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Professional Diploma in Biochemistry 2nd Semester C – 502 Analytical Biochemistry 1  Prepared by Prof. Dr. Elham Y. Hashem 2 Lecture 1 Analytical Biochemistry 4 Course Contents...

Professional Diploma in Biochemistry 2nd Semester C – 502 Analytical Biochemistry 1  Prepared by Prof. Dr. Elham Y. Hashem 2 Lecture 1 Analytical Biochemistry 4 Course Contents Principles and theory of Analytical techniques Affinity Chromatography HPLC Electrophoresis Spectrophotometry Fluorimetry Mass Spectroscopy 5  INTRO DUCTION Analytical chemistry is a branch of chemistry, and its objective is essentially to develop and apply new methodology and instrumentation with the goal of providing information on the nature and composition of matter. Analytical chemistry also allows the determination of a compound’s structure, either partially or totally, in samples. Finally, part of the role of analytical chemistry is to provide an interpretation of the results obtained. 6  Cont……. Analytical chemistry has become indispensable ‫ ال غنا عنة‬in a number of areas beyond those considered traditional such as chemistry or parachemistry, being increasingly present in activities closely associated with mankind ‫البشية‬ ‫ر‬ such as applications in medical sciences (diagnostics), biochemistry, food sciences, environmental sciences. 7  Cont……. Being an analyst requires scientific competence , austerity ‫ ب س ا ط ة‬and honesty. ‫قدرة‬ To undertake these kinds of study the analyst must be well trained in different techniques. A whole range of questions come up, not necessarily in the following order: The sample is of what kind (steel, soil, water, drug ………) ? Does it require a partial or complete analysis of the sample ? Is the analyte a major component (1 to 100 %), minor component (0.01 to 1 %) or trace level component (less than 0.01 %) of the sample ? Are qualified personnel available to conduct the analysis ? 8  Must the analysis be repetitively? Cont……… What is the precision needed? What is the cost of analysis? Must the sample be recovered after measurement? What are the consequences ‫ النتائج‬of a possible error in measurement? How long will the analysis take? What will the reliability ‫الثقة‬ of the results be for the method chosen? 9 Cont…….. The science called chemometrics is aimed at helping to find the best method required for solving an analytical problem according to three different directions: methodology, minimum sampling plan, and data treatment , and interpretation of results. First step: choosing a method of analysis. Second step: choosing a technique. Third step: Choice of sample procedure and the sample preparation method. 10 Separation Method The invention ‫ ا خ ت ر ا ع‬of chromatography started when used filter paper to partially separate substances in solution. Chromatography, the process by which the components of a mixture can be separated, has become one of the primary analytical methods for the identification and quantification of compounds in the gaseous or liquid state. The basic principle is based on )the concentration equilibrium of the components of interest, between two immiscible phases(. 11  General C o n c e p t O F Analytical Chromatography Chromatography is a physico-chemical method of separation of components within mixtures, liquid or gaseous, in the same vein ‫ منوال‬as distillation, crystallization, or the fractionated extraction. A basic chromatographic process may be described as follows: 1- A vertical glass tube (the column) is filled with a suitable finely powdered solid, the stationary phase. 2- At the top of this column is placed a small volume of the sample mixture to be separated into individual components. 3- The sample is then taken up by continuous addition of the mobile phase, which goes through the column by gravity, carrying the various constituents of the mixture along with it. This process is called elution. ‫عملية إستخراج‬ If the components migrate at different velocities, they will become separated from each other and can be recovered, mixed with the mobile phase. 12 GENERAL C O NCEPTS O F ANALYTICAL CHROMATOGRAPHY 13  Cont………… Chromatogram The chromatogram is the representation of the variation, with time (rarely volume), of the amount of the analyte in the mobile phase exiting the chromatographic column. It is a curve that has a baseline which corresponds to the trace obtained in the absence of a compound being eluted. The separation is complete when the chromatogram shows as many chromatographic peaks as there are components in the mixture to be analyzed. 14 In a chromatographic phase system, there are at least three sets of equilibria: solute/mobile phase, solute/stationary phase and mobile phase/stationary phase. 15  Retention Parameters ‫المتبقي‬ Retention times Retention volume (Elution volume) Hold-up volume (dead volume) Retention factor (Capacity) 16 Retention Times Retention time is the time elapsed ‫ إنقضى‬from the sample introduction to the detection of the peak maximum on the chromatogram. In an ideal case, tR is independent of the quantity injected. Hold-up time or )dead time(, t o, is the time required for the mobile phase to pass through the column. adjusted retention time of the compound, tR’ is The difference between the retention time and the hold-up time. 17  Retention Volume (elution volume) The retention volume VR of an analyte represents the volume of mobile phase necessary to enable its migration throughout the column from the moment of entrance to the moment in which it leaves. VR is calculated from the following expression, if the flow rate F is constant: The volume of a peak, Vpeak corresponds to that volume of the mobile phase in which the compound is diluted when leaving the column. 18  H o l d -UP Volume (Dead Vo l u m e ) The volume of the mobile phase in the column (known as the dead volume), VM , corresponds to the accessible ‫ سهل‬interstitial volume ‫فراغا‬. It is often ‫ غالبا‬calculated from a chromatogram, provided a solute not retained ‫المتبقية‬ by the stationary phase is present. The dead volume is deduced from tM and the flow rate F: 19  Retention Factor ( capacity) When a compound of total mass mT is introduced onto the column, it separates into two quantities: ( mM , the mass in the mobile phase) and ( mS , the mass in the stationary phase). During the solute’s migration down the column, these two quantities remain constant. Their ratio, called the retention factor k, is constant and independent of mT: 20  xx Retention Factor (capacity) K is not varied with the flow rate or the column length. K is it not a constant as it depends upon the experimental conditions. This parameter takes into account the ability, great or small, of the column to retain each compound. Ideally, k should be superior ‫ متفوقة‬to 1 but less than 5 , otherwise the time of analysis is unduly elongated ‫ زيادة غير مبررة‬. If we accept that at each instant ‫ لحظة‬, the ratio of the nS molecules fixed upon the stationary phase (mass mS and of the nM molecules present in the mobile phase (mass mM, is the same as that of the times tS and tM spent in each phase for a single molecule, the three ratios will therefore have the same value: 21  xx Retention Factor (Capacity) Knowing that the retention time of a compound tR is such that tR = tM + tS, the value of k is therefore accessible from the chromatogram tS = tR; 22  Separation Factor (Selectivity) The separation factor, α , enables the comparison of two adjacent peaks 1 and 2 present in the same chromatogram. By definition is greater than unity (species 1 elutes faster than species 2): or 23  Resolution Factor (R) To quantify the separation between two compounds 1 and 2. Contrary ‫ عكس‬to the selectivity factor which does not take into account peak widths. the following expression is used to calculate R between two compounds 1 and 2: 24  Resolution Factor (R) 25 Resolution Factor (R) 26  Resolution Factor (R) Chromatograms of a separation. The mobile phase in each trace is a binary mixture water/acetonitrile: (a) 50/50; (b) 55/45; (c) 60/40; (d) 65/35. The arrow indicates the dead time tM (min) 27  Resolution Factor (R) The resolution ( R ) and the elution time ( t ) are the two most important dependent variables to consider. In all optimizations, the goal is to achieve a sufficiently complete separation of the ‫يحقق‬ compounds of interest in the minimum time, though it should not be forgotten that time will be required to readjust the column to the initial conditions to be ready for the next analysis. 28 29