rus
Issue #2/2016
Y.Yashin, A.Vedenin, and A.Yashin
60th Anniversary of chromatographic instrument engineering
60th Anniversary of chromatographic instrument engineering
Due to up-to-date instrumentation design, chromatography became one of main analytical methods in the 20th century. More than 60% of chemical analyses throughout the world are performed now using the chromatographic techniques. This science was rated as one of twenty outstanding achievements of the 20th century for contribution to development of vital areas of science, technology, and industrial production. Today chromatography is not only science, but also one of the most effective analytical techniques and an individual instrument manufacturing industry with annual production capacity of more than 14 billion dollars and annual growth of 6–7%. Advancements in devices for gas, liquid, and ion chromatography as well as gas chromatography combined with mass spectrometry are analyzed. Gained advantages are highlighted and general trends in chromatography techniques and devices development are indicated.
Теги: : gas chromatography ion chromatography liquid chromatography mass spectrometry газовая хроматография жидкостная хроматография ионная хроматография масс-спектрометрия
Introduction
The chromatography was discovered by M.S.Tsvet in 1903. For nearly half a century, it was used to separate individual substances, extract the separated components in isolated form, and purify them. All that time, chromatography technique remained virtually the same as it had been proposed by M.S.Tsvet, namely, there were no detecting systems at the column outlet or forced feed of the mobile phase, this phase flowed under gravitational supply pressure.
In 1955, first serial gas chromatographs had been commercialized that allowed applying the chromatography technique for analytical purposes. It was the revolutionary event. Chromatographs had developed and won more and more spheres of application so rapidly that by 1960 gas chromatographic method had become one of the main ways to analyze complex multicomponent mixtures, especially in the petrochemical industry. Later, first liquid chromatographs were developed in 1968 (DuPont), then ion chromatographs in 1977 (Dionex), and finally gas chromatographs combined with mass spectrometers (GC-MS systems) (Hewlett-Packard) [1–3].
Chromatographs are very versatile in design: there are laboratory, automated industrial, movable, portable, microprocessor-controlled, specialized devices, e.g. chromatographs for space exploration. According to some global estimations, over 3 million chromatographs have been produced around the world. The sales volume of the chromatographic equipment and supplies is estimated at $ 14 billion in 2015. More than 60% of all chemical analyzes in different countries around the world are conducted today using chromatographs, although there are dozens of other methods of analysis.
These successes of analytical chromatography were associated with the development of advanced chromatographic equipment. Mass introduction of a particular chromatography technique into analytical practice was caused by mass production of the relevant chromatograph type. The amino acid analyzer that was invented by Stanford Moore and William H. Stein in 1956 based on ion exchange chromatography, which enabled a mass amino acid analysis, can serve as a most bright example.
Performance of chromatographic instruments has reached a very high level today due to full automation of process control and treatment of results as well as implementation of latest achievements in electronics, precision mechanics, pneumatics, planar technology, and optics. During their 60-year history, chromatographs were sequentially analog, digital, and microprocessor-controlled.
It should be noted that a key role in the analytical capabilities of chromatographs is played by chromatographic columns. In particular, chromatography became usable for separation and analysis of optical isomers in chiral chromatography only due to development of appropriate sorbents and columns. This feature is especially important in modern pharmaceutics since over 70% of all drugs are optically active. At the same time, legislation in many countries requires examination of the impact of both L-isomers and D-isomers of amino acids on a human body.
Chromatography was recognized as the main analytical method of the twentieth century and included into twenty outstanding discoveries of the last century, which had the most impact on human life. In addition, chromatography obviously promoted progress in many other areas of science, technology, and various industries.
The status of chromatographic techniques and instruments was elucidated in scientific collections issued to the centennial chromatography jubilee [4–5], some other latest publications [6], comprehensive encyclopedia of chromatography [7] as well as scientometric review of publications on chromatography for 20 years [8]. As the big book of chromatography [5] states, the chromatography provides the link between science and technologies, especially biotechnologies. The biotechnological enterprises are among most rapidly growing ones (numbering over 5000 in the US and over 5000 in Europe).
Gas chromatographs
First commercial gas chromatographs were delivered in US by three firms, Perkin-Elmer, Burrell Corporation, and Podbielniak in 1955. In 1956, another five US companies presented their developments. The most perfect and successful first commercial model P-E 154 Vapor Fractometer of the company Perkin-Elmer was most popular among consumers during seven years [1, 2].
Thereafter, the development and production of gas chromatographs (GC) grew very rapidly: in 1955 they were produced by 3 companies; in 1956 by 7 companies; in 1961 by 16 companies; in 1962 by 23 companies, and in 2000 by 69 companies (among them, 29 companies in the United States; 10 in USSR or Russia; 9 in UK; 3 in Germany; 3 in Japan; 3 in the Netherlands; 2 in Italy; 2 in China, and one in each of the following countries: France, Spain, Denmark, India, and Ukraine (data from LC-GC Europe 2000/2001, Vol.13, No. 8, P. 586). In Japan, first commercial gas chromatographs were released in 1956–1957 (Shimadzu, and Hitachi). In former USSR, first serial gas chromatographs HT2 and HT2M were produced by All-Union R&D institute for complex automation of oil and gas industry in 1958; advanced models UH-1 and UH-2 appeared in 1960. UK companies also developed gas chromatographs in the years 1956–1958, the most popular being argon gas chromatograph manufactured by Pye Unicam since 1958. Italian company Carlo Erba presented its first gas chromatograph Fractovap with capillary columns in 1960. The first industrial chromatograph RH-1 with a flame ionization detector was released in the former USSR [1, 2].
The fast development of gas chromatograph engineering was assisted by invention of new devices such as highly sensitive ionization detectors, temperature programming modules, and capillary columns.
Key achievements in that period consisted in implementation of electronic modules for gas flow velocity and pressure stabilization and control as well as two-dimensional gas chromatography layouts.
Now commercial gas chromatographs are equipped with various detectors, mainly mass spectrometry, flame ionization, electron capture, flame-photometric, photo-ionizing, thermionic, or thermal conductivity based ones. Less commonly used are chemiluminescent, infrared, atomic emission detectors. Among them, there are either all-purpose (thermal conductivity based, atomic emission, and mass spectrometry) or selective detectors. In particular, the flame ionization detector (FID) is sensitive only to organic compounds; photo-ionizing detector can operate only with compounds having the ionization potential less than about 10–11 eV; electron capture detector has especially high sensitivity to halogen-organic compounds; in contrary, thermionic detector is selective in relation to nitrogen or phosphorus-containing compounds whereas the flame-photometric detector (FPD) to the sulfur or phosphorus-containing compounds while the chemiluminescent detector responds only to sulfur or nitrogen-containing compounds. Selective detectors have a great advantage when analyzing impurities in complex mixtures. It is possible to create conditions such that the impurities were determined whereas the basic substances in the mixture were omitted. For example, the electron capture detector is very convenient to measure organochlorine pesticides in the environment.
To enhance analytical capabilities, gas chromatographs are equipped with optional accessories. Solid-phase, micro-solid-phase, supercritical-liquid, and gas extraction devices are used for sample concentration; special systems perform blowing and trapping; autosamplers, cryo-focusing traps, and gas cocks serve for sample input or injection; heating units are required for pyrolysis; derivatizing systems (in particular, methanators or similar devices) are applied for pre-column or post-column derivatization, etc.
List of commercial domestic and foreign gas chromatographs is presented in Tables 1 and 2.
In China, chromatographs are produced primarily at two major instrument-making plants in Beijing and Shanghai.
In certain periods, chromatographs were produced in former Czechoslovakia ("Chrome" series, the company "Laboratory Instruments"), Finland (the company "Orion Analytics"), as well as Poland, Bulgaria, and the German Democratic Republic (the company Willy-Gide).
By beginning of 21st century, production of a complete gas chromatographs (GC) line was established including laboratory instruments, GC-MS systems, on-line analyzers, multi-dimensional GC, industrial automatic explosion-proof devices, portable and preparative models. Two main trends in the development of gas chromatography Instrumentation are evident at present, miniaturization and specialization for specific applications.
Portable gas chromatographs
Issues of miniaturization are discussed in detail in the review [9]. Last years, new portable gas chromatographs were developed including, for example, FROG-4000 (weighting 2.5 kg) for the determination of volatile compounds and Canary-3 (weighting less than 2 kg) for determining moderately volatile organic compounds (both made by Defiant Techn, USA); Micro C2V-200 (made by "Neochrom", Ukraine), Mars-400 (China). Among Russian portable GC, one can highlight models FGH-1 and AHT-TI ("Microsensor technologies", Moscow) as well as ECHO-V FID and ECHO-EW (Novosibirsk). The latest achievement in the Russian Federation is the gas microchromatograph "PIA" invented by Prof. I.A.Platonov (weighting only 0.8 kg) with the analysis time of 1.5 minutes and power consumption of only 10W.
Specialized gas chromatographs
Chromatographs are universal instruments by nature. One chromatograph can perform thousands or even tens of thousands of different tests by changing only the column and the detector. Today, however, specialized gas chromatographs attract even more interest. Such equipment is produced, in particular, by "Interlab". It includes a poison analyzer "Maestro GC-MS" for the analysis of drugs and narcotics with the built-in mass spectra database as well as microbiological analyzer "Maestro GC-MS II". This instrument was developed in Russia and registered as a new medical technology (Certificate of Federal Service for Surveillance in Healthcare (Roszdravnadzor) No.2010/038 dated 24.02.2010). The method and instrument have passed approval procedure and now are effectively used in many medical institutions. The method is based on precise determination of microorganism markers (fatty acids, aldehydes, alcohols) in any clinical sample using the GC-MS technique. It allows determining 57 microorganisms during 35 minutes.
Leading developers abroad specialize chromatographs to study human metabolism, metabolite libraries (up to 40,000) for early diagnostics of many diseases being already created. Gas chromatographs are adapted for analysis of disease markers, in particular, markers of cancer, in human exhaled air, e.g. OralChromaTM breath analysis devices and similar). The chromatographic analyzers are developed for industrial production control, in particular, gas analyzers of GC866 series. Gas chromatographs can be modified to perform the elemental analyzes.
Thus, GC-controlled software packages for simulated distillation (SIMDIS) calculations are developed to describe carbon, nitrogen, and sulfur (CNS) atoms distribution across fractions of crude oil and oil products with various boiling points.
Industrial gas chromatographs
The specifics of on-line industrial chromatographs are in a fully unmanned operation and explosion-proof design. Now, such devices are produced in our country by many firms (see Table 3). The industrial gas chromatographs are installed directly in plants to monitor and control the manufacturing processes. Of the foreign companies, the most prominent are Yokogawa (Japan), АВВ (USA), and Siemens (Germany). There emerged the portable industrial gas chromatographs as well.
Liquid chromatographs
The first commercial liquid chromatographs had emerged in the years 1968–1970, i.e. the branch of liquid chromatographic instrument engineering is only about 45 years old. The time lag between the liquid chromatography method discovery by M. S. Tsvet in 1903 and start of commercial liquid chromatograph production is hence huge, reaching about 65–67 years.
It is connected with the fact that the demand in analysis of high-molecular biological or synthetic compounds arose only by the seventies due to rapid development of biochemistry, biotechnology, pharmaceutics, biopolymers, and synthetic polymers. Gas chromatography was not suited for this purpose, because it allows analyzing only volatile compounds that do not decompose in vapor phase. These are generally substances with molecular weights of 500–700. Methods of reaction, pyrolysis, or high-temperature gas chromatography were unable to extend analytical capabilities over demanded range of high-molecular compounds [3].
Then specialists had tried to apply the liquid chromatography, but this technique was very slow, the separation lasted for several hours. It was necessary to accelerate both external and internal mass transfer between the grains and inside the intragranular pores. Diffusion in a liquid phase was by 4–5 orders of magnitude slower than in a gas phase. The most natural way was in reducing the sorbent grain size.
However, grain size reduction by orders of magnitude (typically from 100 microns to 5 microns) dramatically increased the hydraulic resistance of columns, thus making necessary introduction of high-pressure pumps. So high-pressure liquid chromatographs (HPLC) first appeared. Performance of columns increased dramatically, and the method was called "High Performance Liquid Chromatography" (HPLC).
Owing to rapid separation, HPLC found a wide use in analytical practice. This period was called the renaissance of liquid chromatography. To provide the highly efficient separation in liquid chromatography, specific conditions were required such as high-pressure pumps able to create the liquid head up to 200–400 bar, detectors with flow-through cells, and mechanically strong sorbents with particle size of 5–10 microns.
First liquid chromatograph was developed by DuPont Company in 1968. However, this company stopped the production after few years due to inability to compete with other companies. Nevertheless, techniques and instrumentation developed with the rate as high as 14% per year. Consequently, the volume of liquid chromatograph production in 30 years twice surpassed the gas chromatograph production volume. HPLC attracted permanently increasing interest. This was not surprising, since HPLC enables to analyze compounds with molecular weights from 50 to several millions, by separating molecules, macromolecules, ions, and microparticles. Areas of HPLC application are much wider than GC. Today these techniques are mutually complementary.
HPLC advanced due to implementation of new detecting and separating techniques as well as new selective sorbents. Up-to-date liquid chromatographs are equipped with kits of various detectors, both all-purpose and selective. Among most widespread are mass-spectrometry (MS) or diode-matrix detectors (DMD). These detectors are 3D and hence permit to identify unknown compounds in mixtures. Other widely applied detectors include spectrophotometric in UV/visible range (190–900 nm) or UV range (190–360, 220, 254, or 280 nm); fluorimetric; electrochemical (amperometric, coulometric, or conductometric); refractometric, and light scattering detectors.
In the case of HPLC, amperometric and coulometric detectors possess a unique combination of high sensitivity (in particular, low detecting threshold) and high selectivity to polyphenols. Under certain conditions, these detectors can selectively determine sugars and amino acids with the high sensitivity. If signals are detected at different potentials, such detectors become three-dimensional, thus allowing to identify unknown compounds. Below are the latest achievements in the development of new detection systems for HPLC:
• MS detectors (triple quadrupole, time-of-flight (TOF), Orbitrap system based, etc.) with limits of detection (LD) as small as femtograms or even attograms (10–15 – 10–18 g);
• Amperometric and coulometric detectors with LD as small as femtograms or even attograms too;
• Light scattering detectors (laser ray in the nuclei condensation mode);
• Charged aerosol based detectors;
• UV-photometric or spectrophotometric (SPP) detectors for "Light-pipe" technology with optical path length of 40–60 mm;
• LED photometric detector in the range of 230–2500 nm with simultaneous registration at 7 wavelengths (UV, visible, near IR) and "Light-pipe" cell technology (Interlab).
One of greatest achievements in the field was development and production of liquid chromatographs for the Ultra HPLC (UHPLC). These instruments use columns filled with microparticles with less than 2 microns in size, which requires application of very high pressure (up to 1000–1500 bar) pumps. Such columns ensure much more rapid (by 2–3 times) separation than columns with 5-micron particles, higher efficiency, and minimal blurring. This is undoubtedly a high technological and scientific achievement, but it requires equipment that is much more expensive. Recently it is shown that UHPLC may be replaced with a conventional HPLC, if 3-micron particles in its column have a surface porosity [11].
Two more achievements consisted in the creation of two-dimensional systems for HPLC as well as micro-HPLC [12] or even nanoHPLC systems [13, 14]; in particular, the splitless nanoflow chromatograph Proxeon Easy HPLC should be mentioned.
Of great interest is HPLC-GC combination. In this case, HPLC plays role of most highly effective "dirty" sample preparation before separation in the GC.
List of commercial domestic and foreign liquid chromatographs is presented in Tables 4 and 5.
Ionic chromatographs
The method of ion-exchange chromatography is known since the thirties. It turned out (after the declassification of secret materials) that this method had been effectively used in the closed Manhattan Project to separate some of isotopes in pure form. Its analytical application started in 1975 after the method of ion chromatography (IC) had been proposed as an analytical version of the ion-exchange chromatography (IOC) [15]. First ion chromatographs were developed as early as in the years 1976–1977 [15, 16]. Main differences of ion chromatography from IOC consist in a faster separation and higher sensitivity. The method of ion-exchange chromatography uses a strong electrolyte as the eluent, which in the case of conductometric detector creates a large background signal. However, real sensitivity in chromatography is determined by the signal-to-noise ratio.
It was therefore proposed by authors [15] to connect in series a second column downstream the main separation column in order to eliminate (or suppress) the background signal and thereby increase the sensitivity. To date, a lot of various background suppression units have been developed, including miniature ones. A review on instrumental and methodological advances in ion chromatography was published by present authors in the International Encyclopedia of Analytical Chemistry [17].
Most great successes in the development of ion chromatographs have been achieved by firms Dionex (USA) and Metrohm (Switzerland).
The UV and amperometric detectors are used in addition to conductometric one. Cations are separated by cation exchange columns while anions by the anion exchange ones.
In the former USSR, the first ion chromatographs Tsvet-3006 and Tsvet-3007 (two-channel) as well as portable HPI-1 were created in 1981–1983 by R&P Association "Khimavtomatika" (Dzerzhinsk). Among them, models Tsvet-3006 and HPI-1 had served to customers during over 20 years. R&P Association "Khimavtomatika" (Moscow) produced ion chromatographs Tsvet-Yauza-02 in the period of 2006–2012. Today, Russian ion chromatographs "Stayer A" are available from CJSC Akvilon (Moscow). This company, along with Metrohm, has developed and now produces industrial ion chromatographs for analysis of aqueous media at nuclear power plants.
The national standard (GOST) has been issued in our country to regulate determination of fluorides, chlorides, nitrates, sulfates, and phosphates in drinking and surface waters using the IC technique.
The latest achievements in the development of new ion chromatographs include:
• Reagentless ion chromatography system Dionex ICS-5000;
• Capillary ion chromatographs Dionex IC 4000;
• Climate change study using ion chromatography technique (ice core analysis);
• Portable ion chromatograph 930 Compact IC;
• Reduction in limits of detection allowing determining ions in ultrapure water.
Preparative chromatographs
Preparative chromatography is used to separate many substances in a pure form. Especially great progress has been made in separation and isolation of biologically active compounds, including optically active isomers. Today, 70% of existing medicaments have optical isomers. In many countries, legislation requires study of L or D optical isomer impact on the human body, for which these isomers must be separated and isolated in pure form.
Key achievements in preparative chromatographs production are as follows:
• Flash systems for fast isolation of commercial-purity substances;
• ContichromTM systems for flexible continuous chromatography; Method for purification of biopharmaceutical substances by multicolumn counter-current gradient chromatography;
• Pilot automated industrial plants "Axioma" (CJSC "BioKhimMak ST", Moscow). The pilot plant "Axioma HP" allows to isolate kilogram-scale quantities of highly purified substances in the mode of isocratic and/or multistage binary gradient elution, spectrophotometric detection, automatic sample injection, and automatic collection of separated fractions (domestic innovation under the guidance of prof. S. M. Staroverov);
• Recycling preparative HPLC system for separation of natural products, fullerenes, and hydrophobic compounds (Japan Analytical Industry Co.). Completely automated preparative liquid chromatography. Models LC-9601, LC-9014, LC-9201, LC-9102/9103. Separation up to 20 g per day. UV or refractometric detector;
• Preparative supercritical fluid chromatographs SFC-MS Prep 15/30/100. Pressure up to 300 bar, temperature up to 90°С. Capacity 15/30/100 mg per hour.
Advances in development of new methods and instruments for chromatography combined with mass spectrometry
According to PITTCON data, the most widely used today are gas and liquid chromatographs combined with mass spectrometers (GC-MS and LC-MS, respectively) [10]. The mass spectrometers were found the ideal detectors for chromatography, because they allow to carry out simultaneously both quantitative and qualitative analysis. Unknown mixture components are identified using mass spectra recorded under standard conditions (mainly electron impact ionization at 70 eV). The following mass spectra libraries for gas chromatography are known:
• Nist Library containing over 240,000 mass spectra and linear retention indexes (LRI) data;
• Wiley Library containing over 390,000 mass spectra;
• Pfleger/ Maurer/ Webel (M.R.W.) Library containing about 7,840 mass spectra of medicaments, narcotics, pesticides, and products of their metabolism;
• Pesticide Library containing 578 mass spectra of pesticides (electron impact ionization) plus 383 mass spectra of pesticides (chemical ionization);
• Metabolomics Library (GC-MS metabolite mass spectra database).
The combination of mass spectra with retention index data increases the accuracy of identification. A large contribution to this field was made by I.A.Zenkevich, professor of St. Petersburg University. In particular, the National Institute of Standards (NIST, USA) used his data to gather the retention index library.
The world’s first desktop gas chromatograph combined with mass spectrometer HP 5992A was been made by Hewlett-Packard company in 1976. At present, mass spectrometers for gas chromatographs are available with the following mass separation systems: quadrupole, ion trap, time of flight, and orbital trap (obrbitrap). Five years ago, some companies began to produce chromatographs combined with mass spectrometers (either gas or liquid) with a triple quadrupole. The first of three sequentially arranged quadrupole cells serves to selectively separate ions with the specified mass for fragmentation; the second cell provides the fragmentation by ions collision, and the third cell plays role of mass analyzer for the fermentation products.
Russian scientists have made a significant contribution into development of chromatography combined with mass spectrometry. In particular, A.A.Makarov invented the orbital ion trap with Fourier transformation (obrbitrap) [Makarov A.A. Mass spectrometers: US Patent 5886346 issued in 1999]. In 2005, US Thermo Scientific Company in cooperation with A.A.Makarov designed a serial mass spectrometer with orbital trap and started production of relevant gas and liquid chromatographs. In 2011, the device was upgraded by adding a high-field trap. Several thousands of these devices were produced [18]. The tribrid MS Orbitrap Fusion (made by Thermo Scientific) combined with the nanoflow chromatograph UltiMate 3000 RLS C demonstrated a record resolution of 450,000. In 2012, the highest award of the International Mass Spectrometry Society, Thomson medal, was awarded to A. A. Makarov for his great achievements in the field of gas chromatography combined with mass spectrometry.
The team headed by A.N.Verenchikov had designed and patented (Verenchikov A.N. et. al. US Patent 7385187) the scheme of multiple reflecting time-of-flight mass spectrometer with planar gridless ion mirrors. The patent was acquired by LECO Company and the multiple reflecting mass spectrometer based on the folded flight path (FFP) principle was designed under the guidance of A.N.Verenchikov. The total flight path length of 20 m provides a resolution of 25,000 while the total flight path length of 40 m yield ultrahigh resolution at the level of 50,000. This time-of-flight mass spectrometer Pegasus GC-HRT was awarded with the Gold Medal at the 2012 Pittsburgh Conference. Previously, A.N.Verenchikov had worked at the Institute for analytical instrumentation of RAS in the group headed by L.N.Gall, which first developed the electrospray ionization method.
I.A.Revelsky, professor of Moscow State University, has developed a method for photochemical ionization at atmospheric pressure. This method allows determining as small quantities of analyte as few femtograms. At present, it finds even more recognition among experts [19].
Russian scientist R.A.Zubarev working now at the Karolinska University in Sweden, was recognized by international scientific community for his achievements in the field of mass spectrometry of biomolecules, development of new ion activation methods and mass spectrometry equipment [19, 20].
Achievements of many other national specialists in the field of gas chromatography combined with mass spectrometry should be noted too, namely:
• Systems for direct analysis in real time with tentacle cation exchange columns (DART+TCX), in which analytes are transferred into the ionized state immediately in thin-layer chromatography (TLC) plate (M. V. Ovcharov, G. A. Kalabin);
• Matrix-assisted laser desorption ionization (MALDI) method immediately in TCX plates as well, in particular for the analysis of pharmaceuticals (D.I.Zhilyaev);
• DART method for direct analysis of pharmaceuticals in real time (E.S.Chernetsova);
• Original method for the selective extraction of antioxidants – phenol carboxylic acids from serum followed by GC-MS determination proposed by A.I.Revelsky;
• Method of polymer- or matrix-assisted laser desorption ionization (PALDI / MALDI) mass spectrometry application for studying the metalliferous cluster ions on various surfaces contributed by A.K.Buryak et al. (A.N.Frumkin Institute of Physical Chemistry and Electrochemistry of RAS);
• Software selection criteria during database search for mass spectra of electron ionization developed by I.A.Revelsky and A. S. Samokhin;
• Method for determining a food production region and revealing counterfeits using ratios of stable isotopes of carbon, nitrogen, oxygen, and hydrogen at the places of their natural concentration suggested by A.Y.Kolesnov (Moscow State University of Food Production).
The liquid chromatographs combined with mass spectrometers were developed much later than GC-MS. Only at the beginning of 1990s, a real breakthrough was made: HPLC chromatograph was combined with a mass spectrometer. Later, advance in HPLC-MS was contributed mainly by new compound ionization methods. Two primary substance ionization methods in LC-MS, electrospray and matrix-assisted laser desorption ionization (MALDI), were developed. The Nobel Prize in Chemistry 2002 was awarded to J.Fenn and K. Tanaka for the development of these methods. But, for the sake of fairness, it should be noted that the electrospray method had been developed by L.N.Gall in Institute for analytical instrumentation of the USSR Academy of Sciences much earlier, as far ago as in 1982. The priority of L.N.Gall in this area was recognized by experts.
Volumes of chromatographic equipment sales
According to data of European directory 2000/2001 (LC-GC Europe 2000, Vol.13, No. 8, P. 586), gas chromatographs were produced by 66 companies and liquid chromatographs by 69 campaigns in those years. Table 5 shows the amounts of sales for certain types of chromatographic equipment, which were taken from the Instrumenta journals (formerly Journal of Analytical Instrument Industry Report).
It is seen that annual sales of gas chromatographs amounted over 30 thousand units during 30 years. It could be said with certainty that about 1.5 million gas chromatographs were produced totally for 60 years.
The number of liquid chromatographs sold for over 45 years was at least 1 million units. Taking into account other chromatograph types such as ionic, portable, industrial, and others, one may assume that the total number of all types of chromatographs produced is about 3 million. The model Hewlett-Packard 5890 should be recognized as the most reliable foreign device since it had been produced with only slight modifications during 20 years and amounted as many as over 10,000 units. Even after production termination, about 6000 of these units operated in European laboratories for a long time.
The most reliable devices in our country were Tsvet-4, Tsvet-100, and the first models Tsvet-500. The instruments Tsvet-100 and Tsvet-500 worked in central factory laboratories (CFL) of various enterprises mainly for over 20–25 years. But the record was established by the model Tsvet-4: it was documented this instrument had been working at Shchelkovo and "Akron" chemical plants for 40 years. In the former USSR, the main consumers of gas chromatographs were chemical research institutes and CFL of chemical enterprises. There were many enterprises, which had applied 100 or more chromatographs. Probably, Kirovo-Chepetsk chemical plant was the champion among them because, according to data of Z.L.Baskin, there were installed at least 500 chromatographs. At least 400– 500 chromatographs operated at many of Bashkirian chemical plants.
Table 6 shows sales of chromatographic equipment, accessories, columns, and services in 2013 and 2014.
It should be noted that chromatographic equipment sales volume is constantly increasing in spite of the crisis processes in the world.
Conclusion
Chromatographs are widely used in all vital areas of human activity: control of environmental pollution and food quality; early detection of diseases and medicament quality control in medicine; forensic medicine, and monitoring of many industrial processes. Only chromatographic methods enable to determine supertoxicants (pesticides, mycotoxins, polycyclic aromatic hydrocarbons, and others) in food products.
Hundreds of standard techniques are issued to regulate determining substances using chromatography.
Main recent results achieved using chromatographic methods
• Decoding of the human genome;
• Separation of multicomponent protein mixtures for purposes of proteomics;
• Analysis of a single cell contents;
• Establishment of a medicament pharmacokinetics;
• Early detection of diseases by analysis of biochemical markers;
• Separation of optical isomers, analysis of enantiomeric purity of medicaments;
• Doping control at the world championships and the Olympic Games;
• Analysis within judicial and forensic examinations; analysis of medicaments and narcotics;
• Monitoring of environmental pollution, including supertoxicants (dioxins, polychlorinated biphenyls, polynuclear aromatic compounds at the ppt level);
• Monitoring of contaminants in food and beverages including most dangerous carcinogenic mycotoxins, nitrosamines, pesticides, etc.;
• Analysis of the components of smells and flavors of foods and drinks;
• Analysis of natural gas, gas condensate, gasoline, kerosene, petroleum products and crude oil, enabling simulated distillation of crude oil;
• Separation and analysis of metals including transuranic elements;
• Full analysis of atmosphere composition in the polluted cities;
• Analysis of chlorofluorocarbons (freons) in stratosphere;
• Analysis of insect pheromones;
• Analysis of soils and atmospheres of other planets (Venus, Mars, lunar soil), and
• Analysis of isotope-substituted compounds.
General trends in the development of chromatography techniques and instruments [21]:
• Improving selectivity of separation;
• Improving performance of columns;
• Improving rapidity of separation;
• Miniaturization of hardware;
• Updating the software for both instrument control and separation and analysis data processing.
The chromatography was discovered by M.S.Tsvet in 1903. For nearly half a century, it was used to separate individual substances, extract the separated components in isolated form, and purify them. All that time, chromatography technique remained virtually the same as it had been proposed by M.S.Tsvet, namely, there were no detecting systems at the column outlet or forced feed of the mobile phase, this phase flowed under gravitational supply pressure.
In 1955, first serial gas chromatographs had been commercialized that allowed applying the chromatography technique for analytical purposes. It was the revolutionary event. Chromatographs had developed and won more and more spheres of application so rapidly that by 1960 gas chromatographic method had become one of the main ways to analyze complex multicomponent mixtures, especially in the petrochemical industry. Later, first liquid chromatographs were developed in 1968 (DuPont), then ion chromatographs in 1977 (Dionex), and finally gas chromatographs combined with mass spectrometers (GC-MS systems) (Hewlett-Packard) [1–3].
Chromatographs are very versatile in design: there are laboratory, automated industrial, movable, portable, microprocessor-controlled, specialized devices, e.g. chromatographs for space exploration. According to some global estimations, over 3 million chromatographs have been produced around the world. The sales volume of the chromatographic equipment and supplies is estimated at $ 14 billion in 2015. More than 60% of all chemical analyzes in different countries around the world are conducted today using chromatographs, although there are dozens of other methods of analysis.
These successes of analytical chromatography were associated with the development of advanced chromatographic equipment. Mass introduction of a particular chromatography technique into analytical practice was caused by mass production of the relevant chromatograph type. The amino acid analyzer that was invented by Stanford Moore and William H. Stein in 1956 based on ion exchange chromatography, which enabled a mass amino acid analysis, can serve as a most bright example.
Performance of chromatographic instruments has reached a very high level today due to full automation of process control and treatment of results as well as implementation of latest achievements in electronics, precision mechanics, pneumatics, planar technology, and optics. During their 60-year history, chromatographs were sequentially analog, digital, and microprocessor-controlled.
It should be noted that a key role in the analytical capabilities of chromatographs is played by chromatographic columns. In particular, chromatography became usable for separation and analysis of optical isomers in chiral chromatography only due to development of appropriate sorbents and columns. This feature is especially important in modern pharmaceutics since over 70% of all drugs are optically active. At the same time, legislation in many countries requires examination of the impact of both L-isomers and D-isomers of amino acids on a human body.
Chromatography was recognized as the main analytical method of the twentieth century and included into twenty outstanding discoveries of the last century, which had the most impact on human life. In addition, chromatography obviously promoted progress in many other areas of science, technology, and various industries.
The status of chromatographic techniques and instruments was elucidated in scientific collections issued to the centennial chromatography jubilee [4–5], some other latest publications [6], comprehensive encyclopedia of chromatography [7] as well as scientometric review of publications on chromatography for 20 years [8]. As the big book of chromatography [5] states, the chromatography provides the link between science and technologies, especially biotechnologies. The biotechnological enterprises are among most rapidly growing ones (numbering over 5000 in the US and over 5000 in Europe).
Gas chromatographs
First commercial gas chromatographs were delivered in US by three firms, Perkin-Elmer, Burrell Corporation, and Podbielniak in 1955. In 1956, another five US companies presented their developments. The most perfect and successful first commercial model P-E 154 Vapor Fractometer of the company Perkin-Elmer was most popular among consumers during seven years [1, 2].
Thereafter, the development and production of gas chromatographs (GC) grew very rapidly: in 1955 they were produced by 3 companies; in 1956 by 7 companies; in 1961 by 16 companies; in 1962 by 23 companies, and in 2000 by 69 companies (among them, 29 companies in the United States; 10 in USSR or Russia; 9 in UK; 3 in Germany; 3 in Japan; 3 in the Netherlands; 2 in Italy; 2 in China, and one in each of the following countries: France, Spain, Denmark, India, and Ukraine (data from LC-GC Europe 2000/2001, Vol.13, No. 8, P. 586). In Japan, first commercial gas chromatographs were released in 1956–1957 (Shimadzu, and Hitachi). In former USSR, first serial gas chromatographs HT2 and HT2M were produced by All-Union R&D institute for complex automation of oil and gas industry in 1958; advanced models UH-1 and UH-2 appeared in 1960. UK companies also developed gas chromatographs in the years 1956–1958, the most popular being argon gas chromatograph manufactured by Pye Unicam since 1958. Italian company Carlo Erba presented its first gas chromatograph Fractovap with capillary columns in 1960. The first industrial chromatograph RH-1 with a flame ionization detector was released in the former USSR [1, 2].
The fast development of gas chromatograph engineering was assisted by invention of new devices such as highly sensitive ionization detectors, temperature programming modules, and capillary columns.
Key achievements in that period consisted in implementation of electronic modules for gas flow velocity and pressure stabilization and control as well as two-dimensional gas chromatography layouts.
Now commercial gas chromatographs are equipped with various detectors, mainly mass spectrometry, flame ionization, electron capture, flame-photometric, photo-ionizing, thermionic, or thermal conductivity based ones. Less commonly used are chemiluminescent, infrared, atomic emission detectors. Among them, there are either all-purpose (thermal conductivity based, atomic emission, and mass spectrometry) or selective detectors. In particular, the flame ionization detector (FID) is sensitive only to organic compounds; photo-ionizing detector can operate only with compounds having the ionization potential less than about 10–11 eV; electron capture detector has especially high sensitivity to halogen-organic compounds; in contrary, thermionic detector is selective in relation to nitrogen or phosphorus-containing compounds whereas the flame-photometric detector (FPD) to the sulfur or phosphorus-containing compounds while the chemiluminescent detector responds only to sulfur or nitrogen-containing compounds. Selective detectors have a great advantage when analyzing impurities in complex mixtures. It is possible to create conditions such that the impurities were determined whereas the basic substances in the mixture were omitted. For example, the electron capture detector is very convenient to measure organochlorine pesticides in the environment.
To enhance analytical capabilities, gas chromatographs are equipped with optional accessories. Solid-phase, micro-solid-phase, supercritical-liquid, and gas extraction devices are used for sample concentration; special systems perform blowing and trapping; autosamplers, cryo-focusing traps, and gas cocks serve for sample input or injection; heating units are required for pyrolysis; derivatizing systems (in particular, methanators or similar devices) are applied for pre-column or post-column derivatization, etc.
List of commercial domestic and foreign gas chromatographs is presented in Tables 1 and 2.
In China, chromatographs are produced primarily at two major instrument-making plants in Beijing and Shanghai.
In certain periods, chromatographs were produced in former Czechoslovakia ("Chrome" series, the company "Laboratory Instruments"), Finland (the company "Orion Analytics"), as well as Poland, Bulgaria, and the German Democratic Republic (the company Willy-Gide).
By beginning of 21st century, production of a complete gas chromatographs (GC) line was established including laboratory instruments, GC-MS systems, on-line analyzers, multi-dimensional GC, industrial automatic explosion-proof devices, portable and preparative models. Two main trends in the development of gas chromatography Instrumentation are evident at present, miniaturization and specialization for specific applications.
Portable gas chromatographs
Issues of miniaturization are discussed in detail in the review [9]. Last years, new portable gas chromatographs were developed including, for example, FROG-4000 (weighting 2.5 kg) for the determination of volatile compounds and Canary-3 (weighting less than 2 kg) for determining moderately volatile organic compounds (both made by Defiant Techn, USA); Micro C2V-200 (made by "Neochrom", Ukraine), Mars-400 (China). Among Russian portable GC, one can highlight models FGH-1 and AHT-TI ("Microsensor technologies", Moscow) as well as ECHO-V FID and ECHO-EW (Novosibirsk). The latest achievement in the Russian Federation is the gas microchromatograph "PIA" invented by Prof. I.A.Platonov (weighting only 0.8 kg) with the analysis time of 1.5 minutes and power consumption of only 10W.
Specialized gas chromatographs
Chromatographs are universal instruments by nature. One chromatograph can perform thousands or even tens of thousands of different tests by changing only the column and the detector. Today, however, specialized gas chromatographs attract even more interest. Such equipment is produced, in particular, by "Interlab". It includes a poison analyzer "Maestro GC-MS" for the analysis of drugs and narcotics with the built-in mass spectra database as well as microbiological analyzer "Maestro GC-MS II". This instrument was developed in Russia and registered as a new medical technology (Certificate of Federal Service for Surveillance in Healthcare (Roszdravnadzor) No.2010/038 dated 24.02.2010). The method and instrument have passed approval procedure and now are effectively used in many medical institutions. The method is based on precise determination of microorganism markers (fatty acids, aldehydes, alcohols) in any clinical sample using the GC-MS technique. It allows determining 57 microorganisms during 35 minutes.
Leading developers abroad specialize chromatographs to study human metabolism, metabolite libraries (up to 40,000) for early diagnostics of many diseases being already created. Gas chromatographs are adapted for analysis of disease markers, in particular, markers of cancer, in human exhaled air, e.g. OralChromaTM breath analysis devices and similar). The chromatographic analyzers are developed for industrial production control, in particular, gas analyzers of GC866 series. Gas chromatographs can be modified to perform the elemental analyzes.
Thus, GC-controlled software packages for simulated distillation (SIMDIS) calculations are developed to describe carbon, nitrogen, and sulfur (CNS) atoms distribution across fractions of crude oil and oil products with various boiling points.
Industrial gas chromatographs
The specifics of on-line industrial chromatographs are in a fully unmanned operation and explosion-proof design. Now, such devices are produced in our country by many firms (see Table 3). The industrial gas chromatographs are installed directly in plants to monitor and control the manufacturing processes. Of the foreign companies, the most prominent are Yokogawa (Japan), АВВ (USA), and Siemens (Germany). There emerged the portable industrial gas chromatographs as well.
Liquid chromatographs
The first commercial liquid chromatographs had emerged in the years 1968–1970, i.e. the branch of liquid chromatographic instrument engineering is only about 45 years old. The time lag between the liquid chromatography method discovery by M. S. Tsvet in 1903 and start of commercial liquid chromatograph production is hence huge, reaching about 65–67 years.
It is connected with the fact that the demand in analysis of high-molecular biological or synthetic compounds arose only by the seventies due to rapid development of biochemistry, biotechnology, pharmaceutics, biopolymers, and synthetic polymers. Gas chromatography was not suited for this purpose, because it allows analyzing only volatile compounds that do not decompose in vapor phase. These are generally substances with molecular weights of 500–700. Methods of reaction, pyrolysis, or high-temperature gas chromatography were unable to extend analytical capabilities over demanded range of high-molecular compounds [3].
Then specialists had tried to apply the liquid chromatography, but this technique was very slow, the separation lasted for several hours. It was necessary to accelerate both external and internal mass transfer between the grains and inside the intragranular pores. Diffusion in a liquid phase was by 4–5 orders of magnitude slower than in a gas phase. The most natural way was in reducing the sorbent grain size.
However, grain size reduction by orders of magnitude (typically from 100 microns to 5 microns) dramatically increased the hydraulic resistance of columns, thus making necessary introduction of high-pressure pumps. So high-pressure liquid chromatographs (HPLC) first appeared. Performance of columns increased dramatically, and the method was called "High Performance Liquid Chromatography" (HPLC).
Owing to rapid separation, HPLC found a wide use in analytical practice. This period was called the renaissance of liquid chromatography. To provide the highly efficient separation in liquid chromatography, specific conditions were required such as high-pressure pumps able to create the liquid head up to 200–400 bar, detectors with flow-through cells, and mechanically strong sorbents with particle size of 5–10 microns.
First liquid chromatograph was developed by DuPont Company in 1968. However, this company stopped the production after few years due to inability to compete with other companies. Nevertheless, techniques and instrumentation developed with the rate as high as 14% per year. Consequently, the volume of liquid chromatograph production in 30 years twice surpassed the gas chromatograph production volume. HPLC attracted permanently increasing interest. This was not surprising, since HPLC enables to analyze compounds with molecular weights from 50 to several millions, by separating molecules, macromolecules, ions, and microparticles. Areas of HPLC application are much wider than GC. Today these techniques are mutually complementary.
HPLC advanced due to implementation of new detecting and separating techniques as well as new selective sorbents. Up-to-date liquid chromatographs are equipped with kits of various detectors, both all-purpose and selective. Among most widespread are mass-spectrometry (MS) or diode-matrix detectors (DMD). These detectors are 3D and hence permit to identify unknown compounds in mixtures. Other widely applied detectors include spectrophotometric in UV/visible range (190–900 nm) or UV range (190–360, 220, 254, or 280 nm); fluorimetric; electrochemical (amperometric, coulometric, or conductometric); refractometric, and light scattering detectors.
In the case of HPLC, amperometric and coulometric detectors possess a unique combination of high sensitivity (in particular, low detecting threshold) and high selectivity to polyphenols. Under certain conditions, these detectors can selectively determine sugars and amino acids with the high sensitivity. If signals are detected at different potentials, such detectors become three-dimensional, thus allowing to identify unknown compounds. Below are the latest achievements in the development of new detection systems for HPLC:
• MS detectors (triple quadrupole, time-of-flight (TOF), Orbitrap system based, etc.) with limits of detection (LD) as small as femtograms or even attograms (10–15 – 10–18 g);
• Amperometric and coulometric detectors with LD as small as femtograms or even attograms too;
• Light scattering detectors (laser ray in the nuclei condensation mode);
• Charged aerosol based detectors;
• UV-photometric or spectrophotometric (SPP) detectors for "Light-pipe" technology with optical path length of 40–60 mm;
• LED photometric detector in the range of 230–2500 nm with simultaneous registration at 7 wavelengths (UV, visible, near IR) and "Light-pipe" cell technology (Interlab).
One of greatest achievements in the field was development and production of liquid chromatographs for the Ultra HPLC (UHPLC). These instruments use columns filled with microparticles with less than 2 microns in size, which requires application of very high pressure (up to 1000–1500 bar) pumps. Such columns ensure much more rapid (by 2–3 times) separation than columns with 5-micron particles, higher efficiency, and minimal blurring. This is undoubtedly a high technological and scientific achievement, but it requires equipment that is much more expensive. Recently it is shown that UHPLC may be replaced with a conventional HPLC, if 3-micron particles in its column have a surface porosity [11].
Two more achievements consisted in the creation of two-dimensional systems for HPLC as well as micro-HPLC [12] or even nanoHPLC systems [13, 14]; in particular, the splitless nanoflow chromatograph Proxeon Easy HPLC should be mentioned.
Of great interest is HPLC-GC combination. In this case, HPLC plays role of most highly effective "dirty" sample preparation before separation in the GC.
List of commercial domestic and foreign liquid chromatographs is presented in Tables 4 and 5.
Ionic chromatographs
The method of ion-exchange chromatography is known since the thirties. It turned out (after the declassification of secret materials) that this method had been effectively used in the closed Manhattan Project to separate some of isotopes in pure form. Its analytical application started in 1975 after the method of ion chromatography (IC) had been proposed as an analytical version of the ion-exchange chromatography (IOC) [15]. First ion chromatographs were developed as early as in the years 1976–1977 [15, 16]. Main differences of ion chromatography from IOC consist in a faster separation and higher sensitivity. The method of ion-exchange chromatography uses a strong electrolyte as the eluent, which in the case of conductometric detector creates a large background signal. However, real sensitivity in chromatography is determined by the signal-to-noise ratio.
It was therefore proposed by authors [15] to connect in series a second column downstream the main separation column in order to eliminate (or suppress) the background signal and thereby increase the sensitivity. To date, a lot of various background suppression units have been developed, including miniature ones. A review on instrumental and methodological advances in ion chromatography was published by present authors in the International Encyclopedia of Analytical Chemistry [17].
Most great successes in the development of ion chromatographs have been achieved by firms Dionex (USA) and Metrohm (Switzerland).
The UV and amperometric detectors are used in addition to conductometric one. Cations are separated by cation exchange columns while anions by the anion exchange ones.
In the former USSR, the first ion chromatographs Tsvet-3006 and Tsvet-3007 (two-channel) as well as portable HPI-1 were created in 1981–1983 by R&P Association "Khimavtomatika" (Dzerzhinsk). Among them, models Tsvet-3006 and HPI-1 had served to customers during over 20 years. R&P Association "Khimavtomatika" (Moscow) produced ion chromatographs Tsvet-Yauza-02 in the period of 2006–2012. Today, Russian ion chromatographs "Stayer A" are available from CJSC Akvilon (Moscow). This company, along with Metrohm, has developed and now produces industrial ion chromatographs for analysis of aqueous media at nuclear power plants.
The national standard (GOST) has been issued in our country to regulate determination of fluorides, chlorides, nitrates, sulfates, and phosphates in drinking and surface waters using the IC technique.
The latest achievements in the development of new ion chromatographs include:
• Reagentless ion chromatography system Dionex ICS-5000;
• Capillary ion chromatographs Dionex IC 4000;
• Climate change study using ion chromatography technique (ice core analysis);
• Portable ion chromatograph 930 Compact IC;
• Reduction in limits of detection allowing determining ions in ultrapure water.
Preparative chromatographs
Preparative chromatography is used to separate many substances in a pure form. Especially great progress has been made in separation and isolation of biologically active compounds, including optically active isomers. Today, 70% of existing medicaments have optical isomers. In many countries, legislation requires study of L or D optical isomer impact on the human body, for which these isomers must be separated and isolated in pure form.
Key achievements in preparative chromatographs production are as follows:
• Flash systems for fast isolation of commercial-purity substances;
• ContichromTM systems for flexible continuous chromatography; Method for purification of biopharmaceutical substances by multicolumn counter-current gradient chromatography;
• Pilot automated industrial plants "Axioma" (CJSC "BioKhimMak ST", Moscow). The pilot plant "Axioma HP" allows to isolate kilogram-scale quantities of highly purified substances in the mode of isocratic and/or multistage binary gradient elution, spectrophotometric detection, automatic sample injection, and automatic collection of separated fractions (domestic innovation under the guidance of prof. S. M. Staroverov);
• Recycling preparative HPLC system for separation of natural products, fullerenes, and hydrophobic compounds (Japan Analytical Industry Co.). Completely automated preparative liquid chromatography. Models LC-9601, LC-9014, LC-9201, LC-9102/9103. Separation up to 20 g per day. UV or refractometric detector;
• Preparative supercritical fluid chromatographs SFC-MS Prep 15/30/100. Pressure up to 300 bar, temperature up to 90°С. Capacity 15/30/100 mg per hour.
Advances in development of new methods and instruments for chromatography combined with mass spectrometry
According to PITTCON data, the most widely used today are gas and liquid chromatographs combined with mass spectrometers (GC-MS and LC-MS, respectively) [10]. The mass spectrometers were found the ideal detectors for chromatography, because they allow to carry out simultaneously both quantitative and qualitative analysis. Unknown mixture components are identified using mass spectra recorded under standard conditions (mainly electron impact ionization at 70 eV). The following mass spectra libraries for gas chromatography are known:
• Nist Library containing over 240,000 mass spectra and linear retention indexes (LRI) data;
• Wiley Library containing over 390,000 mass spectra;
• Pfleger/ Maurer/ Webel (M.R.W.) Library containing about 7,840 mass spectra of medicaments, narcotics, pesticides, and products of their metabolism;
• Pesticide Library containing 578 mass spectra of pesticides (electron impact ionization) plus 383 mass spectra of pesticides (chemical ionization);
• Metabolomics Library (GC-MS metabolite mass spectra database).
The combination of mass spectra with retention index data increases the accuracy of identification. A large contribution to this field was made by I.A.Zenkevich, professor of St. Petersburg University. In particular, the National Institute of Standards (NIST, USA) used his data to gather the retention index library.
The world’s first desktop gas chromatograph combined with mass spectrometer HP 5992A was been made by Hewlett-Packard company in 1976. At present, mass spectrometers for gas chromatographs are available with the following mass separation systems: quadrupole, ion trap, time of flight, and orbital trap (obrbitrap). Five years ago, some companies began to produce chromatographs combined with mass spectrometers (either gas or liquid) with a triple quadrupole. The first of three sequentially arranged quadrupole cells serves to selectively separate ions with the specified mass for fragmentation; the second cell provides the fragmentation by ions collision, and the third cell plays role of mass analyzer for the fermentation products.
Russian scientists have made a significant contribution into development of chromatography combined with mass spectrometry. In particular, A.A.Makarov invented the orbital ion trap with Fourier transformation (obrbitrap) [Makarov A.A. Mass spectrometers: US Patent 5886346 issued in 1999]. In 2005, US Thermo Scientific Company in cooperation with A.A.Makarov designed a serial mass spectrometer with orbital trap and started production of relevant gas and liquid chromatographs. In 2011, the device was upgraded by adding a high-field trap. Several thousands of these devices were produced [18]. The tribrid MS Orbitrap Fusion (made by Thermo Scientific) combined with the nanoflow chromatograph UltiMate 3000 RLS C demonstrated a record resolution of 450,000. In 2012, the highest award of the International Mass Spectrometry Society, Thomson medal, was awarded to A. A. Makarov for his great achievements in the field of gas chromatography combined with mass spectrometry.
The team headed by A.N.Verenchikov had designed and patented (Verenchikov A.N. et. al. US Patent 7385187) the scheme of multiple reflecting time-of-flight mass spectrometer with planar gridless ion mirrors. The patent was acquired by LECO Company and the multiple reflecting mass spectrometer based on the folded flight path (FFP) principle was designed under the guidance of A.N.Verenchikov. The total flight path length of 20 m provides a resolution of 25,000 while the total flight path length of 40 m yield ultrahigh resolution at the level of 50,000. This time-of-flight mass spectrometer Pegasus GC-HRT was awarded with the Gold Medal at the 2012 Pittsburgh Conference. Previously, A.N.Verenchikov had worked at the Institute for analytical instrumentation of RAS in the group headed by L.N.Gall, which first developed the electrospray ionization method.
I.A.Revelsky, professor of Moscow State University, has developed a method for photochemical ionization at atmospheric pressure. This method allows determining as small quantities of analyte as few femtograms. At present, it finds even more recognition among experts [19].
Russian scientist R.A.Zubarev working now at the Karolinska University in Sweden, was recognized by international scientific community for his achievements in the field of mass spectrometry of biomolecules, development of new ion activation methods and mass spectrometry equipment [19, 20].
Achievements of many other national specialists in the field of gas chromatography combined with mass spectrometry should be noted too, namely:
• Systems for direct analysis in real time with tentacle cation exchange columns (DART+TCX), in which analytes are transferred into the ionized state immediately in thin-layer chromatography (TLC) plate (M. V. Ovcharov, G. A. Kalabin);
• Matrix-assisted laser desorption ionization (MALDI) method immediately in TCX plates as well, in particular for the analysis of pharmaceuticals (D.I.Zhilyaev);
• DART method for direct analysis of pharmaceuticals in real time (E.S.Chernetsova);
• Original method for the selective extraction of antioxidants – phenol carboxylic acids from serum followed by GC-MS determination proposed by A.I.Revelsky;
• Method of polymer- or matrix-assisted laser desorption ionization (PALDI / MALDI) mass spectrometry application for studying the metalliferous cluster ions on various surfaces contributed by A.K.Buryak et al. (A.N.Frumkin Institute of Physical Chemistry and Electrochemistry of RAS);
• Software selection criteria during database search for mass spectra of electron ionization developed by I.A.Revelsky and A. S. Samokhin;
• Method for determining a food production region and revealing counterfeits using ratios of stable isotopes of carbon, nitrogen, oxygen, and hydrogen at the places of their natural concentration suggested by A.Y.Kolesnov (Moscow State University of Food Production).
The liquid chromatographs combined with mass spectrometers were developed much later than GC-MS. Only at the beginning of 1990s, a real breakthrough was made: HPLC chromatograph was combined with a mass spectrometer. Later, advance in HPLC-MS was contributed mainly by new compound ionization methods. Two primary substance ionization methods in LC-MS, electrospray and matrix-assisted laser desorption ionization (MALDI), were developed. The Nobel Prize in Chemistry 2002 was awarded to J.Fenn and K. Tanaka for the development of these methods. But, for the sake of fairness, it should be noted that the electrospray method had been developed by L.N.Gall in Institute for analytical instrumentation of the USSR Academy of Sciences much earlier, as far ago as in 1982. The priority of L.N.Gall in this area was recognized by experts.
Volumes of chromatographic equipment sales
According to data of European directory 2000/2001 (LC-GC Europe 2000, Vol.13, No. 8, P. 586), gas chromatographs were produced by 66 companies and liquid chromatographs by 69 campaigns in those years. Table 5 shows the amounts of sales for certain types of chromatographic equipment, which were taken from the Instrumenta journals (formerly Journal of Analytical Instrument Industry Report).
It is seen that annual sales of gas chromatographs amounted over 30 thousand units during 30 years. It could be said with certainty that about 1.5 million gas chromatographs were produced totally for 60 years.
The number of liquid chromatographs sold for over 45 years was at least 1 million units. Taking into account other chromatograph types such as ionic, portable, industrial, and others, one may assume that the total number of all types of chromatographs produced is about 3 million. The model Hewlett-Packard 5890 should be recognized as the most reliable foreign device since it had been produced with only slight modifications during 20 years and amounted as many as over 10,000 units. Even after production termination, about 6000 of these units operated in European laboratories for a long time.
The most reliable devices in our country were Tsvet-4, Tsvet-100, and the first models Tsvet-500. The instruments Tsvet-100 and Tsvet-500 worked in central factory laboratories (CFL) of various enterprises mainly for over 20–25 years. But the record was established by the model Tsvet-4: it was documented this instrument had been working at Shchelkovo and "Akron" chemical plants for 40 years. In the former USSR, the main consumers of gas chromatographs were chemical research institutes and CFL of chemical enterprises. There were many enterprises, which had applied 100 or more chromatographs. Probably, Kirovo-Chepetsk chemical plant was the champion among them because, according to data of Z.L.Baskin, there were installed at least 500 chromatographs. At least 400– 500 chromatographs operated at many of Bashkirian chemical plants.
Table 6 shows sales of chromatographic equipment, accessories, columns, and services in 2013 and 2014.
It should be noted that chromatographic equipment sales volume is constantly increasing in spite of the crisis processes in the world.
Conclusion
Chromatographs are widely used in all vital areas of human activity: control of environmental pollution and food quality; early detection of diseases and medicament quality control in medicine; forensic medicine, and monitoring of many industrial processes. Only chromatographic methods enable to determine supertoxicants (pesticides, mycotoxins, polycyclic aromatic hydrocarbons, and others) in food products.
Hundreds of standard techniques are issued to regulate determining substances using chromatography.
Main recent results achieved using chromatographic methods
• Decoding of the human genome;
• Separation of multicomponent protein mixtures for purposes of proteomics;
• Analysis of a single cell contents;
• Establishment of a medicament pharmacokinetics;
• Early detection of diseases by analysis of biochemical markers;
• Separation of optical isomers, analysis of enantiomeric purity of medicaments;
• Doping control at the world championships and the Olympic Games;
• Analysis within judicial and forensic examinations; analysis of medicaments and narcotics;
• Monitoring of environmental pollution, including supertoxicants (dioxins, polychlorinated biphenyls, polynuclear aromatic compounds at the ppt level);
• Monitoring of contaminants in food and beverages including most dangerous carcinogenic mycotoxins, nitrosamines, pesticides, etc.;
• Analysis of the components of smells and flavors of foods and drinks;
• Analysis of natural gas, gas condensate, gasoline, kerosene, petroleum products and crude oil, enabling simulated distillation of crude oil;
• Separation and analysis of metals including transuranic elements;
• Full analysis of atmosphere composition in the polluted cities;
• Analysis of chlorofluorocarbons (freons) in stratosphere;
• Analysis of insect pheromones;
• Analysis of soils and atmospheres of other planets (Venus, Mars, lunar soil), and
• Analysis of isotope-substituted compounds.
General trends in the development of chromatography techniques and instruments [21]:
• Improving selectivity of separation;
• Improving performance of columns;
• Improving rapidity of separation;
• Miniaturization of hardware;
• Updating the software for both instrument control and separation and analysis data processing.
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