High performance liquid chromatography (HPLC)

Transcription

1 High performance liquid chromatography (HPLC) 1.1 The aim of the practice is to get insight into the basics of a user level High Performance Liquid Chromatographic approach through a pharmaceutical application and to show, how to determine and interpret the principal chromatographic parameters in practice. 1.2 Theory of HPLC HPLC is an acronym, related to the most widely used separation technique that involves: the injection of a small volume of liquid sample into a metal tube packed with tiny particles (3 to 5 μm) called the stationary phase. individual components of the injected sample are forced through the stationary phase with the mobile phase (a liquid) delivered by a pump. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between the molecules and the packing particles. Types of HPLC Based on the chemical nature of the stationary phase, and on the retention mechanism, HPLC can be divided into three types, which cover almost 90% of all chromatographic applications. 1) Adsorption chromatography: the stationary phase is an adsorbent, and the retention mechanism based on repeated adsorption and desorption steps. Normal-phase chromatography: in which the stationary phase (or adsorbent) is more polar than the mobile phase (or eluent). For example, the adsorbent can be polar silica-gel and the solvent can be n-hexane or diethyl-ether. Reverse-phase chromatography: in which the stationary phase is nonpolar or weakly polar and the solvent is more polar (just the opposite as in normalphase chromatography). Usually, the reversed stationary phases are chemically modified silica-gel, which covalently bind alkyl chains. Mobile phases are usually polar mixture of an organic solvent (acetonitrile, methanol, 2-propanol, etc.) and water. 1 unmodified silica gel (normal-phase particle) reversed-phase particle (with free silanol groups) endcapped reversed-phase particle Figure 1. Chemical modification of the silica gel using octadecyl ligands (C18). 2) Ion-exchange chromatography: the stationary phase has a charged surface with opposite charges on it compared to the sample ions. This technique is used almost only

2 for ionic or ionisable samples. The mobile phase is an aqueous buffer with controlled ph and ionic strength. 3) Size exclusion chromatography: the stationary phase is made of a material with precisely controlled pore size. The large molecules rapidly pass through the column, while the smaller ones penetrate into the pores and washed out later by the mobile phase. 1.3 The HPLC instrument Figure 2. Schematic diagram of a typical HPLC instrument. The instrumentation includes flask(s) for the mobile phase storage, pump(s), injector (manual or automated), column, detector and acquisition and display system. The heart of the system is the column where separation occurs. Flasks for the mobile phase storage: Using high pressure pumps to deliver liquid, the solvent have to be gas-free in HPLC experiments. The excess gas in the mobile phase causes several problems during the analysis. Because of the compressibility of the gases, the pump pressure and the flow rate will fluctuate, and it would cause significant disturbance in the detection and in the repeatability of the chromatographic data. Because of these reason, the mobile phase has to be degassed. Degassing may be accomplished by one of the following methods or their combination: Degassing the liquid under vacuum-heavy-walled flask is really important, in this case. Heating the liquid until its boiling occur. Placing the container of liquid in an ultrasonic bath, or inserting an ultrasonic probe in it. Bubbling a fine stream of He through the liquid; He has the unique ability to purge other gases out of solutions. Some instruments are equipped with built-in degasser, but its capacity is not enough in every case. Pumps: The pumps applied in the practice of HPLC have to provide a constant and almost pulse-free flow of the mobile phase through the system. If we use one pump, the composition of the mobile phase is constant during the experiment, which is called isocratic elution. With the aid of two or more pumps, using a time program, the composition of the mobile phase is variable. When the mobile phase composition is changing during the analysis, the technique is called gradient elution.

3 Injector: Injector for liquid chromatographic system should provide the possibility of injecting the liquid sample within a large volume range with high reproducibility and under high pressure (up to 1200 bar). They should also produce minimum band broadening and minimize possible flow disturbances. Generally, the most useful and widely used sampling device for modern LC is the six-port Rheodyne valve. Figure 3. Manual six-port injector with the two position for sample introduction. In the six-port Rheodyne valve the sample is introduced into the sample loop (in LOAD position) using a special syringe. A clockwise rotation of the valve rotor (INJECT position) places the sample-filled loop into the mobile-phase stream, with subsequent injection of the sample onto the top of the column through a low-volume, cleanly swept channel. Columns: The part of the HPLC where the stationary phase is immobilised and the retentions of the compounds take place. Modern HPLC column beds used in adsorption chromatography are small rigid porous particles with high surface area. Figure 4. LC column with different length and diameter.

4 Detector response Nowadays, for the material of the column housing stainless steel is chosen because it offers the best compromise of cost, workability and corrosion resistance. Depending on the chromatographic procedure, the column length and diameter can change in a wide range. In modern instruments column thermostat is used to ensure the constant temperature in the column during separation. Detectors: Detectors equipped with the flow-through cell were a major breakthrough in the development of modern liquid chromatography. Such detection was first applied by the group of Tiselius, in Sweden in 1940, by continuously measuring the refractive index of the column effluent. Current LC detectors have wide dynamic range, and have high sensitivities often allowing the detection of nanograms of material. A few types of detectors: UV/Vis detector Fluorescence detector Conductivity detector Refractive index detector Mass spectrometer (MS) In the last decade there is a significant progress in the development of LC-MS interfacing systems. MS as an on-line HPLC detector is said to be the most sensitive, selective and in the same time the most universal detector. But it is still the most expensive one. 1.4 HPLC parameters The result of the chromatographic experiment is the chromatogram, which is the function of the detector response versus the time. Time (min) Figure 5. HPLC separation of a multi-component mixture

5 Retention time (t R ): is the easiest way to define chromatographic retention. We measure the time between the injection and the maximum of detector response (peak maximum) for the corresponding component. Retention time is inversely proportional to the eluent flow rate. Retention volume (V R ): is a more exact and global retention parameter. It is the product of the retention time and the volumetric flow rate of the mobile phase. Represents the volume of the mobile phase passed through the column while eluting a particular component. where F is the volumetric flow rate of the mobile phase (cm 3 /min) The retention volume of a component can be split into two parts: Dead volume (V M ) (or void volume) is the volume of the mobile phase that passed through the column while the components were moving with the liquid phase. V M is equal to the volume of the liquid phase in the column and it will be the same for any component eluted from the given column. In other words every component spend the same time in the mobile phase, the separation occurs because they spend different time in the stationary phase. Reduced retention volume (V R ) is the volume of the eluent that passed through the column while the component was retained on the surface. This volume is different for every component of the sample. Retention volume is independent of the flow parameters of the particular run, but it depends on the geometrical parameters of the column. Retention factor (k ) is dimensionless and independent of any geometrical parameters of the column or HPLC system. It could be considered to be a thermodynamic characteristic of the adsorbent-compound-eluent system. Theoretical plate number (N): The plate model supposes that the chromatographic column contains a large number of separate layers called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these plates. The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next. It is important to remember that these theoretical plates do not really exist; they are a figment of the imagination that help us to understand the processes at work in the column. They also serve as a way of measuring column efficiency, either by starting the number of theoretical plates in a column.

6 Theoretical plate height (H): where L is the length of the column. 1.5 Practice: Quantitative analysis of active substance of Saridon analgetic Instrumentation: - Eluent reservoir : glass flasks - Degasser: DGU-14A VP (Shimadzu, Japan) - Pumps: LC-10AD VP (Shimadzu, Japan) - Injector: 7125 Rheodyne injector (Rheodyne, USA) - Column: Xterra RP18 (3.9mm 150 mm; 5 μm); - Diode Array Detector: SPD-M10A VP (Shimadzu, Japan) - System Controller: SCL-10A VP (Shimadzu, Japan) - LabSolution software (Shimadzu, Japan). Mobil phase (eluents): eluent A: 0,1 v/v% TFA (trifluoracetic acid) eluent B: 0,1 v/v% TFA Chemicals (samples): thiourea, paracetamol and caffeine standard, Saridon tablet Figure 6. Chemical structures of the two studied compounds; caffeine (a) and paracetamol (b).

7 Tasks: Column characterisation: Determine the dead volume of the applied column with thiourea as a nonretained marker. Fill out the table below: Qualitative analysis of the standard compounds: Determine the retention volume and retention factor of paracetamol and caffeine by injecting standard solution under isocratic conditions. Effect of the experimental parameters on the retention volume of the standard compounds: Determine the effect of the change of methanol ratio of the mobile phase under isocratic conditions on the retention volumes of the studied compounds. Fill out the tables below: CH 3 OH % paracetamol CH 3 OH % caffeine EVALUATE AND SUMMARIZE THE RESULTS IN A FEW SENTECES Calibration curve and quantitative analysis: Dilute three times the standard stock solution and measure the peak area of the standards (three times each concentration). Calculate the average peak area (and standard deviation) of the three injections. Construct a calibration curve of peak area versus concentration. Inject the ten-fold diluted Saridon tablet solution three times and

8 determine the concentration of caffeine and paracetamol based on the average of the measured peak areas and the constructed calibration curve. Calculate the concentration (mg/tablet) of the pharmaceutically active compounds in a single Saridon tablet. 1.6 Questions 1) What is the definition of chromatography? 2) What is liquid chromatography? 3) What is the difference between normal- and reversed-phase chromatography? 4) Define the retention volume and the retention factor? 5) What is the chromatogram? 6) Define the main parts of an HPLC instrument! 7) What is the difference between isocratic and gradient elution technique?

9 Determination of preservatives and vitamin C with capillary zone electrophoresis (CZE) 1.1 Aim Capillary electrophoresis review. Qualitative and quantitative measuring of preservatives and vitamin C in lime juice by capillary electrophoresis method. 1.2 Introduction Separation by electrophoresis is obtained by differential migration of solutes in an electric field. In capillary electrophoresis (CE), electrophoresis is performed in narrow-bore capillaries, typically 25 to 200 µm inner diameter (id), which are usually filled only with buffer (electrolyte). The high electrical resistance of the capillary enables the application of very high electrical fields (100 to 500 V/cm) with only minimal heat generation. Moreover, the large surface area-to-volume ratio of the capillary efficiently dissipates the heat that is generated. The use of the high electrical fields results in short analysis times and high efficiency and resolution. In addition the numerous separation modes which offer different separation mechanisms and selectivities, minimal sample volume requirements (1 to 50 nl), on-capillary detection, and the potential for quantitative analysis and automation, CE is rapidly becoming a premier separation technique. One of the greatest advantages of CE is its diverse application range. Originally considered primarily for the analysis of biological macromolecules, it has proved useful for separations of compounds such as amino acids, chiral drugs, vitamins, pesticides, inorganic ions, organic acids, dyes, surfactants, peptides and proteins, carbohydrates, oligonucleotides and DNA restriction fragments, and even whole cells and virus particles. 1.3 Theory A schematic diagram of a generic capillary electrophoresis system is shown in Figure 1. Briefly, the ends of a narrow-bore, fused silica capillary are placed in buffer reservoirs. The content of the reservoirs is identical to that within the capillary. The reservoirs also contain the electrodes used to make electrical contact between the high voltage (HV) (10 to 30 kv) power supply and capillary. Sample is loaded onto the capillary by replacing one of the reservoirs (usually at the anode) with a sample reservoir and applying either an electric field or an external pressure. After replacing the buffer reservoir, the electric field is applied and the separation performed. Optical detection can be made at the opposite end, directly through the capillary wall. Figure 1. Schematic of CE instrumentation

10 Separation by electrophoresis is based on differences in solute velocity in an electric field. The velocity of an ion can be given by where v = ion velocity [cm/s] v = µ e E µ e = electrophoretic mobility [cm 2 /V s] E = applied electric field [V/cm] The mobility, for a given ion and medium, is a constant which is characteristic of that ion. The mobility is determined by the electric force that the molecule experiences, balanced by its frictional drag through the medium. The electric force can be given by F e = q E and the frictional force (for a spherical ion) by F f = - 6 π η r v where q = ion charge η = solution viscosity r = ion radius v = ion velocity qe = 6πηrv During electrophoresis a steady state, defined by the balance of these forces, is attained. At this point the forces are equal but opposite and F e = F f qe = 6πηrv Solving for velocity and substituting equation (4) into equation (1) yields an equation that describes the mobility in terms of physical parameters μ e = q 6πηr From this equation it is evident that small, highly charged species have high mobilities whereas large, minimally charged species have low mobilities.

11 Electro-osmotic flow (EOF) EOF is the bulk flow of liquid in the capillary and is a consequence of the surface charge on the interior capillary wall. The EOF results from the effect of the applied electric field on the solution doublelayer at the wall (Figure 2). The potential difference created close to the wall is known as the zeta potential. Figure 2. Representation of the double layer at the capillary wall Under aqueous conditions most solid surfaces possess an excess of negative charges. EOF is most strongly controlled by the numerous silanol groups (SiOH) that can exist in anionic form (SiO-) (Figure 3a). Although the exact pi of fused silica is difficult to determine, EOF becomes significant above ph 2.5. Counterions (cations), which build up near the surface to maintain charge balance, form the doublelayer (Figure 3b). When the voltage is applied across the capillary the cations forming the diffuse double-layer are attracted toward the cathode. Because they are solvated their movement drags the bulk solution in the capillary toward the cathode. This process is shown in schematic form in Figure 3c. The magnitude of the EOF can be expressed in terms of mobility by where μ EOF = EOF mobility ζ = zeta potential ε = dielectric constant of electrolyte ε o = dielectric constant of vacuum η = solution viscosity μ EOF = ζεε 0 4πη The zeta potential is essentially determined by the surface charge on the capillary wall. Since this charge is strongly ph dependent, the magnitude of the EOF varies with ph. At high ph, where the silanol groups are predominantly deprotonated, the EOF is significantly greater than at low ph where they become protonated. The zeta potential is also dependent on the ionic strength of the buffer. Increased ionic strength results in double-layer compression,

12 decreased zeta potential, and reduced EOF. Figure 3. Development of the electro-osmotic flow: a) negatively charged fused silica surface (Si-O - ) b) hydrated cations accumulating near surface c) bulk flow towards the cathode upon application of electric field The EOF controls the amount of time solutes remain in the capillary by superposition of flow on to solute mobility. Thus, EOF causes movement of nearly all species, regardless of charge, in the same direction. Under normal conditions (that is, negatively charged capillary surface), the flow is from the anode to the cathode. As depicted in Figure 4, cations migrate fastest since the electrophoretic attraction towards the cathode and the EOF are in the same direction, neutrals are all carried at the velocity of the EOF but are not separated from each other, and anions migrate slowest since they are attracted to the anode but are still carried by the EOF toward the cathode (since the magnitude of the flow can be more than an order of magnitude greater than their electrophoretic mobilities). Figure 4. Differential solute migration superimposed on electro-osmotic flow in CE using uncoated capillary. Modification of capillary wall charge can decrease EOF (even to zero, if the capillary is covalently coated with a polymer). In these circumstances, anions and cations can migrate in opposite directions.

13 A unique feature of EOF in the capillary is the flat profile of the flow, as depicted in Figure 5. Since the driving force of the flow is uniformly distributed along the capillary there is no pressure drop within the capillary, and the flow is nearly uniform throughout. The flat flow profile is beneficial since it does not contribute to the dispersion of solute zones. Peak efficiency, often in excess of 10 5 theoretical plates, is due in part to the plug profile of the electro-osmotic flow. Modes of operation Figure 5. Plug-like flow profile of EOF The basic methods encompassed by CE include capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP). The separation mechanisms of each mode are described in table 1. Mode Basis of separation Capillary zone Free solution electrophoresis mobility (CZE) Micellar Hydrophobic / ionic electrokinetic interactions with chromatography micelle (MEKC) Capillary gel electrophoresis Size and charge (CGE) Isoelectric Isoelectric point focusing (IEF) Isotachophoresis Moving boundaries (ITP) Table 1. Modes of CE CZE is the simplest form of CE, mainly because the capillary is only filled with buffer. Separation occurs because solutes migrate in discrete zones and at different velocities, based on their charge to size ratio. Separation of both anionic and cationic solutes is possible due to electro-osmotic flow (EOF). Neutral solutes do not migrate and all co-elute with the EOF. 1.4 Instrumental aspects of CE Capillary. Ideal properties of the capillary material include being chemically and electrically inert, UV-Visible transparent flexible and robust, and inexpensive. Meeting most of these requirements, fused silica is the primary material employed today. Effective control of capillary temperature is important for reproducible operation. Injection. The two most common sample injection methods are hydrodynamic (by application of pressure) and electrokinetic (by application of low field strength).

14 Detection. UV-Visible absorption is the most widely used detection method. An optical window in the capillary is easily created by removal of a small (1-3 mm) section of the protective polyimide coating. Other detectors are LIF (laser induced fluorescence), conductivity detector, mass spectrometer, etc. Electropherogram The result of an electrophoretic run is an electropherogram (Figure 6). An electropherogram is a plot with migration time on the X axis and absorbance data plotted on the Y axis. Qualitative information of compounds is provided by the position of peaks (migration time), while quantitative information (concentration of compounds) can be obtained from the peak high or peak area. mau MeHg-CYS EtHg-CYS PhHg-CYS CYS Hg(CYS) min 12 Figure 6. Electropherogram of mercury compounds complexed with cystein Mobility and migration time The time required for a solute to migrate to the point of detection is called the migration time, and is given by the quotient of migration distance and velocity. The migration time and other experimental parameters can be used to calculate the apparent solute mobility using μ = l L t U [cm2 / V s] where μ a = μ e + μ EOF U = applied voltage l = effective capillary length (from injection to the detector) L = total capillary length t = migration time

15 In the presence of EOF, the measured mobility is called the apparent mobility, µ a. The effective mobility, µ e, can be extracted from apparent mobility by independently measuring the EOF using a neutral marker (e.g. acetone) that moves at a velocity equal to the EOF. Efficiency The theoretical plate number, N, can be determined directly from an electropherogram, using where t = migration time w 1/2 = temporal peak width at half height N = 5,54 ( t ) w 1/2 N = 16 ( t w ) 2 Theoretical plate number can be related to the HEPT (height equivalent to a theoretical plate), H, by 2 where l = effective capillary length H = l N Resolution (R) Resolution of sample components is the ultimate goal in separation science. Resolution is most simply defined as R = 2 (t 2 t 1 )/(w 1 + w 2 ) where t = migration time w = baseline peak width (in time) 1.5 Practice: Measuring of preservatives and vitamin C in lime juice Turn on the computer, capillary electrophoresis instrument, and start the program. Put the capillary cassette with the capillary in the instrument. Running conditions: -Instrument: -Buffer: -Capillary length (total and effective): -Injection mode: -Temperature: -Voltage: -Polarity: -Detection wavelength:

16 Samples: Benzoic acid, sorbic acid and ascorbic acid (vitamin C) of known concentration (standard solutions) Lime juice. O OH H3C OH OH HO O O O HO OH Sorbic acid (E200) Benzoic acid (E210) Vitamin C (E300) MW = g/mol MW = g/mol MW = g/mol λ max = 255 nm λ max = 225 nm λ max =265 nm Tasks 1. Make the electrophoretic run of a mixture of standards. 2. Identify the peaks (compounds) according to their charge/mass ratio. 3. Determine t (migration time) and w 1/2 (peak width at half height) of the peaks. 4. Calculate µ a (apparent mobility) of each compound. 5. Determine separation efficiency by the calculation of parameters N, H and R. 6. Make the electrophoretic run of lime juice. 7. Identify the peaks (qualitative analysis) and calculate the concentration of compounds (quantitative analysis) by comparing the electropherogram to that of standard mixture. The laboratory notebook should contain the followings: theory of the analytical method parts of the CE settings, working parameters detailed description of the measurement electropherograms calculations results 1.6 Questions 1. What is the principle of separation in capillary zone electrophoresis? 2. Define ion velocity and electrophoretic mobility! 3. What is electro-osmotic flow (EOF)? Describe its benefits and flow-profile! 4. What will be the order of migration of components in an uncoated capillary at ph 9? 5. What are the main parts of a CE instrument? 6. Name two injection modes in CE! 7. What kind of detectors can be applied in CE? 8. What is an electropherogram and how can it be used for quantitative and qualitative analysis? 1.7 Bibliography David Heiger: High performance capillary electrophoresis, 2000 Agilent Technologies

17 Determination of ethanol content in Sinupret (oral drops) samples with gas chromatography (GC) 1.1 Aim Overview of the main parts of GC, the setting of the measuring method used in the practice, manual injection of Sinupret sample and standard samples, qualitative and quantitative analysis of ethanol in Sinupret sample by flame ionization detection, examination of factors affecting gas chromatographic separation. 1.2 Introduction Gas chromatography (GC) is a widely used analytical method to separate thermally stable, volatile organic and inorganic compounds of a complex mixture. The gas chromatography can be used for example in environmental science, brewing, the food industry, perfumery and flavorings analysis, the petrochemical industry, microbiological analyses, the pharmaceutical industry and clinical biochemistry. The advantages include efficiency, selectivity, small volumes of sample, and that the separation of the components are not destroyed, so with even related techniques (eg, gas chromatograph-mass spectrometer) the analysis can be carried further. 1.3 Theory Chromatography methods employ a stationary phase and a mobile phase. Components of a mixture are carried through the stationary phase by the flow of the mobile one; separations are based on differences in migration rates among the sample components. Two types of gas chromatography are encountered: gas-solid chromatography and gas-liquid chromatography. Gas-solid chromatography employs a solid stationary phase, in gas-liquid chromatography the stationary phase is a liquid. The mobile phase is gas. In gas chromatography (Figure 1.) the most common method of sample injection involves the use of a microsyringe to inject a liquid or gaseous sample through a self-sealing, silicone-rubber diaphragm or septum into a flash vaporizer port located at the head of chromatographic column (capillary column) and separated analytes flow through a detector, whose response is displayed on a computer. Injection of the sample may be made manually or using an autosampler. Figure 1. Schematic diagram of a capillary GC system

18 Elution is brought about by the flow of an inert gaseous mobile phase (helium, nitrogen and hydrogen). The choice of gases is often dictated by the type of detector used. In contrast to most other types of chromatography, the mobile phase does not interact with molecules of the analyte; its only function is to transport the analyte through the column. When properly selected for the chromatographic conditions the sample components separated bands from the mobile phase, the separated components and the stationary phase in the reverse order of interaction strength will reach the detector. The chromatographic separation factors are the quality and speed of the carrier gas, the temperature, the length and internal diameter of column, the type and thickness of stationary phase. The detector indicates the separated components, measuring some physical or chemical properties. The detector signal can be employed for qualitative identification and quantitative determination of separated components Basic terms Retention The analyte has been retained because it spends a time (t S ) in the stationary phase. Separation is based on the different times that components spend in the stationary phase. Minimum possible or dead time (t M ) [min] is the time it takes for an unretained species to pass through a chromatographic column. Retention time (t R ) [min] is the easiest way to define chromatographic retention. We measure the time between the injection and the maximum detector respond (peak maximum) for the correspondent compound: t R = t S + t M Adjusted retention time (t R ) [min] for a retained solute is the additional time required to travel the length of the column, beyond the time required by unretained solvent: t R = t R - t M Retention factor (k ) is dimensionless and independent of any geometrical parameters of the column. It could be considered to be a thermodynamic characteristic of the adsorbentcompound-eluent system: tr tm k = t Relative retention (Selectivity factor) (α): =k 2 /k 1 where k 2 > k 1, so The greater the relative retention the greater the separation between two components. Efficiency of Separation: Two factors contribute to how well compounds are separated by chromatography. One is the difference in elution times between peaks: the farther apart, the better their separation. The other factor is how broad the peaks are: the wider the peaks, the poorer their separation. M

19 Resolution (R) of a column tells us how far apart two bands are relative to their widths. The resolution provides a quantitative measure of the ability of the column to separate two analytes. In chromatography, the resolution of two peaks from each other is defined as: tr R= 2* w t R w where w is the peak width at baseline. For quantitative analysis, a resolution > 1.5 is highly desirable. Number of theoretical plate (N): The plate model supposes that the chromatographic column is contains a large number of separate layers, called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next. It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column. They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column. 2 t N=16* R w where w is the peak width at baseline. 2 Plate height / Height Equivalent to a Theoretical Plate (H) is approximately the length of column required for one equilibration of solute between mobile and stationary phases: where L is length of the column. H=L/N The chromatogram is a graph showing the detector response as a function of elution time. It has two main parameters: the retention time (t R ) and the peak area. The retention time is used for the qualitative analysis of components. Quantitative analysis is based on the area of a peak. In the linear response concentration range, the area of a peak is proportional to the quantity of that component. In gas chromatographic analysis an internal standard (IS) is generally added to the sample. Internal standards are similar in analytical behavior to the compounds of interest, and not expected to be found in the samples. Spiking the samples with internal standards helps to compensate imprecisions derived from sample preparation or variable injection volumes. A constant amount of the internal standards added to all samples, and that same amount of the internal standard is also included in each of the calibration standards. The ratio of the peak area of the target compound in the sample to the peak area of the internal standard in the sample is compared to a similar ratio derived for each calibration standard. This ratio is termed the response factor (RF) or relative response factor (RRF), indicating that peak area of the target compound is calculated relative to that of the internal standard: Ax CIS RF = AIS Cx A x -area of the compound c x - concentration of the compound A IS -area of the internal standard c IS - concentration of the internal standard

20 Ax /A IS (Ax 3 /A IS ) 3 (Ax 2 /A IS ) 2 (Ax 1 /A IS ) 1 (Cx 1 /C IS ) 1 (Cx 2 /C IS ) 2 (Cx 3 /C IS )i Cx /C IS Figure 2. Calibration in internal standard method 1.4 The main parts of gas chromatograph (Figure 1.) Gas Systems The mobile phase or carrier gas is helium. The choice of gases is often dictated by the type of detector used. The gases of flame ionization detector are hydrogen and air. Injector The split / splitless injector (Figure 3.) is used in conjunction with capillary column GC. Injection takes place into a heated glass or quartz liner rather than directly onto the column. In the split mode, the sample is split into two unequal portions the smaller of which goes onto the column. This technique is used with concentrated samples. In the splitless mode, the entire sample is introduced onto the column. Figure 3. A split / splitless injector

21 Column Two general types of columns are encountered in gas chromatography, packed and capillary. Capillary columns (Figure 4.) are made from fused silica, usually coated on the outside with polyimide to give the column flexibility. The wall of the column is coated with the liquid stationary phase. The most common type of coating is based on organo silicone polymers, which are chemically bonded to the silanol groups on the wall of the column and the chains of the polymers are further cross-linked. Polyethylene glycol is also commonly used as a polar stationary phase for gas chromatography (wax-column). Important separation factors are the length and internal diameter of column, the type and thickness of stationary phase. The column is ordinarily house in a thermostated oven. The optimum column temperature depends upon the boiling point of the sample and the degree of separation required. The ovens can be programmed to either produce a constant temperature, isothermal conditions or a gradual increase in temperature. Figure 4. A capillary column Detector Detectors used in GC vary in nature depending upon the characteristics of the analyte and the circumstances of its determination. The flame ionization detector (Figure 5.) is the most widely used and generally applicable detector for gas chromatography. The flame is mixed with hydrogen and air. When solute molecules contained in the carrier gas elute from the column and pass into the detector they burn in the flame and in doing so, generate ions which move to the collector electrode, due to the potential difference between the jet and the electrode. The resulting ionization current is amplified and fed to the data system. Figure 5. A typical flame ionization detector

22 1.5 Practice Procedure 1. Preparation of sample Standard and sample - n-propanol (internal standard) - Sinupret (oral drops) - Pipette 200 L of the Sinupret sample into a 10 ml volumetric flask. Add 40 μl internal standard (n-propanol) to the sample and fill to the mark with water! Procedure 2. Setting of the parameters of measuring method HP 5890 gas chromatograph Instrumentation (details of the instrument used settings, working parameters) Gas chromatograph: HP 5890N GC Injector: split-splitless, manual, T=200C, mode: splitless Column: capillary column, WAX (25 m * 0,25 mm * 0,25 m); stationary phase: polyethylene glycol Detector: flame ionization detector T=225C; detector gases: hydrogen, compressed air Temperature 50C (0,5 min)25c /min 150C (0 min) 50C (6,5 min) - isothermal Carrier gas: hydrogen, flow rate 1,5 ml/min Injection volume: 1 L Injection mode: split mode 50:1, manual - Set the parameters on the gas chromatograph with the help of the instructor! - Set the temperature of split-splitless injector! T=200C - Set the temperature of FID detector! T=225C - Set the temperature of oven! 1. We start the measurement on 50C, which is hold for 0,5 min, than we start to increase the temperature with 25C/min to 150C. 2. We start the measurement on 50C, which is hold for 6,5 min.

23 Procedure 3. Determination of dead time Standard for dead time determination: steam of dichloromethane - Inject 5l steam of dichloromethane and record the dead time! dead time, t M (min) steam of dichloromethane How to inject? Rinse the syringe 5 times with solvent (distilled water) and take up 1 L of sample and 1 L of air and push the needle through the rubber septum into the heated injection port of the chromatograph. Push the sample and remove the syringe then start the measuring method with start button! Rinse the syringe with solvent before every injection! Procedure 4. Qualitataive analysis-identification of ethanol in Sinupret sample Standards and sample for qualitative analysis standard solutions for qualitative analysis: - internal standard solution (n-propanol) - standard solution (ethanol) Sinupret (oral drops) samples Compounds Formula Boling point ethanol 78,37 C (analyte) C 2 H 5 OH n-propanol (internal standard-is) C 3 H 7 OH 97 C - Inject 1 l of the Sinupret sample into the gas chromatograph! Record the retention time of components! t R (min) in sample 1. component 2. component - Inject 1 l of standard ethanol solution into the gas chromatograph! Record the retention time of ethanol! - Inject 1 l of the internal standard solution (n-propanol) into the gas chromatograph! Record the retention time of n-propanol! t R (min) in standard solution ethanol n-propanol In the notebook identify the ethanol peak on the chromatogram of Sinupret sample! (Compare the retention times of the components in the Sinupret sample with the retention times in the standard solutions)!

24 Procedure 5. Quantitative analysis-determination the amount of ethanol in Sinupret sample In the notebook construct the calibration curve! Give the ethanol content of Sinupret sample in v/v%! Standards for quantitative analysis Each solution contains fixed mass of internal standard, and various masses of standard analyte, in distilled water. There are two parallel calibration solution on every level. calibration solutions % ethanol (v/v) volume of ethanol (l) volume of internal standard (l) 1. 0, , Calibration solutions A et A IS A et / A IS Mean (A et / A IS) Sinupret sample A et A IS Procedure 6. The examination of the effect of temperature for the separation. -Change the oven temperature from programmed to isothermal! T= 50C (6,5 min)! -Inject the Sinupret sample into the gas chromatograph! Record the retention time of components! 1. component 2. component t R (min) in sample In the notebook compare the recorded chromatogram with the chromatogram recorded with temperature program:50c (0,5 min)25c /min 150C (0 min)! Procedure 7. Characterization of the separation In the notebook calculate the k,r, N, H, R parameters with the help of table below and the chromatogram recorded under the practice! (Use the chromatogram of the Sinupret sample recording with programmed temperature)

25 t M t R t R k w N H R Name of the parameter Calculation ethanol n-propanol Instructions for notebook -The basic concepts of gas chromatographic separation! -The schematic diagram of GC, the main parts, the main tasks. -Sample preparation and experimental procedures! -The chromatographic circumstances! (Type and temperature of injector, detector, temperature programs, type and flow rate of carrier gas, characteristics of column). - Chromatograms recorded during the practice! - Results and discussion! - Identification of the ethanol in the Sinupret sample! (Procedure 4.) - The amount of ethanol (v/v%) in the Sinupret sample! (Procedure 5.) -The effect of temperature for the separation! (Procedure 6.) - Characterization of the separation! (Procedure 7.) 1.6 Questions What is the gas chromatography? What are the main parts of the gas chromatograph? What happens to be the principle of separation of components? How does the flame ionization detector work? What is the chromatogram? What is the retention time? List some factors affecting the separation and explain their effect! 1.7 Bibliography Daniel C. Harris Quantitative Chemical Analysis F. James Holler, Douglas A. Skoog and Stanley R. Crouch Principles of Instrumental Analysis F. James Holler and Stanley R. Crouch Skoog and West s Fundamentals of Analytical Chemistry David Harvey - Modern Analytical Chemistry David G. Watson - Pharmaceutical Analysis

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