When seeing the title of this article, readers of 

 might wonder: again an anniversary? After all, three years ago we already celebrated the centenary of chromatography with one of our "Milestones in Chromatography" columns, discussing the life and activities of Mikhail S. Tswett (1). Why are we repeating it — and why are we placing the name of our technique in quotation marks? 			

It is, of course, true that Tswett first described the principles of his new separation technique in 1903 in Warsaw, in a lecture at a local society of people interested in science and nature (2) that was published two years later in the society's periodical (3). In the lecture, he actually suggested two possible ways for pigment separation: adsorption precipitation and adsorption filtration (terms used by him at that time). In the first case, powdered adsorbent was added to the pigment solution that adsorbed some of the pigments; subsequently, the pigments were selectively dissolved by the use of various solvents. The second was a dynamic method, where the pigment solution was filtrated through the adsorbent, which was packed into the narrow tube of a filter funnel: some pigments passed unadsorbed through the packing, while others were selectively retained in different parts of this "column" (to use our present nomenclature). As we can see, the second method had all the principal features of chromatographic separation. However, at that time Tswett did not decide as yet which should be the preferred method and, reading his 1903 lecture, it seems that he actually favored adsorption precipitation. Also, as of 1903, he had not yet used the term "chromatography." The term was introduced three years later, in his two fundamental papers published in the widely read journal of the German Botanical Society (4,5), making it clear that by then he had decided on the dynamic technique. This maturation of his ideas was the result of the three years of hard work between the 1903 Warsaw lecture and the two publications in 1906. 

Our present column is devoted to the centenary of these two papers first using the term "chromatography." We shall follow Tswett's activities in Warsaw, leading finally to these fundamental publications. However, first a brief summary of his background is needed. 

Mikhail Semenovich Tswett was born on May 14, 1872, in the small Italian town of Asti. His father was a Russian official while his mother was the descendant of Italian settlers in present-day Turkey, most likely around 1300–1400. His parents were traveling from the Italian seaport Genoa to Arona, a resort on the Lago di Maggiore, the beautiful lake at the foothills of the Alps. His mother died soon after childbirth and his father took the infant to Switzerland, where Mikhail grew up. His mother language was French and he learned Russian from his father only as a teenager. (Actually, he had problems with this language even years after moving to Russia: according to recollections of his contemporaries he spoke Russian with a French accent). He also spoke German and Italian, and understood English. 

Young Mikhail went to school in Switzerland, in Lausanne and Geneva, majoring in botany at the University of Geneva, and finished his studies with a Ph.D. degee in 1896. His doctoral thesis dealt with studies of protoplasm and chloroplasts, components of plant cells (6). By that time, his father was a high financial official in Russia on the Crimean Peninsula, and upon graduation, Mikhail decided to join his father. He had great hopes that the Russian scientific establishment would greet him with open arms, with his Swiss doctorate. However, as they say, he fell flat on his face: nobody was interested in him (according to his own words, he was "alien to everybody"). After some time, he was finally permitted to work in a laboratory in St. Petersburg and had a partial teaching position in a girls' school; however, his father had to help him financially for years. Mikhail also found out that his foreign education and degrees were not recognized in Russia: for any academic position he would need at least a Russian Magister's (master's) degree, and for a professorial appointment, a Russian doctor of science degree. He tried to get permission to resubmit his Geneva doctoral thesis to a Russian university, but in vain, as new research and an entirely new thesis were required. He finally finished his new thesis entitled, "The Physical-Chemical Structure of the Chlorophyll Particle," by the spring of 1901 and then submitted it to the University of Kazan'. It was published in a local scientific periodical (7), and he finally earned the master's degree in September 1901. Although Tswett considered this process an unnecessary burden, only needed "to please protectionism reigning in our country" (his own words), the investigations reported in this thesis actually represented the basis of his ideas to improve knowledge about the chlorophyll pigment, and eventually led to the development of chromatography. 

Soon after obtaining the master's degree, Tswett was offered a position at the University of Warsaw, in Russian-occupied Poland, by Dmitrii I. Ivanovskii (1864–1920), the noted virologist and the discoverer of the tobacco mosaic virus. Ivanovskii had just been appointed as the head of the Department of Plant Anatomy and Physiology in Warsaw; previously he had been a Privat-Dozent at St. Petersburg University. (In the 19th century, the Russian education system adopted this German academic title to characterize scholars who were permitted to lecture at a university, but had no full-time professorial appointment. They were not on the regular university payroll, but were paid according to the number of lectures given and number of students attending the lectures.) They knew each other from St. Petersburg and were on friendly terms. Tswett immediately accepted the offer and moved to Warsaw by the end of 1901. 

E. Soczewinski disclosed interesting information about the University of Warsaw (Figure 1) (8). It was started in 1816 as a Polish school but was closed after the 1830 uprising against Russian occupation and reopened only in 1862. In 1869, it was transformed into an Imperial University, now with Russian as the teaching language, and the Polish professors also were forced to teach in Russian or resign. This is the period when great effort was made in the Russification of the various ethnic groups within Imperial Russia. For example, Russian scholars joining Warsaw University as faculty members even received some supplementary payment and the requirements for faculty members were lowered. Professorial appointments for Russians in Warsaw could be obtained with only a master's degree (at the time of his appointment, Ivanovskii also had only this degree; he made his doctorate only a few years later). This forced trend is well reflected in the number of professors with Polish ethnic origin at Warsaw University: in 1870 there were 36; in 1880, 24; and in 1910, only one. 

Tswett's starting position was only that of a laboratory assistant, and to supplement his meager salary, he was also teaching at a secondary school. Finally, by the end of 1902, he was accepted as a Privat-Dozent at the University, enabling him to lecture to students.  

Except, Ivanovskii Tswett did not know anybody in Warsaw. He was a lonely man and this remained so until his marriage in 1907. Every year starting in 1902 he spent a few months in Western Europe (mainly Germany), visiting universities and libraries, or just on holiday. In Warsaw, he spent practically his whole time at the university, and was working alone. It is interesting to note that all of his scientific publications (we know of a total of 58) were single-authored: he never had a co-author, not even an assistant. Until his marriage, he evidently did not even have a home. When his future wife's mother inquired about her prospective son-in-law, she found out that he was actually living at the university and was sleeping on the table in the botanical study (9). 

As soon as he settled down in Warsaw, Tswett continued his investigations of plant pigments, as a follow-up to his master's thesis. His aim was to study them in their native state, separated from the leaves' substrate and from each other. At that time, such studies usually were carried out through chemical treatment, but he felt that such manipulations might alter the original compounds Therefore, his aim was to improve methodology. His philosophy was best summarized in his often cited statement (10): 

"Any scientific advance is an advance of the method." Regretfully, the method is not infrequently the weakest aspect of scientific research. Each generation inherits, as students do, techniques of the previous generation, and without subjecting them to serious criticism, being satisfied by the fact that they are generally accepted. They use them to obtain new results that win recognition by contemporaries, but have no lasting value." 

In his master's thesis, Tswett dealt with the well-known phenomenon that not all solvents can dissolve chlorophyll from leaves. Scientists presented different explanations for the failure of some solvents and the success of others. However, Tswett rejected these hypotheses and pointed to adsorption effects as the reason for the permanent bonds. According to him, the pigments were bound by adsorption to the substrate of the leaves and the strength of adsorption of the individual pigments was different. To override these forces, one needs solvents with sufficient dissolution power, stronger than the adsorption forces. Chlorophyll is strongly adsorbed and one needs a polar solvent, for example, ethanol, for this purpose. At the same time, the carotenes are adsorbed more weakly and, thus, even nonpolar solvents (for example, ligroin or petrol ether) are sufficient for their dissolution from the leaves. 

From this explanation, the validity of which he demonstrated by various experiments, it was only one step to conclude that one should be able to imitate this process and use selective adsorption–desorption, using the proper adsorbent and solvents, for the separation of plant pigments. After systematic work for over a year, he had sufficient data to prepare a report for discussion with his colleagues. This was the paper he presented on March 8, 1903, at the meeting of the Warsaw Society of Natural Scientists, summarizing his results on "a new category of adsorption phenomena and their application to biochemical analysis" (2). However, as I already mentioned, he did not use the term "chromatography" in this paper. Also, he had not yet decided which of the two possible techniques — adsorption precipitation or adsorption filtration — should be preferred. 

Realization of the Chromatographic Technique

Tswett considered his lecture of March 1903 as an interim report. Most likely, this was the reason that he did not publish his results in any German or French scientific journal, not even in a Russian journal distributed nationwide. The text of his lecture was eventually printed in the reports of the Warsaw Society of Natural Scientists (3), a local periodical, published irregularly (the particular issue indicates 1903 as the year, but it was actually published only two years later, in 1905). Thus, apart from his colleagues in Warsaw who attended his lecture (there were a total of 41 present, most likely also including students), nobody else knew about his work. [Tswett's paper was republished in Russian in 1946 in a collection of his papers (11). Finally, in 1954, the chromatography supply house M. Woelm, in Eschwege, Germany, published its English (and German) translation as a company brochure (12)]. 

After his lecture, Tswett continued his work on the new separation process. He systematically tried out 128 substances to see whether they could be used as adsorbents and focused on three as the most suitable: inulin, calcium carbonate, and alumina. He found that they behaved differently and the selection should depend on the pigment mixture to be separated. He also slowly started to favor the dynamic process (adsorption filtration) and gradually introduced the new technique to his investigations of the pigments present in various plants. However, apart from his 1903 lecture, he did not publish anything from 1902 to 1904. 

Meanwhile, politics also interfered with Tswett's activities. The start of the war between Russia and Japan, at the beginning of 1904, was followed by disturbances in Russia, and this also was felt at the universities. By the fall of 1904, the students disrupted classes and, by January 1905, an open revolt broke out in Russia, further disrupting the activities of the schools. Warsaw University was closed by authorities for the school year of 1905–1906 and students were denied access to the university buildings. Professors and instructors still had opportunity to continue their research and work in the laboratories, but the continuous unrest certainly affected their activities. From 1903 to 1907, Tswett spent a few months each year in Germany, going to libraries to check the newest periodicals and books unavailable in Warsaw, and also was active in conducting research there. For example, he spent some time at the University of Kiel, in the laboratory of Professor J. Reinke, collecting algae in the harbor (Figure 2), and also in Berlin, collecting samples of a plant along the Spree River, and was investigating the pigment contents of these plants. But he still did not publish reports on his technique and results. Then, in 1905, the situation changed. 

Figure 2: M.S. Tswett in Kiel, Germany, in 1905.			

At that time, Hans Molisch (1856–1937) was one of the most respected botanists in Europe: between 1894 and 1908 he served as professor and head of the Institute of Plant Physiology at the University of Prague, and from 1908 until his retirement in 1926 in the same position at the University of Vienna (Figure 3). Molisch had just published a paper on the pigments of brown algae (13) and evidently, Tswett (most likely, based upon his investigations in Kiel) disagreed with some of his conclusions. Therefore, he submitted his critical comments to the journal where Molisch's paper was published, stating that his own investigations, using a "new, reliable method," disprove Molisch's results; however, he did not give any further details (14). This paper opened a beehive. Molisch immediately answered it, rejecting all of Tswett's criticisms, and stating that one cannot refer to a method what nobody knows (15). Soon F.G. Kohl (1855–1910), professor of botany at the University of Marburg/Lahn, in Germany, also entered the ring. He questioned how Tswett, a practically unknown upstart, dared to criticize Molisch, an internally known and renowned scientist, without any concrete data (16). Kohl also expressed his indignation that Tswett did not cite his book on carotenes published in 1902 (that had nothing to do with the dispute). Tswett responded to both objections in one paper (17). In this, he repeated his criticisms of Molisch's work and then stated that his own investigations were actually carried out by using two new techniques: a differential method (that is, the adsorption precipitation technique) and a new adsorption method (that is, adsorption filtration). He also stated that until now these methods were only described in a Russian publication (citing his 1903 lecture), but that a German publication was in preparation. 

Figure 3: Hans Molisch (1856â1937).			

It is interesting to see the quick succession of these relatively brief, polemic papers — it appears as if Tswett suddenly awakened and was ready to fight. Indeed, within two months he submitted to the journal of the German Botanical Society his two fundamental papers, the centenary of which we are celebrating (4,5): they were received by the editorial office on June 21 and July 21, respectively, and published in the fall in two consecutive issues of the journal (Figure 4). 

In most publications on the history of chromatography, the second paper (5), entitled "Adsorption Analysis and the Chromatographic Technique: Application to the Chemistry of Chlorophyll" is emphasized, because that contained the detailed description of chromatography, the apparatus used and the results of separation when a pigment solution was analyzed. However, the first paper, entitled "Physico-Chemical Studies of Chlorophyll: The Adsorptions" (4) is even more interesting, because it gives the background, outlines the way how Tswett reached his conclusions, and his hypothesis on selective adsorption and the effect of various solvents. The following quotation from this paper gives a wonderful summary of chromatographic separation: 

"There is a definite adsorption sequence according to which the substances can displace each other. The following important application is based on this law. When a chlorophyll solution in petrol ether is filtered through the column of an adsorbent (I am mainly using calcium carbonate, tightly packed into a narrow glass tube), then the pigments will be separated from the top down in individual colored zones, based on this adsorption sequence, according to which the pigments which are adsorbed stronger will displace those which are retained more weakly. This separation will become practically complete if, after the pigment extract was passed through the adsorbent column, the latter is washed with pure solvent." 

				 Continuing, Tswett then had his most famous statement on the name of the new technique (italics mine): 			

"Like light rays in the spectrum, the different components of a pigment mixture, obeying a law, are separated on the calcium carbonate column and can thus be qualitatively and quantitatively determined. 

I call such a preparation a chromatogram and the corresponding method the chromatographic method.

There have been some speculations why Tswett selected this name for the technique. "Chromatography" is composed from two Greek words, 

 meaning "to write." Thus, the word means "color writing." Historically, it had not been unusual to coin Greek words to define some scientific term, and because chromatography of a pigment mixture indeed produces individual color rings in the column (Tswett's second 1906 paper actually showed an illustration of a chromatographic column with the multicolored rings), the term seems to be logical. However, just two paragraphs later, Tswett emphasized that the technique is not restricted to the separation of colored pigments, it also can be used equally well to separate colorless chemical compounds; in other words, the "colored rings" are not a quintessential characteristics of the technique.  

				 Some time ago, another explanation also was suggested for Tswett's selection of this name (18). The interesting fact is, namely, that Tswett's surname is identical to the Russian word for "color" (

) (the spelling "Tswett" generally used by him corresponds to the German orthography). Thus, "chromatography" also can be interpreted as "Tswett's writing." Howard Purnell also mentioned this possibility 40 years ago, adding that it would be nice to think that Tswett, through the double meaning of chroma = tsvet, "took advantage of the opportunity to indulge his sense of humor" (19). 			

Our aim was to point out the gradual steps in Tswett's work that eventually led to the development of chromatography and to the two fundamental papers the centenary of which we are celebrating this year. Tswett's further activities in the subsequent decade are not our subject, there is plenty of literature dealing with it. Still, we cannot finish our discussion without pointing to the difficulties Tswett had with the acceptance of his work.  

Figure 5: Leon Marchlewski (1869â1946).			

One would think that the logical and clear explanations in Tswett's papers convinced his opponents about the advantages of the new technique, particularly because the following year, he personally demonstrated the chromatographic technique twice, at the May 30 and June 28 meetings of the German Botanical Society, in Berlin, to a wide audience. This, however, did not calm down his critics; in fact, newer and even stronger voices joined the camp of his opponents. In his papers, Tswett (mildly) criticized previous work of Marchlewski and Schunck and he obtained an immediate (and very acerbic) rebuttal by Leon Marchlewski (1869–1946), at that time professor at the Jagiellonian University of Cracow, in the part of Poland belonging to the Austro-Hungarian Monarchy (Figure 5). Marchlewski studied at the Federal Technical University in Zurich, Switzerland, and worked for years in England with E. Schunck, an early researcher of the chemistry of chlorophyll. In a polemic answer to Tswett's papers, he ridiculed the new technique, and warned Tswett that he should not believe that "a simple filtration experiment" would be enough "to swing himself to the height of a reformer of chlorophyll chemistry" (20). (It is an open question how much Marchlewski's antagonism was due to vanity, feeling of personal insult, or patriotism. Within the Austro-Hungarian Monarchy, the University in Cracow was a Polish school, with wide autonomy, and Marchlewski was a very patriotic Pole, naturally opposing Tswett as a Russian, in the Russian-occupied part of Poland, where the Polish language and national spirit was suppressed.) An even more important — although more dignified — opponent of Tswett was R. Willstätter (1872–1942), at that time probably the most respected German organic chemist who at that time felt that he was the authority in chlorophyll research; in 1915, he indeed received the Chemistry Nobel Prize for this work (Figure 6). Willstätter called chromatography "an odd way" to perform pigment research and stated, that chromatographic separation was not reliable because chemical changes were occurring during it (21). [Richard Kuhn, one of Willstätter's students, admitted 50 years later that they simply used the wrong adsorbent (22).] In the first decade after Tswett's publication, chromatography was accepted only by a couple of scientists, most notably by Charles Dhéré, in Switzerland, and Leroy S. Palmer, in the United States. It took one generation and needed change in the philosophy of organic and biochemical research until it could become a universally accepted and used technique. 			

Finally, a few words about Tswett's personal life. As already mentioned, he married in 1907; in that year he left Warsaw University, becoming associated with the Veterinary Institute and then next year, with the Warsaw Polytechnic Institute. In 1910, he published his major book, summarizing the results of his research (23) for which he received a Russian scientific prize. Slowly, Tswett's health started to decline and he was forced to reduce his activities. In 1915, he had to leave Warsaw before the advancing German troops, moving temporary to Moscow and Nizhnii Novgorod. Finally, in 1917, he obtained an appointment as a full professor at the highly respected University of Tartu (in present-day Estonia; then part of Russia). He moved to Tartu, but within a year he was again on the road because of German occupation of the Baltic area. By then Tswett's health further deteriorated and he died on June 26, 1919, in Voronezh. His memory is, however, alive through chromatography, the method he created, which is one of the major scientific accomplishments of the 20th Century. 

Figure 6: Richard Willstatter (1872â1942).

I would like to express my appreciation to Dr. E. Soczewinski, Professor Emeritus at the Medical Academy, Lublin, Poland, for providing the contemporary picture post card shown in Figure 1. I received the photograph of Tswett shown in Figure 2 from the late K.I. Sakodynskii (1930–1996) who in turn obtained it from E.A. Lyashenko, the niece of M.S. Tswett. The other three photographs are from my own collection.  

From 1988 to 2004, "Milestones in Chromatography" editor Leslie S. Ettre was associated with the Chemical Engineering Department of Yale University (New Haven, Connecticut), first as an adjunct professor and then as a research fellow. Previously, he had been with the Perkin-Elmer Corporation for 30 years. He is currently a member of LCGC's editorial advisory board. Direct correspondence about this column to "Milestones in Chromatography," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

(2) M.S. Tswett, On a New Category of Adsorption Phenomena and their Application to Biochemical Analysis. Lecture at the March 8 (21), 1903, meeting of the Biological Section of the Warsaw Society of Natural Scientists. 

Trudy Varshavskogo Obshchestva Estestvoispytatelei: Otdelenie Biologii 

Trudy Obshchestva Estestvoispytatelei pri Imperiatorski Kazanskom Universitet 

Michael Tswett, the Creator of Chromatography

 (Russian Academy of Science, Moscow, 1997), p. 103. 

Chromatographic Adsorption Analysis: Selected Works of M.S. Tswett

Articles

Milestones in Chromatography

This month's "Milestones in Chromatography" column commemorates the centenary of the two publications of M.S. Tswett first using the term "chromatography."

The Centenary of "Chromatography"

		  Because most samples are usually unsuitable for direct analysis, extraction is perhaps one of the most important sample preparation techniques for environmental samples. The most common types of extraction techniques include solid phase extraction (SPE), Soxhlett/liquid–liquid extraction (LLE), and advanced extraction techniques. The method used for a particular application depends on the nature of a sample. 

 North America Market for Environmental Extraction Techniques			

Solid phase extraction is an important method of prechromatographic sample treatment. The versatility of SPE allows it to be used for many different purposes such as purification and concentration of trace elements in a sample, among others. SPE comes in a variety of formats including columns, cartridges, microplates, and pipette tips. LLE and Soxhlet are similar techniques and are used typically for preparing liquid matrices. LLE operates on the basis of mass transfer of a component between two immiscible liquid phases that are brought into contact with each other. In contrast to LLE, Soxhlet extraction methods are used most often when solid samples must be extracted. 

While SPE and Soxhlet/LLE represent the majority of the market demand, the use of advanced extraction techniques, including accelerated or pressurized solvent extraction, microwave-assisted extraction, and supercritical fluid extraction, also have  become commonplace. These systems provide faster, more efficient extraction than traditional methods. Demand for advanced extraction techniques is fueled by soil and pollution analyses. 

In 2005, the North American market for extraction techniques for environmental applications accounted for about $52 million. Although growth in North America is expected to be modest, new EPA regulations and approved methods, especially for advanced extraction techniques, provide a stimulus for continued market expansion. 

The foregoing data was extracted from Strategic Directions International, Inc. Market Analysis and Perspectives (MAP) Report. For more information, contact Glenn Cudiamat, vice president of Research Services, Strategic Directions International, Inc., 6242 Westchester Parkway, Suite 100, Los Angeles, CA 90045; tel. (310) 641-4982, fax (310) 641-8851, e-mail: 

Departments

Because most samples are usually unsuitable for direct analysis, extraction is perhaps one of the most important sample preparation techniques for environmental samples.

Chromatography Market Profile: Extraction Techniques for Environmental Samples

The gradient linearity and step tests are two of the most useful performance tests that can be made for a liquid chromatography (LC) system. These check the linearity of gradient generation and the accuracy of mobile phase proportioning. These tests, and examples of problems detected as a result of these tests, have been the subject of at least seven "LC Troubleshooting" columns (1–7) over the last 18 years. We strongly recommend that every LC system undergo these tests at least on an annual basis, and preferably semiannually. When a new and different example of a problem detected by these tests is discovered, it is hard to bypass the opportunity to share it with our readers. So this month, you get yet another example of how an LC system can fail. 			

Just as a reminder, the test is quite simple. Replace the column with a piece of capillary tubing (for example, 1 m of 0.005-in. i.d. PEEK). Place water in the A-reservoir and water plus 0.1% acetone in the B-reservoir. Set the UV detector to 265 nm. Adjust the flow rate so that there is sufficient backpressure (≈35 bar) for good check-valve operation, such as 1–2 mL/min. The first test is a gradient linearity test — just run a blank gradient and record the baseline. This will give you information about gradient linearity, and you can use the results to calculate the system dwell-volume (also called the gradient holdup volume). The second test is a step test, in which isocratic steps are programmed between 0 and 100% B, generally at 5% or 10% increments. More details about these tests can be found in references 6 and 7, which you can access on-line (8).  

We run the two gradient tests as a part of a semiannual instrument performance test suite. These are programmed to run unattended, and data are analyzed after the tests are complete. First, we ran the gradient linearity test as a linear gradient of 0–100% B over 15 min at 1 mL/min. The results for the initial test are shown in Figure 1a. The second test is the step test. We run this with steps at 0, 10, 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, and 100% B. We add the 5% steps near 50% B, because, in our experience, if problems are going to occur, they occur near 50% B more often than not, especially with low-pressure-mixing systems. (A good example of low-pressure-proportioning-valve failure was presented in reference 6.) The program is set up to generate each step for 4 min and is run at 2 mL/min. The results of the initial step test are shown in Figure 2a. 

A well-behaved linearity test should appear similar to that shown in Figure 1b. Clearly there is something wrong with the run of Figure 1a (dashed line added for reference). The initial and final isocratic sections appear to be OK, but the center portion is quite nonlinear. Remember that the apparent isocratic hold at the beginning of the gradient linearity test is due to the dwell volume of the system, approximately 2.3 mL for this system (see reference 6 for determining dwell volume from such plots). 

A step-test plot similar to that shown in Figure 2b was expected, with even step heights and the same degree of rounding for each step. There is obviously something wrong in the run of Figure 2a too. Because the linearity and step-test experiments are different ways of measuring mobile phase proportioning, it is not surprising that both tests fail — it would be more unusual if one test passed and the other failed. 

To quantify the pump performance, the step heights are measured and converted to %B values. Just determine the difference in UV absorbance between the 0 and 100% B baselines and use this to determine the height of each step. Table I shows the data for Figure 2a as test 

. The actual values differ by as much as 16% from the values set in the program, with an average deviation of 10.6%. Definitely unacceptable! 

This is a high-pressure mixing system comprising two dual-piston pumps; there are eight traditional ball-type check valves. In our experience, the check valves are the single most likely source of problems in today's LC systems (for more on check valves, see reference 8). One way to check for flow through the check valves is to pump an air bubble through the system. Set the pump(s) to deliver 50% B at 1 mL/min, then briefly lift the inlet line out of the A-reservoir so that a bubble approximately 2-cm long is drawn into the tubing. You should be able to watch the bubble flow through the transparent inlet line tubing up to the point where the flow stream splits just before the pump. If both pump heads are working properly, the bubble will split evenly between the pump heads. The bubble will be drawn into one head during its fill stroke, then, when the second pump head fills, the bubble will be diverted to the second head. (If you are using an in-line degasser, you might have to shut off the degasser during this test so that the bubble reaches the pump.) Repeat the test for the B-pump. In our case, the bubble split, as expected, for the A-pump, but only entered the left head of the B-pump. When the right head executed its fill stroke, the bubble was stationary. This told us that the problem was related to the right head.  

We sonicated and replaced the inlet check-valve for the right head of the B-pump and repeated the tests. The results now were those shown in Figures 1b and 2b — passing in both cases. The quantitative results of the Figure 2b step-test are shown in the right-hand two columns of Table I (test 

). No step deviated by more than 1% (our acceptance criteria) and the average step was off by approximately 0.6%. 

As an academic exercise, it is interesting to determine the theoretical impact on mobile phase proportioning if one check valve is not working. Table II summarizes these data. For a high-pressure-mixing system, the various mobile phase proportions are obtained by varying the flow rate of the two pumps during the gradient (or step test). The settings for each step are shown as the %A and %B set points in the left two columns. The flow rates should be shared equally for the two pump heads of each pump, thus, at the 45% B setting, and 2 mL/min total delivery, each head of the A-pump should deliver 0.55 mL/min and each B-pump head should deliver 0.45 mL/min. If one check valve fails completely, its pump head will not deliver any solvent. For this example, we assumed that the right pump head of the B-pump was not working, so we assigned a flow rate of 0 mL/min for that head, as shown in the flow rate values of Table II. Now simply express the total %B delivered as a percent of the total flow rate and you get the calculated %B; the difference between this and the set point gives us the calculated error. The average calculated error in Table II (11.19%) is very close to the experimental value (10.60) for the run of Figure 2a shown in Table I. The last step is to calculate the difference for each step between the theoretically calculated error and the actual value. The deviation between these two values is less than 1% in all cases, confirming what we suspected by observation — one head on the B-pump wasn't working. (Note that the similarity of the numbers in the right-hand columns of Tables I and II is probably due to the same contributions to error of the same three pump heads in both tests — we think . . . .) 

Table II: Comparison of theoretical failure vs. experimental data			

This is an interesting case study, because it allows us to get to the same answer from several different approaches. Either the step-test or the linearity test would have brought this problem to our attention. The bubble test and our calculated pump delivery values also would have led us to the same answer. Most of the time, however, we would have suspected problems before running the tests. The tests would merely confirm our suspicions. For example, with the present problem we would observe that retention times were too long in our system suitability test, which would alert us to look for the problem source.  

It is important to realize that system failure can occur at any time, and sometimes without any apparent initiating factor. In the present case, a pump flow rate accuracy test and a pressure bleed-down test (which evaluates the performance of the outlet check valves) passed earlier on the same day the step and linearity tests failed. This reminds us how important it is to run a system suitability test at the beginning of each batch of samples so that you know the system is working properly when you are about to use it. Consistent retention times and peak areas for quality control samples run during and at the end of the batch will give added confidence in the LC system performance.  

This brings up a question about the value of periodic performance tests — if the system can work well before the test and fail during the test, what is the point of running such tests? The current case study is a rather dramatic example of the complete failure of a system component, a check valve. More commonly, however, a partial failure will occur, and these can be harder to recognize and diagnose. For example, the source can be pump calibration (1), degassing and air bubbles (2,4), pump seals and check valves (5), the mobile phase proportioning valves (7), or other problems. In the case of the proportioning valve failure (7), it is unlikely that the subtle failure would have been recognized by examination of the system suitability tests for a method. The gradient performance tests, such as those described in reference 6, will help you isolate and identify the source of the problem. 

The bottom line is that we feel strongly enough about the importance of running periodic LC system performance tests that we go to the trouble and expense of testing every LC system twice a year. At a bare minimum, you should run these tests once a year during the annual preventive maintenance session. One of the basic concepts that we teach in our LC troubleshooting classes is that you are going to have a hard time figuring out what is wrong with your LC system if you do not know how it works when everything is working properly. The semiannual performance test gives you a good set of reference data for comparative purposes when something does go wrong.  

 "LC Troubleshooting" Editor John W. Dolan is Vice-President of LC Resources, Walnut Creek, California; and a member of 

's editorial advisory board. Direct correspondence about this column to "LC Troubleshooting," 

, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

For an ongoing discussion of LC trouble-shooting with John Dolan and other chromatographers, visit the Chromatography Forum discussion group at http://

Jon Gilroy is an analyst at BASi Northwest Laboratory, McMinnville, Oregon. He specializes in LC instrument troubleshooting, and his research interests are in LC column selectivity.

LC Troubleshooting

The gradient linearity and step tests are two of the most useful performance tests that can be performed for a liquid chromatography (LC) system.

Gradient Performance Problems - A Case Study

Chromatographers need to use appropriate fittings and ferrules to get the most out of their instruments; proper fittings help ensure the best separation and detection performance while getting the most out of gas tanks and filters. Connections at the tank regulator, the instrument bulkhead, the column, and finally the detector all have specialized requirements and assembly procedures that should be followed. Not doing so risks wasting expensive gases and exhausting in-line filters prematurely, as well as causing excessive peak band broadening and distortion, shortened column life, and poor detector performance. Problems with gas fittings fall into two categories: leaks and band-broadening. 			

Leaking connections cause a number of significant problems, including poor retention time and peak area reproducibility, loss of accuracy, drifting detector baselines, high detector noise, shortened column life, and increased gas consumption. Hydrogen leakage from flame-ionization detectors or carrier-gas supplies can be a potential explosion hazard.  

Contrary to intuition, a leak is not just a wasteful one-way path from the high pressures inside the tubing to the atmosphere; oxygen can diffuse from the atmosphere back into areas where its partial pressure is lower, which is always the case with gas chromatography (GC) gases except for detector fuel air. High oxygen levels in the carrier gas can cause excessive stationary phase bleed and greatly reduced column life, as well as increased detector or mass spectrometry (MS) noise and background signal levels. 

Column connections require specialized low-dead-volume fittings. Internal areas in a fitting that are exposed to carrier gas but are not actively swept by carrier flow — so-called dead volumes — can cause peak broadening and tailing, especially at the low flow rates of narrow-bore columns with inner diameters under 320 μm. GC instrument manufacturers and consumable suppliers make available the specific types of fittings and ferrules for each inlet system and detector that will minimize peak broadening and tailing. It might be tempting to substitute less expensive fittings and ferrules or to keep only a few types on hand, but doing so almost compromises instrument performance. 

Chromatographers always should choose nuts and ferrules that are in good condition, not worn or distorted. Proper assembly and tightening are required, and the procedures are different for new parts than for reassembly of used parts. Fittings that employ graphite or polymer composites need to be checked and maintained periodically because the soft ferrule material can deform and loosen. And finally, leak-checking is always necessary when making up new or reused fittings. As I have repeated more times than I can count, never use a soap solution to check for leaks. Liquid can flow back into a leak just as easily as can oxygen; commercial electronic leak checkers are mandatory in my lab. In the few cases where an electronic device will not respond, such as for detector air, use a pure liquid such as water or propanol. 

Three kinds of tubing material are found in gas chromatographs: copper, stainless steel, and plastic. Plastic tubing should be used only in noncritical areas where small leaks and some contamination can be tolerated, such as for the air supply to valve actuators or other pneumatically driven devices. Other than this, plastic tubing has no place in a gas chromatograph: the main problem is that plastic tubing can contaminate a gas stream with plasticizers and residual volatile chemicals, as well as with oxygen that diffuses in through the tubing walls. 

All detector gases, including air, need to be provided from dedicated supplies. Otherwise, besides a contamination risk, the large air pressure fluctuations that occur upon valve actuations will not affect the detectors' outputs. For the most part, internal tubing in GC systems is made from stainless steel. Stainless steel tubing is best for the connections from gas tanks as well, although copper is just as acceptable if the fittings are not to be opened and remade more than a few times.  

The predominant type of GC gas connector is the swaged-ferrule fitting, which works by applying mechanical force with a threaded nut on a conical ferrule that is seated against the inside of a compression fitting (see Figure 1a). The ferrule is compressed inward slightly by longitudinal forces and its leading edge grips the tubing, forming a seal between the fitting and the tubing that holds the assembly in place. An additional back ferrule is often used with stainless steel or copper tubing to decouple the rotational force that is applied while tightening the nut, so that the front ferrule does not turn. This helps compensate for any slight out-of-roundness in the tubing. 

The most familiar application of swaged fittings is the connection of carrier- and detector-gas lines to the chromatograph. Several 1/4-in. and 1/8-in. fittings usually can be found between carrier- and detector-gas supply tanks and the gas chromatograph. A number of such fittings also are employed between bulkhead connections on the back or side of a gas chromatograph and internal pressure regulators, flow controllers, or needle valves. 

A second type of connection is the threaded or "pipe" fitting, shown in Figure 1b. Pipe fittings also are found at the outlet of gas tank pressure regulators. One end of the fitting screws into a threaded pressure regulator or needle valve body, while the other generally has a swage-type connection. PTFE tape wrapped around the threads seals the fitting, so no ferrules are used. These connections can be opened and resealed many times, so long as the sealant tape is renewed each time the connection is assembled. Pipe fittings also are found inside the GC system in pressure regulators and flow controllers. Sealing tape never should be used on a swaged fitting, neither on the threads, which do not form part of the seal, nor on the ferrules themselves. 

A third type of commonly used fitting is found in the high-pressure connections between gas tanks and pressure regulators. These are another form of compression fitting that relies solely on tightening forces to compress one or two soft brass sealing surfaces together; there are no ferrules. Figure 1c illustrates this type of arrangement. Gas tank fittings are specific to the type and classification of gas in use, as well as the country of origin, and they are designed so that it is not possible to connect regulators and tanks that are incompatible.  

One misapplication of sealing tape that I sometimes see is its use on such fittings. At best this is not effective — the tape will introduce more of a leak than it can fix — and at worse this practice is dangerous because it can lead to larger leaks of flammable hydrogen. If a regulator will not seal properly to a cylinder then replace the regulator and return the cylinder for a replacement. 

Types of Swaged Connections for Capillary GC

Capillary columns are a special case for swaged fittings. Fused-silica columns cannot use conventional metallic ferrule made of steel or brass because the column will be crushed. A huge number of different-size ferrules made of plastic, composites, and a newer soft metal alloy are available instead. Among these, pure graphite is sometimes used because of its high temperature limit and easy conformity to a fitting's internal contours. Ease of removal from a column or fitting makes ferrules of polyimide or its composites with graphite the best choices for most applications. Pure polyimide ferrules are used in low-temperature applications, other than in the column oven. Graphite–polyimide composites are best used in situations that require an extended temperature range plus the ability to be reused several times. More recently, ferrules of a soft metal alloy have become available that combine high temperature limits and very low leak rates; these ferrules are especially well-suited for MS applications. 

Capillary column ferrules are available in sizes for 1/8- and 1/16-in. fittings, drilled with one or more inner holes to match the column outer diameter. The 1/8-in. size ferrules are often termed reducing ferrules because they have an inner diameter that is significantly reduced from the nominal 1/8-in. size. These ferrules connect capillary columns to nominally 1/8-in. size swage-type fittings, as shown in Figure 2a. One familiar use is in a packed-column inlet adaptor for 530-μm i.d. columns. Simultaneous separations on two different fused-silica columns can be accomplished by connecting the columns to one inlet with a two-hole reducing ferrule. Reducing ferrules also conveniently connect 1/16- or 3/32-in. metal tubing to 1/8-in. standard swaged fittings on internal pressure regulators or flow controllers. Common reducing ferrule sizes and matching capillary column inner diameters are given in Table I. 

Table I: Recommended ferrule sizes for capillary columns			

Figures 2b and 2c show how 1/16-in. ferrules are applied when connecting to a capillary inlet or detector. This type of fitting can accommodate the ferrule in a forward (Figure 2b) or a reverse (Figure 2c) orientation. In the reverse orientation, the nut is drilled out, tapered, and threaded, and the ferrule goes into the nut instead of into the fitting. The ferrule is installed on the column in the reverse direction, because the conical part of the compression fitting is now inside the nut. These ferrules generally fit a nominal 1/16-in. size fitting, again with inner holes drilled for various column outer diameters.  

GC users can assemble these various types of connections leak and dead-volume free by following a few simple steps. First, prepare the tubing. Cut the end of 1/8- or 1/4-in. metal tubing using a sharp plumber's cutting tool. Smaller 1/16- or 3/32-in. tubing should be cut with a rotating wheel tubing cutter designed for this purpose — they are available from several chromatography suppliers. Wire cutters never should be used. Make sure that the cut end of metal tubing is clean, square, and free of burrs. Check that the tubing shape has not been distorted by cutting. Also inspect the outside of the tubing near the end for any longitudinal scratches that could allow gas to leak between the ferrule and the tubing. If the tubing is damaged, make another cut rather than trying to force the tubing into the connection. Thread the nut and ferrules onto the metal tubing, making sure that they are in the correct orientation and order. In most cases, the tubing should be inserted into the fitting as far as it will go. 

A word on ferrule composition for metal tubing: try to match similar materials whenever possible. Use softer brass ferrules and fittings with copper tubing, and use stainless steel ferrules and fittings with stainless steel tubing or with copper if necessary. Using brass fittings with stainless steel tubing is not recommended because the tubing will tend to resist taking a set at the ferrule's contact annulus. 

Thread the nut and ferrule onto fused-silica tubing before cutting the tubing. Otherwise, you can push some ferrule pieces into the column as you thread it through the ferrule. Then cut the fused-silica column tubing with a diamond, sapphire, or ceramic cutting tool designed specifically for cutting fused-silica capillary columns. Be sure that the ferrule faces the right direction; it is easy to put a small ferrule on backwards. For critical applications such as installation in an on-column injector, it is a good idea to inspect the end of fused-silica tubing under a magnifying lens for a rough cut or small particles. Do not hesitate to make several tries until the end is clean and square. Some manufacturers' instructions specify the distance the column should be positioned past the end of the nut. Measure and mark the specified distance with a small piece of tape so that you can accurately position the column in the connection. Do not forget to remove the tape before heating the GC oven. 

Now insert the tubing into the fitting. Make sure the ferrules slide easily into position, and then gently start the nut onto the threads. If you cannot manually turn the nut for one turn or so, stop and inspect both threads for damage. Be sure that you have used the correct ferrules. If the threads are damaged, you should replace the damaged part rather than trying to force the connection together. Fittings with damaged threads will almost never make a leak-free connection. 

After the parts are in position, hand-tighten the nut until it stops. Always use two wrenches if possible, one on the nut and one on the fitting, inlet, or detector, and begin to tighten. Hold the wrench on the stationary part steady to prevent it from turning, and turn the nut with the other wrench. Manufacturers of swaged fittings provide instructions on how far to tighten their specific sizes and types of fitting. The instructions are available over the Internet, upon request, and with packages of fittings. Lacking specific instructions, a new fitting on metal tubing should be tightened from 1/2-turn for 1/16-in. sizes, 3/4-turn for 1/8-in. sizes and one to one-and-a-half turns for larger diameters. Relying on a torque wrench to tighten swaged fittings is not recommended, because the required torque will vary too much depending upon the metals and lubricants used. An old fitting that already has the ferrule in place should be tightened only about one-quarter turn beyond finger tight; just enough to reseat it. Be careful when moving from one fitting to another with the same nut and ferrule — check carefully for leaks and be prepared to tighten slightly more. 

For fused-silica columns, tighten the nut on a new ferrule just until the tubing no longer can be moved, then pressurize and check for leaks. Most column fittings, except for the newer metallic types, should be retightened and leak-checked once after the first oven heating cycle. If you reuse a fused-silica connection tighten it slightly beyond finger-tight, about 1/4-turn and leak check again. 

All carrier- or detector-gas connections should be tested for leaks. The extra time involved is well worth it for the resultant reduced troubleshooting time and improved results. Chromatographers can best leak test connections with a leak-test device or with some isopropanol or water if an electronic device is not available. Never use soap or a leak-check solution containing surfactants or other nonvolatile chemicals; they will contaminate a capillary column or detector system. Electronic leak-checking devices are very sensitive for helium and hydrogen carrier or combustion gas, and they are preferred for capillary column work because leak checking with liquids is not sensitive enough at capillary flow rates. Such a device is mandatory for an MS detector. 

Turn on the gas chromatograph, pressurize the system to a minimum of 10 psig, heat the inlets and detectors, but leave the column oven off. Now operate the leak detector and set its zero with the probe held in air, away from the GC system. Place the probe near each fitting at the point the column or tubing exits the ferrule, and at the other end of the threads and wait for as long 10 s for the response. A small response on the most sensitive setting is acceptable. For MS systems even this small response should be minimized. Slight leaks often can be remedied by tightening the fitting another 1/8-turn. If the leak persists after two or three tries, check to be sure that an adjacent fitting is not leaking severely and giving you a false reading. If you cannot seal the leak satisfactorily, then the ferrules must be renewed. If you cannot get a leak-free connection with new ferrules and newly cut tubing, then the fitting must be replaced. 

During routine operation, keep an eye on retention times and baseline stability. Establish an expected level of drift and instability based upon previous experience with a leak-free system. Larger than normal shifts in retention time, or noisy or drifting detector baselines indicate a leak at the inlet, the detector, the combustion gases, or the makeup gas. Tighten suspect ferrules 1/8-turn and observe the results. If the symptoms persist, then look for other causes such as unstable inlet pressure or an exhausted detector- or carrier-gas filter. Factory-installed internal connections seldom leak if left undisturbed. 

Leak-free connections are required for the best possible GC performance. Leaking fittings can cause baseline noise and drift, unstable retention times, reduced column life, and inaccurate results. Chromatographers can ensure a leak-free system by using new ferrules, and if necessary new fittings, whenever setting up a GC column or setting up the instrument. If there is any doubt, take the time to leak check the system before beginning to run analyses. A complete record of observed baseline noise and drift, column bleed, and retention time stability is invaluable for making an informed comparison when a problem develops. 

"GC Connections" editor John V. Hinshaw is senior staff engineer at Serveron Corp., Hillsboro, Oregon, and a member of 

s editorial advisory board. Direct correspondence about this column to "GC Connections," 

For an ongoing discussion of GC issues with John Hinshaw and other chromatographers, visit the Chromatography Forum discussion group at http://

GC Connections

This month, John Hinshaw discusses how to make, maintain, and troubleshoot various types of connections used in gas chromatographs.

Making a Great Connection

Sample preparation for total drug analysis in blood can be achieved by a variety of techniques, including centrifugal precipitation, liquid–liquid extraction and solid-phase extraction (SPE). Plasma proteins are precipitated using organic solvents (such as acetonitrile) and the precipitate is centrifuged into a pellet (1–3). For reproducible and accurate results, good protein removal by solvent precipitation and separation of the drug containing supernatant is critical. This separation typically is achieved with either long or multiple centrifugations. Protein precipitation ("crashing") is the preferred method for early animal studies. The increased number of samples created the need for higher throughput, multiwell techniques. In addition, the trend toward smaller plasma volumes combined with much lower centrifugal forces (~2000

 for microfuge tubes) presents an obstacle to obtaining high quality, reproducible, liquid chromatography–mass spectrometry (LC–MS) ready samples 

Efforts to improve sample preparation throughput have been made using 96-well filtration plates (4–7). Validation of the protein precipitation filtration method for the drug salbutamol using one of three different prototype filter plates gave acceptable results using vacuum filtration, but the method still involved multiple steps (8). This article describes the use of a 96-well, solvent-resistant filtration plate for total drug analysis in blood. Samples are prepared in the same manner as the centrifugal pelleting method but instead of pelleting and transferring the supernatant to obtain protein-free drug solution, the precipitated protein and the drug are separated by centrifugal-driven filtration. Pipetting the supernatant is eliminated and clean, protein-free drug solution is collected in a 96-well plate below the filter plate. This is particularly difficult for small plasma volumes. The polytetrafluorethylene (PTFE) membrane in the filter plate has high precipitated protein retention and is very clean with extremely low extractable content. The result is high sample recovery and no interfering contaminants. Together with detection by LC–MS-MS, this approach drives a high-throughput total drug analysis method that is faster and simpler than existing methods. 

Comparison of the centrifugal pellet and centrifugal filter-plate methods was conducted using five discovery screening programs. Thirteen compounds (A, B, C, D, E, F, G, H, I, J, K, L, and M) were evaluated in one of five matrices: rat plasma, rat serum, dog plasma, mouse serum, and cyno monkey plasma. Validation of the bioanalytical methods, precision, and accuracy all were explored in the five programs. 

In experiment I, bioanalytical assay validation in rat plasma with compounds A and B was compared using both the pellet and the filter-plate methods. In experiments II–IV, standard curves as well as quality control (QC) sample performance in running actual program samples were compared from both methods (side by side) for the drugs and animal models indicated. In experiment V, only the filter-plate method was run. The following is a summary of the five experiments that were performed: 

Filtration versus Pellet Protein Precipitation

 Pharmacokinetic study compounds C, D, E, and F. 

III. Standard curves in rat and mouse serum. 

 Biology study compounds G, H, I, J, and K. 

IV. Standards and QC samples in dog plasma. 

				  Comparison of (two) injections of same set of compound M in cyno monkey plasma calibration curve and QC samples at beginning and end of an assay (7-h run). 			

Plasma and serum samples were supplied by BMS Discovery Biology, Pharmacokinetics, and Toxicity departments. 

Compounds were supplied by BMS Discovery Chemistry department. 

HPLC-grade acetonitrile was obtained from Burdick & Jackson (Muskegon, Michigan). 98% GR formic acid was obtained from EMD (Germany). Deionized water (18.2 ΩÂ·cm) was purified by a Milli-Q water purification system from Millipore Corporation (Billerica, Massachusetts). 

				 A) Standard Protein Precipitation (Pellet) Method 			

Standard protein precipitation sample preparations were performed in a 96-well plate format with a Quadra-96 automated liquid handler from Tomtec (Hamden, Connecticut). Aliquots of 25 μL of the plasma/serum sample were treated with 50 μL of acidified (0.1% formic acid) acetonitrile containing 0.5 μg/mL of the internal standard followed by vortex mixing for 10 min. The supernatant was then separated from the precipitated proteins by a 30-min centrifugation (;1480

) and was transferred to a second 96-well plate. Typically, the volume of pellet and the level of solution in each vial were not consistent. Often, another 10-min centrifugation step was required to concentrate protein particles to avoid their transfer into the final aliquot for analysis. An aliquot of 10 μL of the clear supernatant was analyzed by LC–MS-MS.  

B) Filter-Plate Protein Precipitation Method

Sample preparations were performed in a 96-well plate format with a Tomtec Quadra-96 automated liquid handler. First, 50 μL of acidified (0.1% formic acid) acetonitrile containing 0.5 μg/mL of the internal standard was added to a 96-well MultiScreen Solvinert plate from Millipore Corporation (Billerica, Massachusetts). 25 μL aliquots of serum–plasma samples from the animal studies were pipetted into the wells followed by vortex mixing for 3 min. The filter plate was stacked over a 0.5-mL, 96-well collection plate with a narrow-diameter 0.2-mL, 96-well plate insert (VWR) and centrifuged for 5 min at ;1480

. An aliquot of 10 μL was injected onto a column for analysis. The standard curves and QC samples were prepared in the same fashion as the samples.  

A high performance liquid chromatography (HPLC) system consisted of two LC-10ADvp pumps with a SCL-10Avp System Controller from Shimadzu (Columbia, Maryland) and a CTC Analytics HTS PAL autosampler from LEAP Technologies (Carrboro, North Carolina) equipped with a cooling stack autosampler tray maintained at 5 °C. The column used was a 50 mm × 2.1 mm, 5-μm Atlantis C18 column from Waters Corporation (Milford, Massachusetts) maintained at 40 °C. The mobile phase, consisting of 10 mM ammonium formate, 0.1% formic acid, and 0.2% acetonitrile in water (A) and acetonitrile with 0.1% formic acid (B), was run at a flow rate of 0.3 mL/min. The initial mobile phase composition was 95% A and 5% B. After sample injection, the mobile phase composition was held constant for 0.5 min and then changed linearly to 5% A and 95% B over 1.5 min and held at that composition for an additional 1.0 min. The mobile phase was then returned to initial conditions over 0.1 min, and the column was reequilibrated for 1.5 min. The total analysis time was 4.6 min.  

The HPLC system was interfaced to a Quattro Premier LC–MS-MS tandem mass spectrometer from Waters/Micromass, (Manchester, UK) equipped with an electrospray interface operating in the positive ionization mode. Ultrahigh pure (UHP) nitrogen was used as the cone and desolvation gases at flow rates of 80 L/h for cone and 1000 L/h for desolvation. The desolvation temperature was 300 °C and the source temperature was 150 °C. Data acquisitions were via selected reaction monitoring (SRM). Ions representing the (M+H)

  species for each BMS compound. Only 12 compounds were mentioned in the introduction.] and the internal standard were selected in MS1 and collisionally dissociated with UHP argon at a pressure of 2 × 10

  mbar to form specific product ions which were subsequently monitored by MS2.  

LC–MS-MS SRM analyte peak area response to internal standard peak area response ratio was used for quantitation. 

Most centrifugal precipitation methods use only one centrifugation step. However, it is important to recognize that a more rigorous, two-step method was used in all the comparisons reported in this study to achieve better results. The time of centrifugation in the pelleting method for a 0.5-mL, 96-well plate varied from 10 to 30 min. We performed a 30-min centrifugation to ensure the quality of protein precipitation and the bioanalytical assay in our routine. 

 Table I: Assay summary of experiment I			

Results of the validation study (Experiment I) are shown in Table I. The standard curve consisted of four replicates at each of seven concentration levels ranging from 0.5 to 1000 ng/mL. QC samples were prepared in eight replicates at five concentration levels from 0.5 to 800 ng/mL. Sample preparation using the filter-plate method gave comparable results to the regular pellet method for compounds A and B in rat plasma. Both standard curves are linear over the 0.5–1000 ng/mL range and percent standard deviation (%SD) is acceptable for both methods, though somewhat lower for the filter plate. Using both methods, QC samples were run (n=8) at 0.5, 1, 8, 80, and 800 ng/mL for compound A. Results are summarized in Table II and are very comparable, indicating that precision and accuracy are similar for both methods. Internal standard responses also were consistent throughout the run for both pellet method and filter-plate method. 			

Standard curves at 10 concentration levels from 0.5 to 1000 ng/ml in duplicates using both methods were generated for compounds C, D, E, F, G, H, I, J, and K (Experiments II and III). Overall %SD data from standard curves for all compounds A–K are summarized in Table III. Except in the case of compound I in mouse serum, %SD is approximately the same or lower in the filter-plate method compared to the pellet method. The overall %SD is 7.5% for the pellet method and 5.7% for the filter-plate method. The filtration-plate method reduces bioanalytical assay variability at low end of standard curves.  

Table II: Precision and accuracy of QC samples for Compound A in Experiment I

In Experiment IV, the pellet and filter-plate methods were compared in a study of compound L in dog plasma. These results demonstrated a 20.9% overall standard deviation compared with the 5.1% observed for the filter-plate method on the same samples. Although improved precision and accuracy were observed in the majority of filter-plate samples (Table III), the reasons for dramatically improved Experiment IV filter-plate performance is still under investigation. 

Table III: Comparison of % standard deviation of standard curves for pellet versus filter plate methods for compounds AâK in each program.			

To determine the stability of samples over multiple injections using the filter-plate method, a reinjection study was performed using compound M in Experiment V. The standard curve and QC samples (8, 80, and 800 ng/mL) were run in duplicate at the beginning and end of the study. When using the pellet method, the second injection of a sample is often not reproducible following a long run. Comparison of peak areas for first injection and second injection of the same samples indicate excellent reproducibility using the filter-plate method (see Figure 1). This improved reproducibility is most likely due to the increased homogeneity of the filtered sample and the decrease in the amount of insoluble protein. Furthermore, the correlation coefficients of the standards and QC samples for the first and second injection of the filtered samples were calculated to be 0.9993 and 0.9997, respectively. 

The filter-plate method is fast and simple. The total sample preparation time was reduced from 50 min for the pellet method to less than 10 min using the filter plate. Because a double centrifugation method was used for comparison, a significant time reduction was achieved. Due to the small sample volume for many animal study samples, longer centrifugation is needed to ensure a concentrated pellet that will not be disturbed during decanting. Therefore, a shorter centrifugation can impact sample homogeneity and specifically affect both reproducibility and column life. When compared with protocols with shorter centrifugation steps, even more significant improvement in data quality should be seen with the filter-plate method.  

Preparing serum or plasma samples for total drug analysis using the 96-well, solvent-resistant filter plate offers comparable or better results to the traditional centrifugal pelleting method. Drug concentrations of samples analyzed in the filter plate method were consistent with the pellet method, however, the filter plate method improved precision, accuracy, and reproducibility of re-injection of samples. The cleaner and more homogenous extracts afford better quality data and longer column life.  

(1) F. Neilsen-Kudsk, I. Magnussen, and P. Jakobsen, Acta Pharmacol, Toxicol. 42, 226 (1978). 

(2) G.P. Butrimovitz and V.A. Raisys, Clin Chem. 25, 1461 (1979). 

(3) R.L. Nation, G.W. Peng, V. Smith, and W.L. Chiou, J. Pharm. Sci. 67, 805 (1978). 

(4) X. Ding, H. Luo, J. Bleecker, Castles, and P. Rudewicz, Proc. 52th ASMS Conf. Mass Spectrom. & Allied Top., MP219 (2004). 

(5) M. Cleeve, R. Calverley, G. Davies, S. Merriman, A. Howells, J. Labadie, and C. Desbrow, Proc. 53th ASMS Conf. Mass Spectrom. & Allied Top., WP192 (2005). 

(6) C. Lindermuth, J. Thomas, and D. Goykhman, Proc. 53th ASMS Conf. Mass Spectrom. & Allied Top., WP194 (2005). 

(8) R.A. Biddlecombe and S. Pleasance, J. Chrom. B 734, 257–265 (1999). 

Peer-Reviewed Article

This article describes the use of a 96-well, solvent-resistant filtration plate for total drug analysis in blood plasma.

LC–MS-MS Total Drug Analysis of Biological Samples Using a High-Throughput Protein Precipitation Method

 published a special supplement that focused on the sample preparation of solids (1). At that time, we covered general methods — supercritical fluid extraction (SFE), pressurized fluid extraction–accelerated solvent extraction (PFE–ASE), microwave-assisted extraction (MAE), and automated Soxhlet extraction. In the ensuing six years, these modern extraction techniques have continued to evolve. In some cases, systems have been further miniaturized and automated. A wider variety of applications has been achieved in such diverse areas as environmental, pharmaceutical, natural product, food, and polymer chemistry. In this installment of "Sample Preparation Perspectives," I will update the earlier coverage and make a note of some of the instrumental advances and a few applications that have spurred the growth of these extraction techniques. Although the literature abounds with applications examples, this review will focus on the technology. Websites of the various manufacturers, textbooks, and review articles are great sources of information on applications examples. 			

Modern liquid–solid extraction has its roots in the shake–filter method. Basically, the shake–filter extraction method involves the intimate contact between a solid material, usually finely divided, and a solvent that has an optimum solubility for the analyte of interest and a minimum solubility for the matrix. The technique works well for porous matrices where the solvent can diffuse into the pores and extract analytes. Sometimes mechanical agitation and heating is used to speed up the extraction process. At the conclusion of the experiment, insoluble materials are removed by filtration or centrifugation. For some samples, the extract can immediately be injected into a chromatograph for further separation. More often, there is some additional sample preparation required to isolate the critical analytes from other extractables that can interfere with the chromatography step. 

Table I: Most modern solid extraction techniques use increased temperature and pressure

For more intimate contact between solvent, the adsorbed (absorbed) analyte and matrix, other sample preparation techniques might be required. Several methods that can achieve this intimate contact include homogenization, sonication, emulsification, or vortexing. For biological samples, various cell disruption techniques can be employed. All of these methods require the application of an additional external force (or forces) to the sample in suspension. 

Most modern liquid–solid extraction techniques use increased temperature or pressure to increase the rate of extraction. Table I summarizes the influence of these parameters on analyte extraction. 			

Soxhlet extraction is by far the most widely used method for solid sample pretreatment. In fact, it is the de facto standard with which all other extraction procedures are compared and contrasted. Soxhlet extraction is simple, effective, and relatively inexpensive. Generally, it provides good analyte recovery but its traditional operation is slow and uses copious amounts of solvent. Although classical Soxhlet extractions can last as long as 18–24 h, once set up and running, the experiment requires very little user involvement. Small-volume Soxhlet systems are available, but the sample size often is dictated by the analyte concentration, the necessary mass to obtain a representative sample, and the chromatography detector sensitivity, all of which could combine to require a larger sample size. 

Traditional Soxhlet extraction has been around for over 100 years, having been invented by Franz von Soxhlet in 1879. The extractor apparatus used in the traditional method is depicted in Figure 1. Typically, dry sample material is placed inside a "thimble" made from cellulose filter paper, which is loaded into the extractor. The extractor is attached to a flask containing a solvent and a condenser. The solvent is heated, causing it to evaporate. The hot solvent vapor travels up to the condenser, where it cools and drips down onto the solid material in the thimble. The chamber containing the sample slowly fills with warm solvent until, when it is almost full, it is emptied by a siphoning action back into the solvent flask. This cycle is allowed to repeat many times. During each cycle, a portion of the analyte (and perhaps some of the matrix) dissolves in the solvent. However, once the extracted analyte reaches the solvent heating flask, it stays there and does not participate in the extraction cycle any further. Only clean, warm solvent is used to extract the solid in the thimble, a key advantage of classical Soxhlet extraction. This procedure prevents the possibility of solvent becoming saturated with extractable material and increases the efficiency of the extraction when compared to simple shake–filter method. 

In 1974, Edward Randall made a major improvement in the Soxhlet extraction technique that reduced the extraction time dramatically (1). In his method, the sample was totally immersed in the boiling solvent. Compared with the condensed solvent in the classical Soxhlet method where the solvent temperature is below the boiling point, the Randall method was faster because analytes are more soluble in hot solvent than in cold-to-warm solvent. The apparatus used to perform the Randall adaptation is shown in Figure 2. The operation of the Randall adaptation is depicted in Figure 3. First, the thimble is lowered into the boiling solvent until the appropriate extraction takes place. Then, to flush residual extract from the sample, a rinse step follows. In this second stage, the thimble is raised above the boiling solvent for a period of time until residual extract is removed from the solid material by the condensed solvent, just as is performed in the original Soxhlet experiment. Finally, the drying step removes the solvent from the solvent flask and concentrates the analyte for further processing. In this step, by closing a solvent return valve, the condensed solvent is redirected away from the sample and boiling solvent and collected in a reservoir for possible reuse or disposal. In some systems, there is a fourth step in which the sample cup is lifted from the heat source and allowed to evaporate further without the chance of sample overheating, boiling dry, or potential oxidation. The Randall approach can decrease the extraction time by as much as a factor of 10 compared with traditional Soxhlet extraction. 

The Randall approach has been adopted by a number of companies with full automation including Foss-Tecator (Eden Praire, Minnesota), Buchi Laboritechnik (Flawil, Switzerland), and C. Gerhardt GmbH (Bonn, Germany). Table II outlines some of the main features of these systems. All of these systems can run totally unattended, offer temperature programming, solvent reclamation, and numerous safety features. Most solvents can be used, although diethyl ether is not recommended for obvious reasons. Automated Soxhlet extraction is approved by the U.S. Environmental Protection Agency (EPA) for the extraction of organic analytes from soil, sediment, sludge, and waste solids (Method 3541). 

Table II: Automated soxhlet extraction products			

There is no doubt that because Soxhlet extraction is the de facto standard in liquid–solid extractions it will continue to be used due to its simplicity, relatively low cost, extraction efficiency, and ease of use. With the advent of fully automated systems, much of the drudgery of classical Soxhlet extraction has been reduced. Nevertheless, some interesting approaches to improve on the technique have been proposed. Focused microwave-assisted Soxhlet extraction could even speed the extraction time even further with certain microwave absorbing solvents for environmental solid samples (2,3). The further application of using water as an extraction solvent makes the process much more environmentally friendly (4). When pollutants are present in aged soils, they are more difficult to extract by Soxhlet extraction than when present in spiked soils. An in situ derivatization technique was found to more easily release pentachlorophenol from sandy soil (5). Using the acetylation agents triethylamine–acetic anhydride and pyridine–acetic anhydride, the authors found that the yields were four times greater and the extraction time shorter with in situ derivatized pentachlorophenol in soil than by the traditional Soxhlet extraction of nonderivatized pentachlorophenol in the same soil.  

When first introduced in the early 1990s, supercritical fluid extraction (SFE) was thought to be the panacea to solve all sample extraction problems for solid samples. The potential arguments were convincing — a safe, solvent-free, easily removed extractant (carbon dioxide) with a tunable solvent power that diffused like a gas and had the solvation power of a liquid. Quite a number of analytical instrument suppliers (around 12 if my memory serves me correctly) jumped on the bandwagon and instruments were introduced with full automation, variable restrictors, sample collection, and many features albeit at a relatively high price tag for most. The EPA was quite keen on supporting development of SFE because there was a strong desire to decrease the use of copious amounts of solvents used in conventional Soxhlet and ultrasonic extractions. There was a vision that SFE could be the universal method to be used to extract all analytes from solid environmental matrices — soil, sand, sludge, fly ash, and so on. Food analysts saw SFE as a universal extraction technology for the extraction of toxic analytes from virtually any type of food sample. Polymer chemists believed that SFE might be able to extract additives from any type of plastic, rubber, or other solid synthetic material.  

Today, there are less than a handful of SFE suppliers, mainly smaller companies with dedicated instruments. As of last count, only Applied Separations (Allentown, Pennsylvania), Thar Technologies (Pittsburgh, Pennsylvania), LECO Corporation (St. Joseph, Michigan) and Jasco (Easton, Maryland) still supply analytical extraction units. In 2004 Teledyne Isco (Lincoln, Nebraska) discontinued the supply of SFE equipment, and the Supelco SFE-400 (Bellefonte, Pennsylvania) is no longer available. 

Still, even today, new applications of SFE are still being reported but from a commercial viewpoint, no new instruments or major accessories for existing instruments have appeared on the market since our original report in 1999 (1). So, for commercial purposes, the analytical-scale SFE technique appears to be stagnant. Nevertheless, from a preparative and industrial-scale viewpoint, SFE is still widely used for such applications as the decaffeination of coffee and extraction of fragrances for perfumes. 

Why didn't SFE achieve the potential that was purported in the early 1990s? Let us briefly examine some of the reasons where SFE went awry. First, supercritical carbon dioxide, by far the most popular SFE extractant, in its densest state (0.95 g/mL) provides the extraction power equivalent to toluene or perhaps ethyl acetate. Thus, supercritical carbon dioxide could not always quantitatively extract even moderately polar analytes, especially in aged soils or in ground, solid polymer beads. For example, the EPA Method 3561 polynuclear aromatic hydrocarbons (PAHs) that are relatively nonpolar molecules cannot be extracted from an inert matrix with pure supercritical carbon dioxide (6). To extract the larger, less volatile, fused-ring PAH aromatics such as benzo[α]pyrene and benzo[

]perylene, an organic modifier must be added. With this organic solvent addition, the SFE method now becomes two separate extraction procedures where the results must be combined to get the complete composition. This approach adds to the complexity and requires an SFE instrument that has an additional pump for organic modifier addition. 

Indeed, most SFE methods require the addition of an organic modifier to perform satisfactorily. The amount of modifier added is sometimes small perhaps only 5–10% of the amount of carbon dioxide by volume. However, in a 30-min extraction, the amount of organic solvent still could amount to a much as 10 mL of organic solvent, which negates one of the biggest advantages of SFE: the need for little or no organic solvent. Still, the amount of organic solvent used in SFE is still reduced compared with the classical extraction methods (and even some of the other modern extraction methods). 

Second, the method development in SFE was less straightforward than initially thought. The big culprits were matrix effects and effect of moisture. Even for the same analyte, a different method was often required depending upon the solid material to be extracted. These matrix effects caused the biggest problem in getting SFE methods through the EPA approval process. If a PAH was in fly ash, clay, sand, loam, peat, or another soil matrix, a different procedure was required for a quantitative extraction. Environmental samples are not always so easily controlled. In addition, the EPA wanted more standardized methodology that can be placed in the hands of contract testing laboratories. The effect of moisture could be overcome often by the addition of a drying agent to the sample but necessitated another step in the extraction process. 

Similarly, sample preparation chemists attempting to use the new technique found it to be unfamiliar relative to their training and background. They had to learn new technology that was more difficult to understand and cookbook methods were not readily available for their analyte–matrix pairs. To aid in the education of the users, SFE training courses were held, symposia were arranged, books were published, application notes could be had, and experienced help was available, primarily through the instrument companies who supplied SFE instrumentation. Instruments had powerful software to help in making the method development process easier. Nevertheless, the complexity of SFE method development was a hindrance to its acceptance. Contrast the more rapid acceptance of accelerated solvent extraction–pressurized fluid extraction (next section), especially by the EPA, because that technique retained the simple principles of classical liquid–solid extraction. 

Third, SFE turned out to be a fairly expensive sample preparation technique. The initial price of an automated SFE unit often was more than the price of the analytical instrument that it served. First time purchasers had a hard time justifying replacing an inexpensive bank of Soxhlet extractors in their fume hood, even if the breakeven point was less than a year if one ran a reasonable number of samples per day. Liquid carbon dioxide required to perform SFE could be purchased for a reasonable cost and the amount of organic solvent required was low relative to the Soxhlet technique. Thus, the operational cost of chemicals was not prohibitive. Where the higher operating costs came into the picture was the labor content where a high level of skill was needed to successfully development methods, in maintaining the instrument, and in troubleshooting problems and understanding the corrective actions when extractions did not go as planned. Today, the SFE instruments that are remaining on the market are not as sophisticated as they were in the 1990s so they are not quite as expensive.  

Fourth, early SFE instruments fell short on performance and left the impression that the technique was difficult to use. Early round-robin interlaboratory testing gave inconsistent results (7). Instruments from different manufacturers performed differently and therefore methods could not be easily duplicated from lab to lab. Second-generation instruments came out with higher levels of automation such as autosamplers, automated collection devices, and variable restrictors for better control over the supercritical fluid conditions but the damage had already been done.  

Nevertheless, SFE has had (and continues to have) many success stories as a selective sample preparation procedure in food chemistry, pesticide residues, and environmental applications. It appears to be best when the analytes are nonpolar or possess relatively low polarity and the matrices are of nonfatty composition. General reviews (8,9) and entire books (10–12) are dedicated to SFE. 

Pressurized Fluid Extraction–Accelerated Solvent Extraction 

Originally introduced by Dionex (Sunnyvale, California) as ASE, the technique was generalized to PFE by the U.S. EPA. The ASE technique was trademarked by Dionex and specific products are not generally endorsed by the EPA. Nevertheless, the PFE technique is now well accepted as an alternative to Soxhlet extraction. EPA Method 3545A, entitled "Pressurized Fluid Extraction," is a recognized alternative procedure for extracting water-insoluble or slightly water-soluble organic compounds from soils, clays, sediments, sludge, and waste solids. This method is applicable to the extraction of semivolatile organic compounds, organophosphorus pesticides, organochlorine pesticides, chlorinated herbicides, polychlorinated biphenyls (PCBs), and polychlorinated dibenzodioxins and dibenzofurans (PCDDs and PCDFs), which can then be analyzed by a variety of chromatographic procedures. PFE, sometimes referred to as pressurized solvent extraction (PSE), uses elevated temperatures (100–180 °C) and pressures (1500–2000 psi) to achieve analyte recoveries equivalent to those from Soxhlet extraction but using less solvent and taking significantly less time. The idea is that a solvent heated above its boiling point in a closed system becomes a very potent extraction solvent. Extractions by PFE generally require only 10–20 min. Sample requirements vary but generally 1–30 g is required depending upon the sensitivity needed. Sample extraction cells can be as small as 1 mL or as large as 100 mL. 

Relative to other new technologies, the EPA adopted the PFE method in less than three years while the first SFE method took nearly eight years to go through the promulgation process. Similar to other modern liquid–solid extraction technologies, PFE uses less solvent, is faster and is more automated than traditional Soxhlet extraction. The beauty of the technique is that it uses the same solvents as classical Soxhlet and sonication methods. Method development is quite easy: one chooses a solvent that is a good solvent for the analyte(s) but a poor solvent for the matrix. The instrumentation of PFE is similar to that of SFE but instead of pumping liquid carbon dioxide into the extraction chamber, conventional extraction solvents are used. Figure 4 shows a general schematic of the Dionex ASE instrument. The sample, usually in a finely ground state, is placed into an extraction cell much like the thimble in an SFE instrument. A pump transfers solvent from one or more reservoirs into the extraction cell, located in an oven. The oven heats (by conventional convection heating) the sample for a finite period of time (;10–20 min), usually determined during the method development operation. Extractions can be performed in static or dynamic modes. After the extraction is completed in the static mode, a nitrogen purge is used to transfer the extract to a collection vial. In the PFE process, the sample is contained in a large volume of solvent and additional steps are needed to concentrate the analyte of interest. If the analytes are low-to-mid polarity and the extraction solvent has high water content, passage of the extract through a reversed-phase solid-phase extraction cartridge can serve as a simple concentration device. Then a small amount of water-miscible organic solvent (acetonitrile or methanol) can elute the analyte into a vial for later injection into a high performance liquid chromatography (HPLC) or gas chromatography instrument. 

Although the Dionex instrument pictured in Figure 4 is serial and only one sample at a time can be extracted, the ASE 200 instrument has an autosampler that can handle as many as 24 samples for unattended operation. If higher throughput is required, Applied Separation's fast PASE system is a parallel PFE system that allows the simultaneous extraction of as many as six samples. However, the PASE does not have an autosampler which means that will require manual involvement every six samples. 

Table III: Extraction conditions for active ingredients in natural products			

The range of applications of PFE–ASE is as vast as that of the other classical and modern extraction techniques. Because convection heating is used in this technique, just about any solvent can be used for extraction. Table III provides an example of the conditions and time required for a PFE extraction of active ingredients from natural products (13). The samples were commercially available nutritional supplements, specifically hypericin from 

 (goldenseal root). The former is used to treat kidney diseases, depression, and other conditions while the latter is an immune system booster. Samples, in capsule form, were opened, the ground material collected and 1–3 g was loaded into the extraction cells with cellulose filter in the cell outlet. Several extraction solvents were investigated and acetonitrile, being compatible with HPLC, was used for final extraction. Using the Dionex ASE-200 equipped with 11-mL extraction cells, both sets of samples were extracted. Analysis was performed by reversed-phase HPLC using external calibration. Table IV shows the results for four capsule extractions for each of the two supplements (13). The results were comparable to the industry standard methods for this type of extraction (Soxhlet and sonication) but were achieved much faster, used less solvent and were fully automated thereby increasing productivity of laboratory personnel. 

Table IV: Results on PFEâASE extractions of natural products			

The use of microwaves in the laboratory was accepted long ago. Microwave digestions have been in use for over 20 years and are widely used for metal analysis in soils and other solids. Microwave-assisted extraction (sometimes referred to an microwave-accelerated solvent extraction) of organic analytes is a more recent technique. An excellent review article was included in the 1999 

 supplement (14). Compared with the traditional liquid–solid extractions described earlier, the use of microwaves as a heat source has some marked differences: the extraction solvent is heated internally rather than by convective heating; the temperature in the extraction container is dictated by the solvent used for extraction and not by the set point of an external oven or heater; and as the temperature of the solvent in the closed container rises, the pressure inside rises above atmospheric pressure. The fact that the solvent itself is heated causes a more rapid response to begin the extraction process faster than with convection heating. 

Of course, microwave extractions can take place in an open vessel just as you heat hot water in a cup while preparing tea. While water has a high dielectric constant and readily absorbs microwave energy, organic solvents such as hydrocarbons do not absorb the microwave energy and therefore are not heated. For microwave extractions with these types of organic solvents via indirect heating, a microwave-absorbing device must be placed inside of the extraction vessel. These devices are usually rods or powder of a microwave-absorbing material such as carbon black coated with an inert polymeric substance (for example, PTFE) that will not contaminate the sample. Figure 5 compares the heating rate of hexane (nonmicrowave absorbing solvent), acetone (microwave absorbing solvent) and hexane with a carbon-black filled fluoropolymer heating insert, Carboflon, a product of CEM Corporation (Mathews, North Carolina). 

Figure 6 illustrates the advantage of using a microwave-absorbing insert for achieving a cleaner MAE. The experiment was to extract the hydrocarbon pollutants out of a soil sample that could be accomplished by toluene extraction alone. In Figure 6a, the extraction solvent was a mixture of toluene and methanol. The polar methanol was required to absorb microwave energy so that the extraction could be viable for MAE. Note the noisy baseline and doublet peaks for the anthracene and phenanthrene. Also note that some polar analytes also were extracted due to the stronger polarity of this extraction solvent. Compare the cleaner chromatogram of Figure 6b where toluene only was used as the extract solvent combined with the use of a Weflon insert, an inert fluorocarbon microwave absorbing device of Milestone (Sorisole [BG], Italy). The anthracene and phenanthrene peak shape is better and there is an absence of unwanted polar compounds because the aromatic nonpolar hydrocarbon toluene was the only extraction solvent. 

In microwave-assisted extraction, special nonmicrowave absorbing, inert extraction cells are required. These cells must be able to withstand high temperatures (as high as 300 °C) and high pressures (as high as 200 psi). All parts of the cells must be non-microwave absorbing and the parts touching the sample must be made of totally inert substances. The cells are constructed of glass or perfluoroalkoxy (PFA) Teflon with PFA Teflon seal covers, glass-filled polyetherimide sleeves for radial strength, and polpropylene support frames with polyether ether ketone (PEEK) load disks and sealing screws for longitudinal (up–down) strength. Also, ceramic materials and fiber-reinforced PEEK are used for microwave transparent extraction cells.  

Because one is dealing with organic solvents heated above their boiling points, there are many built-in safety features for commercial microwave-extraction systems. These safety features are found in both the extraction cells and in the microwave cavity of the oven. The extraction cells have safety vents in case of excessive pressure buildup. Solvent sensors are installed inside of the microwave oven in case a leak of solvent does occur. The extraction vessels that are commercially available from various suppliers can be temperature controlled so that the extraction temperature inside of the cell can be optimally set. In some units, an infrared sensor is available for contactless temperature monitoring. In addition, because stirring is desirable to ensure intimate contact between the finely divided samples and the extraction solvent, vessels also can be stirred using a stirring bar. Oven doors also can be safety protected in case of a catastrophic internal pressure buildup in the oven itself.  

Because multiple samples can be simultaneously processed in a microwave oven, the sample throughput can be very high compared with some of the other liquid–solid extraction techniques. For example, both Milestone's Ethos high throughput system and CEM MARS systems have the capability for as many as 24 or 40 extraction vessels/batch, respectively. 

Recent developments in MAE have included a new approach to control the microwave energy inside of an extraction vessel as the dielectric media changes. In the new automated ExplorerPLS small-scale microwave sample prep system from CEM Corp., the single-mode self-tuning microwave cavity compensates for changes in physical properties of the sample as it changes during the application of the microwave field. The mechanical design of the cavity allows the microwave to enter the cavity wherever it wants, dictated by the sample in the cavity that draws the microwave from a series of windows cut into the circular waveguide inner wall. Originally designed as a CEM microwave synthesis system, the ExplorerPLS sample prep system for MAE uses disposable high-pressure 10-mL extraction tubes with 0.1–7 g working volume for applications for samples as small as 100-mg.  

Microwave extraction is now widely accepted as an alternative to Soxhlet, PFE–ASE and other liquid–solid extraction techniques. For example, similar to the EPA Method 3545 described earlier for PFE–ASE, EPA Method 3546 is an MAE procedure for extracting water insoluble or slightly water soluble organic compounds from soils, clays, sediments, sludges, and solid wastes. This method is applicable to the extraction of semivolatile organic compounds, organophosphorus pesticides, organochlorine pesticides, chlorinated herbicides, phenoxyacid herbicides, substituted phenols, PCBs, and PCDDs and PCDFs. The method uses microwave energy to produce elevated temperature and pressure conditions (that is, 100–115 °C and 50–175 psi) in a closed vessel containing the sample and organic solvent(s) to achieve analyte recoveries equivalent to those from Soxhlet extraction (Method 3540) using less solvent and taking significantly less time than the Soxhlet procedure.  

Comments about the Use of Modern Liquid–Solid Extraction Procedures

With the exception of SFE, the use of the modern liquid–solid extraction procedures is a variation of a theme. They all use the same types of solvents and can accomplish the same types of applications. Each of them uses a slightly different approach in achieving the same results. For example, PFE–ASE and MAE both use high temperature and high pressure to achieve faster extractions than traditional or automated Soxhlet extraction but the results achieved are the same. In fact, most modern extraction techniques treat the traditional Soxhlet extraction as the de facto standard. Because the extraction conditions are better controlled in automated systems, the relative standard deviations can be somewhat better than the traditional methods. However, the cost of the newer automated systems is considerably higher than the equipment needed to perform traditional Soxhlet extraction. The biggest cost saving argument for the modern approaches is the reduction of solvent usage and also in the reduction in manual labor. Also, most of the systems offer a lower degree of exposure of workers to organic solvents. On the other hand, with the modern liquid–solid approaches, the operation of organic solvent extractions at high pressure and high temperature requires extraordinary safety features to be built into the equipment that, in turn, also causes a purchase price increase. For a high throughput laboratory requiring daily extractions of many samples of solid materials such as soils, food samples, plastic materials, and natural products, the added investment in automated systems should pay for itself in a relatively short period of time. 

SFE has lost some of its luster, with only a few companies offering this technology. However, it still has its place in niche applications and is a safe extraction technique that is considered to be environmentally friendly. The operating cost is among the lowest since the use of organic solvents with associated purchase and disposal costs is minimal. 

In this installment of "Sample Prep Perspectives," Ron Majors examines modern sample preparation methods for solids that often involve increased temperature and higher pressure to speed up the extraction process.

LCGC North America-07-01-2006

The Centenary of "Chromatography"

Chromatography Market Profile: Extraction Techniques for Environmental Samples

Gradient Performance Problems - A Case Study

Making a Great Connection

LC–MS-MS Total Drug Analysis of Biological Samples Using a High-Throughput Protein Precipitation Method

Modern Technique for the Extraction of Solid Materials - An Update

Peaks of Interest