HPLC method description. High performance liquid chromatography
Introduction.
The rapid development of liquid chromatography in the last 10 years is mainly due to the intensive development of theoretical foundations and the practical use of its highly effective version, as well as the creation and industrial production of the necessary sorbents and equipment.
A distinctive feature of high-performance liquid chromatography (HPLC) is the use of sorbents with a grain size of 3-10 microns, which ensures rapid mass transfer with very high separation efficiency.
Currently, HPLC has taken first place among instrumental methods in terms of development rates, even surpassing gas chromatography. The most important advantage of HPLC compared to gas chromatography is the ability to study almost any object without any restrictions on their physicochemical properties, for example, boiling points or molecular weight.
Today, HPLC is a well-designed instrumental method that is widely used in a wide variety of fields of science and technology. Its importance is especially great in such critical areas as biochemistry, molecular biology, environmental pollution control, as well as in the chemical, petrochemical, food and pharmaceutical industries.
since it is necessary to take into account a number of very specific requirements due to the following features of the methodology.
A. HPLC columns are packed with very small particle diameter media. As a result, at the solvent volumetric velocities required for rapid sample separation, high pressure is created on the column.
b. Detectors used in HPLC are sensitive to fluctuations in eluent flow and pressure (noise). Moreover, when using concentration detectors, even higher stability of the eluent volumetric velocity is required.
V. The process of chromatographic separation is accompanied by a number of antagonistic effects, for example, dispersion of the sample in the mobile phase leads to mixing of the separated components and reduces the maximum concentration of the substance in the eluted peak (in the detector). Dispersion is observed in all areas of the system from the sample injection point to the detector.
d. Solvents acting as a mobile phase can often cause corrosion of equipment. This primarily applies to solvents used in reverse phase chromatography, which is preferred in biochemical HPLC applications.
The specifics of HPLC as an instrumental technique must be taken into account during the development, creation and operation of these systems. It took more than ten years of search and research to create commercial samples of chromatographic systems and their components that are sufficiently reliable, simple and safe to operate with an acceptable ratio between price and technical characteristics. The recent trends towards reducing columns (both length and diameter) force new demands on instruments.
1.1. EFFICIENCYANDSELECTIVITY
Chromatography is a method of separating the components of a mixture based on the difference in their equilibrium distribution between two "immiscible phases, one of which is stationary and the other is mobile. The components of the sample move along the column when they are in the mobile phase, and remain in place when they are in the stationary phase. The greater the affinity of the component for the stationary phase and the less for the mobile phase, the slower it moves through the column and the longer it is retained in it. Due to the difference in the affinity of the components of the mixture for the stationary and mobile phases, the main goal of chromatography is achieved - separation an acceptable period of time of the mixture into individual bands (peaks) of the components as they move along the column with the mobile phase.
From these general concepts, it is clear that chromatographic separation is possible only if the components of the sample, entering the column when the sample is introduced, firstly, are dissolved in the mobile phase and, secondly, interact (retained) with the stationary phase . If, when introducing a sample, any components are not in the form of a solution, they will be filtered and will not participate in the chromatographic process. Likewise, components that do not interact with the stationary phase will pass through the column with the mobile phase without separating into their components.
Let us accept the condition that some two components are soluble in the mobile phase and interact with the stationary phase, i.e. the chroiatographic process can proceed without disturbances. In this case, after passing the mixture through the column, you can obtain chromatograms of the form a, b or V(Fig. 1.1). These chromatograms illustrate chromatographic separations that differ in efficiency (A and b) with equal selectivity and selectivity (b And V) with equal efficiency.
The narrower the peak obtained at the same retention time, the higher the column efficiency. Column efficiency is measured by the number of theoretical plates (NPT) N:
the higher the efficiency
Rice. 1.2. Chromatographic peak parameters and calculation of the number of theoretical plates:
t R - peak retention time; h - peak height; Wj/j - peak width at half its height
Rice. 1.1. Type of chromatogram depending on the efficiency and selectivity of the column:
A- normal selectivity, reduced efficiency (fewer theoretical plates); b - usual selectivity and efficiency; V - normal efficiency, increased selectivity (higher ratio of component retention times)
efficiency, the greater the FTT, the smaller the broadening of the peak of the initially narrow band as it passes through the column, and the narrower the peak at the exit of the column. PTT characterizes the number of steps in establishing equilibrium between the mobile and stationary phases.
Knowing the number of theoretical plates per column and the length of the column L (µm), as well as the average sorbent grain diameter d c (µm), it is easy to obtain the values of the height equivalent to a theoretical plate (HETT), as well as the reduced height equivalent to a theoretical plate (RHETT):
BETT = L/ N
PVETT =B3TT/d c .
Having the values of FTT, HETT and PHETT, one can easily compare the efficiency of columns of different types, different lengths, filled with sorbents of different nature and granularity. By comparing the PTT of two columns of the same length, their efficiency is compared. When comparing HETP, columns with sorbents of the same grain size and different lengths are compared. Finally, the PVETT value allows for any two columns to evaluate the quality of the sorbent, firstly, and the quality of filling the columns, and secondly, regardless of the length of the columns, the granulation of the sorbents of its nature.
Column selectivity plays a large role in achieving chromatographic separation.
The selectivity of a column depends on many factors, and the skill of the experimenter is largely determined by the ability to influence the selectivity of separation. For this, three very important factors are in the hands of the chromatographer: the choice of the chemical nature of the sorbent, the choice of the composition of the solvent and its modifiers, and taking into account the chemical structure and properties of the separated components. Sometimes a change in the temperature of the column, which changes the distribution coefficients of substances between the mobile and stationary phases, has a noticeable effect on selectivity.
When considering and evaluating the separation of two components in a chromatogram, resolution is an important parameter. R s, which relates the output times and peak widths of both separated components
Resolution as a parameter characterizing peak separation increases as selectivity increases, reflected by an increase in the numerator, and efficiency increases, reflected by a decrease in the denominator value due to a decrease in the width of the peaks. Therefore, the rapid progress of liquid chromatography led to a change in the concept of “high pressure liquid chromatography” - it was replaced by “high resolution liquid chromatography” (while the abbreviated form of the term in English was preserved HPLC as the most correctly characterizing the direction of development of modern liquid chromatography).
Thus, column washout is reduced and efficiency is increased when a finer sorbent is used, more uniform in composition (narrow fraction), more densely and uniformly packed in the column, using thinner graft phase layers, less viscous solvents and optimal flow rates.
However, along with the blurring of the chromatographic zone band during the separation process in the column, it can also be washed out in the sample introduction device, in the connecting capillaries injector - column and column - detector, in the detector cell and in some auxiliary devices (microfilters for trapping mechanical particles from samples installed after the injector, pre-columns, coil reactors, etc.) - The greater the extra-column volume compared to the retained volume of the peak, the greater the erosion. It also matters where the dead volume is located: the narrower the chromatographic signal, the greater the blurring of the dead volume. Therefore, special attention should be paid to the design of that part of the chromatograph where the chromatographic zone is the narrowest (the injector and devices from the injector to the column) - here extra-column erosion is most dangerous and has the greatest impact. Although it is believed that in well-designed chromatographs the sources of additional extra-column dilution should be reduced to a minimum, nevertheless, each new device, each modification of the chromatograph must necessarily end with testing on a column and comparison of the resulting chromatogram with the passport one. If peak distortion or a sharp decrease in efficiency is observed, capillaries and other devices newly introduced into the system should be carefully inspected.
Off-column washout and its misjudgment can lead to a significant (over 50%) loss of efficiency, especially in cases where relatively old chromatographs are attempted to be used for high-speed HPLC, microcolumn HPLC, and other variants of modern HPLC that require microinjectors, connecting capillaries with internal with a diameter of 0.05-0.15 mm of minimum length, columns with a capacity of 10-1000 µl, detectors with microcuvettes with a capacity of 0.03-1 µl and with high speed, high-speed recorders and integrators.
1.2. SOLVENT RETENTION AND STRENGTH
In order for the analytes to be separated on the column, as mentioned above, the capacity coefficient k" must be greater than 0, i.e. substances must be retained by the stationary phase, the sorbent. However, the capacity factor should not be too high to obtain an acceptable elution time. If for a given mixture of substances a stationary phase is selected that retains them, then further work on developing an analysis technique consists in choosing a solvent that would ideally provide different for all components, but acceptably not very large k". This is achieved by changing the elution strength of the solvent.
In the case of adsorption chromatography on silica gel or aluminum oxide, as a rule, the strength of a two-component solvent (for example, hexane with the addition of isopropanol) is increased by increasing the content of the polar component (isopropanol), or decreased by decreasing the isopropanol content. If the containing polar component becomes too small (less than 0.1%), it should be replaced with a weaker elution force. The same is done, replacing either a polar or a non-polar component with others, even if this system does not provide the desired selectivity with respect to the components of interest in the mixture. When selecting solvent systems, both the solubility of the components of the mixture and the eluotropic series of solvents compiled by different authors are taken into account.
The strength of the solvent is selected in approximately the same way when using grafted polar phases (nitrile, amino, diol, nitro, etc.), taking into account possible chemical reactions and excluding solvents dangerous to the phase (for example, ketones for the amino phase).
In the case of reverse phase chromatography, the solvent strength is increased by increasing the content of the organic component in the eluent (methanol, acetonitrile or THF) and decreased by adding more water. If it is not possible to achieve the desired selectivity, they use another organic component or try to change it using various additives (acids, ion-pair reagents, etc.).
In separations using ion exchange chromatography, the strength of the solvent is changed by increasing or decreasing the concentration of the buffer solution or changing the pH; in some cases, modification with organic substances is used.
However, especially in the case of complex natural and biological mixtures, it is often not possible to select the strength of the solvent in such a way that all sample components elute within an acceptable time. Then you have to resort to gradient elution, i.e., use a solvent whose elution strength changes during the analysis process so that it constantly increases according to a predetermined program. This technique makes it possible to achieve the elution of all components of complex mixtures in a relatively short period of time and their separation into components in the form of narrow peaks.
1.3. SORBENT PARTICLE SIZE, PERMEABILITY AND EFFICIENCY
Considering the erosion in the column, we indicated that the efficiency of the column (HETT) depends on the size of the sorbent particles. To a large extent, the rapid development of HPLC over the past 10-12 years was due, firstly, to the development of methods for producing sorbents with particle sizes from 3 to 10 microns and with a narrow fractional composition, providing high efficiency with good permeability, and secondly, the development methods for filling columns with these sorbents and, thirdly, the development and serial production of liquid chromatographs with high-pressure pumps, injectors and detectors with small-volume cuvettes capable of recording small-volume peaks.
For well-packed slurry-packed columns, the reduced equivalent theoretical plate height (LPHE) can be 2 regardless of whether 3, 5, 10, or 20 μm particles are used for packing. In this case, we will receive respectively columns (with a standard length of 250 mm) with an efficiency of 41670, 25000, 12500 and 6250 t.t. It seems natural to select the most efficient column packed with 3 µm particles. However, this efficiency will come at the cost of very high pressure operation and relatively low separation speed, since the existing pump will most likely be able to pump solvent through such a column at a high volumetric velocity. Here we are faced with the question of the relationship between the particle size of the sorbent, the efficiency and permeability of the columns.
If we express the column resistance factor from here - a dimensionless quantity, we obtain the following equation:
The resistance factor for columns packed with microparticles of the same type using the same method varies slightly and is the following values:
Particle type "... Irregular Spherical
form form
Dry packaging. . . . . 1000-2000 800-1200
Suspension packaging. . . 700-1500 500-700
The column inlet pressure is proportional to the linear flow velocity, column drag factor, solvent viscosity, and column length and inversely proportional to the square of the particle diameter.
Applying this relationship to the above-described columns with particles with diameters of 3, 5, 10 and 20 µm and assuming constant linear flow rate, column resistance factor and solvent viscosity, we obtain an inlet pressure ratio of 44:16:4:1 for columns of equal length. Thus, if for a reverse-phase sorbent with a particle size of 10 μm when using methanol solvent systems - . water (70:30) usually on a standard column at a solvent flow rate of 1 ml/min, the pressure at the entrance to the column is 5 MPa, then for particles of 5 μm - 20 MPa and for 3 μm - 55 MPa. When using silica gel and a less viscous solvent system, hexane - isopropanol (100:2), the values will be significantly lower: 1, 4 and 11 MPa, respectively. If in the case of a reversed-phase sorbent the use of particles with a size of 3 μm is very problematic, and 5 μm is possible, but not on all devices, then for a normal-phase sorbent there are no problems with pressure. It should be noted that modern high-speed HPLC typically uses a higher solvent flow rate than in the example above, so the pressure requirements increase even more.
However, in cases where a certain number of theoretical plates is required for separation and it is desirable to carry out rate analysis, the picture changes somewhat. Since the lengths of columns with sorbents with grain sizes of 3, 5, 10 microns, with equal efficiency, will be 7.5, respectively; 12.5 and 25 cm, then the pressure ratio at the inlet to the columns will change to 3:2:1. Accordingly, the duration of analysis on such columns of equal efficiency will be in the ratio 0.3:0.5:1, i.e., when moving from 10 to 5 and 3 microns, the duration of analysis will be reduced by 2 and 3.3 times. This faster analysis comes at the cost of proportionately higher pressure at the column inlet.
The data presented are valid for those cases where sorbents of different grain sizes have the same particle size distribution curves, the columns are packed in the same way and have the same column resistance factor. It should be borne in mind that the difficulty of obtaining narrow fractions of the sorbent increases as the particle size decreases and that. Fractions from different manufacturers have different fractional compositions. Therefore, the column resistance factor will vary depending on the grain size, sorbent type, column packing method, etc.
CLASSIFICATION OF HPLC METHODS BY SEPARATION MECHANISM
Most separations carried out by HPLC are based on a mixed mechanism of interaction of substances with a sorbent, providing greater or lesser retention of components in the column. Separation mechanisms in a more or less pure form are quite rare in practice, for example, adsorption when using absolutely anhydrous silica gel and anhydrous hexane to separate aromatic hydrocarbons.
With a mixed retention mechanism for substances of different structures and molecular weights, it is possible to evaluate the contribution to retention of adsorption, distribution, exclusion and other mechanisms. However, for a better understanding and understanding of the separation mechanisms in HPLC, it is advisable to consider separations with a predominance of one or another mechanism as relating to a certain type of chromatography, for example, ion exchange chromatography.
2.1.1 ADSORPTION CHROMATOGRAPHY
Separation by adsorption chromatography is carried out as a result of the interaction of a substance with adsorbents, such as silica gel or aluminum oxide, which have active centers on the surface. The difference in the ability to interact with the adsorption centers of different sample molecules leads to their separation into zones during movement with the mobile phase along the column. The zone separation of the components achieved in this case depends on the interaction with both the solvent and the adsorbent.
Sorption on the surface of an adsorbent containing hydroxyl groups is based on a specific interaction between the polar surface of the adsorbent and polar (or polarizable) groups or sections of molecules. Such interactions include dipole-dipole interaction between permanent or induced dipoles, the formation of a hydrogen bond, up to the formation of r-complexes or charge transfer complexes. A possible and quite frequent occurrence in practical work is the manifestation of chemisorption, which can lead to a significant increase in retention time, a sharp decrease in efficiency, the appearance of decomposition products or irreversible sorption of the substance.
Adsorption isotherms of substances have a linear, convex or concave shape. With a linear adsorption isotherm, the peak of the substance is symmetrical and the retention time does not depend on the sample size. Most often, adsorption isotherms of substances are nonlinear and have a convex shape, which leads to some asymmetry of the peak with the formation of a tail.
The most widely used in HPLC are silica gel adsorbents with different pore volumes, surface areas, and pore diameters. Aluminum oxide is used much less frequently and other adsorbents, widely used in classical column and thin-layer chromatography, are used extremely rarely. The main reason for this is the insufficient mechanical strength of most other adsorbents, which does not allow them to be packaged or used at high pressures characteristic of HPLC.
The polar groups that cause adsorption and are located on the surface of silica gel and aluminum oxide are similar in properties. Therefore, usually the order of elution of mixtures of substances and the eluotropic series of solvents are the same for them. However, the difference in the chemical structure of silica gel and aluminum oxide sometimes leads to differences in selectivity - then preference is given to one or another adsorbent that is more suitable for a given specific task. For example, alumina provides greater selectivity for the separation of certain polynuclear aromatic hydrocarbons.
The preference usually given to silica gel compared to aluminum oxide is explained by a wider choice of silica gels in terms of porosity, surface and pore diameter, as well as the significantly higher catalytic activity of aluminum oxide, which often leads to distortion of analysis results due to the decomposition of sample components or their irreversible chemisorption .
2.1.2 Disadvantages of adsorption chromatography that limit its use
The popularity of adsorption chromatography gradually decreased as the HPLC method developed; it was increasingly replaced and continues to be replaced by other options, such as reverse-phase and normal-phase HPLC on sorbents with a grafted phase. What are the disadvantages of adsorption chromatography that led to this?
First of all, this is the long duration of the processes of equilibrating adsorbents with solvents containing water in trace quantities, the difficulty of preparing such solvents with a certain and reproducible humidity. This results in poor reproducibility of retention, resolution, and selectivity parameters. For the same reason, it is impossible to use gradient elution - the return to the initial state is so long that it significantly exceeds the time gained by using a gradient.
Significant disadvantages of adsorbents, especially aluminum oxide, associated with frequent cases of rearrangements of compounds sensitive to catalysis, their decomposition, and irreversible sorption, are also well known and have been repeatedly noted in the literature. Irreversibly sorbed substances, accumulating at the initial section of the column, change the nature of the sorbent and can lead to an increase in the resistance of the column or even to its complete clogging. The last drawback can be eliminated by using a pre-column, which By- As resistance and clogging increases, it is replaced with a new one* or refilled with a new sorbent. However, irreversible sorption, which also occurs in this case, results in a chromatogram in which sample components sensitive to sorption or catalytic decomposition are completely or partially absent.
2.2. DISTRIBUTION CHROMATOGRAPHY
Partition chromatography is a variant of HPLC in which the separation of a mixture into components is carried out due to the difference in their distribution coefficients between two immiscible phases: a solvent (mobile phase) and a phase on the sorbent (stationary phase). Historically, the first were sorbents of this type, which were obtained by applying liquid phases (oxydipropionitrile, paraffin oil, etc.) onto porous supports, similar to how sorbents were and are prepared for gas-liquid chromatography (GLC). However, the disadvantages of such sorbents were immediately revealed, the main of which was the relatively rapid rinsing of the phase from the carrier. Due to this, the amount of phase in the column gradually decreased, the retention times also decreased, and adsorption centers not covered by the phase appeared in the initial section of the column, causing the formation of peak tails. This drawback was combated by saturating the solvent with the applied phase before it entered the column. Entrainment was also reduced when more viscous and less soluble polymer phases were used, but in this case, due to the difficulty of diffusion from thick polymer films, column efficiency was markedly reduced.
It turned out to be logical to graft the liquid phase onto the carrier through chemical bonds in such a way that its removal becomes physically impossible, i.e., to turn the carrier and the phase into one - into the so-called grafted-phase sorbent.
Subsequent efforts of researchers were aimed at searching for reagents whose grafting would proceed fairly quickly and completely, and the bonds formed would be as stable as possible. Such reagents were alkylchlorosilanes and their derivatives, which made it possible, using a similar technology, to obtain graft-phase sorbents of various types and with different polar and non-polar groups on the surface.
The successful application of the latter type of sorbents for HPLC has contributed to the growth of their production by a wide variety of manufacturers. Each company produced such sorbents, as a rule, based on its own type of silica gel and using its own technology, which usually constitutes production “know-how”. As a result, a large number of sorbents, chemically called exactly the same (for example, octadecylsilane-grafted silica gel), have very different chromatographic characteristics. This is due to the fact that silica gel can have wider or narrower pores, a different surface, porosity, its surface before grafting can be hydroxylated or not, mono-, di- or trichlorosilanes can be grafted, grafting conditions can give monomeric, polymeric or mixed layer phase, different methods are used to remove residual reagents, additional deactivation of silanol and other active groups may or may not be used.
The complexity of the technology for grafting reagents and preparing raw materials and materials, its multi-stage nature, leads to the fact that even batches of sorbents obtained using the same technology from one manufacturing company can have slightly different chromatographic characteristics. This is especially true in cases where such sorbents are used for the analysis of multicomponent mixtures containing substances that differ markedly in the number and position of functional groups, and the type of functionality.
Taking into account the above, one should always strive to ensure that when using the analysis technique described in the literature, the same sorbent and the same operating conditions are used. In this case, the likelihood that the work will not be reproduced is minimal. If this is not possible, but a sorbent from another company with a similar grafted phase is taken, you need to be prepared for the fact that it will take a long time to rework the technique. At the same time, there is a possibility (and it should be taken into account) that with this sorbent, even after long development, the required separation may not be achieved. The presence in the literature of many described separation techniques on old sorbents that have been produced for a long time stimulates their further production and use for this reason. However, in cases where it is necessary to move on to the development of original methods, especially in relation to substances prone to decomposition, chemisorption, rearrangements, it is advisable to start working on sorbents that have been developed recently and produced using new, improved versions of the technology. New sorbents have a more uniform fractional composition, more uniform and complete surface coverage with the grafted phase, and more advanced final stages of sorbent processing.
2.3. ION EXCHANGE CHROMATOGRAPHY
In ion exchange chromatography, the separation of mixture components is achieved through the reversible interaction of ionizing substances with the ionic groups of the sorbent. Preservation of the electrical neutrality of the sorbent is ensured by the presence of counterions capable of ion exchange located in close proximity to the surface. The ion of the introduced sample, interacting with the fixed charge of the sorbent, exchanges with the counterion. Substances with different affinities for fixed charges are separated on anion exchangers or cation exchangers. Anion exchangers have positively charged groups on the surface and sorb anions from the mobile phase. Cation exchangers accordingly contain groups with a -negative charge that interact with cations.
As a mobile phase, aqueous solutions of salts of acids, bases and solvents such as liquid ammonia are used, i.e., solvent systems with a high dielectric constant e and a greater tendency to ionize compounds. They usually work with buffer solutions that allow the pH value to be adjusted.
During chromatographic separation, ions of the analyte compete with ions contained in the eluent, tending to interact with oppositely charged groups of the sorbent. It follows that ion exchange chromatography can be used to separate any compounds that can be ionized in some way. It is possible to analyze even neutral sugar molecules in the form of their complexes with borate ion:
Sugar + VO 3 2 - = Sugar -VO 3 2 -.
Ion exchange chromatography is indispensable for the separation of highly polar substances, which cannot be analyzed by GLC without conversion to derivatives. These compounds include amino acids, peptides, and sugars.
Ion exchange chromatography is widely used in medicine, biology, biochemistry, for environmental monitoring, in the analysis of the content of drugs and their metabolites in the blood and urine, pesticides in food raw materials, as well as for the separation of inorganic compounds, including radioisotopes, lanthanides, actinides, etc. Analysis of biopolymers (proteins, nucleic acids, etc.), which usually took hours or days, using ion exchange chromatography is carried out in 20-40 minutes with better separation. The use of ion exchange chromatography in biology has made it possible to observe samples directly in biological media, reducing the possibility of rearrangement or isomerization, which can lead to incorrect interpretation of the final result. It is interesting to use this method to monitor changes occurring in biological fluids. The use of porous weak anion exchangers based on silica gel allowed the separation of peptides. V
The ion exchange mechanism can be represented in the form of the following equations:
for anion exchange
X-+R+Y- h ->■ Y-+R+X-.
for cation exchange |
X+ + R-Y+ h=* Y++R-X+.
In the first case, the X~ sample ion competes with the mobile phase ion Y~ for the R+ ion centers of the ion exchanger, and in the second case, the X+ sample cations compete with the Y+ ions of the mobile phase for the R~ ionic centers.
Naturally, sample ions that weakly interact with the ion exchanger will be weakly retained on the column during this competition and will be the first to be washed out from it, and, conversely, more strongly retained ions will be the last to elute from the column. Typically, BTqpH4Hbie interactions of a nonionic nature occur due to adsorption or hydrogen bonds of the sample with the nonionic part of the matrix or due to the limited solubility of the sample in the mobile phase. It is difficult to isolate “classical” ion exchange chromatography in its “pure” form, and therefore some chromatographers proceed from empirical rather than theoretical principles in ion exchange chromatography.
The separation of specific substances depends primarily on the choice of the most suitable sorbent and mobile phase. Ion exchange resins and silica gels with grafted ionogenic groups are used as stationary phases in ion exchange chromatography.
2.4. SECTION EXCLUSION CHROMATOGRAPHY
Exclusion chromatography is an option! liquid chromatography, in which separation occurs due to the distribution of molecules between the solvent located inside the pores of the sorbent and the solvent flowing " between its particles.
Unlike other HPLC options, where separation coming Due to the different interactions of the components with the surface of the sorbent, the role of the solid filler in size exclusion chromatography is only to form pores of a certain size, and the stationary phase is the solvent that fills these pores. Therefore, the use of the term “sorbent” to these fillers is to a certain extent arbitrary.
A fundamental feature of the method is the ability to separate molecules according to their size in solution in the range of almost any molecular weight - from 10 2 to 10 8, which makes it indispensable for the study of synthetic and biopolymers.
Traditionally, the process carried out in organic solvents is still often called gel permeation chromatography, and in aqueous systems - gel filtration chromatography. In this book, a single term is adopted for both options, which comes from the English “Size Exclusion” - exclusion by size - and most fully reflects the mechanism of the process.
A detailed analysis of existing ideas about the very complex theory of the size exclusion chromatography process is carried out in monographs.
Total volume of solvent in the column Vt (this is often called the total volume of the column, since Vd does not take part in the chromatographic process) is the sum of the volumes of the mobile and stationary phases.
The retention of molecules in an exclusion column is determined by the probability of their diffusion into the pores and depends on the ratio of the sizes of molecules and pores, which is shown schematically in Fig. 2.15. Distribution coefficient Ka, as in other variants of chromatography, it is the ratio of the concentrations of a substance in the stationary and mobile phases.
Since the mobile and stationary phases have the same composition, then Kd a substance for which both phases are equally accessible is equal to unity. This situation is realized for molecules C with the smallest sizes (including solvent molecules), which penetrate into all pores (see Fig. 2.15) and therefore move through the column most slowly. Their retention volume is equal to the total volume of the solvent Vt-
Rice. 2.15. Model of molecular separation by measure in size exclusion chromatography
All molecules whose size is larger than the size of the sorbent pores cannot enter them (complete exclusion) and pass through the channels between the particles. They elute from the column with the same retention volume equal to the volume of the mobile phase V 0 - The partition coefficient for these molecules is zero.
Molecules of intermediate size, capable of penetrating only some of the pores, are retained in the column according to their size. The distribution coefficient of these molecules varies from zero to one and characterizes the fraction of the pore volume accessible to molecules of a given size. Their retained volume is determined by the sum of V o and the accessible portion of the pore volume.
QUALITATIVE ANALYSIS
A chromatographer entering the field of high-performance liquid chromatography must become familiar with the fundamentals of qualitative analysis. Qualitative analysis is used to identify a known product, obtained in a new way or found in a mixture with other products." It is necessary when isolating various components from complex biological and chemical mixtures, which is especially important in medicine, forensics, ecology, for monitoring the presence of some medicinal chemical products and their metabolites in bioml.ter.ials.. "Familiarity with the basics of qualitative" analysis will help to avoid common mistakes, for example, distinguishing impurities in a sample from impurities in a solvent or checking the purity of a substance at more than one wavelength spectrophotometer, but on different ones, etc.
Before proceeding with the analysis, it is necessary to determine whether the entire sample is eluted from the column by a given solvent system or not. To be sure of complete elution, it is necessary to collect all the liquid flowing from the column, evaporate the solvent, weigh the residue and find the degree of sample recovery.
Identification of components in HPLC can be done in three ways: 1) using retention information; 2) examine the zones obtained during separation in a liquid chromatograph column using spectral or chemical analysis methods; 3) directly connect the spectrum analyzer to the column.
Retention volume is used to record peaks in chromatography. V R or retention time t R. Both quantities are characteristic of a substance in a given chromatographic system. Since the retention time of the substance being separated consists of the interaction time in the column and the time of passage of the empty sections of the tube, it varies from instrument to instrument. It is convenient to have a substance not retained by a given column, taking it as a standard whose retention time and volume t 0 , V o. Chromatography of the substance and the standard must be carried out under the same conditions (pressure and flow rate). When identified by retention data, known individual substances that may be present in the samples are separated in the same chromatographic system and values are obtained for them t R. Comparing these values t R with the retention time of the unknown peak, it can be found that they either coincide, in which case it is likely that the peaks correspond to the same substance, or t R known substance does not correspond t R unknown zone. Then an approximate estimate of the values is still possible t R substances that are not available for direct measurement of the degree of their retention. Let's consider both options.
In the first case, a preliminary study of the sample is obviously necessary to postulate the presence of specific substances in it. When working with simple mixtures, it is not difficult to determine whether the degree of retention of the zones of the sample and known substances coincides or not, i.e. the values tB same or different. In the case of complex mixtures, several substances may elute with similar values t R, and the zones actually obtained during chromatographic separation overlap. As a result, obtaining accurate values t R becomes impossible for different zones. Reliability of identification increases with increasing resolution, more careful control of separation conditions, and repeated measurement of values t R and averaging the found values. In this case, the chromatographic separation of known and unknown substances must alternate. When separating complex mixtures, the value t R substances can change under the influence of the matrix of the sample itself. This effect is possible at the beginning of the chromatogram and when peaks overlap; It is also possible to tighten the zones, as has already been mentioned.
In such cases, the standard should be added to the sample in a 1:1 ratio. If the substances are identical, the value t R the starting material does not change, and only one peak is obtained in the chromatogram. If you have a device with a cyclic chromatography system, then for reliable identification it is advisable to pass the mixture through the column several times.
Information on retention rates can also be found in the literature, but the value of this information is limited. Since columns from even the same batch give poor reproducibility, literature values do not always correspond to the true value t R on this column. For adsorption chromatography, however, it is possible to predict t R based on literature data. Another difficulty associated with the use of literary meanings t R, - the difficulty of finding them in the specialized literature, although bibliographic reviews published in the Jornal of chromatography have an updated index by type of substance.
In the second case, when the retention times of known compounds and sample zones do not coincide, it is possible to predict the retention time of the unknown component. Predictions of relative retention based on structure data in steric exclusion chromatography are quite reliable. They are less accurate in adsorption and partition chromatography, and especially when working on a chemically bound phase. For ion and ion-pair chromatography of substances with known p Ka Only approximate determinations of values are possible tR. It is always easier to predict relative retention or *x values than absolute values k". Relative values t R easier to assess for related compounds or derivatives, such as substituted alkylcarboxylic acids or benzene derivatives.
When isocratically separating homologues or oligomers, the following pattern is sometimes observed:
\ gk" = A + Bn,
Where A And IN- constants for a number of selected samples and for a given chromatographic system (on the same column, with the same mobile and stationary phases); P- the number of identical structural units in the sample molecule.
Introduction of a functional group / into the sample molecule will lead to a change k" in the first equation by some constant coefficient a/ in a given chromatographic system. It is possible to obtain group constants a/ for various substituent groups/, the values of which will increase with increasing polarity of functional groups in all types of chromatography, except reverse phase, where the values of the constants will decrease with increasing polarity.
Some group constants a/ for various substituent groups/ are given in table. 9.1.
In adsorption chromatography, the first equation is not always applicable, since it is valid provided that all isomers have the same value k", which is not always observed. It is possible, however, to plot the logfe" of the same compounds on one column versus the logfe" of the same compounds on a different column or versus corresponding characteristics in thin layer chromatography, for example, log[(l- Rf) IRf].
Capacity coefficient values can be used when comparing retention data k", because unlike him t R the speed of the mobile phase and the geometric features of the column are not affected.
Chemically bound phase separations are similar to partition chromatography separations with similar phases, and therefore steady state extraction data can be used to predict retention times.
In ion exchange chromatography, three factors influence the degree of retention: the degree of ionization of acids and bases, the charge of the ionized molecule, and the ability of the substance from the aqueous mobile phase used in ion exchange chromatography to migrate into the organic phase. The latter depends on the molecular weight of the compound and its hydrophobicity. Therefore, stronger acids or bases are more strongly retained during anion-exchange or cation-exchange separation. When decreasing pK a of an individual acid included in the sample, the retention increases when a number of acids are separated due to anion exchange, and with an increase in p/C o, the retention of bases increases when they are separated due to cation exchange.
Thus, the coincidence of the retention times of a known substance with the observed one makes it possible to assume their identity. The reliability of identification increases if the chromatograms of a known substance and an unknown component are compared under different conditions. If substances behave identically in adsorption and reverse phase or ion exchange and size exclusion chromatography, the reliability of identification increases. If the reliability of identification with equal relative retention is 90%, then when studying the behavior of the same substances under significantly different conditions, the reliability of identification is already 99%.
A valuable characteristic of a substance used in identification is the ratio of the signals obtained for a given substance on two different detectors. The analyzed substance, after leaving the column, passes first through the first detector, then through the second, and the signals coming from the detectors are recorded simultaneously using a multi-pen recorder or on two recorders. Typically, a series connection of an ultraviolet detector (more sensitive, but selective) with a refractometer, or an ultraviolet with a fluorescence detector, or two ultraviolet detectors operating at different wavelengths is used. The relative response, i.e. the ratio of the refractometer signal to the photometer signal, is a characteristic of the substance, provided that both detectors operate in their linear range; this is tested by administering different amounts of the same substance. Qualitative information can be obtained by working with photometric detectors equipped with a stop flow device that allows the spectrum of the peak emerging from the column to be recorded while it is in the flow cell, comparing it with the spectrum of a known compound.
Modern, still expensive, spectrophotometers with a diode array are of significant interest in identification.
A completely unknown substance cannot be identified using high-performance liquid chromatography alone; other methods are also necessary.
QUANTITATIVE ANALYSIS
Quantitative liquid chromatography is a well-developed analytical method that is not inferior in accuracy to quantitative gas chromatography and significantly exceeds the accuracy of TLC or electrophoresis. Unfortunately, in HPLC there is no detector that would have close sensitivity for compounds of different chemical structures (like a katharometer in GLC) Therefore, to obtain quantitative results, calibration of the device is mandatory.
Quantitative analysis consists of the following stages: 1) chromatographic separation; 2) measurement of peak areas or heights; 3) calculation of the quantitative composition of the mixture based on chromatographic data; 4) interpretation of the results obtained, i.e. statistical processing. Let's consider all these stages.
4.1. CHRMATOGRAPHIC SEPARATION
Errors may be made during sample collection. It is especially important to avoid error and to obtain an adequate representative sample of heterogeneous solids, volatile or unstable substances, and agricultural products and biomaterials. Heterogeneous samples, for example food products, are thoroughly mixed and quartered. By performing this operation many times, sample homogeneity is achieved.
Errors and losses of substances can be made at the stage of extraction, isolation, purification, etc.
Samples must be completely dissolved and their solutions prepared to an accuracy of ±0.1%. It is advisable to dissolve the sample in the mobile phase, which will eliminate the possibility of its precipitation after introduction into the chromatograph. If dissolution in the mobile phase is not possible, then a solvent miscible with it should be used and sample volumes (less than 25 µl) should be introduced into the chromatograph.
Significant errors can occur during sample injection due to sample fractionation, leakage, and peak smearing. The blurring of peaks causes the formation of tails, leading to partial overlap of peaks and, as a consequence, to errors in detection. Loop valve devices are preferable to syringes for sample introduction in quantitative analysis due to higher accuracy and less operator dependency.
When chromatographic separation of substances, complications can also arise that lead to distortion of data: quantitative analysis. There may be decomposition or conversion of the sample during the chromatographic process or irreversible adsorption of the substance onto the column. It is important to ensure the absence of these undesirable phenomena and, if necessary, regenerate the column or replace it. Peak overlap and tailing can also be reduced by varying the chromatographic conditions.
Peaks with false or unclear shapes, as well as peaks whose release time is close to to, since their separation may not be sufficient. Typically, peaks with a value d"^0.5 are used. The highest efficiency of the column is achieved by introducing 10~ 5 -10~ 6 g of dissolved substance per 1 g of sorbent. When introducing large quantities of sample, the dependence of the peak height on the load may turn out to be nonlinear and a quantitative assessment is required by peak areas.
Errors associated with detection or amplification lead to significant distortion of the results of chromatographic separation. Each detector is characterized by specificity, linearity and sensitivity. Selectivity testing is especially important when analyzing trace impurities. The response of UV detectors can vary by a factor of 104 to substances with similar functional groups. It is necessary to calibrate the detector response for each analyte. Naturally, substances that do not absorb in the UV region will not give a signal to the recorder when used as a photometer detector. When using a refractometer, negative peaks may appear. In addition, this detector must be thermostated, which is not required for the UV detector.
The linearity of the detector determines the size of the injected sample. It is important to remember that column flow rate, column and detector temperatures, and detector design all affect the accuracy of the quantitative analysis. Errors in the transmission of an electrical signal to an output device (recorder), integrator or computer can arise due to noise induction, lack of grounding, voltage fluctuations in the network, etc.
4.2. MEASUREMENT OF PEAK AREA OR HEIGHT
Peak height h (Fig. 10.1) is the distance from the top of the peak to the baseline; it is measured linearly or by counting the number of divisions on a recorder. Some electronic integrators and computers provide information on peak heights. The position of the baseline of the shifted peaks is found by interpolating the ordinate values corresponding to the beginning and end of the peak (peak 1 and 3 see fig. 10.1). To improve accuracy, it is necessary to have a flat, stable baseline. In the case of unsplit peaks, the baseline is drawn between the start and end of the peak rather than replaced by a zero line. Because peak heights are less dependent on the influence of adjacent overlapping peaks, peak height estimation is more accurate and is almost always used in trace trace analysis.
Peak area can be determined in various ways. Let's look at some of them.
1. The planimetric method involves tracing the peak with a hand-held planimeter, which is a device that mechanically determines the area of the peak. The method is accurate, but labor-intensive and poorly reproducible. This method is not recommended.
2. Paper silhouette method - the peak is cut out and weighed. The method is highly reproducible, but labor-intensive, and the chromatogram is destroyed. Its applicability depends on the uniformity of the chart strip. The method also cannot be widely recommended.
4. The triangulation method consists of constructing a triangle by drawing tangents to the sides of the peak. The top of the triangle is higher than the top of the peak. Increasing the area formed by this extended vertex will be consistent throughout the chromatogram and will not greatly affect the accuracy. In addition, some area lost when drawing tangents will be compensated. The base of the triangle is determined by the intersection of the tangents with the base line, and the area is determined by the product of 7 g of the base and the height. This method is the best for determining the areas of asymmetric peaks. However, the reproducibility when constructing tangents by different operators is different and therefore; low.
5. The disk integrator method is based on an electromechanical device attached to a recorder. The pen attached to the integrator moves along a strip at the bottom of the tape at a speed proportional to the movement of the recorder pen.
As with manual measurements, the peak should remain on the recorder scale, but adjustments to compensate for baseline shifts and incomplete separation of adjacent peaks reduce reliability and increase analysis time.
The method is more accurate than manual measurement methods, especially for asymmetric peaks, and offers a speed advantage. In addition, it provides a permanent quantitative record of the analysis.
6. Methods using electronic integrators that determine peak area and print information about that area and retention times may include baseline shift correction and determine the area of only partially separated peaks. The main advantages are accuracy, speed, independence of action from the operation of the recorder. Integrators have memory and can be programmed for a specific analysis using a pre-installed program. The advantages of the integrator include its ability to use correction factors for the detector response when recalculating the original data on peak areas, compensating for differences in the sensitivity of the detector to different substances. Such systems save time, improve analytical accuracy, and are useful for routine analytical analysis.
7. In liquid chromatography, computers are widely used to measure peak areas. They print a complete message, including the names of the substances, peak areas, retention times, detector response correction factors, and abundance (wt %) for the various sample components.
GENERAL PHARMACOPOEIAN ARTICLE
Instead of Art. GF XI
High-performance liquid chromatography (high-pressure liquid chromatography) is a column chromatography method in which the mobile phase is a liquid moving through a chromatography column filled with a stationary phase (sorbent). Columns for high-performance liquid chromatography are characterized by high hydraulic resistance at the inlet.
Depending on the mechanism of separation of substances, the following variants of high-performance liquid chromatography are distinguished: adsorption, partition, ion exchange, exclusion, chiral, etc. in accordance with the nature of the main intermolecular interactions that occur. In adsorption chromatography, the separation of substances occurs due to their different abilities to be adsorbed and desorbed from the surface of a sorbent with a developed surface, for example, silica gel. In partition high-performance liquid chromatography, separation occurs due to the difference in the distribution coefficients of the substances being separated between the stationary phase (usually chemically grafted to the surface of a stationary carrier) and the mobile phase.
Depending on the type of mobile and stationary phase, normal-phase and reverse-phase chromatography are distinguished. In normal-phase high-performance liquid chromatography, the stationary phase is polar (most often silica gel or silica gel with grafted NH 2 or CN groups, etc.), and the mobile phase is non-polar (hexane, or mixtures of hexane with more polar organic solvents - chloroform, alcohols, etc.). The retention of substances increases with increasing polarity. In normal phase chromatography, the elution ability of the mobile phase increases with increasing polarity.
In reversed-phase chromatography, the stationary phase is non-polar (hydrophobic silica gels with grafted groups C4, C8, C18, etc.); mobile phase – polar (mixtures of water and polar solvents: acetonitrile, methanol, tetrahydrofuran, etc.). The retention of substances increases with increasing hydrophobicity (non-polarity). The higher the organic solvent content, the higher the elution ability of the mobile phase.
In ion exchange chromatography, the molecules of a mixture of substances, dissociated in solution into cations and anions, are separated when moving through a sorbent (cation exchanger or anion exchanger) due to the different strength of interaction of the determined ions with the ionic groups of the sorbent.
In size exclusion (sieve, gel permeation, gel filtration) chromatography, molecules of substances are separated by size due to their different ability to penetrate the pores of the stationary phase. In this case, the largest molecules that are able to penetrate into the minimum number of pores of the stationary phase are the first to leave the column, and substances with small molecular sizes are the last to leave.
Chiral chromatography separates optically active compounds into individual enantiomers. The separation can be performed on chiral stationary phases or on achiral stationary phases using chiral mobile phases.
There are other options for high performance liquid chromatography.
often separation occurs not by one, but by several mechanisms simultaneously, depending on the type of mobile and stationary phases, as well as the nature of the compound being determined.
Application area
High-performance liquid chromatography has been successfully used for both qualitative and quantitative analysis of medicinal products in the tests “Identity”, “Foreign Impurities”, “Dissolution”, “Dosage Uniformity”, “Quantitative Determination”. It should be noted that chromatography allows you to combine several tests in one sample, including “Authenticity” and “Quantitative Determination”.
Equipment
To carry out the analysis, appropriate instruments are used - liquid chromatographs.
A liquid chromatograph usually includes the following main components:
— mobile phase preparation unit, including a container with the mobile phase (or containers with individual solvents included in the mobile phase) and a mobile phase degassing system;
— pumping system;
— mobile phase mixer (if necessary);
— sample introduction system (injector), can be manual or automatic (autosampler);
— chromatographic column (can be installed in a thermostat);
— detector (one or several with different detection methods);
— chromatograph control system, data collection and processing.
In addition, the chromatograph may include: a sample preparation system and a pre-column reactor, a column switching system, a post-column reactor and other equipment.
Pumping system
Pumps supply the mobile phase to the column at a given speed. The mobile phase composition and flow rate can be constant or varied during analysis. In the case of a constant composition of the mobile phase, the process is called isocratic, and in the second - gradient. A modern liquid chromatograph pumping system consists of one or more computer-controlled pumps. This allows you to change the composition of the mobile phase according to a specific program during gradient elution. Pumps for analytical high-performance liquid chromatography allow you to maintain the flow rate of the mobile phase into the column in the range from 0.1 to 10 ml/min at a pressure at the inlet of the column up to 40 MPa. Pressure pulsations are minimized by special damper systems included in the design of the pumps. The working parts of the pumps are made of corrosion-resistant materials, which allows the use of aggressive components in the mobile phase.
Faucets
In the mixer, a single mobile phase is formed from individual solvents supplied by pumps, if the required mixture has not been prepared in advance. Mixing of the mobile phase components in the mixer can occur both at low pressure (before the pumps) and at high pressure (after the pumps). The mixer can be used for mobile phase preparation and isocratic elution.
Mixer volume can affect the retention time of components during gradient elution.
Injectors
Injectors can be universal, with the ability to change the volume of the injected sample, or discrete for injecting only a certain volume of sample. Both types of injectors can be automatic ("autoinjectors" or "autosamplers"). The injector for introducing the sample (solution) is located directly in front of the chromatographic column. The design of the injector allows you to change the direction of flow of the mobile phase and carry out preliminary introduction of the sample into a dosing loop of a certain volume (usually from 10 to 100 μl) or into a special dosing device of variable volume. The volume of the loop is indicated on its marking. The discrete injector design usually allows for loop replacement. Modern automatic injectors can have a number of additional functions, for example, perform the function of a sample preparation station: mix and dilute samples, carry out a pre-column derivatization reaction.
Chromatographic column
Chromatographic columns are usually tubes made of stainless steel, glass or plastic, filled with sorbent and closed on both sides with filters with a pore diameter of 2–5 µm. The length of the analytical column can be in the range from 5 to 60 cm or more, the internal diameter can be from 2 to 10 mm. Columns with an internal diameter of less than 2 mm are used in microcolumn chromatography. There are also capillary columns with an internal diameter of about 0.3–0.7 mm. Columns for preparative chromatography can have an internal diameter of 50 mm or more.
Short columns (pre-columns) can be installed in front of the analytical column to perform various auxiliary functions, the main of which is to protect the analytical column. Typically, analysis is carried out at room temperature, but to increase separation efficiency and reduce analysis time, column thermostatting can be used at temperatures up to 80 - 100 °C. The possibility of using elevated temperatures during separation is limited by the stability of the stationary phase, since its destruction is possible at elevated temperatures.
Stationary phase (sorbent)
The following are usually used as sorbents:
- silica gel, aluminum oxide, used in normal phase chromatography. The retention mechanism in this case is usually adsorption;
- silica gel, resins or polymers grafted with acidic or basic groups. Area of application – ion exchange and ion chromatography;
- silica gel or polymers with a specified pore size distribution (size exclusion chromatography);
- chemically modified sorbents (sorbents with grafted phases), most often prepared on the basis of silica gel. The retention mechanism is adsorption or distribution between the mobile and stationary phases. The scope of application depends on the type of grafted functional groups. Some types of sorbents can be used in both reverse and normal phase chromatography;
- chemically modified chiral sorbents, for example, cellulose and amylose derivatives, proteins and peptides, cyclodextrins, chitosans, used for the separation of enantiomers (chiral chromatography).
Sorbents with grafted phases can have varying degrees of chemical modification. The most commonly used grafted phases are:
– octadecyl groups (sorbent octadecylsilane (ODS) or C 18);
– octyl groups (sorbent octylsilane or C 8);
– phenyl groups (phenylsilane sorbent);
– cyanopropyl groups (CN sorbent);
– aminopropyl groups (NH 2 sorbent);
– diol groups (sorbent diol).
Most often, the analysis is performed on non-polar grafted phases in a reversed-phase mode using a C 18 sorbent.
Sorbents with grafted phases obtained on the basis of silica gel are chemically stable at pH values from 2.0 to 7.0, unless otherwise specifically stated by the manufacturer. Sorbent particles can have spherical or irregular shapes and varied porosity. The particle size of the sorbent in analytical high-performance liquid chromatography is usually 3–10 µm, in preparative high-performance liquid chromatography – 50 µm or more. There are also monolithic columns in which the sorbent is a monolith with through pores that fills the entire volume of the column.
High separation efficiency is ensured by the high surface area of sorbent particles (which is a consequence of their microscopic size and the presence of pores), as well as the uniform composition of the sorbent and its dense and uniform packing.
Detectors
High performance liquid chromatography uses a variety of detection methods. In the general case, the mobile phase with components dissolved in it after the chromatographic column enters the detector cell, where one or another of its properties is continuously measured (absorption in the ultraviolet or visible region of the spectrum, fluorescence, refractive index, electrical conductivity, etc.). The resulting chromatogram is a graph of the dependence of some physical or physicochemical parameter of the mobile phase on time.
The most common detectors in high-performance liquid chromatography are spectrophotometric. During the elution of substances, the optical density of the eluate is measured in a specially designed microcuvette at a pre-selected wavelength. The detector's wide range of linearity allows the analysis of both impurities and the main components of the mixture in a single chromatogram. A spectrophotometric detector allows detection at any wavelength in its operating range (usually 190-600 nm). Multiwave detectors are also used, allowing detection at several wavelengths simultaneously, and diode array detectors, allowing optical density to be recorded simultaneously over the entire operating wavelength range (usually 190-950 nm). This makes it possible to record the absorption spectra of components passing through the detector cell.
A fluorimetric detector is used to determine fluorescent compounds or non-fluorescent compounds in the form of their fluorescent derivatives. The operating principle of a fluorimetric detector is based on measuring the fluorescent emission of absorbed light. Absorption is usually carried out in the ultraviolet region of the spectrum, the wavelengths of fluorescent radiation exceeding the wavelengths of absorbed light. Fluorimetric detectors have very high sensitivity and selectivity. The sensitivity of fluorescence detectors is approximately 1000 times higher than the sensitivity of spectrophotometric ones. Modern fluorescent detectors make it possible not only to obtain chromatograms, but also to record the excitation and fluorescence spectra of the analyzed compounds.
To determine compounds that weakly absorb in the ultraviolet and visible regions of the spectrum (for example, carbohydrates), use refractometric detectors (refractometers). The disadvantages of these detectors are their low (compared to spectrophotometric detectors) sensitivity and significant temperature dependence of the signal intensity (the detector must be thermostated), as well as the impossibility of using them in the gradient elution mode.
Principle of operation evaporative laser light scattering detectors is based on the difference in vapor pressure of chromatographic solvents included in the mobile phase and the analyzed substances. The mobile phase at the outlet of the column is introduced into the atomizer, mixed with nitrogen or CO 2 and, in the form of a fine aerosol, enters a heated evaporation tube with a temperature of 30–160 °C, in which the mobile phase evaporates. An aerosol of non-volatile particles of the analyzed substances scatters the light flux in the dispersion chamber. By the degree of dispersion of the light flux, one can judge the amount of the compound being determined. The detector is more sensitive than a refractometric one; its signal does not depend on the optical properties of the sample, on the type of functional groups in the substances being determined, on the composition of the mobile phase, and can be used in the gradient elution mode.
Electrochemical detectors (conductometric, amperometric, coulometric, etc.). An amperometric detector is used to detect electroactive compounds that can be oxidized or reduced on the surface of a solid electrode. The analytical signal is the magnitude of the oxidation or reduction current. The detector cell has at least two electrodes - a working electrode and a reference electrode (silver chloride or steel). A working potential is applied to the electrodes, the value of which depends on the nature of the compounds being determined. Measurements can be carried out both at a constant potential and in a pulsed mode, when the profile of changes in the potential of the working electrode is set during one cycle of signal recording. An amperometric detector uses working electrodes made of carbon materials (most often glassy carbon or graphite) and metal ones: platinum, gold, copper, nickel.
A conductometric detector is used to detect anions and cations in ion chromatography. The principle of its operation is based on measuring the electrical conductivity of the mobile phase during the elution of the substance.
A mass spectrometric detector, which has high sensitivity and selectivity, is extremely informative. The latest models of liquid chromatography mass spectrometers operate in the m/z mass range from 20 to 4000 amu.
In high-performance liquid chromatography, Fourier-IR detectors, radioactivity and some others are also used.
Data collection and processing system
A modern data processing system is a personal computer interfaced with a chromatograph with installed software that allows you to record and process a chromatogram, as well as control the operation of the chromatograph and monitor the main parameters of the chromatographic system.
Mobile phase
The mobile phase in high-performance liquid chromatography performs a dual function: it ensures the transport of desorbed molecules along the column and regulates the equilibrium constants, and, consequently, retention as a result of interaction with the stationary phase (sorbed on the surface) and with the molecules of the substances being separated. Thus, by changing the composition of the mobile phase in high-performance liquid chromatography, it is possible to influence the retention times of compounds, the selectivity and efficiency of their separation.
The mobile phase can consist of one solvent, often two, and, if necessary, three or more. The composition of the mobile phase is indicated as the volume ratio of its constituent solvents. In some cases, the mass ratio may be indicated, which must be specifically stated. Buffer solutions with a certain pH value, various salts, acids and bases and other modifiers can be used as components of the mobile phase.
Normal-phase chromatography usually uses liquid hydrocarbons (hexane, cyclohexane, heptane) and other relatively non-polar solvents with small additions of polar organic compounds that regulate the elution force of the mobile phase.
In reverse phase chromatography, water or aqueous-organic mixtures are used as the mobile phase. Organic additives are usually polar organic solvents (acetonitrile and methanol). To optimize separation, aqueous solutions with a certain pH value can be used, in particular buffer solutions, as well as various additives to the mobile phase: phosphoric and acetic acids when separating acidic compounds; ammonia and aliphatic amines when separating basic compounds, and other modifiers.
The chromatographic analysis is greatly influenced by the purity of the mobile phase, so it is preferable to use solvents produced specifically for liquid chromatography (including water).
When using a UV spectrophotometric detector, the mobile phase should not have significant absorption at the wavelength selected for detection. The transparency limit or optical density at a certain wavelength of a particular solvent manufacturer is often indicated on the packaging.
The mobile phase and analyzed solutions should not contain undissolved particles and gas bubbles. Water obtained in laboratory conditions, aqueous solutions, organic solvents pre-mixed with water, as well as analyzed solutions must be subjected to fine filtration and degassing. For these purposes, vacuum filtration is usually used through a membrane filter with a pore size of 0.45 μm, inert with respect to a given solvent or solution.
List of chromatographic conditions to be specified
The pharmacopoeial monograph must contain: the full commercial name of the column indicating the manufacturer and catalog number, the dimensions of the column (length and internal diameter), the type of sorbent indicating the particle size, pore size, column temperature (if thermostatting is necessary), the volume of the injected sample (volume loops), composition of the mobile phase and method of its preparation, feed rate of the mobile phase, type of detector and detection conditions (if necessary, parameters of the detector cell used), description of the gradient mode (if used), including the stage of re-equilibration to the initial conditions, chromatography time, detailed description of the calculation method and formula, description of the preparation of standard and test solutions.
If pre-column derivatization is used in an autosampler, information about the autosampler program is provided. If post-column derivatization is used, the feed rate of the derivatizing reagent, the volume of the mixing loop and its temperature are indicated.
Modified types of high performance liquid chromatography
Ion pair chromatography
One of the varieties of reversed-phase high-performance liquid chromatography is ion pair chromatography, which allows the determination of ionized compounds. To do this, hydrophobic organic compounds with ionic groups (ion pair reagents) are added to the traditional reverse-phase high-performance liquid chromatography mobile phase. Sodium alkyl sulfates are usually used to separate bases; tetraalkylammonium salts (tetrabutylammonium phosphate, cetyltrimethylammonium bromide, etc.) are used to separate acids. In the ion-pair mode, the selectivity of the separation of nonionic components will be limited by the reverse-phase retention mechanism, and the retention of bases and acids will increase markedly, while the shape of the chromatographic peaks will improve.
Retention in the ion-pair mode is due to rather complex equilibrium processes that compete with each other. On the one hand, due to hydrophobic interactions and the effect of displacement of the polar environment of the mobile phase, the sorption of hydrophobic ions on the surface of alkylsilica gel is possible in such a way that the charged groups face the mobile phase. In this case, the surface acquires ion-exchange properties, and retention obeys the laws of ion-exchange chromatography. On the other hand, it is possible to form an ion pair directly in the volume of the eluent, followed by its sorption on the sorbent according to the reversed-phase mechanism.
Hydrophilic interaction chromatography ( HILIC chromatography)
Hydrophilic interaction chromatography is used to separate polar compounds that are weakly retained in reverse phase high performance liquid chromatography. In this version of chromatography, water-acetonitrile mixtures with the addition of salts, acids or bases are used as the mobile phase. Stationary phases, as a rule, are silica gels modified with polar groups (amino, diol, cyanopropyl groups, etc.). More polar compounds are held more strongly. The elution ability of the mobile phase increases with increasing polarity.
Ion exchange and ion high performance liquid chromatography
Ion exchange chromatography is used for the analysis of both organic (heterocyclic bases, amino acids, proteins, etc.) and inorganic (various cations and anions) compounds. The separation of the components of the analyzed mixture in ion exchange chromatography is based on the reversible interaction of the ions of the analyzed substances with the ion exchange groups of the sorbent. These sorbents are mainly either polymer ion exchange resins (usually copolymers of styrene and divinylbenzene with grafted ion exchange groups) or silica gels with grafted ion exchange groups. Sorbents with groups: -NH 3 +, -R 3 N +, -R 2 HN +, -RH 2 N +, etc. are used to separate anions (anion exchangers), and sorbents with groups: -SO 3 –, -RSO 3 – , –COOH, -PO 3 – and others for the separation of cations (cation exchangers).
Aqueous solutions of acids, bases and salts are used as a mobile phase in ion exchange chromatography. Typically, buffer solutions are used to maintain certain pH values. It is also possible to use small additions of water-miscible organic solvents - acetonitrile, methanol, ethanol, tetrahydrofuran.
Ion chromatography – a variant of ion exchange chromatography, in which a conductometric detector is used to detect the compounds (ions) being determined. For highly sensitive determination of changes in the electrical conductivity of the mobile phase passing through the detector, the background electrical conductivity of the mobile phase must be low.
There are two main variants of ion chromatography.
The first of them, two-column ion chromatography, is based on suppression of the electrical conductivity of the mobile phase electrolyte using a second ion exchange column or a special membrane suppression system located between the analytical column and the detector. As it passes through the system, the electrical conductivity of the mobile phase decreases.
The second variant of ion chromatography is single-column ion chromatography. This option uses a mobile phase with very low electrical conductivity. Weak organic acids are widely used as electrolytes: benzoic, salicylic or isophthalic.
Size exclusion high performance liquid chromatography
Size exclusion chromatography (gel chromatography) is a special version of high-performance liquid chromatography based on the separation of molecules by their size. The distribution of molecules between the stationary and mobile phases is based on the size of the molecules and partly on their shape and polarity.
Two limiting types of interaction of molecules with a porous stationary phase are possible. Molecules larger than the maximum pore diameter are not retained at all and elute first, moving simultaneously with the mobile phase. Molecules with sizes smaller than the minimum pore diameter of the sorbent freely penetrate the pores and are the last to be eluted from the column. The remaining molecules, having intermediate sizes, are partially retained in the pores and, during elution, are divided into fractions in accordance with their sizes and, partially, penetrate into the pores of the sorbent depending on their size and partially depending on their shape. As a result, substances elute with different retention times.
Ion exclusion chromatography
The mechanism of ion exclusion chromatography is based on the effect as a result of which compounds in ionized form are not retained on the ion exchange sorbent, while compounds in molecular form are distributed between the stationary and aqueous phases inside the pores of the ion exchange sorbent and the mobile phase migrating in the space between the sorbent particles. Separation is based on electrostatic repulsion, polar and hydrophobic interactions between dissolved compounds and the sorbent.
Anionic groups on the surface of the sorbent act as a semi-permeable “membrane” between the stationary and mobile phases. Negatively charged components do not reach the stationary mobile phase, since they are repelled by similarly charged functional groups and elute in the “dead” (free) volume of the column. The components in molecular form are not “rejected” by the cation-exchange sorbent and are distributed between the stationary and mobile phases. The difference in the degree of retention of nonionic components of the mixture is dictated by a combination of polar interactions of nonionic components with the functional groups of the cation-exchange sorbent and hydrophobic interactions of nonionic components with the non-polar sorbent matrix.
Chiral chromatography
The goal of chiral chromatography is the separation of optical isomers. Separations are performed on chiral stationary phases or on conventional achiral stationary phases using chiral mobile phases. As chiral stationary phases, sorbents with a modified surface, groups or substances having chiral centers (chitosans, cyclodextrins, polysaccharides, proteins, etc. (chiral selectors) are used. In this case, the same phases as in normal-phase or reversed-phase chromatography. When using achiral stationary phases, to ensure the separation of enantiomers, chiral modifiers will be added to the mobile phases: chiral metal complexes, neutral chiral ligands, chiral ion-pair reagents, etc.
Ultraperformance liquid chromatography
Ultraperformance liquid chromatography is a variant of liquid chromatography that is more efficient than classical high-performance liquid chromatography.
A feature of ultra-performance liquid chromatography is the use of sorbents with particle sizes from 1.5 to 2 microns. Chromatography column sizes typically range from 50 to 150 mm in length and 1 to 4 mm in diameter. The volume of the injected sample can range from 1 to 50 µl. The use of such chromatographic columns can significantly reduce analysis time and increase the efficiency of chromatographic separation. However, in this case, the pressure on the column can reach 80–120 MPa, the required frequency of detector data collection can increase to 40–100 hertz, and the extracolumn volume of the chromatographic system must be minimized. The chromatography equipment and columns used in ultra-performance liquid chromatography are specially adapted to meet the requirements of this type of chromatography.
Equipment designed for ultra-performance liquid chromatography can also be used in the classic version of high-performance liquid chromatography.
Introduction
Chromatographic analysis is a criterion for the homogeneity of a substance: if the analyzed substance is not separated by any chromatographic method, then it is considered homogeneous (without impurities).
The fundamental difference between chromatographic methods and other physicochemical methods of analysis is the possibility of separating substances with similar properties. After separation, the components of the analyzed mixture can be identified (identify the nature) and quantified (mass, concentration) by any chemical, physical and physicochemical methods.
Chromatography is widely used in laboratories and industry for qualitative and quantitative analysis of multicomponent systems, production control, especially in connection with the automation of many processes, as well as for the preparative (including industrial) isolation of individual substances (for example, noble metals), separation of rare and scattered elements.
In accordance with the state of aggregation of the eluent, gas chromatography (GC, GC) and liquid chromatography (HPLC, HPLC) are distinguished.
High-performance liquid chromatography (HPLC) is used for the analysis, separation and purification of synthetic polymers, drugs, detergents, proteins, hormones and other biologically important compounds. The use of highly sensitive detectors makes it possible to work with very small quantities of substances (10 -11 -10 -9 g), which is extremely important in biological research.
The HPLC method is carried out on various liquid chromatographs. Modern liquid chromatographs are designed to separate complex mixtures of substances into individual components and carry out qualitative and quantitative analysis of the components of the separated mixture.
high performance liquid chromatography propyphenazone
In connection with the introduction of GMP into the practice of pharmaceutical production in Russia. The importance of using modern unified methods of analysis is increasing, both at manufacturing enterprises and in the system of state control of the quality of medicines. The basic method for analyzing the quality of substances and finished drugs in countries with a developed pharmaceutical industry (USA, England, Japan, EU countries) is high-performance liquid chromatography (HPLC). This method according to its characteristics meets the requirements for quantitative analysis of about 80-90% of drugs.
The technique for performing any chromatographic determinations has some general requirements. First of all, it is necessary to note those of them that raise the most questions among novice specialists.
1. Air conditioning of the room. There should be no sudden temperature fluctuations in the room where the liquid chromatograph is installed.
Changing the temperature can change the retention, efficiency, and even selectivity of the separation.
In the summer heat in unconditioned rooms, working with normal-phase, low-boiling mobile phases becomes very difficult. During the day, their gradual evaporation occurs, which leads to a change in the composition of the eluent.
At low temperatures, problems arise when working with eluents enriched with water and/or containing alcohols. The viscosity of such eluents increases sharply with decreasing temperature, which leads to an increase in pressure in the system.
The effect of small temperature fluctuations on separation can be eliminated by thermostatting the chromatographic column, or the entire liquid system (which is not possible for all chromatographs).
2. Power quality. Most modern chromatographs are equipped with power stabilization systems, however, the quality of the on-site power supply must also be high. If the power supply is insufficient, any start of a series of determinations in automatic mode may fail due to a malfunction.
3. Purity of solvents. To prepare mobile phases, especially pure solvents should be used.
In general, the requirements for mobile phase purity depend on the detection method, the elution method (isocratic or gradient), the sensitivity of the detector to the target analyte and its concentration.
When using UV detection, the requirements for the purity of solvents increase when moving to the short-wave range, less than 230-240 nm. For isocratic elution during UV detection at wavelengths greater than 220-240 nm, “high purity” solvents can be used. and distillate water. All reagents added to the mobile phase must also be of sufficient purity; It may be useful to recrystallize crystalline reagents before use.
For gradient elution, it is necessary to use “for liquid chromatography” grade solvents and double distillate water. Special requirements in the gradient elution method (in reversed-phase chromatography) are placed on the purity of the aqueous buffer and water in particular. First of all, this is due to the fact that at the initial stage of elution, the adsorbent absorbs contaminant components from the mobile phase enriched in aqueous buffer, which are subsequently eluted and appear on the chromatogram in the form of “humps”, “thresholds” and individual peaks, which greatly complicate the selection of useful analyte signals .
The purest solvents are required for group determinations of trace amounts of substances in the gradient elution mode.
To perform determinations in gradient elution mode, as well as precision determinations in isocratic mode, the mobile phase must be used once, that is, the eluate must be discarded or discarded.
In isocratic elution, if there are no particular sensitivity problems, the spent eluent can be reused. A system in which the eluate, after passing through the detector, is returned to the container containing the mobile phase is called a “recycle system.” Such a system is especially useful in the case of carrying out a large number of routine isocratic determinations on standard columns (250x4.6, 150x4.6) at a volumetric flow rate of about 1 ml/min. In these cases, the recycle system provides savings of up to 200-300 ml of organic solvent per day. This economical system allows the use of very pure, expensive solvents for analysis. The issue of saving expensive solvents is less acute in the case of using microcolumns (80x2, 100x2), since separation requires an order of magnitude smaller volume of the mobile phase.
4. Degassing of solvents. Solvents used in chromatography to prepare mobile phases usually contain dissolved air. Water contains especially a lot of air.
When working with non-degassed eluents, air bubbles enter various components of the liquid system: pump, column, capillaries, detector. When air enters a liquid system, high, periodic noise appears on the chromatogram due to pressure fluctuations in the liquid system. This leads to a sharp decrease in the sensitivity of the analysis.
To remove air from the eluent, it is degassed. As a rule, only eluents for reverse phase separations are degassed - since aqueous-organic mixtures contain significant amounts of dissolved air. Degassing must be carried out especially carefully in the case of gradient elution, as well as when using fluorimetric detection.
During gradient elution in a reversed-phase mode, mixing of two eluents occurs - aqueous-organic mixtures of different compositions. Mixing non-degassed eluents leads to intense release of dissolved air, which is critical for the determination as a whole (in the chromatogram, air bubbles are recorded as sharp “emissions” on the zero line).
The sensitivity of fluorimetric detection decreases with a high content of dissolved air in the mobile phase (fluorescence quenching occurs). Therefore, when using fluorimetric detection, special attention must be paid to degassing the eluent.
There are three main methods for degassing mobile phases for liquid chromatography.
A. Degassing by vacuum - the eluent is kept in a Claisen flask under the vacuum of a water jet pump for several minutes. When carrying out degassing, boiling of the eluent should be avoided.
b. Thermal degassing is used for degassing aqueous-organic eluents with a high proportion of water. The mobile phase is placed in a flask, which is not hermetically sealed with a stopper, and left in a water bath at a temperature of about 50°C. After 10-15 minutes, the flask is sealed with a stopper and cooled under running water to room temperature.
V. Ultrasound degassing. The mobile phase is treated with ultrasound for several minutes and then allowed to settle for 10-15 minutes. This method is often not effective enough for degassing aqueous-organic eluents.
Modern pumping systems for liquid chromatography are equipped with automatic degassing systems. However, when carrying out gradient analyses, it is better to degas both mobile phases first and “manually”, using one of the given methods.
5. Filtration of the mobile phase. To ensure uninterrupted operation of the pump, it is advisable to filter the mobile phase under vacuum using a membrane filter.
6. Washing the column and components of the liquid system. After working with aqueous-organic mobile phases containing salts and acids, the entire liquid system (including the column) should be washed with distilled water with the addition of 5-10% organic solvent. This washing is done so that during non-working hours the components of the chromatograph liquid system and the stationary phase itself do not wear out additionally.
Failure to carry out such washing leads, first of all, to the fact that after stopping the pump, salts are deposited from the eluent on its parts and on the walls of the detector cuvette. This, in turn, leads to unstable operation of the device as a whole, as well as premature wear of the moving parts of the pump. Regular failure to flush the system from salt- and acid-containing eluents can lead to a reduction in the lifetime of the stationary phase.
Adding some organic solvent to the wash water is necessary to prevent biological contamination of the fluid system.
7. Transition to a new mobile phase that does not mix with the previous one. This transition is carried out through an intermediate solvent that is indefinitely miscible with both mobile phases - usually through isopropanol or acetone.
To switch from an aqueous eluent to a non-polar eluent, the liquid system should be washed with water with the addition of an organic solvent, then remove the chromatographic column, wash the system with isopropanol (acetone), wash the system with a non-polar eluent, and install a new column.
For the reverse transition, remove the chromatographic column, wash the liquid system with isopropanol (acetone), then with an aqueous eluent, then install a new column.
When switching from an aqueous eluent to a non-polar one, make sure in advance that the pump seal material is designed to work with non-polar solvents.
8. Sample filtration. If the analyzed sample contains undissolved suspended matter, then it is advisable to filter it by passing the sample through a membrane filter connected to a syringe. Unfortunately, if the sample is small, less than a milliliter, then filtering it in this way becomes almost impossible.
When regularly analyzing samples containing suspended matter, the inlet filter on the column (frit) may become clogged, which will primarily lead to an increase in pressure in the system. In this case, it is better to replace the inlet filter, and if there is no replacement, rinse it in an organic solvent with ultrasonic treatment for 10-15 minutes.
The most optimal solution to the problem is to use an in-line filter in front of the column. The in-line filter contains a replaceable frit - the same as on the column. Replacing frit on an in-line filter is a routine operation that can be performed quite often.
9. Application of pre-columns. With regular analysis of “dirty” samples, the chromatographic column quickly becomes dirty and loses its separation ability. A well-known alternative to careful sample preparation in this case is the use of a pre-column, which protects the main column from contamination.
Sometimes it is advisable not to carry out sample preparation at all, but to place an in-line filter and pre-column in line in front of the main column. The advantages of this scheme are simplicity and rapidity of analysis with less labor and reagents.
10. Preservation of chromatographic columns. Before long-term storage, chromatographic columns are washed and filled with a solvent that is quite specific for each type of stationary phase.
Thus, chromatographic columns for operation in normal-phase systems are usually filled with a high-boiling hydrocarbon, for example, isooctane. The reversed phases are washed with water and filled with acetonitrile, or at low feed speed with isopropanol. Phases intended for working with aqueous buffers are filled with water with a small addition of sodium azide (bacteriostatic).
Instructions for storing the column may be indicated in its passport.
11. Storage of water buffers. In the case of routine determinations, it is quite convenient to immediately prepare a large volume of aqueous buffer for preparing the mobile phase. Unfortunately, the aqueous buffer cannot be stored for more than a few days unless sodium azide, a bacteriostatic agent, is added to it. Mobile phases based on phosphate buffer are very poorly stored.
Sometimes a large volume of aqueous buffer is prepared in order to “increase the reproducibility of the analysis.” Generally speaking, with this approach the reproducibility of the analysis does not increase, but problems with buffer storage inevitably arise.
Generally speaking, the answer to the question is whether to prepare a water buffer for a week or for one day? - determined solely by the principle of convenience.
12. Regularity of calibration. As a rule, calibration against the standard is carried out every day, or every time a new eluent is prepared.
Calibration is carried out when the chromatographic system reaches a steady state; The readable parameters are the retention time of the standard peak, its area (in case of spectrophotometric detection - at the reference wavelength), spectral ratios (in case of using a scanning or diode-array spectrophotometric detector).
At the beginning of work, the standard can be analyzed twice to confirm retention time reproducibility.
1. Determination of the components of the drug "BICILLIN-3" by HPLC
Bicillin-3 is a long-acting penicillin and is a mixture of sodium, novocaine and benzathine salts of benzylpenicillin (BP). According to the current VFS 42-3034-98, the determination of BP in the drug is carried out using HPLC, novocaine is determined spectrophotometrically, and benzathine (N,N1-dibenzylethylenediamine) is extracted with ether from an aqueous solution saturated with sodium chloride. After evaporation of the ether, benzathine is determined by titration with perchloric acid.
In the European Pharmacopoeia, the content of BP and benzathine in the benzathine salt of BP is determined using gradient HPLC in a mixture of methanol with sodium phosphate solution at pH 3.5.
The purpose of the work is to develop an HPLC method in isocratic mode for the determination of components in bicillin-3.
experimental part
We used bicillin-3 produced by AKO Sintez (Kurgan). The study was carried out on a chromatograph from Waters (USA) with a pump model 510, a UV detector model 481 and an injector model 7125 (Rheodyne) with a dosing loop with a capacity of 50 μl. For detection, a wavelength of 214 nm was used, at which all analyzed compounds are well detected. Registration of chromatograms and calculation of peak areas and main retention parameters were carried out using a personal computer with an analog-to-digital converter and the Multichrome program.
A reverse-phase version of the HPLC method was studied on a Luna C18 (2) column measuring 250 x 4.6 mm from Phenomenex (USA), since the column had previously proven itself to be relatively cheap with improved symmetry of the output of organic amine peaks. For the same purpose, a mixture of acetonitrile with a buffer solution containing triethylamine as one of the components and having a pH of 5.0 was used as a mobile phase.
The initial solution for preparing the mobile phase is a 2.5 M solution of phosphoric acid, which was titrated with triethylamine to pH 5.0. A buffer solution for HPLC was prepared by diluting the initial solution with water 10 times. 750 ml of the resulting buffer solution was mixed with 250 ml of acetonitrile. At the same time, the apparent pH value of the mobile phase increased to 5.7. The mobile phase speed is 1 ml/min. Chromatography was carried out at room temperature. Analysis time 20 min.
Since the components included in the drug differ in acid-base properties - BP is an acid, and novocaine and benzathine are bases, with increasing pH their retention times in the pH range where their ionization changes shift in different directions. Therefore, by changing the pH it is easy to select a convenient retention of the analyzed components. However, an increase in pH leads to a noticeable deterioration in the shape of the benzathine peak, and a decrease leads to insufficient resolution of novocaine and BP hydrolysis products. The separation of bicillin-3 components under the above conditions is shown in the figure. The retention times of novocaine, benzathine and BP were 4.2, 11.6 and 14.8 min, respectively.
What is significant is the output of the novocaine peak between 2 peaks, which are products of BP hydrolysis. In this regard, for better separation of components, it is recommended to add small amounts of 2.5 M solutions of phosphoric acid or triethylamine to the mobile phase and control the separation by chromatography of a mixture of novocaine and BP, the solution of which was stored for about a day at room temperature.
For quantitative determination, 20-25 mg of bicillin-3 was added to a 100 ml volumetric flask and dissolved in a 20% aqueous solution of acetonitrile. The use of methanol or its solutions for dissolution led to partial methylation of BP. An increase in the concentration of acetonitrile led to a broadening of the novocaine peak. The upper limit of drug concentration is limited by its solubility. Calibration curves for BP and benzathine were obtained using the sodium salt of BP and benzathine diacetate after appropriate conversion. The calibration graph for BP is linear in the region of 0.1-0.5 mg/ml, for benzathine and novocaine - in the region of 0.01-0.05 mg/ml. The results of determining the components in 5 series of the drug are presented in Table 1, where each value is the average of 5 determinations. The relative standard deviation was 1.6% for novocaine, 3.4% for benzathine and 1.4% for BP.
From Table 1 it follows that the results of quantitative determination using HPLC fall within the permissible limits regulated by the ND.
To confirm the correctness of the proposed method, the components of bicillin-3 were analyzed in model mixtures prepared by mixing the sodium salt of BP, benzathine diacetate and novocaine. The results are shown in Table 2. The results were recalculated to the original components.
Each value in the "Found" column of Table 2 is the average result of 3 determinations. The average relative deviation was 2.2% for benzathine, 0.9% for novocaine and 0.8% for BP, which correlates with the relative average standard deviations found when analyzing components in real samples. For benzathine, the scatter of results is slightly higher than for other components, which is explained by the low height and irregular shape of the peak and, accordingly, a larger integration error. Another reason for relatively large errors in the determination of benzathine may be the injector memory effect when analyzing highly adsorbed substances. However, even such a scatter, slightly larger than that accepted for analyzes using the HPLC method, is quite acceptable for the determination of benzathine.
conclusions
1. A method for the detection and quantitative determination of components in the preparation "Bicillin-3" has been developed.
2. The method has been tested on a number of batches of the drug and confirmed by analysis of model mixtures of known composition.
2. HPLC in the analysis of drugs containing propyphenazone
Propyphenazone (4-isopropyl-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one; isopropylantipyrine) is a non-narcotic analgesic of the pyrazolone series and is part of combination over-the-counter drugs. Currently, Caffetin tablets (composition: propyphenazone - 0.21 g, paracetamol - 0.25 g, caffeine - 0.05 g, codeine phosphate - 0.01 g) and saridon (paracetamol - 0.25 g) are widely used in medical practice. g, propyphenazone - 0.15 g, caffeine - 0.05 g). According to existing regulatory documentation, chromatography in a thin layer of sorbent is used to confirm the authenticity of caffetin tablets. Quantitative determination is proposed to be carried out in separate portions of the drug using different methods for each component: using spectrophotometry, titrimetry, a combination of thin layer chromatography and spectrophotometry. In the regulatory documentation for saridon, paracetamol, caffeine and propyphenazone are determined by HPLC on 12.5 cm long columns with Merck Lichrospher C18 sorbent and a mobile phase of the composition: methanol-0.01 M phosphoric acid in a ratio of 30: 70.
The purpose of this work is to develop methods for the detection and quantitative determination of the components of caffetin and saridon tablets using HPLC.
The work used tablets of caffetine produced by Alkaloid Skopje (Republic of Macedonia), saridon produced by Laboratories Roche Nicholas S.A., Gaillard (France) and the substances of the components included in their composition. The study was carried out on a domestic microcolumn liquid chromatograph "Milichrome-4" with a UV spectrophotometric detector, an 8 cm long column with reverse-phase Separon-C18 sorbent as a stationary phase. The polar nature of the analyzed compounds and their good solubility in water and acetonitrile determined the choice of water-acetonitrile mixtures in different ratios as the mobile phase. Mobile phases were tested: acetonitrile - water in ratios 9: 1; 7:3; 6:4; 8:2 and acetonitrile-water-diethylamine (3:2:0.2). The use of mobile phases with a volume fraction of organic solvent exceeding 80% was avoided in order to exclude normal-phase interactions, which complicate further control of the composition of the mobile phase. A solvent volume fraction of less than 5% leads to functional instability of the mobile phase and irreproducible retention times. The introduction of diethylamine into the mobile phase as a modifier made it possible to achieve the separation of all 4 components of caffetin tablets. It is known that on the surface of octadecyl silica gel there is a significant amount of residual silanol groups capable of ion exchange interaction. Diethylamine eliminates silanol groups from the chromatographic process, improves peak shape, reduces analysis time and adjusts the pH on the silica gel surface. The measurement was carried out under the following conditions: recording scale 2.0, retention time 0.8 s, eluent flow rate 50 μL/min, injection volume 3 μL. Peak detection was carried out at 2 wavelengths - 238 and 276 nm.
Identification was carried out according to retention parameters, which were determined previously using standard solutions of the substances under study.
The components of caffetine tablets are separated using the mobile phase acetonitrile-water-diethylamine (3:2.2:0.2). The retention time for paracetamol was 3.08 minutes, propyphenazone - 5.73 minutes, caffeine - 4.0 minutes, codeine - 4.67 minutes.
The components of saridon can also be separated using a mobile phase of acetonitrile-water (8:2). Retention time for paracetamol - 3.9 minutes, propyphenazone - 5.11 minutes, caffeine - 4.44 minutes.
For quantitative determination, the absolute calibration method was used. A direct proportional relationship between the concentration of the substance and the peak height was observed for paracetamol in the range of 50-200 µg/ml, for propyphenazone - 25-128 µg/ml, caffeine - 20-50 µg/ml, codeine - 59-234 µg/ml.
The HPLC method has some limitations in the analysis of complex mixtures. With the simultaneous presence of substances in macro- and microquantities in the mixture, the column is overloaded, which affects the quality of separation and the shape of the emerging peaks. In caffetine, the content of codeine phosphate in relation to paracetamol and propyphenazone is 21-25 times less, so liquid extraction is recommended to separate codeine from the remaining components of the tablets. We have previously established that paracetamol, propyphenazone and caffeine are extracted during a single extraction with ethyl acetate from aqueous solutions at pH 2.0 in amounts of 87.43, 87.29 and 87.84%, respectively, and codeine completely remains in the aqueous solution and for its extraction and concentration it is necessary to use chloroform at pH 9.0-10.0.
Method for the quantitative determination of paracetamol, propyphenazone and caffeine in saridon and caffetin tablets. 20 tablets are ground in a mortar into a fine homogeneous powder, about 0.01 g (exactly weighed) of the ground tablet powder is weighed and placed in a 25 ml volumetric flask, 10 ml of acetonitrile is added. and mix thoroughly.
The contents of the flask are brought to the mark with acetonitrile, mixed and filtered. The solution is injected into the chromatograph column in a volume of 3.0 μl. The content of paracetamol, propyphenazone and caffeine is determined by the absolute calibration method. The results of the determination are given in Tables 1 and 2, from which it is clear that the data obtained fall within the permissible content limits according to regulatory documentation (ND).
Method for the quantitative determination of codeine phosphate in caffetine tablets. About 0.2 g of crushed tablets (exactly weighed) are dissolved in 20 ml of water, mixed well until a homogeneous solution is obtained, filtered through a filter for fine and very fine sediments, the filter is washed with 10 ml of purified water. The solution is acidified with a 10% sulfuric acid solution to pH 2.0. Extract three times with ethyl acetate in 10 ml portions. The extracts are discarded. A 25% ammonia solution is added to the aqueous solution to pH 9.0-10.0. Extract three times with chloroform in 10 ml portions. The combined chloroform extracts are placed in porcelain cups and evaporated at room temperature. The dry residues are dissolved in acetonitrile, transferred to a 25 ml volumetric flask and diluted to the mark with the same solvent. 3 μl of the resulting solution is introduced into a chromatograph column and codeine is determined under the described conditions. The results of the determination are given in Table 3.
To evaluate the accuracy of the proposed methods and check the reproducibility of the results, model mixtures were prepared and studied. Data based on the example of saridon tablets are given in Table 4. As can be seen from Table 4, the relative error of determination does not exceed ±1.19% for paracetamol, ±1.16% for propyphenazone, and ±1.63% for caffeine.
Method for quantitative determination of components of saridon tablets in model mixtures. Weigh out precise portions of paracetamol (about 0.08 g), propyphenazone (about 0.05 g) and caffeine (about 0.016 g), transfer to a 50 ml volumetric flask, dissolve in a small volume of acetonitrile and make up to the mark with the same solvent. Take an aliquot of 2.5 ml and transfer it to a 25 ml volumetric flask, adjust the volume to the mark with the same solvent, mix and filter. The solution is injected into the chromatograph column in a volume of 3 μl.
conclusions
1. A method has been developed for detecting the components of caffetin and saridon tablets using HPLC.
The retention time for paracetamol was 3.08 minutes, for propyphenazone - 5.73 minutes, caffeine - 4 minutes and codeine - 4.67 minutes.
2. An HPLC method was proposed for the quantitative determination of the components of caffetin and saridon tablets.
The relative error of determination was ±1.19-1.21% for paracetamol, ±1.16-1.71% for propyphenazone, ±1.22-1.63% for caffeine and ±2.95% for codeine.
3. Standardization of the drug "Adanol"
The pharmaceutical company Polisan has developed a number of complex drugs with metabolic effects that stimulate metabolic processes in the brain, including Cytoflavin (injections, tablets) and Adanol. "Adanol" has pronounced antihypoxic and anti-ischemic properties and is a promising drug for the treatment of patients with the consequences of stroke. It is a tablet dosage form coated with an enteric coating.
It contains succinic acid (SA), piracetam (Pc), riboxin (Rb), nicotinamide (NA), pyridoxine hydrochloride (PG), riboflavin mononucleotide (RF).
The purpose of the work is to develop a method for the qualitative and quantitative determination of UC in complex multicomponent mixtures using the example of the drug "Adanol".
The work used a high-pressure liquid chromatograph from Shimadzu (Japan) with a UV detector and a Hypersil BDS C18 column from Supelco Inc. grain size 5 microns, length 250 mm with internal diameter 4.6 mm. The mobile phase is an aqueous-organic phase based on a phosphate buffer (pH 2.6-7.0). Detection wavelength 206 nm. Analysis mode isocratic, elution rate 500 µl/min; sample volume 20 µl. UV spectra were recorded on a UV mini-1240 spectrophotometer from Shimadzu.
For the quantitative determination of most of the substances included in the drug, spectrophotometric methods of analysis have been proposed. However, a comparison of the spectral characteristics of the components of the drug showed that the absorption regions of YAK, PG, NA, Pb and Pc in the UV zone overlap each other (Fig. 1).
In this regard, their content in the mixture cannot be determined by direct spectrophotometry and in this case it is advisable to use the HPLC method. Only RF has a specific absorption region of more than 350 nm (lmax=373, E1% 1cm=202 and lmax=445 nm, E1% 1cm=243), therefore a qualitative and quantitative analysis by spectrophotometric method has been proposed for it.
Based on the data obtained, the optimal working wavelength for the analysis of 5 substances was selected, the component lopt = 206 nm (see Fig. 1). This value is the maximum of the UV spectrum of UC, which has the lowest specific absorption (lmax = 206 nm E1% 1cm = 5.8) compared to the other components of the mixture (see table).
Since all substances included in the preparation are ionic, it is advisable to use reverse-phase chromatography for their analysis using non-polar stationary and polar mobile phases. In reverse-phase chromatography of ionic compounds, the pH value is one of the factors that significantly affects the efficiency of separation of individual substances. When working with modern reverse-phase sorbents, buffer solutions with pH ranging from 2.0 to 8.0 are usually used in the mobile phase. Since the selected optimal operating wavelength is 206 nm, it is best to use a phosphate buffer because it does not have a position in the UV wavelength range greater than 200 nm.
To study the behavior of UC at different pH values, its spectra were recorded in phosphate buffer solutions pH 7.0 and 2.6 (Fig. 2). At pH 2.6, a hypochromic effect of the molecular form of UC is observed - absorption decreases by 3 times compared to the spectrum at pH 7.0 (at this pH value, the dissociation of UC is completely suppressed). Taking this into account, the optimal pH value of the mobile phase is 7.0. Next, the influence of the composition and pH of the mobile phase on the efficiency of separation of drug components was studied. At pH 2.6, there was no complete separation of the mixture into individual peaks - PC, Na and PG are not divided and come out first as one peak, followed by YaK and Pb in the form of individual peaks. At pH 5.5 there was also no complete separation of the components. At pH 7.0, complete separation of the mixture components occurred. All substances appeared in the form of separate peaks in the following sequence: YAK, Pc, PG, NA and Pb. However, the separation process is long - 45-50 minutes.
To ensure greater elution force of the mobile phase and speed up the separation process, it is necessary to introduce a less polar organic solvent into its composition. Of the solvents most often used as eluents in HPLC, according to the transparency limit in UV light at the selected working wavelength (lopt = 206 nm), we could use acetonitrile and methanol, whose transparency limits are 195 and 205 nm, respectively.
When 2% methanol was introduced into the mobile phase, the process time was reduced, but the NA peak had a high asymmetry and the Pb peak overlapped the tail of the NA peak. After reducing the methanol concentration to 1%, the peaks of Pb and NA did not overlap, but the asymmetry of the NA peak increased. In order to reduce it, acetonitrile was introduced into the mobile phase containing 1% methanol.
As a result of the experiments, a mobile phase was selected - an aqueous-organic phase consisting of a phosphate buffer pH 7.0 containing 1% methanol and 0.5% acetonitrile, which ensured the effective separation of all 5 components (Fig. 3). The total chromatography time in this system was 35 minutes, and the retention times of the components were approximately (in minutes): YA - 5.3, Ps - 15, PG - 19.3, NA - 26, Rb - 31.
Quantitative determination was carried out by the external standard method using solutions of standard samples of individual components.
The metrological characteristics of the proposed quantitative determination method were studied using model mixtures in 5 replicates and are presented in the table.
As follows from the table, using the developed method in the drug "Adanol" it is possible to determine all 5 components with a relative error of no more than 3% with a confidence probability of 95%.
In addition to the peaks of the main components of the drug, the chromatogram obtained under the conditions described above identifies peaks of hypoxanthine and nicotinic acid, present in the original substances, as well as those formed during hydrolysis from Pb and NA, respectively. Thus, the developed method allows us to qualitatively and quantitatively determine these foreign impurities in the preparation.
conclusions
1. The optimal conditions for the quantitative analysis of succinic acid using the HPLC method in a multicomponent mixture were determined.
2. A method has been developed for qualitative and quantitative analysis of the components of the drug "Adanol", including impurities, with a relative error of determination of no more than 3%.
List of used literature
1. M.A. Kazmin, A.V. Mikhalev, A.P. Arzamastsev "Determination of the components of the drug "BICILLIN-3" by HPLC" // Pharmacy - No. 5 - 2002 - p. 5-6.
2. T.H. Vergeichik, N.S. Onegova "HPLC in the analysis of drugs containing propyphenazone" // Pharmacy - No. 6 - 2002 - pp. 13-16.
3. A.Yu. Petrov, S.A. Dmitrichenko, A.L. Kovalenko, L.E. Alekseeva “Standardization of the drug “Adanol” // Pharmacy - No. 5 - 2002 - pp. 11-13.
Additional
1. Baram G.I., Fedorova G.A. // Application of chromatography in the food, microbiological and medical industries: Mat. All Conf. - Gelendzhik, 1990 - pp. 43-44.
2. Krichkovskaya L.V., Chernenkaya L.A. // Application of chromatography in the food, microbiological and medical industries: Mat. All Conf. - Gelendzhik, October 8-12, 1990, M., 1990. - P.49.
3. Chromatography: Practical application of the method: In 2 parts. Part 2 - M.: Mir, 1986. - 422 p.
High-performance liquid chromatography (HPLC) is a column chromatography method in which the mobile phase (MP) is a liquid moving through a chromatography column filled with a stationary phase (sorbent). HPLC columns are characterized by high hydraulic pressure at the column inlet, which is why HPLC is sometimes called "high pressure liquid chromatography".
Depending on the mechanism of separation of substances, the following HPLC options are distinguished: adsorption, partition, ion exchange, size exclusion, chiral, etc.
In adsorption chromatography, the separation of substances occurs due to their different abilities to adsorb and desorb from the surface of an adsorbent with a developed surface, for example, silica gel.
In partition HPLC, separation occurs due to the difference in the distribution coefficients of the substances being separated between the stationary phase (usually chemically grafted to the surface of a stationary carrier) and the mobile phase.
Based on polarity, PF and NP HPLC are divided into normal-phase and reverse-phase.
Normal-phase is a variant of chromatography that uses a polar sorbent (for example, silica gel or silica gel with grafted NH 2 or CN groups) and a non-polar PF (for example, hexane with various additives). In the reverse-phase version of chromatography, non-polar chemically modified sorbents (for example, non-polar alkyl radical C 18) and polar mobile phases (for example, methanol, acetonitrile) are used.
In ion exchange chromatography, the molecules of a mixture of substances, dissociated in solution into cations and anions, are separated when moving through a sorbent (cation exchanger or anion exchanger) due to their different rates of exchange with the ionic groups of the sorbent.
In size exclusion (sieve, gel permeation, gel filtration) chromatography, molecules of substances are separated by size due to their different ability to penetrate the pores of the stationary phase. In this case, the largest molecules (with the highest molecular weight) capable of penetrating into the minimum number of pores of the stationary phase are the first to leave the column, and substances with small molecular sizes are the last to leave.
often separation occurs not through one, but through several mechanisms simultaneously.
The HPLC method can be used to control the quality of any non-gaseous analyte. To carry out the analysis, appropriate instruments are used - liquid chromatographs.
A liquid chromatograph usually includes the following main components:
– PF preparation unit, including a container with the mobile phase (or containers with individual solvents included in the mobile phase) and a PF degassing system;
– pumping system;
– mobile phase mixer (if necessary);
– sample introduction system (injector);
– chromatographic column (can be installed in a thermostat);
– detector;
– data collection and processing system.
Pumping system
Pumps supply PF to the column at a given constant speed. The composition of the mobile phase may be constant or vary during analysis. In the first case, the process is called isocratic, and in the second - gradient. Filters with a pore diameter of 0.45 µm are sometimes installed in front of the pumping system to filter the mobile phase. A modern liquid chromatograph pumping system consists of one or more computer-controlled pumps. This allows you to change the composition of the PF according to a specific program during gradient elution. Mixing of PF components in a mixer can occur both at low pressure (before the pumps) and at high pressure (after the pumps). The mixer can be used to prepare the PF and during isocratic elution, however, a more accurate ratio of components is achieved by pre-mixing the PF components for the isocratic process. Pumps for analytical HPLC make it possible to maintain a constant flow rate of PF into the column in the range from 0.1 to 10 ml/min at a pressure at the column inlet of up to 50 MPa. It is advisable, however, that this value should not exceed 20 MPa. Pressure pulsations are minimized by special damper systems included in the design of the pumps. The working parts of the pumps are made of corrosion-resistant materials, which allows the use of aggressive components in the PF composition.
In high-performance liquid chromatography (HPLC), the nature of the processes occurring in the chromatographic column is generally identical to the processes in gas chromatography. The only difference is in the use of liquid as a stationary phase. Due to the high density of liquid mobile phases and the high resistance of columns, gas and liquid chromatography differ greatly in instrumentation.
In HPLC, pure solvents or mixtures thereof are usually used as mobile phases.
To create a stream of pure solvent (or mixtures of solvents), called an eluent in liquid chromatography, pumps included in the hydraulic system of the chromatograph are used.
Adsorption chromatography is carried out as a result of the interaction of a substance with adsorbents, such as silica gel or aluminum oxide, which have active centers on the surface. The difference in the ability to interact with the adsorption centers of different sample molecules leads to their separation into zones during movement with the mobile phase along the column. The zone separation of the components achieved in this case depends on the interaction with both the solvent and the adsorbent.
The most widely used in HPLC are silica gel adsorbents with different volumes, surface areas and pore diameters. Aluminum oxide and other adsorbents are used much less frequently. The main reason for this:
insufficient mechanical strength, which does not allow packaging and use at high pressures characteristic of HPLC;
silica gel, compared to aluminum oxide, has a wider range of porosity, surface area and pore diameter; The significantly greater catalytic activity of aluminum oxide leads to distortion of analysis results due to the decomposition of sample components or their irreversible chemisorption.
Detectors for HPLC
High-performance liquid chromatography (HPLC) is used to detect polar non-volatile substances that, for some reason, cannot be converted into a form suitable for gas chromatography, even in the form of derivatives. Such substances, in particular, include sulfonic acids, water-soluble dyes and some pesticides, for example phenyl-urea derivatives.
Detectors:
UV detector on a diode matrix. A “matrix” of photodiodes (more than two hundred of them) constantly registers signals in the UV and visible regions of the spectrum, thus providing recording of UV-B spectra in scanning mode. This allows you to continuously record, at high sensitivity, undistorted spectra of components quickly passing through a special cell.
Compared to single wavelength detection, which does not provide information about peak purity, the ability to compare full spectra of a diode array provides a much higher degree of confidence in the identification result.
Fluorescence detector. The great popularity of fluorescent detectors is due to their very high selectivity and sensitivity, and the fact that many environmental pollutants fluoresce (eg polyaromatic hydrocarbons).
Electrochemical detector used to detect substances that are easily oxidized or reduced: phenols, mercaptans, amines, aromatic nitro- and halogen derivatives, aldehydes, ketones, benzidines.
Chromatographic separation of a mixture on a column due to the slow progress of the PF takes a lot of time. To speed up the process, chromatography is carried out under pressure. This method is called high-performance liquid chromatography (HPLC)
Modernization of equipment used in classical liquid column chromatography has made it one of the most promising and modern methods of analysis. High-performance liquid chromatography is a convenient method for the separation, preparative isolation and qualitative and quantitative analysis of non-volatile thermolabile compounds with both low and high molecular weight.
Depending on the type of sorbent used, this method uses 2 chromatography options: on a polar sorbent using a non-polar eluent (direct phase option) and on a non-polar sorbent using a polar eluent - the so-called reverse-phase high-performance liquid chromatography (RPHPLC).
During the transition from eluent to eluent, equilibrium under HPLC conditions is established many times faster than under conditions of polar sorbents and non-aqueous PFs. As a result of this, as well as the convenience of working with aqueous and aqueous-alcohol eluents, OFVLC has now gained great popularity. Most HPLC analyzes are carried out using this method.
Detectors. The output of an individual component from the column is recorded using a detector. For registration, you can use a change in any analytical signal coming from the mobile phase and associated with the nature and quantity of the mixture component. Liquid chromatography uses analytical signals such as light absorption or light emission of the output solution (photometric and fluorimetric detectors), refractive index (refractometric detectors), potential and electrical conductivity (electrochemical detectors), etc.
The continuously detected signal is recorded by a recorder. A chromatogram is a sequence of detector signals recorded on a recorder tape, generated when individual components of a mixture leave the column. If the mixture is separated, individual peaks are visible on the external chromatogram. The position of the peak in the chromatogram is used for the purpose of identifying the substance, the height or area of the peak - for the purpose of quantitative determination.
