现代分离技术习题（Modern separation technologyexercises）现代分离技术习题(Modern separation technology exercises)The twentieth chapter, HPLCThinking questions and exercises1. the main similarities and differences between HPLC and gas chromatography are briefly described.The same point: both high efficiency, high speed, high selectivity chromatography method, both separation and analysis function, can be detected onlineDifference:Analysis of objects and ranges, selection of mobile phases,operating conditionsGC has the advantages of good gasification, good thermal stability and low boiling point, and occupies 20% of the machine. The mobile phase is a limited number of "inert" gas, which only acts as a carrier, and has little effect on the composition, and operates at atmospheric pressureHPLC can be made into solution after the dissolution of the sample, high boiling point, high molecular weight, difficult to gasification, ionic stability or unstable compounds, occupy 80% of the machine, the liquid phase for the liquid or a variety of liquid mixture. In additionto its carrying capacity, it can also be controlled and improved by solvent. At room temperature and under high pressure2. what is the chemical bonding phase? What are the common chemical bonding phases? Which liquid chromatography are used?A chemical reaction is used to bond the fixed liquid to the surfaceof the carrier and the resulting packing is called a chemically bonded phase. The utility model has the advantages that the use process is not lost, the chemical property is stable, the thermal stability is good,and the utility model is suitable for gradient elution.The commonly used Si-O-Si-C type bonding phases are classified into three kinds: non-polar, medium polarity and polarity according to polarity. The nonpolar bonded phase: such as the common ODS bonded phase, both the distribution and the adsorption effect, is widely used for the analysis of non-polar or weak polar compounds; the middle of the bonded phase: a common ether bonded phase, the bonded phase can be positive or reversed-phase chromatographic stationary phase, mobile phase polarityas the polarity: the bonded phase: common amino and cyano bonded phase, are used as chromatographic stationary phase, phase separation of sugar or amino bonded stationary phase is the most commonly used.3. what is called normal phase chromatography? What is reverse phase chromatography? Which compounds are applied separately?Normal phase chromatography: a method of chromatography having a polarity of mobile phase less than the polarity of the stationary phase.A molecular substance used to separate polar and moderately polarsubstances dissolved in organic solvents; used in the separation of substances containing differentfunctional groups.Reversed-phase chromatography: a method for chromatography having a polarity of mobile phase greater than the polarity of the stationary phase. A molecular compound used to separate nonpolar to moderatelypolar compounds.4. briefly describe the separation mechanism of reversed-phasebonded phase chromatography.A typical reversed-phase bond chromatography is a system consistingof nonpolar stationary phases and polar mobile phases. Stationary phases are commonly used in the eighteen alkyl (ODS or C18) bonded phase; the mobile phase is usually methanol water or acetonitrile water. Anatypical reversed-phase chromatography system consists of a weak polaror moderately polar bonding phase and a mobile phase having a polarity greater than the stationary phase.The surface of the antiphase bonded phase has nonpolar alkylfunctional groups and an amorphous silanol group. The silanol group has the adsorption properties, and the amount of the remaining silanolgroups depends on the coverage. For the separation mechanism ofreversed-phase chromatographic retention mechanism at present, there is no consensus, there are two views, one that belongs to the distribution of chromatography, another that belongs to the adsorption chromatography. The mechanism of the partition chromatography is that the polar organicsolvent with mixed polar solvent (water, ten organic solvent) is adsorbed on the non-polar alkyl coordination group surface, The component molecules are assigned in the mobile phase with the liquid phase adsorbed by the non-polar alkyl ligand. The mechanism of adsorption chromatography can be explained by the theory of solvent extraction. The theory treats nonpolar alkyl linkage phases as molecular hairs covered with a bound eighteen alkyl group on the surface of silica gel, which has strong hydrophobic properties. When the polar solvent water and organic solvent composition of the mobile phase to separate organic compounds, on the one hand, the non-polar part of the nonpolar component molecules or component molecules, due to hydrophobic interaction, will be "squeeze out from the water, produce association between hydrophobic alkyl stationary phase. As a result, the component molecules are retained in the fixed phase. On the other hand, the polar component of separated material under the action of the polarity of mobile phase, make it from the fixed phase, reduce the retention, obviously this dissociation process, namely, the two force difference, determines the retention behavior of molecules inchromatography. Generally speaking, on a stationary phase with alkyl nonpolar part of the surface area of the large base or isolated molecules in the mobile phase or the surface tension and the dielectric constant is bigger, the stronger association, k'distribution ratio is greater, the greater the value of reserves. It is not difficult tounderstand that in reversed-phase bonded chromatography, the polar components first flow out, and the less polar components flow out.What are the differences between the 5. ion chromatography,Reversed-phase Ion Pair Chromatography and ion suppression chromatography?Ion chromatography (Ion Chromatography): the ion exchange resin is the stationary phase, and the electrolyte solution is the mobile phase.A conductivity detector is used as a universal detector. The reaction principle of the sample component on the separating column and the restraining column is the same as that of the ion exchange chromatography. Ion chromatography is the best method for the analysis of anions in solution. It can also be used for the analysis of cations.Reversed-phase Ion Pair Chromatography (IPC or PIC): in reversed-phase chromatography, an ion pair reagent is added to the polar mobile phase to form a neutral ion pair between the measured component and its counter ion, increasing K and tR to improve separation. Apply to stronger organic acids and bases.Reversed-phase ion suppression chromatography: in reversed phase chromatography, the buffer solution is used to adjust the pH value of the mobile phase, so as to inhibit the dissociation of the group and increase its K and tR, so as to improve the separation. Apply to extremely weak acid and base matter (pH=3~7 weak acid, pH=7~8 weak base, amphoteric compound)What is the separation mechanism of 6. affinity chromatography? What are the characteristics?Conventional wisdom holds that affinity chromatography is based on ligand ligand affinity reaction and uses differential migration theory of chromatography to achieve separation of target molecules, and is only a selective filtration method for component separation. However, this theory is based on ligandligand affinity reactions that are homogeneous reactions and macroscopic equilibrium thermodynamics. However, in fact, the partition coefficients of the target molecules in the two phase are too large, and the adsorption isotherms on the stationary phase are mostly linear. Therefore, the retention mechanism of biological macromolecules in affinity chromatography and the mathematical model of chromatography process have been a relatively weak link, which needs to be further improvedAffinity chromatography has high specificity, the separation, purification, concentration after biological samples have high purity, greatly reduce the subsequent determination (such as HPLC) when the background noise,Thus, the subsequent determination has very high sensitivity.What are the similarities and differences between the 7. rate theoretical equations in HPLC and those in GC? How to guide theselection of HPLC experimental conditions?Solution: the main factors causing chromatographic peaks in liquid chromatography are eddy current diffusion, mobile flow phase mass transfer, retained mobile phase mass transfer, and off column effect.In gas chromatography, radial diffusion is often significant, while the effect of radial diffusion in liquid chromatography is weak and can often be neglected. In addition, liquid retention, mass transfer and off column effects are more prominent in liquid chromatography.In high performance liquid chromatography, the complete expressionfor liquid-liquid partition chromatography, Van, and Deemter equationsisTherefore, the experimental conditions of HPLC should be as follows: 1. Small grain size and uniform spherical chemical bonding phase; the low viscosity mobile phase should not flow too fast; and the column temperature is appropriate.8., try to discuss the factors that affect the separation degree of HPLC, how to improve the separation degree?(1) chromatographic filling propertiesThe performance of liquid chromatographic column separation is determined by the three parameters: the size of the stationary phase, the length of the column, the column pressure and the pressure drop determined by the filling condition. The three parameter determines the sample component retention time, K thermodynamic factors not only with the retention time of chromatographic process, and also directly decide on the viscosity parameters and flow performance of column columnefficiency and the degree of separation phase, these parameters are important factors affecting the chromatographic separation process dynamics. But in high-performance liquidchromatography, the preparation of separation column is a very demanding technical work, usually the purchase of commercial products, rarely prepared by itself.(2) the polarity of mobile phase and mobile phaseIn liquid chromatography, changing the composition and polarity of eluent is the most direct factor to improve the separation. Liquid chromatography is unlikely to improve mass transfer by increasing column temperature. Therefore, most of them are constant temperature analysis. The mobile phase selection is especially important in liquid chromatography. The mobile phase can significantly change the separation of components.(3) flow velocityWhen the velocity is greater than 0.5 cm/s, the curve from H to u is a straight line with little slope. The column efficiency is not greatly improved by decreasing the flow velocity. But in actual operation, the flow is still an important optional parameter for adjusting the separation and the peak time.9., discuss the choice of the separation conditions of reversed-phase HPLC.Reverse phase HPLC is a liquid chromatographic separation model with surface non-polar carrier as stationary phase and solvent with strongerpolarity than stationary phase as mobile phase. The retention values of samples in reversed-phase HPLC chromatography are mainly determined by the fixed surface area, the type and concentration of bonding phases,and the retention values usually increase with the increase of chain length or the hydrophobicity of the bonded phase. The solute retention value is directly proportional to the surface area of the fixed surface, and when other conditions are the same, the solute has a short retention value on the low surface area chromatographiccolumn. The retention value of the sample can also be adjusted by changing the composition of the mobile phase or the strength of the solvent. The strength of the solvent depends on the nature of theorganic solvent and its concentration in the mobile phase.10. is the intensity of the mobile phase the same in the positiveand reverse phase HPLC?In normal phase chromatography, because the stationary phase is polar, the stronger the solvent polarity is, the stronger the elution capacity is, i.e., the strong solvent is a strong solvent. In reversed-phase chromatography, as the stationary phase is nonpolar, the strength of the solvent increases with decreasing polarity of the solvent,A solvent with a weak polarity is a strong solvent.11. what is called gradient elution? What are the similarities and differences between it and GC?In an analysis period, according to certain procedures for changing the mobile phase composition or concentration, known as gradient elution.It is an important method to improve the separation of liquid chromatography.Gradient elution and gas chromatography in the temperature programmed is similar, but the former is the continuous change of the mobile phase polarity, pH or ionic strength, and temperature change. Programmed temperature is also an important method to improve gas chromatographic separation.12. what are the principles and characteristics of evaporative light scattering detectors?Evaporative light scattering detector (Evaporative Light-scattering Detector) is a universal detector that can detect organic substances such as ginsenosides and Huang Qijia glycosides that have no UV absorption.One, ELSD principleConstant velocity (high performance liquid chromatograph, countercurrent chromatography and high performance capillary electrophoresis) eluent after entering the detector, first by high-pressure gas atomization, droplet atomization formed into the evaporation chamber (drift tube, drift tube), mobile phase and low boiling components by evaporation, the remaining small droplets of high boiling components divided into the scattering cell, passing through the scattering cell by scattering, light scattering by photoelectric tube receiving form an electrical signal, electric signal through theamplifying circuit, analog-to-digital conversion circuit, a digital computer - chromatogram signal chromatography workstation.Two, characteristics1., the eluent needs to be atomized, so the purity and pressure ofthe atomization will affect the signal-to-noise ratio of the detector.2., the mobile phase to evaporate, so can not use volatilesubstances to regulate the pH value of the mobile phase. The components of the lower boiling point of the material can be vaporizedby adjusting the evaporation temperature. In the absence of evaporationof the material under test, the higher the temperature, the morecomplete the evaporation of the mobile phase, the better the baseline of the chromatogram, and the higher the signal-to-noise ratio. If the measured material is close to or below the boiling point flowevaporation temperature phase, however, is unable to detect; 100% of the water as the mobile phase, temperature is only the evaporation chamberis set to 150 degrees Celsius, organic matter lower boiling point than water can be separated by gas chromatography detection. Since the flow phase and solvent evaporates, the chromatogram collected by the ELSD detector generally has no solvent peak; and the gradient elution has no refractive parallax effect and generally does not exhibit baseline drift.3. to detect light scattering changes, all materials entering the scattering pool can be detected, and the response value is only relatedto the amount of matter.The 4. concentration is not linear with the peak area, and followsthe logarithm of the natural logarithm.13. what is the commonly used method of quantitative analysis of HPLC? Which methods need correction factor to correct peak area? Which methods do not have to correct the factor?The commonly used methods for quantitative analysis of HPLC are:External standard method: external standard work curve method, external standard point method, external standard two point method, etc.Internal standard method: internal standard work curve method,internal standard point method, internal standard two point method, internal standard comparison method, etc.The calibration factor is not necessary when using the standardcurve method of internal standard and external standard, and other methods need correction factor to correct peak area14. point out the elution sequence of benzene, naphthalene and anthracene in reversed-phase chromatography and explain the reason.The order of polarity of the three is from large to small, benzene, naphthalene and anthracene,Therefore, the elution order in reverse phase chromatography is benzene, naphthalene and anthracene, and benzene is the first peak.15. which HPLC method should be used to separate the following substances?(1) ethanol and butanol; (2) Ba2+ and Sr2+; (3) pentanoic acid and butyric acid; (4) high molecular weight glucoside.(1) positive phase bonded phase chromatography(2) ion exchange chromatography(3) ion pair chromatography(4) steric exclusion chromatography;"Chromatography analysis" exercisesChapter 1 Introduction to chromatography analysisJudgment question:1 the following conditions will increase the performance of the chromatographic column:A reduces the velocity of the mobile phase; ()B particles uniformly packed; ()C increases the diameter of the stationary phase particles; ()D increases the column temperature. ()Question 1: Please select the most suitable chromatographic analysis method for the following samples.The thermal instability of the sample. (2) low boiling aromatic hydrocarbons. () the complex, multiple groups of samples. ()A gas chromatography.B liquid chromatography.2. points out which of the following parameter changes will cause an increase in relative retention values?(1) the column length increased; (2) the ratio increased; (3) the column temperature decreased; (4) the phase velocity of mobile phase decreased.3. it is pointed out that the following conditions can reduce the height of theoretical plate:(1) increasing the column length; (2) decreasing the Dt value; (3) decreasing the speed of the flow line; (4) decreasing the diameter of the fixed phase particles,(5) increasing column temperature. Explain why.Simple answer:1. What is the balance of chromatographic distribution? How do the partition coefficients, the capacity factor, the relative rate, and the distribution temperature line measure the distribution balance of the components in the two phases?2, what factors do the theoretical equations of the plate derive from? What is their significance?3, try to explain the function and shortcomings of the plate theory in the interpretation of chromatographic separation.4, try to explain the theoretical basis of Van Deemter equation and the implications of its expressions.What is the practical significance of the 5 and Van Deemterequations? What is the reason for the deformation of the coupled curve?6. Test the basis and classification of chromatography.7. Briefly describe the types and characteristics of chromatographic columns.8. The separation principle and characteristics of chromatographyare briefly described.9. The characteristics and application scope of gas chromatography (OLC, GSC, CGC), high performance liquid chromatography (HPlC) and supercritical fluid chromatography are briefly described.10. Besides the factors discussed in the Van Deemte equation, what are the factors that affect the broadening of chromatographic peaks? How to overcome it in actual work?11, select the plate height (H) what is the color spectrumseparation index according to the operating conditions?12, try to explain the meaning of A, B and C constants in Van Deemter equation, and the unit and its calculation method.13, a gas chromatography column marked: OV - 101 column, the column efficiency of n is higher than 1200, which marked the correct way? Why?14, compare the chromatography (GC, HPLC) and chemical analysis, mass spectrometry, infrared spectroscopy, the similarities and differences between fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, atomic spectrometry, electrochemical analysis method, analysis method and how to improve the development of chromatography?15. Mark the name of each parameter in the chromatogram (see below) and point out the parameters of the qualitative and quantitative chromatographyFixed film thickness 16, higher than the B A column column, other conditions completely different, is two on the optimal linear velocity (uopt) value of the same?17, there is a lower boiling paraffin homologen samples withnonpolar stationary liquid chromatographic column, low column temperature and low liquid loading ratio, or high high liquid column temperature, load ratio (good to complete the same time analysis)? Why?Calculation problem:1. there is a liquid chromatography column, t, M = 4 points, and now the A and B, C, D, four components of the retention value and peak width as follows:Please calculate the n value and N eff value of each component.2. on a chromatographic column, the peak time of air peak is 0.5 minutes, the sample peak time is 2.44 minutes, and the peak width is 9.7 seconds. Assuming that the chromatographic peaksare normally distributed and the chromatographic column is 1 meters long, the theoretical plate number of the column, the theoretical height of the tray, the number of effective plates and the height of the effective plate are calculated.3. A and B two components are separated at column 100. The partition coefficient of A is 110, and the partition coefficient of B is 120. How long will it take to separate the two from 1.1? The theoretical plate height is 0.1mm.The second chapter gas chromatography analysisJudgment question:1 using polar column (GC) analysis of samples, the first out of the peak component is:A boiling high component; ()B large molecular weight component; ()C polar component; ()Choice question:1 select the most suitable gas chromatography stationary phase (or fixative) according to the sample.The n-pentane, octane. (2) N 2, O 2. ()A zeolite.B methyl silicone rubber SE-30.C polyethylene glycol PEG -20M.Simple answer:By experiment, do you think a newly prepared gas liquid packed column needs aging before use? Why?Please list at least four qualitative methods commonly used in chromatography.What is the quantitative correction factor? Why is correction factor introduced in chromatographic quantitative analysis?4. after the analysis of the experiment, we will estimate what type of fixative or stationary phase should be used.(1) determination of SO2 and H2S in air; (2) determination of moisture in several hundred ppm grades in acetone;(3) determination of trace organic phosphorus andorganochlorine pesticides residues in vegetables and fruits;(4) determination of trace amounts of benzene, toluene and xylene in styrene in the production section of styrene;(5) analysis of C2 - C4 hydrocarbon in petroleum cracking gas; (6) determination of ortho - and para - cresol in cresol wastewater;(7) analysis of C4 - C2 alcohols.5. what are the common carrier gas? How to select and purify carrier gas?6. gas system leakage may occur what? Why?What are the similarities and differences between 7. gas liquid chromatographic stationary phases and gas solidchromatographic stationary phases?8. what are the requirements for chromatographic fixative? How are they classified?What is the relationship between the order of the 9. components and the intermolecular force?10. briefly describe the meanings and characteristics of WCOT, SCOT and PLOT column abbreviations.11. why 0.2mm fine bore capillary column must be split during operation12., why does capillary gas chromatography have to be combined witha tail blowing device, such as no tail blowing operation?13. compare the difference between capillary gaschromatography and packed column gas chromatography.Calculation problem:1. the content of fatty acids in eggs was analyzed by chromatography. The relative correction factor of 0.43 palmitic acid, oleic acid area correction factor is 0.52, the relative correction factor of 0.37stearic acid, linoleic acidrelative correction factor was 0.41; palmitic acid peak area of 80mm 2, the peak area is 100mm oleic acid 2, stearic acid peak area for the50mm 2, the peak area of Asia oleic acid is 70mm 2. Please calculate the percentage of palmitic acid in eggs by normalization method2. there are four chromatographic peaks on the color spectrum. The distance from the introduction to the highest point of each group is as follows:Air 2.50 min; heptane 16.4 min; toluene 19.2 min; octane 31.5 min.Please calculate the retention index of toluene.3. a mixture of ethanol, heptane, benzene and ethyl acetate was analyzed. The measured peak area of them was 5, 9, 4, and 7.0cm 2, by manual check their relative weight correction factor of FW were 0.64,0.70, 0.78 and 0.79 respectively, according to the normalization method for weight percent concentration of them.The third chapter is the analysis of HPLCJudgment question:1, to improve the selection performance of columns, which of the following most effective measures should be adopted?:A uses a highly selective stationary phase; ()B reduces theaffinity of the relative component of the flow; ()C increase column length; ()Choice question:1 if you wish to analyze the following compounds by liquid chromatography, please select the optimum separation type.The determination of halogenated alkane isomers. (2) glutathione S-transferase separation in animal liver. ()Separation and determination of proteins and molecular weight in the serum. () the separation of chicken ovomucoid occurred in the ionization under alkaline conditions. (A) liquid liquid partition chromatography, B affinity chromatography, C, volume exclusion chromatography, D ion exchange chromatographyE liquid solid adsorption chromatographyFill in the:In reversed-phase ion pair chromatography, equilibrium ion concentration increases with retention values (); equilibrium ions increase in hydrophobicity, retention values (); mobile phase The ratio of water to water decreases, the organic solvent increases, and the retention value (); if the component is acid, when the pH value of the mobile phase decreases, the retention value (...).Simple answer:1. point out the elution sequence of n-hexane and acetic acid in reverse liquid liquid partition chromatography and explain the reason.2. what is the chemical bonding phase? What are the characteristics?3., compare the HPLC and GC separation principle, instrument structure and application methods of similarities and differences.4. How does high-performance liquid chromatography achieve efficient and high-speed separations (compared with classical column chromatography)?5. Briefly describe the main influencing factors and improvement methods of chromatographic peak broadening of HPLC.6. What are the classes of HPLC methods? What is their essence?7. Describe the characteristics of positive Reversed-phase Ion Pair Chromatography and the factors affecting the separation of components.8. What are the effects of the following conditions on the separation and detection of components?(1) when using an ultraviolet absorption detector, the mobilephase is hexane containing impurities (Fang Ting)(2) in liquid solid chromatography, by containing a small amount of polar impurities (such as water) of n-hexane as mobile phase(3) in the gradient elution, the non-polar mobile phase with a trace of polar impurities is used to replace the more volatile mobile phase.The fourth chapter is thin layer chromatography analysisBrief answer: 1.. Compared with liquid chromatography analysis,what's special about thin layer chromatography?2. briefly describe the working process of thin layer chromatography analysis.。
