Chromatography

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1. PRESENTED BY Khondaker Afrina Hoque ID:1114015, Reg:900048 Department of Chemistry Comilla University

2. PRESENTATION ON CHROMATOGRAPHIC METHODS OF ANALYSIS

3. Content Chromatography Principle Chromatogram Classification Configuration Gas Chromatography Principle Instrumentation Application

4. HPLC Principle Instrumentation Application Gel permeation Principle Instrumentation Application Electrophoresis Principle Instrumentation Application

5. Chromatography Greek word: Chroma means color Graphein means to write. Invention: 1850s, Schönbein used filter paper to partially separate substances in solution. Goppelsröder (in Switzerland) In 1861 he wrote ‘ I am convinced that this method will prove to be very practical for the rapid determination of the nature of a mixture of dyes, especially if appropriately chosen and characterised reagents are used ’. shortly after 1900s Michael S. Tswett assign the invention of modern chromatography.

6. INTRODUCTION Chromatography is the separation of the components of a mixture based on the different degrees to which they interact with two separate material phases. Two phases: mobile phase can be either a gas or a liquid. stationary phase can be either a liquid or solid .

7. General principle A basic chromatographic process may be described as follows: 1. A vertical hollow 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. Figure : A basic experiment in chromatography. (a) The necessary ingredients (C, column; SP, stationary phase; MP, mobile phase; and S, sample); (b) introduction of the sample; (c) start of elution; (d) recovery of the products following separation.

8. 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. If the components migrate at different velocities, they will become separated from each other and can be recovered, mixed with the mobile phase. 4.The interaction between mobile and stationary phases include the physico chemical principles such as the adsorption, ion exchange, molecular sieving and affinity.

9. 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. The separation is complete when the chromatogram shows as many chromatographic peaks as there are components in the mixture to be analysed . Figure: A real chromatogram.

10. CLASSIFICATION classification has been established by consideration of the physical nature of the two phases involved:

11. CONFIGURATION Chromatography techniques can be further classified according to configuration :

12. GAS CHROMATOGRAPHY The father of modern gas chromatography is Nobel Prize winner John Porter Martin, who also developed the first liquid-gas chromatograph (1950).

13. PRINCIPLE of GC In separation techniques (GLC), THREE main components are important: i . Stationary phase - column ii. Mobile phase – carrier gas iii. Mixture of components – vapour pressure (volatility) and solubility . Figure : A GC installation Schematic of a gas chromatograph.

14. The mobile phase that transports the analytes through the column is a gas referred to as the carrier gas. The column is usually a narrow-bore tube depending upon the type and the contents of the stationary phase. At the end of the column, the mobile phase (carrier gas), passes through a detector before it exits to the atmosphere. Classification- depending on stationary phase

15. Gas Liquid Chromatography (G.L.C ) The mobile phase - gas such as helium The stationary phase - high boiling point liquid adsorbed onto a solid.

16. INSTRUMENTATION Instrumentation • Tank of carrier gas • Flow regulator and flow meter • Injection port • Column • Temperature controlled device • Detector • Microprocessor/recorder Figure: GLC Chromatography.

17. The Mobile Phase (Carrier Gas) An inert gas such as He or N2 Function is to transport sample vapors through column No chemical interaction with sample Typical parameters Column inlet pressure: 10-50 psi (above ambient) Flow rate: 25-50 mL /min (packed column) Precise control of carrier gas flow rate is critical to obtaining reproducible retention times. Requirements of a carrier gas Inertness Suitable for the detector High purity Easily available Cheap Should not cause the risk of fire Should give best column performance.

18. Sample Injection Sample is injected using a syringe into a flowing stream of hot mobile phase: High temperature (at least 50 °C above boiling point of sample) causes vaporization of sample. Introduces a narrow plug of sample vapor onto the column. Various designs: For packed columns, inject 1 to 5µL of sample. For capillary columns, a split valve is used to introduce a small fraction of sample onto column . Figure: Injector.

19. Columns Capillary columns Typical length 30m. 0.1 – 0.5mm inside diameter. Glass, stainless steel. Coating inside the surface. Produce narrow peaks Allow separation of complex mixtures. Sample size needed normally less than micro liter. Packed columns 1.5 – 10 m long 2-4 mm diameter Made of Teflon. Solid packing, to max the area and min the thickness. High sample capacity. Two types: 1. Packed column. 2. Capillary column

20. Column Tubing Possible tubing material includes: SS-durable, but relatively reactive surface may cause component loss. Glass-fragile, usually required treatment to deactivate the surface Fused silica-used only in capillary columns, inert and robust. The most preferable material to be used. Column Efficiency The column is important to produce narrow, well-separated peaks from multi component sample. High efficiency-narrow peaks. Low efficiency Efficiency is determined by the column construction (small tubing diameter and thin stationary phase layer is the best) and the carrier gas flow rate.

21. DETECTORS The requirements of an ideal detector are- Applicability to wide range of samples Rapid response time High sensitivity Linearity Response should be unaffected by temperature, flow rate… Non destructive technique Simple & inexpensive Stable and predictable response

22. Classification of Detector:

23. Thermal conductivity detector Element is electrically heated at constant power. • Temperature depends on thermal conductivity of surrounding gas. • Measure conductivity with respect to a reference. • When analyte comes off, filament temperature goes up, resistance goes down .

24. Mechanism: A detector cell contains a heated filament with an applied cell, change in the filament current occurs. The current change is compared against current in reference cell. The difference is measured and a signal is generated. Sensitivity : 5-20 ng . Selectivity : All compounds. Gases : Hydrogen, Helium. Temperature : 150-2500°C.

25. Advantages of Catharometer Linearity is good Applicable to most compounds Non destructive Simple & inexpensive Disadvantages Low sensitivity. Affected by fluctuations in temperature and flow rate Biological samples cannot be analyzed.

26. Flame Ionization Detector Column effluent is passed through a H2-air flame produces ions and electrons. Charged particles are accelerated by voltage applied between jet and collector results in current. Less sensitive to non hydrocarbon groups. Insensitive to H 2 O,CO 2 ,SO 2 . Flame ionization detector

27. Mechanism : Compounds are burned in a hydrogen-air flame. Carbon containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Sensitivity : 0.1-10 ng . Selectivity : compounds with C-H bonds. Gases : combustion –hydrogen and air, makeup-He,N 2 . Temperatur e: 250-3000°C .

28. Advantages: μg quantities of the solute can be detected. Stable. Responds to most of the organic compounds. Linearity is excellent. Disadvantages : destroy the sample.

29. Electron capture detector Figure: Electron capture Detector Carrier gas (and analyte) passes over β- emitter, resulting in ionization and electron production. Produce current between electrodes. Most commonly used for halogenated organics.

30. Mechanism: Electrons are supplied from a 63 Ni foil lining the detector cell. A current is generated in the cell. Electronegative compounds capture electrons resulting in a reduction in the current. The amount of current loss is indirectly measured and a signal is generated. • Sensitivity : 0.1-10 ng . • Selectivity : Halogens, nitrates. • Gases : Nitrogen or argon. • Temperature : 300-4000°C. Advantages Highly sensitive. Disadvantages Used only for compounds with electron affinity.

31. Nitrogen phosphorous detector Mechanism: compounds are burned in a plasma surrounding rubidium bead supplied with hydrogen and air. Nitrogen and phosphorous containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. sensitivity : 0.1 and 0.4 pg/s. Selectivity : Nitrogen phosphorous containing compounds. Gases : combustion- hydrogen ,make up – helium.

32. Recorder Recorder is a device that draws the chromatogram that results from a chromatographic process onto chart paper. The device can have a full scale deflection (FSD) voltage that commonly ranges from 1mv to 10v . The time scale of the chart movement normally ranges from about 1 cm per second to 1 cm per hour.

33. Factors affecting separation Particle size and surface area. Carrier gas flow rate. Type and amount of stationary phase. Column length. Column diameter. Column temperature

34. Advantages of GLC Both qualitative and quantitative analysis are possible. Instrument is simple ,time of analysis is short. High sensitivity. The method is applicable to about 60% of organic compounds. Very small samples sizes can be used. Analysis can be highly accurate and precise.

35. HPLC - High Performance Liquid Chromatography Invention : 1964- Csaba Horvarth and colleague S.R Lipsky at Yale University – “High pressure liquid chromatography”. Present name – “high performance liquid chromatography”. HPLC : “HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC is characterized by the use of high pressure to push a mobile phase solution through a column of stationary phase allowing separation of complex mixtures with high resolution”. • Mobile phase is Liquid. • Stationary phase is Solid or Liquid.

36. Principle -HPLC The process involves the interaction of the compounds in the analyte or sample across an immobile surface (stationary phase). The compounds bind at specific regions of stationary phase based on certain physical and chemical properties. These bound molecules are then eluted with a suitable buffer and the same are collected with time. The properties are – Polarity Charge Molecular weight Present of functional group

37. Two criteria should be met: Firstly, Packing should be finely divided and have spherical regularity. Secondly, Stationary phase should be in the form of a thin uniform film with no stagnant pools. Figure: Basic components of HPLC.

38. Instrumentation of HPLC Solvent (mobile phase) Solvent Delivery System (Pump) Injector Sample Column (stationary phase) Detectors (Diode Array) Waste Collector Recorder (Data Collection )

39. Solvent (mobile phase) In normal phase typically non polar solvents such as hexane, heptane, isooctane are used in combination with slightly more polar solvents such as isopropanol, ethyl-acetate or chloroform. In reverse phase applications water is usually the base solvent. Other polar solvents such as Methanol, Acetonitrile or Tetrahydrofuran are added in fixed or varying proportions. pH is adjusted by buffers to modify separations of ionizable solutes.

40. Solvent Delivery System (Pump) Role: 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). During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient). 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).

41. Advantages of reciprocating pump : Small internal volume Capable of high output pressure Can readily be used for gradient elution Provides constant flow rates Independent of solvent viscosity or column back pressure.

42. Injector Role : 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 up to 6000 psi of the liquid system. An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical . Figure: Sample loop injector.

43. Column (Stationary phase) 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 back pressure 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 .

44. Several Column Types Normal phase Reverse phase Size exclusion Ion exchange

45. Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample. phase partition chromatography makes use of a polar liquid stationary phase chemically bonded to these polar particles, which typically consist of silica, Si–O–, bonding sites . normal phase bonded phases are those in which a cyano group (–CN), an amino group (–NH2), or a diol group (–CHOH–CH2OH) are part of the structure of the bonded phase. Mobile - hexane, cyclohexane, carbon tetrachloride, chloroform, benzene, and toluene.

46. Components elute in increasing order of polarity. Most polar…….Least polar Silica or alumina possess polar sites that interact with polar molecules.

47. Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) the solvents are polar aqueous organic mixtures such as methanol-water or acetonitrile -water.

48. If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly with non-polar molecules. Components elute in decreasing order of polarity. Most non-polar…….Least non-polar

49. Size exclusion In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules . Size exclusion gels separate on the basis of molecular size . Individual gel beads have pores of set size, that restrict entry to molecules of a minimum size. Larger molecules…….Smaller molecules

50. Ion exchange In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites. CATION EXCHANGE Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged). +ve charged species adhere to the support and are later eluted with acid (H+) Most +ve…………..Least +ve

51. ANION EXCHANGE: Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged). -ve charged species adhere to the support and are later eluted with acid (H+) Most –ve…….………...Least –ve Anions exchange Cl -

52. Modes of High Performance Liquid Chromatography

53. Materials of construction for the tubing : Stainless steel (the most popular; gives high pressure capabilities) Glass (mostly for biomolecules). PEEK polymer (biocompatible and chemically inert to most solvents. Packing material : The packing material is prepared from SILICA particle, ALUMINA particle and ion exchange RESIN. Porous plug of stainless steel or Teflon are used in the end of the columns to retain the packing material. According to the mode of HPLC , they are available in different size , diameters, pore size or they can have special materials attached

54. Types of columns in HPLC Guard Column. Fast Column. Preparative( i.d . > 4.6 mm; lengths 50 –250 mm ). Capillary( i.d . 0.1 -1.0 mm; various lengths ). Nano ( i.d . < 0.1 mm, or sometimes stated as < 100 μm ). Analytical [ internal diameter ( i.d .) 1.0 -4.6-mm; lengths 15 –250 mm ].

55. Guard Column These are placed anterior to the separating column. This serves as protective factor. They are dependable columns designed to filter or remove : Particles that clog the separation column Compounds and ions that could ultimately cause “ Baseline drift ”, decreased resolution, decreased sensitivity and create false peaks. These columns must be changed on a regular basis in order to optimize their protective function. Capillary Column it is also known as micro columns. It has a diameter much less than a millimeter and there 3 types: Open tubular Partially packed Tightly packed They allow the user to work with nano liter sample volume , decreased flow rate and decreased solvent usage volume , led to cost effectiveness.

56. Fast Column One of the primary reasons for using these column is to obtain improved sample output ( amount of compound per unit time ). Fast column are designed to decrease the time of chromatographic analysis. Here internal diameter is same but length is short and packed with smaller particles , that are 3 μm diameter. Advantages- Increased sensitivity Decreased analysis time Decreased mobile phase usage Increase reproducibility Preparatory Column It is used when objective is to prepare bulk ( milligrams ) of sample for laboratory preparatory application. It has usually a large column diameter , which is designed to facilitate large volume injections into the HPLC system.

57. Detector: Function : to examine the solution that elutes from the column and output an electronic signal proportional to the concentrations of individual components present. Common HPLC Detectors : UV Absorption Diode Array Fluorescence Refractive Index Electrochemical

58. UV Absorption Detector Instrument: it consists of light source, a wavelength selector, and a phototube Figure: The HPLC variable-wavelength UV detector

59. Advantages : Much better sensitivity (about 0.01ppm) Is not temperature sensitive Relatively inexpensive Can be used with gradient elution. sensitive to large number of organic compounds . Disadvantages: It cannot be used with Solvent s that have significant absorption in the UV Or with sample components that do not absorb in the UV.

60. Fluorescence Detector Figure: The HPLC fluorescence detector Instrumentation: consists of, a light source a wavelength selector (usually a filter) for creating and isolating a desired wavelength, a sample compartment, a second wavelength selector (another filter) a phototube detector for isolating and measuring the fluorescence wavelength. Advantages : More selective than UV detector. More sensitive than UV detector.

61. Refractive index detector . FIGURE: An illustration of a refractive index detector. Measure the refractive index of liquids and liquid solutions . Detects concentration of about 10 -5 to 10 -6 g/ml.

62. Advantages: The major advantage of this detector is that it is almost universal. All substances have their own characteristic refractive index. Thus, the only time that a mixture component would not give a peak is when it has a refractive index equal to that of the mobile phase, a rare occurrence. Disadvantages : it is not very sensitive . It is very sensitive to temperature changes. Also, it is difficult to use this detector with the gradient elution method because it is sensitive to changes in the mobile phase composition

63. 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: 1. determine the time of elution (retention time) of the sample components (qualitative analysis) and 2. the amount of sample ( quantitative analysis) .

64. Application There are mainly two types of application: Qualitative Analysis Quantitative Analysis

65. Qualitative Analysis QUALITATIVE ANALYSIS HPLC is used for identification of compound: Here comparison of retention time of test sample with the reference compound carried out. Checking purity of compound : Purity of compound checked by comparison of chromatogram of test with the reference standard. Presence of impurities : If impurities present into sample we observed additional peak when we compared the chromatogram of test with the reference standard. QUANTITATIVE ANALYSIS HPLC is used for assay of many drugs like cephalosporin, furosemide .

66. It is also used into drug mixture determination. Biopharmaceutical and pharmacokinetic study. Stability study Purification of some compound of natural or synthetic origin. Investigation of biological material such as gastric content, blood, urine sample etc. are done by HPLC. Many poisonous substances can also be investigated by HPLC. INGORGANIC CHEMISTRY Chromatographic separation of anion like I-,IO-, ClO-4 can be done effectively by using ion exchange chromatography.

67. BIOCHEMICAL ANALYSIS LIPID Separation of reference mixtures of glycerides , fatty acid can be done on silica column. AMINO ACID HPLC is widely used for analysis and separation of amino acid and protein. For this size exclusion chromatography is used. CARBOHYDRATE HPLC is suitable for analysis of carbohydrates. HPLC is used for determination of sugar content of soya bean extracts, daily products. NUCLEIC ACID HPLC is used into all area of nucleic acid research. HPLC is used for analysis of nucleic acid, separation of starting material, intermediates, separation and purification of nucleic acid.

68. VITAMINS HPLC is used for analysis of vitamins in variety of food products and animal feeds. ISOLATION OF NATURAL PHARMACEUTICAL ACTIVE COMPOUNDS : Some plant containing alkaloid and glycosides. From that alkaloid and glycosides can be isolated by means of HPLC. PETROCHEMICAL INDUSTRY HPLC is used for rapid separation of petroleum products and coal products with a wide boiling range using silica gel as stationary phase. ENVIRONMENTAL POLLUANTS HPLC is used for detection of level pesticides and carcinogens in air, water, food supplies. Organophosphate like Parathion, Melathion can be analysed by HPLC.

69. GEL PERMEATION Introduction: Also known as, Gel permeation chromatography Gel chromatography Size exclusion chromatography Gel filtration Molecular-sieve chromatography Invention: 1951-1955 the SEC concept was first recognised but was not clearly formulated. 1963- Jim Waters build the first commercial GPC equipment based on John Moore’s design. 1979- Yau , Kirkland and Bly published the first definitive text on SEC which contributed to the success of SEC and a complete revised edition was publish in 2009.

70. Definition: This is a technique in which the separation of components is based on the difference in molecular weight or size, and is one of the effective methods used to isolate and analyze the biomacromolecular substances.

71. Principle of separation It’s a technique that separates dissolved molecules on the basis of their size by pumping these molecules through specialized columns containing a micro porous packing material(gel). Stationary phase is a porous polymer matrix whose pores are completely filled with the solvent to be used as the mobile phase. The pore size is highly critical, since the basis of the separation is that molecules above a certain size are totally excluded from the pores, and the interior of the pores is accessible, partly or wholly, to smaller molecules. The flow of mobile phase will cause larger molecules to pass through the column unhindered, without penetrating the gel matrix, whereas smaller molecules will be retarded according to their penetration of the gel .

72. Instrumentation GPC components 1.Stationary Phase 2. The Mobile Phase 3. The Columns 4. The Pump 5. Detectors

73. 1. Stationary phase: Composed of semi-permeable, porous polymer gel beads with well defined range of pore sizes . Properties of gel beads: 1- Chemically inert 2- Mechanically stable 3- Has ideal and homogeneous porous structure (wide pore size give low resolution). 4- Uniform particle and pore size. 5- The pore size of the gel must be carefully controlled. Examples of gel Dextran ( Sephadex ) gel: An α 1-6- polymer of glucose natural gel. Agarose gel: A 1,3 linked β- D- galactose and 1,4 linked 3,6-anhydro- α, L- galactose natural gel. Acrylamide gel: A polymerized acrylamide , a synthetic gel.

74. 2. The Mobile Phase Composed of a liquid used to dissolve the biomolecules to make the mobile phase permitting high detection response and wet the packing surface.

75. 3. Columns Commercially Available Columns include Analytical column- 7.5–8mm diameters. Preparative columns-22–25mm Usual column lengths-25, 30, 50, and 60 cm. Narrow bore columns- 2–3mm diameter have been introduced. 4. The pump Are either syringe pumps or reciprocating pumps with a highly constant flow rate.

76. 5. Detectors Concentration sensitive detectors Bulk Property Detectors- Refractive Index (RI) Detector. Solute Property Detectors- Ultraviolet (UV) Absorption Detector. Evaporative Detectors- Evaporative Light Scattering Detector (ELSD). Molar mass sensitive detectors 1 . Light Scattering Detectors Low Angle Light Scattering (LALS) Detectors Multi angle Light Scattering (MALS) detectors 2. Viscosity Detectors- Differential Viscometers

77. Advantages and disadvantages Advantages: Short analysis time. Well defined separation. Narrow bands and good sensitivity. There is no sample loss. Small amount of mobile phase required. The flow rate can be set. Disadvantages: Limited number of peaks that can be resolved within the short time scale of the GPC run. Filtrations must be performed before using the instrument to prevent dust and other particulates from ruining the columns and interfering with the detectors. The molecular masses of most of the chains will be too close for the GPC separation to show anything more than broad peaks.

78. Applications of GPC Proteins fractionation Purification Molecular weight determination. Separation of sugar, proteins, peptides, rubbers and others on the basis of their size. This technique can be use to determine the quaternary structure of purified proteins.

79. Electrophoresis An electric field is an electrically charged region of space, such as between a pair of electrodes connected to a power supply. The technique utilizes the varied rates and direction with which different organic ions (or large molecules with charged sites) migrate while under the influence of the electric field. This technique is called electrophoresis . FIGURE :An illustration of the concept of electrophoresis.

80. Principle of electrophoresis The principles of separation are : 1) ions of opposite charge will migrate in different directions andbecome separated on that basis, and 2) ions of like charge, while migrating in the same direction, become separated due to different migration rates. Factors influencing migration rate are charge values and different mobilities . The mobility of an ion is dependent on the size and shape of the ion as well as the nature of the medium through which it must migrate . medium used: electrolyte soaked cellulose sheet (paper electrophoresis), a thin gel slab (gel electrophoresis), or a capillary tube (capillary electrophoresis). The nature of the electrolyte solution used and its pH are also variable .

81. Instrumentation-capillary electrophoresis Electrophoresis in buffer filled, narrow-bore capillaries. Each capillary is about 25-100µm in internal diameter. Small cross-sectional area, long length leading to high resistance, low currents. Vmax = 20-100KV. N= 100,000-10,000,000 high resolution.

82. Column The Capillaries are normally made of fused silica as in the case of Gas Chromatography open columns. The column is coated ith polyimide to improve the durability. Glass and Teflon have also been used but silica provides best functionality. These may be made hydrophobic by the incorporation of alkyl groups which would shield the silanol groups and reduce electrosmotic flow. Injector Remove anode end capillary from buffer. Place end of capillary in sample Injection volume ranges from 5-50nl. While values as low as 100pl have been reported. Apply field for short time. Replace in buffer.

83. Detector UV/Visible absorption Fluorescence Radiometric Mass spectrometry Indirect detection

84. Application Separation of Molecular Species Separation of Small Ions CE in DNA Sequencing Properties of Amphiprotic Compounds Separation of Amphiprotic Species

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