers. This in turn means that, unlike the aforementioned techniques, terahertz pulsed imaging (TPI) nondestructively provides spatially resolved information from below the surface of the sample (tablet). A nondestructive alternative for characterizing only the density variations within the tablet, and therefore only physical properties of a sample, is X-ray microtomography. However, long acquisition times, computational requirements, and the possibility of radiation-induced strain in the sample are limitations of this technique. Consequently, terahertz measurements are arguably more suited than any of the aforementioned techniques to monitor key quality attributes of the pharmaceutical solid dosage forms during manufacturing. 

 The 3D TPI measurements discussed in this column were performed using a TPI imaga 2000 system (TeraView, Cambridge, UK). 

The method employed to generate terahertz pulsed radiation is based upon illuminating a biased photoconductive switch (antenna) by a femtosecond laser pulse with a photon energy greater than the bandgap of a gallium arsenide substrate. This process will lead to production of photocarriers — electrons and holes in the conduction and valence bands, respectively. Fast varying density of the photocarriers and their acceleration in the external field of the applied bias will generate an electromagnetic field radiating into free space in the terahertz frequency range. The nature of terahertz pulse is broadband (typically 0.06–3 THz, corresponding to 2–100 cm

The detection of the incident terahertz pulse is also based upon a photoconductive antenna. Assuming that an above-band gap optical gating pulse is focused onto a photoconductive antenna-based detector, it will produce photocarriers in the antenna substrate. An incoming terahertz beam will induce a transient bias voltage across the antenna gap between the electrodes. If the incident terahertz electric field and induced photocarriers are both present, the photocurrent will flow across the gap. This current is proportional to the terahertz electric field. By varying the optical path length between terahertz and optical pulses, the entire terahertz time-domain signal can be sampled with both the amplitude and phase of the terahertz pulse available. This is in contrast to most spectroscopic techniques that yield only the signal intensity.  

The components of the TPI system are shown in Figure 1. A Ti:sapphire laser provides a train of laser pulses that are split into a pump beam for terahertz generation and a probe beam for detection, both based upon photo-excitation of a gallium arsenide antenna. These two optical pulses are launched into separate optical fibers that link the TPI core unit with the tablet scanning unit. After leaving the output from the optical fibers in the tablet scanning unit, the pump and probe beams are focused onto the gaps of the photoconductive emitter and detector, respectively, using a silicon lens. A modulated bias was applied across the emitter electrodes and the detector gated output signal was fed to a lock-in amplifier. Reference for the lock-in is provided by the electric modulation of the emitter. A dynamic range of greater than 70 dB was demonstrated using time-gated and lock-in detection. To record the time evolution of the terahertz electric field, a rapid scan delay line is incorporated in the probe beam that systematically varies the delay between probe and pump beam at the detector. The generated terahertz signal from the emitter is focused by a silicon lens to a diffraction-limited spot on the tablet surface. The terahertz radiation reflected from the tablet is collected by the same silicon lens and focused onto the detector.  

Prior knowledge of the tablet shape profile allows a six-axis robot arm to position the solid dosage form under investigation in focus and always normal to the incident terahertz beam. In this manner, a 3D terahertz image of the solid dosage form is acquired. When the terahertz pulse is applied on a tablet, part of the incident terahertz pulse is reflected from the tablet's outer surface. The other portion passes into the tablet and is partially reflected every time it encounters a new subsurface within the tablet, characterized by a different index of refraction. The resulting terahertz waveform thus consists of multiple pulses arriving at the detector at delayed times, depending upon the index of refraction and the thickness of the traversed layer. 

3D TPI analysis of tablets identified the following key metrics (Figure 2) that could possibly be used to assess the variations in the individual batch average dissolution behavior of tablets produced using different weight gains or by changing some of the coating process parameters. 

Terahertz electric field strength of the reflection peak from air–enteric coating interface depends on the outer coating refractive index value. The peak amplitude can be affected by the density of the coating of the surface. 

Coating thickness is determined as the ratio of the peak position difference between the surface reflection and the interface reflection, divided by the refractive index value of the tablet coating. The coating thickness maps were created by plotting the coating thickness at each point of the tablet surface. They show the distribution and uniformity of the coating layer over the tablet surface and can be used to identify inhomogeneities or defects in individual tablet coatings. 

The interface index is defined as the peak height ratio of the interface reflection and the surface reflection. The interface index gives a measure of the strength of reflection from buried surface and can give an indication of a change in physical composition at the interface.  

As previously mentioned, the resulting terahertz waveform consists of multiple pulses (reflections) arriving at the detector at delayed times. These times depend upon the index of refraction and the thickness of the traversed layers. 

The positive reflection peak at 0.00 mm time delay arises due to the reflection of the terahertz pulse from the tablet surface (that is, the air–enteric coating interface).  

The positive reflection peak at approximately 0.15 mm originates from the enteric coating–subcoating interface — this moves to longer times as the weight gain of the applied enteric coating increases. 

The negative reflection peak at approximately 0.20 mm results from the subcoating–tablet core interface — this moves to longer times with the enteric coating weight gain increase. 

Formation of either positive or negative reflection peak corresponds to either positive or negative change that refractive index undergoes between the adjacent layers. 

The illustration of the formation of different terahertz reflections is shown in Figure 3. Each time the terahertz pulse encounters a new interface (that is, a physical–chemical change within the solid dosage form) that is associated with a change in the refractive index, it is reflected back to the detector. Because of the high signal-to-noise ratio of the terahertz technique, it is possible to detect very small changes in the refractive index and therefore detect various interfaces within the solid dosage form.  

When the photoconductive antenna is illuminated with a laser pulse, it generates a train of pulses with decreasing amplitudes. These pulses (echo signals) arise due to the additional reflections from the antenna–air and detector–air interfaces. As the terahertz pulse propagates through the sample, it becomes modified by the sample response as well as the echoes. As a consequence, the resultant terahertz signal reflected from the sample is the convolution of the system response (terahertz signal emitted from the biased photoconductive antenna with contribution of any echo signals arising from the instrument itself — reflections from the photoconductive antenna and the detector) and the sample response to this signal. Depending upon the thickness of the semiconductor devices and the sample material, the unwanted additional signals can overlap with the main terahertz pulses, which further complicate the signal interpretation. To recover only the sample response and calculate the key metrics, the resultant terahertz waveform (raw data) is deconvolved with the system response by dividing the raw terahertz waveform, in frequency domain, with the reference waveform recorded with a metallic mirror. The deconvolution process is performed in such a way that high-frequency noise is suppressed by applying a numerical bandpass filter with a specified cutoff frequency. This procedure provides an axial resolution of approximately 38 μm. The actual value will depend upon the sample refractive index. Deconvolved data can be processed further to calculate coating thickness values at each pixel of the sample surface as well as the interface index values.  

To give an example, enteric-coated tablets were analyzed using terahertz pulsed imaging. For two observed coating layers, the time-of-flight calculation was employed to calculate the thickness of each layer. 

The deconvolved terahertz time-domain waveforms (Figure 4) carry information about physicochemical properties of coatings and layers within two different tablets of different weight gains. 

The formation of the positive reflection peak at around 0.20 mm (Figure 4) is indicative of the positive change in the refractive index at the transition from the enteric to the subcoating. This position varies with the increase of the weight gain of the applied enteric coating layer. 

To highlight the existence of the additional features within tablets, Figure 5 shows a cross-sectional map of a 10% tablet (Figures 5a and 5b) and tablets from the batch that exhibit the internal structure at 10% (Figures 5c and 5d) and 16% (Figures 5e and 5f) weight gain. These tablets were from the same coating run taken from the coating pan at different times. The importance here is to understand whether this internal structure affects the performance of the drug release.  

The image in Figure 6 clearly shows the effect of the fast coat on the enteric coating–subcoating interface in terms of uneven surfaces and discontinuity across the length. In some areas of the tablet the subcoating was "washed off" the tablet surface. 

The time delay between the two reflection peaks, either reflection peaks between the outer tablet surface and the interface between the enteric and subcoating or between the enteric–subcoating interface and subcoating–tablet core, was used to calculate the thickness of either the enteric or the subcoating for every recorded pixel (Figure 7). 

The terahertz images as well as the statistical analysis (Table I) reveal that the mean coating thickness (averaged over all three tablet surfaces) increases as the weight gain of the enteric coating increases.  

Table I: Results from the statistical analysis   

3D TPI reveals a change in the interface between the enteric and subcoating as the process conditions change (that is, coating viscosity and spray rate). This difference in the interface potentially indicates a change in adhesion between the two coating layers. 

As seen in Figure 8, a significant correlation exists between the nondestructive 3D TPI determined coating thickness and mean dissolution time determined for each individual tablet. Thus, 3D TPI reveals the positive correlation between mean dissolution time and the mean enteric coating thickness (averaged over all three tablet surfaces). 

As observed in Figure 8, the tablet identified as Tablet 7 is an obvious outlier. The observed behavior might be explained by the following: Tablet 7 shows the thinnest subcoating. Elements of the additional structure within the enteric coating are less evident for this particular tablet. 

TPI can be used to determine the coating thicknesses of pharmaceutical products. The thickness can then be correlated to not only the mean dissolution time but also to the onset of dissolution, which can be important in enteric-coated products. Weight gain, traditionally used as a nondestructive indicator of product performance, failed to provide enough information about the performance of the analyzed tablets. However, terahertz coating thickness could be correlated with the actual product performance. 

This investigation has highlighted the potential of TPI to be used as a method for investigating tablet coating uniformity and integrity and coating–coating interface variations to establish process or formulation changes. Because TPI can be applied to tablets relatively quickly and nondestructively, it has the potential to be used as an online tool to aid understanding of the changes that could occur during development and manufacturing, and therefore ensure optimal performance of the finished pharmaceutical product.  

 is with TeraView Limited, Cambridge, U.K.

Columns: Laser and Optics Interface

In this column, Guest Columnist Philip Taday explores the use of terahertz spectroscopy for tablet coating analysis.

Terahertz Pulsed Imaging for Nondestructive Testing of Pharmaceutical Products

United States Pharmacopoeia (USP) general chapter 1058 on analytical instrument qualification (AIQ) has finally become official and was released in August 2008 in the first supplement to the USP XXXI (1). In this collumn, I'll revisit this approach to AIQ and discuss the impact that this will have in the regulated spectroscopy laboratory.

In my 2006 column (2), I wrote about the draft USP General Chapter 1058, or USP <1058> on analytical instrument qualification (AIQ). Now that USP <1058> is effective from August 2008, I want to revisit the subject and look in more detail at the approach and the impact this will have on the regulated spectroscopy laboratory. 

As a reminder, USP <1058> originated at a conference organized by the AAPS (American Association of Pharmaceutical Scientists) in 2003 entitled "Analytical Instrument Validation." The first thing to bite the dust was "validation" because the attendees agreed that instruments are qualified and processes, methods, and computer systems are validated. Thus, analytical instrument qualification was born. Prior to this it had humble beginnings as simply equipment qualification (EQ). The debate about EQ versus AIQ should be reserved for a bar with copious amounts of alcohol because you will be talking about the same process except the name has changed. Either term means that any instrument is fit for its intended use — nothing more and nothing less. 

AAPS published a white paper of the conference in 2004 (3), incorporation as a potential USP general chapter followed in 2005, and review cycles followed until it was finally adopted. It is worth remembering at this point that USP general chapters between <1> and <999> are requirements (you must comply or your product may be deemed adulterated) and those between <1000> and <1999> are informational in nature (alternative approaches are OK if they are justified).  

Take care, however. Although <1058> is informational, it implicitly refers to other USP GENERAL CHAPTERS pertinent to specific techniques (for example, <21> Thermometers or <41> Weights and balances that are requirements). Therefore, be warned and careful in your overall approach to qualification. 

At first sight, USP <1058> seems fine: it appears to be a logical and practical approach to qualification of equipment and instrumentation. It is only when you dig into the details and explore the implications that you find that there are potential problems that could catch you out if you don't think things through and interpret the general chapter sensibly. As we go through this column, I will point out areas that will need special care in your interpretation and approach. 

Positioning AIQ versus Other Laboratory Quality Checks

A common debate with analysts who do not understand the problem is that if we validate an analytical method it does not matter which instrument we run it on: a spectrometer is a spectrometer, isn't it? Unfortunately, this is not the case, and a hierarchy of quality checks is described succinctly in <1058>. 

The foundation of all quality analytical work is the qualification of the instrument: so you do undertake AIQ first (ideally during the purchase and installation and before you use the instrument). This establishes that the instrument is fit for use around the operating parameters that you test against.  

Similar to building a house, the next stage is to develop and validate analytical methods for your work within the parameters you have qualified — don't exceed them, as your instrument is not qualified.  

Next, when you apply the method, you'll check that the instrument works before you commit samples for analysis. This could be a balance check versus a known calibrated weight to see correct operation or that a spectral library can identify known standards. This is the equivalent of a system suitability test, or SST, which is typically performed on the day you analyze the samples. 

Finally, you will include quality control and perhaps blank samples to check that the instrument readings are OK and give you confidence in the method's operation on the day. 

This is shown diagrammatically in the USP chapter as a triangle with AIQ at the base and QC samples at the apex. 

So the bottom line is if you don't qualify the instrument or do it incorrectly, all the other work you do is potentially wasted. So that's the easy bit, but the problem with <1058> is that it is written from the perspective of the laboratory. But do you develop and manufacture your own instruments? Probably not, so let's look in more detail at the basics of the instrument qualification process described in <1058>. In doing so, we'll revisit the hierarchy of the quality checks again but in an expanded form.  

The process for instrument qualification follows the 4Qs model for the specification, installation check, and monitoring of ongoing instrument performance. 

: Define the functional and operational specifications of the instrument and any associated software for the intended purpose. To be performed before purchase of a new instrument. 

: Establish that an instrument is delivered as designed and specified and that it is properly installed in the selected environment. To be performed at installation of the system on new and old or existing systems (please see my comments below).  

: Document that the instrument will function according to its operational specification in the selected environment (so what happened to defined intended purpose?). 

: Document that the instrument consistently performs according to specification and intended use. I will not discuss PQ further in this column. 

However, there are a few little problems with this simplistic approach. 

I'm lazy — let's get the vendor to do the DQ. 

This is one of the major causes of trouble with this approach. Under DQ the chapter states that, "DQ may be performed not only by the instrument developer or manufacturer but also may be performed by the user." Wrong! Wrong!! Wrong!!! A manufacturer's specifications have a number of purposes, such as to show that their instrument is better than the competition, to limit the vendor's liability, and to be the benchmark to compare against when the instrument is installed. Furthermore, in some cases, a manufacturer's specifications may be totally irrelevant and meaningless to your intended use. As a specific example, a benchtop centrifuge had a rotor speed specification of 3500 ± 1; however, on further investigation this was measured without a rotor. Therefore, what manufacturer's instrument specifications are not useful for is your intended use specification or to be the sole component of an instrument's DQ. This is the total abrogation of the user's responsibility. 

You, the laboratory user, are responsible by law under the GMPs to define intended purpose for your instruments. Furthermore, you, the laboratory user, are responsible to your company to purchase instruments that meet your business need. Never rely on a manufacturer's specification to specify your requirements; however, it can be used to enhance or change your specification.  

More Detail of the Quality Checks Hierarchy

The <1058> triangle that I mentioned earlier must be modified so that you can have a better understanding of my approach to the 4Qs model. My take on this is shown in Figure 1, and it is shown as a time sequence running from left to right. The elements of data quality described in <1058> are shown as the top four layers. I have expanded the AIQ layer to show how it relates to the instrument vendor's processes, and we will concentrate on these two layers in more detail. 

In the beginning was an instrument that was specified, designed, manufactured, and supported by a vendor to perform one or a series of analytical tasks. This instrument sits at the vendor's facility until a salesperson visits, or you flick through an issue of 

, drop into an exhibition, or decide to purchase an instrument to do a specific task.  

STOP. Do not pass go and do not collect $200. Also, do not get seduced by the salesperson slithering over the floor toward you. Here is where you have to do some work and define what you want the instrument to do, especially if it is a new technique for your laboratory. Do some leg work and research and define the following:  

Sample presentation to the instrument: liquid, solid, semi-solid?  

Numbers of samples you expect: do you need an autosampler? 

Services: Power supply required and any other services? 

Environment: Will you be analyzing toxic substances, or can the system sit in the laboratory? 

Now write it down: this is your specification. Then approve it, refine it, and update it because it's a living document. 

GO. Now check your specification versus the instrument from a vendor because you have the basis on which you can make the decision: your specification. This is why <1058> is wrong to suggest that the bulk of the work is a vendor's; it is not. Only a user can undertake a full DQ. A vendor might supply their specification, but that is only to compare with yours. Ignoring your specification is a high business risk, but you have never bought an instrument that did not work, have you?  

Look outside the pharmaceutical industry for a moment to other quality standards. ISO 17025 has a footnote to laboratory equipment that most people gloss over, but it is very important in this context. The footnote states that laboratory and manufacturer's specifications might be different; you might want the system to do something that a manufacturer has not considered or work in a very narrow range of the spectrum that the instrument is capable of. Hence, the need to define your instrument and system requirements, otherwise you will have problems in that the purchased system might not meet your needs. The message for the DQ phase of AIQ is for the user to ask what they want the instrument or system to do and document it, regardless of the statements in USP <1058> that are designed to dump the responsibility on the vendor and not the user.  

The section of USP <1058> on installation qualification mentions that IQ also applies to "any instrument that exists on site but has not been previously qualified." I disagree. What this means is that you take your functioning system apart and rebuild it? Now that's an added-value activity that will impress your boss at appraisal time. Consider the following approach as an alternative: what you need to do is establish control over the existing instrument and all its constituent components and show that it works as intended.  

First, list all the major components of the system, take the model and serial numbers of the instruments, and details of any computer and the software (application, database, and operating systems and the respective service packs). Of course you might already have this information in a log book, in which case check that the items are correct and update anything that needs it.  

Second, collate all documentation associated with the system: installation and service records, and establish, if you have not done so, an instrument/system log book. 

Third, put the system under change control and do not change or update any component unless you have gone through a change request. 

Fourth, document the fact that no retrospective IQ will be performed because you will be taking a working system to pieces and rebuilding it. 

Fifth, carry out a full operational qualification to demonstrate that it performs its intended purpose against your specification. 

Columns: Focus on Quality

In this column, Bob McDowall discusses the impact of USP general chapter 1058 on the spectroscopy laboratory.

USP <1058> Analytical Instrument Qualification and the Laboratory Impact

Issue PDF

Click the title above to open the Spectroscopy April 2009 regular issue, Vol 24 No 4, in an interactive PDF format.