CHEM 202 2nd Term 2013-2014 Organic Chemistry II Instructor: Prof. Khaled S. Abdel Halim [email protected] http://faculty.uoh.edu.sa/k.abdulhalem/ CHEM 202 Lab Manual Manual Outlines This manual will provide an introduction to basic spectroscopic techniques, laboratory synthesis of organic chemicals and multistep synthesis. The manual will introduce the fundamentals of organic spectroscopic equipments such as FTIR and UV-vis. Furthermore, the manual will discuss how HPLC (High performance liquid chromatography) is a powerful tool in analysis and how to carry experiments on HPLC. Finally, a tutorial for synthesis, purification and identification of organic compounds will be introduced. It is important for chemical engineer to have some background in organic spectroscopy and organic synthesis tests to understand how dealing with different types of organic compounds. Lab. I Spectroscopy I: theoretical approach (IR) Lab. II Spectroscopy II: UV-vis fundamentals Lab.III FTIR tutorial & polystyrene test Lab.IV FTIR- Aspirin analysis Lab.V UV-vis - Demo Lab. VI UV – spectrum test Lab.VII HPLC- tutorial Lab. VIII HPLC test Lab. IX Synthesis of organic compounds- tutorial Lab. I & II Organic Spectroscopy Theoretical approach Organic Spectroscopy In this chapter, we shall study the following topics: •Spectroscopic methods. •Infrared Spectroscopy. •Ultraviolet and visible spectra. Overview * Modern spectroscopic methods have largely replaced chemical tests as the standard means of identifying chemical structures (particularly in organic chemistry). Spectroscopic techniques are fundamentally based on the molecules absorbing energy then monitoring the affect this has on the molecule. Spectroscopic methods Infra red (IR) Ultra-violet / visible (UV-VIS) Nuclear magnetic Resonance (NMR) Mass Spectrometry (MS) Electromagnetic Radiation (EMR) : “Visible light is the common type of EMR but this is just a small portion of all possible types. Electromagnetic radiation has both particle and wave properties A particle of energy is called a photon. Each photon has a discrete amount of energy : a quantum, ∆E = h ν= h c / λ (h = Planck's constant) Infrared Spectroscopy Electromagnetic radiation is energy that transmitted through space in the form of waves such as radio waves, ultraviolet, infrared, visible ,…etc. Each type is characterized by wavelength and frequency. Wavelength is the distance from the crest of one wave to the crest of the next wave while frequency is the number of complete cycle per second. Wave number is the number of cycles per cm. Wavelength and frequency are inversely proportional while energy and frequency are directly proportional. . The amount of energy is determined by the intensity or brightness of the radiation. The intensity is proportional with the number of photons. Feature of a spectrum Spectrum is a graph of wavelength (or frequency) versus % of transmission (%T) or absorbance (A). For IR spectra, wavelength or frequency is usually plotted with %T while ultraviolet spectra is usually represented as graphs of A versus wavelength. The absence of absorption by a compound at a specific wavelength is recorded as 100% T (ideal). When a compound absorb radiation at specific wavelength, the intensity of radiation being transmitted decreases, so %T will decrease and appear in spectrum as a dip called absorption peak or absorption band. 100 % T is base line is recorded at the top of IR spectrum. Atoms form covalent bonds undergo vibrations or oscillations (like two balls attached by a spring). When molecules absorb IR, the amplitude of vibration increased (excited vibration state). The absorbed energy is dissipated as heat when molecule returns to ground state. The wavelength of absorption by a given bond depends on the energy of difference ground state and excited state. So, different types of bonds like C-H or C=O absorb IR at different and characteristics wavelength. Nonpolar bonds do not absorb IR (no change in bond moment when vibrate). Relatively nonpolar bond as C-C or C-H give weak absorption while O-H or C=O exhibt strong absorption. IR spectrum IR spectrophotometer is the instrument used to measure the absorption of IR radiation. A diagram obtained from IR spectrophotometer is called IR spectra. Modern IR instrument used fast Fourier analysis called Fourier transform infrared spectrometer (FTIR). IR bands can be classified according to intensity into strong (s), medium (m), weak (w) and shoulder (sh). Shoulder is given for overlapping stronger and weaker bands. Single OH group produces strong absorption (polar) while single C-H gives weak absorption. Multiply C-H bonds gives medium or even strong absorption Analysis of IR spectra Typical ranges of wavelengths of absorption for each functional group were determined and summarized in correlation charts . IR Spectra of compounds C-C and C-H IR of Haloalkanes C-X bond falls in the fingerprint region of IR spectra 500-1430 cm-1 Alcohols & amines IR Spectra of ethers C-O bond absorption is usually strong because of highly electronegativity of O atom. C-O stretching band in the fingerprint region at 1050-1260 cm-1. Sp3 C-H C-C C-O at 2800-2900 cm-1 at 1380 cm-1 at 1110 cm-1 IR spectra of carbonyl compounds Providing that you have the following IR data, answer the following questions: (i) A compound of formula C4H8O2, which structure shown below is consistent with the following IR spectrum? (ii) A molecule with formula C6H12O shows a strong peak at 1725 cm-1 and no peak at 3300-3600 cm-1. Circle all possible structures that are consistent with this data. (iii) The following IR Spectrum best represents which of the following compounds? (1) Ultraviolet and visible spectra The visible spectrum spans from about 400 nm (violet) to 750 nm (red), while the ultraviolet spectrum ranges from 100 to 400 nm. The quantity of energy absorbed by a compound is inversely proportional to the wavelength of the radiation. Both UV and visible radiation are of higher energy than infrared radiation. Absorption of UV or visible light results in electronic transition (from lower to higher energy level). These transition require 40-300 kcal/mole. The wave length of UV or visible light absorbed depends on electronic transition, molecules require more energy for transition absorb at shorter wavelength. Compounds that absorb light in the visible region (colored compounds) have more easily promoted electron than compounds that absorb at shorter UV wavelengths. Modern UV spectrometer sample cell UV Spectrum The UV Spectrum: 1. The x-axis of the spectrum is in wavelength; 200-350 nm for UV, 200-700 for UVVIS determinations 2. Due to the lack of any fine structure, spectra are rarely shown in their raw form, rather, the peak maxima are simply reported as a numerical list of “lamba max” values or lmax lmax = 206 nm 252 317 376 Absorbance in UV spectroscopy The absorption of energy in UV is recorded as absorbance (not transmittance as in IR spectra). The absorbance depends on the electronic structure of the compound and also on the concentration of the sample and the length of the sample cell. So , the actual absorbance is defined as molar absorptivity. Mechanism of electron absorption in UV spectroscopy There are 3 types of electron transitions give rise to UV or visible spectra. The most useful region of UV spectrum at wavelength longer than 200 nm. Energy s* Unoccupied levels p* Atomic orbital n Atomic orbital Occupied levels p p* s Molecular orbitals s* Energy n p s s s* alkanes s p* carbonyls p p* unsaturated cmpds. n s* O, N, S, halogens n p* carbonyls Absorption arising from transitions of n electrons Acetone for example contain both pi bond and n electrons exhibits both http://chemistry.boisestate.edu/people/richardbanks/organic/mc/vol8/mcquestions317h.htm http://chemistry.boisestate.edu/people/richardbanks/organic/mc/vol5/mcquestions317e.htm http://www.chem.ucalgary.ca/courses/350/Carey5th/Ch13/ch13-ir-1.html http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Questions/problems/exam6.htm Lab.III FTIR tutorial & polystyrene test Introduction to Fourier Transform Infrared Spectrometry © 2001 Thermo Nicolet Corporation All rights reserved, worldwide. I N T R O D U C T I O N What is FT-IR? FT-IR stands for Fourier Transform InfraRed, the preferred method of infrared spectroscopy. In infrared spectroscopy, IR radiation is passed through a sample. Some of the infrared radiation is absorbed by the sample and some of it is passed through (transmitted). The resulting spectrum represents the molecular absorption and transmission, creating a molecular fingerprint of the sample. Like a fingerprint no two unique molecular structures produce the same infrared spectrum. This makes infrared spectroscopy useful for several types of analysis. So, what information can FT-IR provide? • It can identify unknown materials • It can determine the quality or consistency of a sample • It can determine the amount of components in a mixture This booklet is an introduction to the concepts behind FT-IR spectroscopy. It covers both the basic theory of FT-IR and how it works as well as discussing some the practical aspects of FT-IR use. We hope that it gives you a good understanding of the importance and usefulness of this powerful technique. 2 T H E O R Y O F F T - I R Why Infrared Spectroscopy? Infrared spectroscopy has been a workhorse technique for materials analysis in the laboratory for over seventy years. An infrared spectrum represents a fingerprint of a sample with absorption peaks which correspond to the frequencies of vibrations between the bonds of the atoms making up the material. Because each different material is a unique combination of atoms, no two compounds produce the exact same infrared spectrum. Therefore, infrared spectroscopy can result in a positive identification (qualitative analysis) of every different kind of material. In addition, the size of the peaks in the spectrum is a direct indication of the amount of material present. With modern software algorithms, infrared is an excellent tool for quantitative analysis. Older Technology The original infrared instruments were of the dispersive type. These instruments separated the individual frequencies of energy emitted from the infrared source. This was accomplished by the use of a prism or grating. An infrared prism works exactly the same as a visible prism which separates visible light into its colors (frequencies). A grating is a more modern dispersive element which better separates the frequencies of infrared energy. The detector measures the amount of energy at each frequency which has passed through the sample. This results in a spectrum which is a plot of intensity vs. frequency. Fourier transform infrared spectroscopy is preferred over dispersive or filter methods of infrared spectral analysis for several reasons: • It is a non-destructive technique • It provides a precise measurement method which requires no external calibration • It can increase speed, collecting a scan every second • It can increase sensitivity – one second scans can be co-added together to ratio out random noise • It has greater optical throughput • It is mechanically simple with only one moving part 3 Why FT-IR? Fourier Transform Infrared (FT-IR) spectrometry was developed in order to overcome the limitations encountered with dispersive instruments. The main difficulty was the slow scanning process. A method for measuring all of the infrared frequencies simultaneously, rather than individually, was needed. A solution was developed which employed a very simple optical device called an interferometer. The interferometer produces a unique type of signal which has all of the infrared frequencies “encoded” into it. The signal can be measured very quickly, usually on the order of one second or so. Thus, the time element per sample is reduced to a matter of a few seconds rather than several minutes. Most interferometers employ a beamsplitter which takes the incoming infrared beam and divides it into two optical beams. One beam reflects off of a flat mirror which is fixed in place. The other beam reflects off of a flat mirror which is on a mechanism which allows this mirror to move a very short distance (typically a few millimeters) away from the beamsplitter. The two beams reflect off of their respective mirrors and are recombined when they meet back at the beamsplitter. Because the path that one beam travels is a fixed length and the other is constantly changing as its mirror moves, the signal which exits the interferometer is the result of these two beams “interfering” with each other. The resulting signal is called an interferogram which has the unique property that every data point (a function of the moving mirror position) which makes up the signal has information about every infrared frequency which comes from the source. This means that as the interferogram is measured, all frequencies are being measured simultaneously. Thus, the use of the interferometer results in extremely fast measurements. Because the analyst requires a frequency spectrum (a plot of the intensity at each individual frequency) in order to make an identification, the measured interferogram signal can not be interpreted directly. A means of “decoding” the individual frequencies is required. This can be accomplished via a well-known mathematical technique called the Fourier transformation. This transformation is performed by the computer which then presents the user with the desired spectral information for analysis. 90 Polystyrene run as film 80 70 60 50 FFT Calculations % T 40 30 20 10 0 4000 3500 3000 2500 2000 1500 Wavenumbers (cm-1) Interferograms 4 CPU Spectrum 1000 500 The Sample Analysis Process The normal instrumental process is as follows: 1. The Source: Infrared energy is emitted from a glowing black-body source. This beam passes through an aperture which controls the amount of energy presented to the sample (and, ultimately, to the detector). 2. The Interferometer: The beam enters the interferometer where the “spectral encoding” takes place. The resulting interferogram signal then exits the interferometer. 3. The Sample: The beam enters the sample compartment where it is transmitted through or reflected off of the surface of the sample, depending on the type of analysis being accomplished. This is where specific frequencies of energy, which are uniquely characteristic of the sample, are absorbed. 4. The Detector: The beam finally passes to the detector for final measurement. The detectors used are specially designed to measure the special interferogram signal. 5. The Computer: The measured signal is digitized and sent to the computer where the Fourier transformation takes place. The final infrared spectrum is then presented to the user for interpretation and any further manipulation. Spectrometer 1. Source 2. Interferometer 90 Polystyrene run as film 80 70 60 50 % T 40 30 3. Sample 20 10 0 4000 3500 3000 2500 2000 1500 1000 500 Wavenumbers (cm-1) Interferogram FFT 5. Computer Spectrum 4. Detector Because there needs to be a relative scale for the absorption intensity, a background spectrum must also be measured. This is normally a measurement with no sample in the beam. This can be compared to the measurement with the sample in the beam to determine the “percent transmittance.” This technique results in a spectrum which has all of the instrumental characteristics removed. Thus, all spectral features which are present are strictly due to the sample. A single background measurement can be used for many sample measurements because this spectrum is characteristic of the instrument itself. 5 A Simple Spectrometer Layout 6 Advantages of FT-IR Some of the major advantages of FT-IR over the dispersive technique include: • Speed: Because all of the frequencies are measured simultaneously, most measurements by FT-IR are made in a matter of seconds rather than several minutes. This is sometimes referred to as the Felgett Advantage. • Sensitivity: Sensitivity is dramatically improved with FT-IR for many reasons. The detectors employed are much more sensitive, the optical throughput is much higher (referred to as the Jacquinot Advantage) which results in much lower noise levels, and the fast scans enable the coaddition of several scans in order to reduce the random measurement noise to any desired level (referred to as signal averaging). • Mechanical Simplicity: The moving mirror in the interferometer is the only continuously moving part in the instrument. Thus, there is very little possibility of mechanical breakdown. • Internally Calibrated: These instruments employ a HeNe laser as an internal wavelength calibration standard (referred to as the Connes Advantage). These instruments are self-calibrating and never need to be calibrated by the user. These advantages, along with several others, make measurements made by FT-IR extremely accurate and reproducible. Thus, it a very reliable technique for positive identification of virtually any sample. The sensitivity benefits enable identification of even the smallest of contaminants. This makes FT-IR an invaluable tool for quality control or quality assurance applications whether it be batch-to-batch comparisons to quality standards or analysis of an unknown contaminant. In addition, the sensitivity and accuracy of FT-IR detectors, along with a wide variety of software algorithms, have dramatically increased the practical use of infrared for quantitative analysis. Quantitative methods can be easily developed and calibrated and can be incorporated into simple procedures for routine analysis. Thus, the Fourier Transform Infrared (FT-IR) technique has brought significant practical advantages to infrared spectroscopy. It has made possible the development of many new sampling techniques which were designed to tackle challenging problems which were impossible by older technology. It has made the use of infrared analysis virtually limitless. 7 5225 Verona Road • Madison, WI 53711-4495 • U.S.A. TEL: 800-201-8132, 608-276-6100 • FAX: 608-273-5046 E-MAIL: [email protected] • www.thermonicolet.com A Thermo Electron business P/N 169-707500 2/01 Lab. IV FTIR Analysis of Aspirin lab. V Shimadzu UV-V Shimatzu 1800 UV-vis Spectrophotometer “Demo” SPECTROPHOTOMETRIC ANALYSIS OF ASPIRIN Introduction: A colored complex is formed between aspirin and the iron (III) ion. The intensity of the color is directly related to the concentration of aspirin present; therefore, spectrophotometric analysis can be used. A series of solutions with different aspirin concentrations will be prepared and complexed. The absorbance of each solution will be measured and a calibration curve will be constructed. Using the standard curve, the amount of aspirin in a commercial aspirin product can be determined. The complex is formed by reacting the aspirin with sodium hydroxide to form the salicylate dianion. O O C CH 3 C O- O (s) + 3OH- (aq) C O- OH O (aq) + CH3C O - (aq) + 2H2O(l) O The addition of acidified iron (III) ion produces the violet tetraaquosalicylatroiron (III) complex. + O- O Fe(H2O)4 +3 - + [Fe(H2O)6 ] C O C O O O + + H2O + H3O Purpose: The purpose of this lab is to determine the amount of aspirin in a commercial aspirin product. This lab may also be used to determine the purity of the aspirin produced in the Microscale Synthesis of Acetylsalicylic Acid lab. Equipment / Materials: 6 - 125 mL erlenmeyer flasks commercial aspirin product or aspirin the student has made 10 mL graduated cylinder acetylsalicylic acid 250 mL volumetric flask 1 M NaOH 100 mL volumetric flask 0.02 M iron (III) buffer 5 mL pipet spectrophotometer 2 cuvettes DI water analytical balance (opt.) Safety: Always wear goggles and an apron in the lab. Be careful while boiling the sodium hydroxide solution. NaOH solutions are dangerous, especially when hot. Procedure Part I: Making Standards. 1. Mass 400 mg of acetylsalicylic acid in a 125 mL Erlenmeyer flask. Add 10 mL of a 1 M NaOH solution to the flask, and heat until the contents begin to boil. 2. Quantitatively transfer the solution to a 250 mL volumetric flask, and dilute with distilled water to the mark. 3. Pipet a 2.5 mL sample of this aspirin standard solution to a 50 mL volumetric flask. Dilute to the mark with a 0.02 M iron (III) solution. Label this solution "A," and place it in a 125 mL Erlenmeyer flask. 4. Prepare similar solutions with 2.0, 1.5, 1.0, and 0.5 mL portions of the aspirin standard. Label these "B, C, D, and E." Part II: Making an unknown from a tablet. 1. Place one aspirin tablet in a 125 mL Erlenmeyer flask. Add 10 mL of a 1 M NaOH solution to the flask, and heat until the contents begin to boil. 2. Quantitatively transfer the solution to a 250 mL volumetric flask, and dilute with distilled water to the mark. 3. Pipet a 2.5 mL sample of this aspirin tablet solution to a 50 mL volumetric flask. Dilute to the mark with a 0.02 M iron (III) solution. Label this solution "unknown," and place it in a 125 mL Erlenmeyer flask. Part III: Making an unknown from the product of the Microscale Synthesis of Acetylsalicylic Acid lab. 1. Mass all of the acetylsalicylic acid product and record the mass in the data section. Place it in a 125 mL Erlenmeyer flask. Add 10 mL of a 1 M NaOH solution to the flask, and heat until the contents begin to boil. 2. Quantitatively transfer the solution to a 250 mL volumetric flask, and dilute with distilled water to the mark. 3. Pipet 10 mL sample of this aspirin solution to a 50 mL volumetric flask. Dilute to the mark with a 0.02 M iron (III) solution. (When adding 0.02 M iron (III) solution, the color should be obviously purple. If it is not, stop adding the 0.02 M iron (III) solution, and alert your instructor.) Label this solution "unknown," and place it in a 125 mL Erlenmeyer flask. Lab. VI UV Analysis of Aspirin Part IV: Testing the Solutions. 1. Turn on the spectrophotometer by twisting the front left knob clockwise. Allow the spec to warm up for 15 minutes. 2. Adjust the wavelength to 530 nm using the large knob on top of the spec. 3. With the sample compartment empty, set the instrument to 0%T using the zero control knob (front left knob). 4. Using a Kimwipe, wipe off the cuvet containing the blank (0ppm), and place this cuvet in the sample compartment, being sure to properly align it. (The line on the cuvet should match up with the notch on the instrument.) Close the cover. 5. Set the mode to absorbance. Using the trans/abs control knob (front right knob) set the absorbance to 0.000. 6. Record the absorbance of the 0ppm solution – this should be a cuvet of iron buffer. 7. Obtain absorbance readings for each of the other standard solutions. Record the results on the data sheet. 8. Measure and record the absorbance of the unknown. Record it on the data sheet. Data: Solution A B C D E unknown Concentration (mg/L) Absorbance For commercial Product: amount of aspirin in unknown mg accepted value mg percent error % For microscale lab product: mass of impure product mg mass of aspirin in product mg % purity of product % Lab. VII Chromatography measurements HPLC HPLC Basics Fundamentals of Liquid Chromatography (HPLC) Courtesy of Agilent Technologies, Inc. Detector Compound B Column and column oven Control and data processing Compound A Compound C 0 Pump Injector 2 4 6 8 10 12 14min Chromatogram HPLC Basics Page 1 Fundamentals of High Performance Liquid Chromatography (HPLC) This course will enable you to: • Explain the general principles of HPLC analyses • Know the major application areas of HPLC • Identify the major components of an HPLC system and explain their principles of operation HPLC Basics Page 2 Chromatographic Separation Techniques Which separation technique for which compound? HPLC Basics Page 3 First, What is Liquid Chromatography? • Liquid chromatography is a separation technique that involves: • the placement (injection) of a small volume of liquid sample • into a tube packed with porous particles (stationary phase) • where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity. • The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. • The separated components are collected at the exit of this column and identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount. Note: The modern form of liquid chromatography is now referred to as “flash chromatography” Note: Look for the comparison with HPLC on page 7 HPLC Basics Page 4 Principles of Liquid Chromatography Load sample Add solvent Column containing stationary phase Collect components Time HPLC Basics Page 5 Then, What is HPLC? • HPLC is an abbreviation for High Performance Liquid Chromatography (It has also been referred to as High Pressure LC) • HPLC has been around for about 35 years and is the largest separations technique used • The history of HPLC: • Beginning of the 60’s: start of HPLC as High Pressure Liquid Chromatography • End of the 70’s improvements of column material and instrumentation – High Performance Liquid Chromatography HPLC in 1973 • Since beginning of the 80’s: “boom” in HPLC started • Since 2006 new terms popped up like UPLC, RRLC, UFLC, RSLC, ….. HPLC in 2009 HPLC Basics Page 6 What is HPLC? • HPLC is a separation technique that involves: • the injection of a small volume of liquid sample • into a tube packed with tiny particles (3 to 5 micron (µm) in diameter called the stationary phase) • where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump. • These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. • These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”. In principle, LC and HPLC work the same way except the speed, efficiency, sensitivity and ease of operation of HPLC is vastly superior. Note: Compare this description to that on page 4 about “Liquid Chromatography” HPLC Basics Page 7 What Does a Liquid Chromatogram Look Like? Compound B These are called chromatographic peaks and each one represents a separated compound Compound C Point of sample injection into the column 0 2 Compound A 4 6 8 10 12 14 min Time after injection This is the chromatogram resulting from the injection of a small volume of liquid extracted from a vitamin E capsule that was dissolved in an organic solvent. Modern HPLC separations usually require 10- to 30-minutes each. HPLC Basics Page 8 What does a high pressure LC look like? (1) Describing the 5 major HPLC components and their functions … Solvent reservoirs and degassing 1 2 5 3 4 1. Pump: • The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL/min). • Normal flow rates in HPLC are in the 1- to 2-mL/min range. • Typical pumps can reach pressures in the range of 6000-9000 psi (400- to 600-bar). • During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient). 2. Injector: • The injector serves to introduce the liquid sample into the flow stream of the mobile phase. • Typical sample volumes are 5- to 20-microliters (µL). • The injector must also be able to withstand the high pressures of the liquid system. • An autosampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical. HPLC Basics Page 9 (2) Describing the 5 major HPLC components and their functions … Solvent reservoirs and degassing 1 2 5 3 4 3. Column: • Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters. • The small particles inside the column are what cause the high backpressure at normal flow rates. • The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph. 4. Detector: • The detector can see (detect) the individual molecules that come out (elute) from the column. • A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components. • The detector provides an output to a recorder or computer that results in the liquid chromatogram (i.e., the graph of the detector response). 5. Computer: • Frequently called the data system, the computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis). HPLC Basics Page 10 What is HPLC used for? Separation and analysis of non-volatile or thermally-unstable compounds HPLC is optimum for the separation of chemical and biological compounds that are non-volatile NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline, etc.), gas chromatography is a better separation technique. Typical non-volatile compounds are: Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol) Salts like sodium chloride and potassium phosphate Proteins like egg white or blood protein Organic chemicals like polymers (e.g. polystyrene, polyethylene) Heavy hydrocarbons like asphalt or motor oil Many natural products such as ginseng, herbal medicines, plant extracts Thermally unstable compounds such as trinitrotoluene (TNT), enzymes HPLC Basics Page 11 What is HPLC used for? Qualitative analysis The identification (ID) of individual compounds in the sample; • the most common parameter for compound ID is its retention time (the time it takes for that specific compound to elute from the column after injection); • depending on the detector used, compound ID is also based on the chemical structure, molecular weight or some other molecular parameter. Retention time of compound B Retention time of compound A Injection point (time zero) 0 2.5 5 7.5 10 12.5 15 min Time after injection HPLC Basics Page 12 What is HPLC used for? Quantitative Analysis The measurement of the amount of a compound in a sample (concentration); meaning, how much is there?; There are two main ways to interpret a chromatogram (i.e. perform quantification): 1. determination of the peak height of a chromatographic peak as measured from the baseline; 2. determination of the peak area (see figure below); In order to make a quantitative assessment of the compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area is measured. In many cases, there is a linear relationship between the height or area and the amount of sample. A B Peak area of Compound A Peak height of Compound A C Peak area of Compound A D Peak area of Compound A Peak area of Compound A 0 2 4 6 8 10 12 14 min HPLC Basics Page 13 What is HPLC used for? Preparation of Pure Compound(s) - By collecting the chromatographic peaks at the exit of the detector, - and concentrating the compound (analyte) by removing/evaporating the solvent, - a pure substance can be prepared for later use (e.g. organic synthesis, clinical studies, toxicology studies, etc.). This methodology is called preparative chromatography. Start Stop Collect pure peak from preparative chromatography column Concentrate by evaporation Preparative LC Columns Use for animal testing HPLC Basics Page 14 What is HPLC used for? Trace analysis A trace compound is a compound that is of interest to the analyst but it’s concentration is very low, usually less than 1% by weight, often parts per million (ppm) or lower; • the determination of trace compounds is very important in pharmaceutical, biological, toxicology, and environmental studies since even a trace substance can be harmful or poisonous; • in a chromatogram trace substances can be difficult to separate or detect; • high resolution separations and very sensitive detectors are required. These are the main substances in the sample These are the trace substances in the sample 0 2 4 6 8 10 12 14 min Time after injection HPLC Basics Page 15 Examples of Different Instruments and Configurations Modular HPLC System – basic configuration with isocratic pump, manual injector, variable wavelength detector, and hand-held controller Modular HPLC System – high-end configuration with quaternary pump, autosampler, column thermostat, diode array detector, and computer with control and data analysis SW Integrated HPLC System “all parts in one box” – different configurations possible, here with gradient pump, autosampler, column oven, VWD, and computer with control and data analysis SW (not shown on picture) HPLC Basics Page 16 Introduction HPLC Applications High performance liquid chromatography (HPLC) is an important tool for the analysis of pharmaceutical drugs, for drug monitoring and for quality assurance. The method enables complex mixtures, for example, herbal medicine plant extracts, to be separated into individual compounds, which can be identified and quantified by suitable detectors and data handling systems. Separation and detection occurs at ambient temperature or slightly above. Therefore, the method is ideally suited for compounds of limited thermal stability. State-of-the-art HPLC equipment can automate HPLC separations, using automatic samplers, injectors, microprocessor-controlled analytical conditions and ChemStations for data evaluation. Important requirements for automation are: • • • • • excellent precision of the liquid chromatography system, data evaluation with report printouts, the possibility to store chromatograms and results, the possibility to detect leaks and other errors for safety reasons, and implemented OQ/PV tools in the HPLC system. Automation not only increases the sample throughput in pharmaceutical laboratories and companies, but also the precision of the results by eliminating human errors. An overview of the pharmaceutical drugs used in this guide is presented on pages 5-6. 3 Agilent ChemStation Database The client/server database for the Agilent ChemStation, for example, the Agilent ChemStore C/S is a data organization system which provides a solution to organize, manange and report chromatographic results, as well as safeguard data. It supports important end-user tasks such as reviewing and summarizing results for statistical evaluation, archiving and restoring data and creating control charts and crosssample reports. These services also aid users in validating their methods and doing on-going system suitability testing. Two examples using the database for the Agilent ChemStation are described on pages 56-66. Agilent 1100 Series Combinatorial Chemistry Analysis System The new Agilent 1100 Series combinatorial chemistry analysis system is a fully automated analysis solution designed to speed up the process of drug discovery. The combination Agilent 1100 Series HPLC, Agilent 1100 Series LC/MSD detector and the Agilent 220 micro plate sampler offers the full potential of well plate technology and is the ideal tool for analyzing complex libraries. Details can be found on page 67. 4 Application Overview Group Examples Analgesic drugs Antipyrine, Hydroxyantipyrine, Acetaminophen 7 Androgen drugs Testosterone Acetate, Testosterone 8 Antianginal drugs Verapamil 9 Antiarrythmic drugs Quinidine, Disopyramide, Procainamide, N-Acetylprocainamide 10 Antiasthmatic drugs Antibacterial drugs Penicillin-like Tetracyclines Miscellaneous Caffeine, Theophylline, Enprofylline, Theobromine 11 Ampicillin, Amoxicillin, Penicillin G, Penicillin V Minocycline, Tetracycline, Doxycycline Hydroxybenzotriazole, Chloramphenicol, Trimethoprim Sulfamethoxazole, Furazolidone, Nalidixic Acid 12 13 Anticoagulant drugs Warfarin 17 Antidepressant drugs Antiepileptic drugs Antiestrogen drugs Antihistaminic drugs Bupropion, Trazodone, Maprotiline 18 Caffeine, Phenytoin, Methylphenylsuccinimide, Phenylethylmalonamide, Carbamazepinepoxide, Ethosuximide, Phenobarbital, Carbamazepine, Primidone 20 Tamoxifen 26 Tetracaine, Promethazine, Chlorpheniramine, Tripelenamine 27 Antihypertensive drugs Enalapril, Captopril Antiinflammatory drugs Naproxen 29 Antiprotozoal drugs Metronidazole 30 Antitumor drugs Paclitaxel (Taxol) 31 Antitussive drugs Catecholamines Dextromethorphan 33 Norepinephrine, Epinephrine, Dihydroxybenzylamine, Dopamine 34 Beclomethasone Dipropionate, Prednisolone, Prednisolone Acetate, Betamethasone, Betamethasone Valerate, Hydrocortisone, Hydrocortisone Acetate 35 Glucocorticoid drugs H2-Antagonists Hypnotic drugs Keratolytic drugs Muscle-relaxing drugs Sedative drugs Sulfa drugs Therapeutic peptides Tricyclic antidepressant drugs Vitamins Fat soluble Water soluble Ranitidine, Cimetidine Barbital, Allobarbital, Phenobarbital, Butabarbital, Butalbital, Amobarbital, Mephobarbital, Flunitrazepam Salicylic Acid, Phtalic Acid, Benzoic Acid Papaverine Diazepam, Oxazepam, Clonazepam, Flunitrazepam Sulfanilamide, Sulfadiazine, Sulfathiazole, Sulfamerazine, Sulfamethazine Angiotensin II, Angiotensin I, Insulin, Oxytocin Protriptyline, Nortriptyline, Doxepin, Imipramine, Amitriptyline, Trimipramine Vitamins A1, D3, E Aminobenzoic Acid, Biotin, Folic Acid, Niacinamide, Pantothenic Acid, Pyridoxal, Pyridoxamine, Pyridoxine, Riboflavine, Thiamine, Thiotic Acid Page 14 28 37 38 41 42 43 44 45 46 47 48 5 Introduction HPLC Applications High performance liquid chromatography (HPLC) is an important tool for the analysis of pharmaceutical drugs, for drug monitoring and for quality assurance. The method enables complex mixtures, for example, herbal medicine plant extracts, to be separated into individual compounds, which can be identified and quantified by suitable detectors and data handling systems. Separation and detection occurs at ambient temperature or slightly above. Therefore, the method is ideally suited for compounds of limited thermal stability. State-of-the-art HPLC equipment can automate HPLC separations, using automatic samplers, injectors, microprocessor-controlled analytical conditions and ChemStations for data evaluation. Important requirements for automation are: • • • • • excellent precision of the liquid chromatography system, data evaluation with report printouts, the possibility to store chromatograms and results, the possibility to detect leaks and other errors for safety reasons, and implemented OQ/PV tools in the HPLC system. Automation not only increases the sample throughput in pharmaceutical laboratories and companies, but also the precision of the results by eliminating human errors. An overview of the pharmaceutical drugs used in this guide is presented on pages 5-6. 3 Agilent ChemStation Database The client/server database for the Agilent ChemStation, for example, the Agilent ChemStore C/S is a data organization system which provides a solution to organize, manange and report chromatographic results, as well as safeguard data. It supports important end-user tasks such as reviewing and summarizing results for statistical evaluation, archiving and restoring data and creating control charts and crosssample reports. These services also aid users in validating their methods and doing on-going system suitability testing. Two examples using the database for the Agilent ChemStation are described on pages 56-66. Agilent 1100 Series Combinatorial Chemistry Analysis System The new Agilent 1100 Series combinatorial chemistry analysis system is a fully automated analysis solution designed to speed up the process of drug discovery. The combination Agilent 1100 Series HPLC, Agilent 1100 Series LC/MSD detector and the Agilent 220 micro plate sampler offers the full potential of well plate technology and is the ideal tool for analyzing complex libraries. Details can be found on page 67. 4 Application Overview Group Examples Analgesic drugs Antipyrine, Hydroxyantipyrine, Acetaminophen 7 Androgen drugs Testosterone Acetate, Testosterone 8 Antianginal drugs Verapamil 9 Antiarrythmic drugs Quinidine, Disopyramide, Procainamide, N-Acetylprocainamide 10 Antiasthmatic drugs Antibacterial drugs Penicillin-like Tetracyclines Miscellaneous Caffeine, Theophylline, Enprofylline, Theobromine 11 Ampicillin, Amoxicillin, Penicillin G, Penicillin V Minocycline, Tetracycline, Doxycycline Hydroxybenzotriazole, Chloramphenicol, Trimethoprim Sulfamethoxazole, Furazolidone, Nalidixic Acid 12 13 Anticoagulant drugs Warfarin 17 Antidepressant drugs Antiepileptic drugs Antiestrogen drugs Antihistaminic drugs Bupropion, Trazodone, Maprotiline 18 Caffeine, Phenytoin, Methylphenylsuccinimide, Phenylethylmalonamide, Carbamazepinepoxide, Ethosuximide, Phenobarbital, Carbamazepine, Primidone 20 Tamoxifen 26 Tetracaine, Promethazine, Chlorpheniramine, Tripelenamine 27 Antihypertensive drugs Enalapril, Captopril Antiinflammatory drugs Naproxen 29 Antiprotozoal drugs Metronidazole 30 Antitumor drugs Paclitaxel (Taxol) 31 Antitussive drugs Catecholamines Dextromethorphan 33 Norepinephrine, Epinephrine, Dihydroxybenzylamine, Dopamine 34 Beclomethasone Dipropionate, Prednisolone, Prednisolone Acetate, Betamethasone, Betamethasone Valerate, Hydrocortisone, Hydrocortisone Acetate 35 Glucocorticoid drugs H2-Antagonists Hypnotic drugs Keratolytic drugs Muscle-relaxing drugs Sedative drugs Sulfa drugs Therapeutic peptides Tricyclic antidepressant drugs Vitamins Fat soluble Water soluble Ranitidine, Cimetidine Barbital, Allobarbital, Phenobarbital, Butabarbital, Butalbital, Amobarbital, Mephobarbital, Flunitrazepam Salicylic Acid, Phtalic Acid, Benzoic Acid Papaverine Diazepam, Oxazepam, Clonazepam, Flunitrazepam Sulfanilamide, Sulfadiazine, Sulfathiazole, Sulfamerazine, Sulfamethazine Angiotensin II, Angiotensin I, Insulin, Oxytocin Protriptyline, Nortriptyline, Doxepin, Imipramine, Amitriptyline, Trimipramine Vitamins A1, D3, E Aminobenzoic Acid, Biotin, Folic Acid, Niacinamide, Pantothenic Acid, Pyridoxal, Pyridoxamine, Pyridoxine, Riboflavine, Thiamine, Thiotic Acid Page 14 28 37 38 41 42 43 44 45 46 47 48 5 Fat-Soluble Vitamins Retinol (A1) Absorbance [mAU] 1 Retinol (A) 2 Cholecalciferol (D3) 3 a-Tocopherol (E) 3 2 800 600 1 400 200 OH 0 HO a- Tocopherol 0 2.5 5 7.5 12.5 10 15 17.5 20 Time [min] Analysis of fat-soluble vitamins O Column Mobile phase Flow rate Gradient Column wash UV detector Column compartment temperature Stop time Post time Injection volume 4.6 x 75 mm Zorbax Eclipse XDB-C18, 3.5 µm A = water, B = methanol 1.0 ml/min at 0 min 90 % B at 15 min 100 % B at 20 min 100 % B at 21 min 90 % B variable wavelength detector 210 nm, standard cell 20 °C 21 min 5 min 5 µl HPLC method performance Limit of detection <4.0 mg/l (5-µl injection), S/N=2 Precision of RT 10 runs, 1000 mg/l< 0.10 % H Precision of area 10 runs, 1000 mg/l< 0.2 % HO Instrumentation: see configuration example 2 on page 77 Cholecalciferol (D3) 47
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