Project Title: e-laboratories for Gas chromatography 1. Experiment Category: Chemistry >> chromatography 2. Experiment Name: Gas Chromatography 3. Date and Issue number: 4. Instructor Name: 5. Institution: Ain Shams University Page 1 of 15
Table of Contents 1. Experiment Overview 2 2. Intended Learning Outcomes (ILOs) 2 3. Introduction: Theoretical Background 3 4. References 6 5. Relation to Course Contents and Topics 6 6. Needed Equipment 6 7. Experiment Output 6 8. Experiment Steps 6 9. Interface Instructions/Help 14 1. Experiment Overview The main objective of this experiment is to familiarize students with Gas chromatography technique as one of the most commonly used Chromatographic techniques. Students will be able to explain the concept of separation and to make quantitative analysis from the chromatogram outcomes. Concept of qualitative analysis will be understood from data given. 2. Intended Learning Outcomes (ILOs) Upon completion of this experiment, students should be able to: 1- Identify components of GC instrument. 2- Describe the analysis of drugs by GC technique. 3- Demonstrate methods of calculations. 4- Analyze, evaluate and interpret the obtained data. 5- Apply qualitative and quantitative analytical of pharmaceutical preparations. 6- Conduct standard pharmaceutical laboratory procedures and instrumentation. 7- Handle and dispose chemicals and pharmaceutical preparations safely. (3.2)* Page 2 of 15
3. Introduction: Theoretical Background 3.1. Theory of Gas Chromatography: Gas chromatography is an analytical technique used to separate volatile organic compounds. In the most generic form, chromatography is based on the separation of compounds (or ions) present in a sample matrix. Chromatography is a separation method in which the components of a sample partition between two phases: One of these phases is a stationary bed with a large surface area, and the other is a gas that percolates through the stationary bed. The sample is vaporized and carried by the mobile gas phase (the carrier gas) through the column. Samples partition (equilibrate) into the stationary liquid phase, based on their solubilities at the given temperature. The components of the sample (called solutes or analytes) separate from one another based on their relative vapor pressures and affinities for the stationary bed. This type of chromatographic process is called elution. The official definitions of the International Union of Pure and Applied Chemistry (IUPAC) are: Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Elution chromatography is a procedure in which the mobile phase is continuously passed through or along the chromatographic bed and the sample is fed into the system as a finite slug. The most familiar detectors to us today are the flame ionization detector (FID), nitrogen-phosphorus detector (NPD), electron capture detector (ECD), flame photometric detector and the mass spectrometer. The coupling of GC with a suitable detection system makes it a very powerful tool in the quality control pharmacists arsenal of analytical techniques. Quality control is a very wide and varied subject that covers drug and herbal products analysis. Depending upon the detector used, GC can provide both qualitative and quantitative data. The main instrumental components of a gas chromatograph are (Figure 1): Gas supply Sample introduction system Column oven Column Detector Read-out device, typically a computer with appropriate software that allows, as a minimum, integration and display of peak area and peak height. Figure 1:Schematic diagram of a gas chromatograph Page 3 of 15
The separation of the compounds is influenced by a series of operating conditions, some of which you can alter as part of the normal GC conditions, while others are not so available (during routine operation of the GC). The typical operating condition that can be altered is: Temperature within the GC oven that influences the so-called column temperature. In practical terms the column can be operated under isothermal conditions or temperature-programmed conditions. In the case of the former, the column temperature remains fixed (e.g., 100 C) throughout the GC run. In the case of the latter, the temperature is varied, at a fixed rate, during the GC run (e.g., 80 C for 2 min, then a ramp rate of 10 C/min, to 200 C with a hold of 3 min). Operating conditions that are generally fixed and not available for change during routine operation include: Choice of carrier gas and its flow rate (i.e., while the choice of carrier gas is important, it is not often practical to change; for example, nitrogen is used as the carrier gas for GC-FID while helium is used for GC-MS). Choice of column (i.e., column length, internal diameter, and stationary phase). 3.2. The chromatogram and terms obtained from it: The basic separation process occurs and the resultant output the so-called chromatogram represents the appearance of the organic solvent and compounds (Figure 2). The chromatogram is therefore a plot of the amount (concentration) of the compounds present as a function of time. Figure 2: A chromatogram: a plot of signal (relative abundance; on the y-axis) versus retention time (minutes; on the x-axis). Within the chromatogram it is possible to define some specific terms and measurements (Figure 3); specifically, the following terms are identified: t o = the time of elution (minutes) of the unretained compound from the point of sample injection. This is often the time, from sample injection, when the organic solvent appears in the chromatogram (see Figure 35). t r = the time of elution (minutes) of each compound from the point of sample injection (or retention time) to the center of the peak. In the case of the example (see Figure 35), two compounds elute from the column; therefore, we can refer to t r1 (the time of elution, from the point of injection, of compound 1) and t r2 (the time of elution, from the point of injection, of compound 2). Page 4 of 15
h = the peak height (in units that are representative of the y-axis on the chromatogram, e.g., microvolts). This is the height of the peak measured from the baseline. A = the peak area (in units that are representative of the y-axis on the chromatogram and the duration of the peak on the x-axis, i.e., time, e.g., μv.s). w b = the width of the peak at its extrapolated base. (Note: w b1 refers to the width of the peak base for compound 1 and w b2 is the width of the peak base for compound 2.) w 1/2 = the width of the peak at half its height. In practical terms this is done by halving the peak height and measuring the width of the peak at this position. k = capacity factor. (Sometimes it is defined by use of a small letter k with a prime, i.e., k or simply k. It has no units.) N = column efficiency. L = column length (the dimension needs to be defined in appropriate units, e.g., meters, centimeters or millimeters). HETP = height equivalent to a theoretical plate, expressed as column efficiency (N), in units of millimeters. A s = asymmetry factor. R = a measure of the degree of separation of adjacent compound peaks. 3.3. Selected chromatographic terms: 1. Capacity Factor Figure 1 Selected chromatographic terms. In order to be able to compare the elution time of one compound between one gas chromatograph and another (whether in the same laboratory or not), the capacity factor for that compound must be calculated. k = (t r t o )/t o (3.1) Page 5 of 15
2. Column Efficiency The number of theoretical plates is therefore a measure of column efficiency (N). the concept of the number of theoretical plates is a useful measure for GC because it gives a practical numerical value that indirectly provides a measure of the peak narrowness. In principle, therefore, the narrower the peak shape is the more peaks (or compounds) can be separated. N = 2π ((t r. h)/a) 2 3. Resolution Resolution is the ability to separate two adjoining compounds such that their peak bases are distinguishable from each other (i.e., a separation exists between the two compound peak shapes). The resolution can be calculated as follows: R = (t r2 t r1 )/(0.5 (w b1 + w b2 )) 4. References [1].I.F.Cui,T.Zhou,J.Zhang,Z.Lou;Phytochemical Analysis,vol(2),116-119,(1991). [2]. Robert L. Grob, Eugene F. Barry, Modern Practice of Gas Chromatography, John Wiley & Sons, 2004 [3]. Harold M. McNair, James M. Miller, Basic Gas Chromatography, John Wiley & Sons, 2011. [4]. Michelle Groves Carlin, John Richard Dean, Forensic Applications of Gas Chromatography, CRC Press, 2013. 5. Relation to Course Contents and Topics This experiment is taught to Fourth Year pharmacy students under the name of "Phytotherapy and Quality Control of Herbal medicine" 6. Needed Equipment No hardware equipment needed 7. Experiment Output The followings are the expected output that student should get by the end of the experiment: 1- Task (1) With a given retention t i m e for each component in a mixture, student should identify each one. 2- Task (2) Student will be able to apply analytical method using GC, prepare working solutions and calculate concentrations according to data got from chromatogram. Finally student should observe the variation of peak areas of each component which are related to concentration. 8. Experiment Steps 1. You are provided with 5 compounds named Ephedrine, Pseudoephedrine, Norephedrine, Norpseudoephedrine & methylephedrine in five different volumetric flasks. 2. You will make a mixture containing any combination of these different compounds but you must withdraw from 1 to 6 mls (at least 1 ml and maximum 6 mls) from each of the five compounds to a 10-ml volumetric flask i.e. your mixture may contain any combination of the 5 compounds (2, 3, 4 or all the 5 compounds). Page 6 of 15
3. Then you will complete to the mark with acetone (if the volumetric is not completed to the mark in the previous step), then mix well. Page 7 of 15
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4. After preparing the mixture, filter it using a syringe filter. Page 9 of 15
5. Read carefully the different parameters of your GC instrument (Temperature, flow rate of carrier gas). 6. Inject 1 ul from your mixture into the GC injection port. Page 10 of 15
7. Each compound will be separated on GC column and will appear as a peak according to its affinity to the stationary phase, they will appear in a chromatogram where the first peak is the largest one and is called the solvent front, Thus they will be eluted with different retention times according to the following table. Page 11 of 15
Name of Cpd Ephedrine Pseudoephedrine Norephedrine Norpseudoephedrine Methylephedrine Retention time (min) 15 16 14 13 17 8. You will identify each compound from its retention time (Qualitative). 9. By clicking inside each peak, you will get its peak area. 10. From the peak areas, get the concentrations of each compound in the final mixture from the following table (Quantitative): Name of Cpd Ephedrine Pseudoephedrine Norephedrine Norpseudoephedrine Methylephedrine Regression Equation Y=1.307 X-0.280 Y=1.050 X-0.280 Y=0.680 X-0.025 Y=0.540 X-0.081 Y=1.357 X-0.036 The regression equation (Y= ax+b) is a relation between X and Y: Where X: concentration of each compound in the final mixture Y: peak area, a: slope b:intercept 11.You will get the concentration of each compound in its volumetric flask before dilution using the following equation. Before Dilution CV After Dilution =C'V' C'=X (from regression equation) V'=10 ml V=volume taken from each volumetric flask C=unknown concentration Page 12 of 15
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9. Interface Instructions/Help Drag these tools using the left click and mouse to work with them. Click with the mouse right click on them to make actions like withdrawing the liquids. Page 14 of 15
Click with the mouse right click on them to make actions like withdrawing the liquids. Click with the mouse left click on them to make actions like mixing the compounds. Page 15 of 15