Liquid Chromatography

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Rosanna Cameron
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1 Liquid Chromatoraphy An LC instrument consists of the solvent system, pump, injector, column and detector (Fi 28-4 or R5 Fi 5.7). Solvent system Solvent reservoirs are equipped with a means of removin dissolved ases (to avoid bubble formation) (i.e. vacuum pumpin system, sparin by inert ases,e.. He), and particulates (i.e. filter). Several reservoirs are available for mixed solvent isocratic elution, stepwise and radient elution. Expensive hih-purity solvents are often required. umpin system An HLC pumpin system has the followin strinent requirements: 1. eneration of pressures of up to 6000 psi 2. pulse-free output 3. flow rates rane from 0.1 to 10 ml/min 4. ood flow control to provide flow reproducibility of 0.5% or better 5. corrosion-resistant components There are 3 main types of HLC pumps: 1. reciprocatin pump 90% usae. The solvent is drawn into a small chamber (when the solvent chec valve is open) and pumped out of it (when the column chec valve is open) by the bac and forth motion of a motor-driven piston (Fi 28-6 or R5 Fi 6.9). The pump has the advantaes of (a) small solvent chamber volume ( µl), (b) hih output pressure (up to 10,000 psi), (c) ready for use in radient elution, (d) constant flow rate which is independent of column bac pressure and solvent viscosity, but disadvantae of a pulsed flow which must be smoothed out usin a pulse damper. 2. Displacement (or syrine) pump The solvent is pumped out of a lare chamber by a pluner (R5 Fi 6.10). The pump produces a pulse-free flow which is also independent of column bac pressure and solvent viscosity. But it has a limited solvent capacity (~250 ml) and requires refillin of solvent chamber for continual use. 3. neumatic (or constant pressure) pump: use a compressed as tan. Sample injection system It is convenient to use a sample loop injector (of size µl) so that the sample can be introduced without depressurin the system. In addition, injection reproducibility is hih (better than 2%) (Fi 28-7 and Fi 27-4). Analytical column Because of pressure limitations, the most LC columns are straiht, have lenth of cm and inside diameters of 4-10 mm, and are paced with particles of size 5-10 µm in diameter. Microbore columns (i.d mm) are sometimes used. Smaller column diameter will result in reater sensitivity because sample dilution within a column decreases as column i.d. decreases (R5 Fi. 6.55). Guard column A short uard column is usually placed before the expensive analytical column to protect it from damae due to (1) particulate matter, (2) irreversible bindin of contaminant to the stationary phase (3) bleedin. But loss in resolution may result (R1* Fi 19.11). Chem 316/. Li/HLC/.1

2 Extra-column band broadenin This occurs in tubins connectin between the column and other parts, such as injection system, the detector reion, etc. The smaller is the radius of the connectin tube, the less is the extra-column band broadenin. 2 πr u Hex = 24DM This effect is neliible in GC because D M is lare. Column heater Better chromatorams may be obtained by increasin column temperature (R1* Fi 19.2). Column pacin ellicular particles: A thin porous layer (e.. silica) is deposited on the surfacd of non-porous beads (e.. lass of larer diameter of µm) (R5 Fi 6.32). These are usually used in uard columns, with a loss in resolution (see R1* Fi ). orous particles: small-sized porous particles (e.. silica of diameter of 3-10 µm) are used in analytical columns. Detector Unlie GC, LC has no universal detectors such as FID and TCD. In addition to the requirements for an ideal GC detector (except temperature rane), LC detectors should have minimal internal volume to reduce extra-column band broadenin. LC detectors are classified as bul property detectors (e.. refractive index) and more selective solute property detectors (e.. UV absorbance, fluorescence). UV absorbance detector The detector volume must be small (1-10 µl) to reduce extra-column band broadenin. This detector withstands a pressure only up to 600 psi and thus requires a pressure reduction device. These detectors are usually double-beam devices in which one beam passes throuh the eluent cell and the other throuh a filter. Matched photoelectric cells are used to compare the 2 beam intensities. The chromatoram consists of a plot of the lo of the ratio of the 2 transduced sinals as a function of time. The intense line of a H lamp (254 nm) isolated by filters is commonly used as the UV source. Deuterium or tunsten filament sources with interference filters are also used (Fi 28-9 & R5 Fi 6.17). A spectrophotometer with ratin optics in the scannin monochromator is a more sophisicated source. The most powerful UV spectrophotometric detectors are diode-array instruments which permit collection of an entire spectrum in about 1 s as each chromatoraphic pea is eluted (Fi and R1* Fi 19.16). Fluorescence detector The excitation sources rane from the H lamp with filters to Xe lamp with ratin monochromator, and to tunable lasers (Fi 15-4). The fluorescence method typically has a hiher sensitivity (by more than an order of manitude) than the absorbance method. Often the samples are treated (or derivatized) with fluoroenic reaents (e.. dansyl chloride) to form fluorescent derivatives. This can be either pre-column or post-column derivatization. Refractive index detector If the solute and solvent in the 2 halves of the flow chamber differ in refractive index, the liht beam incident on the chamber will be bent (fi or R5 Fi 6-26). This results in the beam displacement, Chem 316/. Li/HLC/.2

3 and variation in the output sinal of the detector. As compared to FID and TCD in GC, the RI detector responds to nearly all solutes. But it is less sensitive and is hihly temperature-dependent. Electrochemical detector ossible oxidation or reduction of many oranic functional roups can be exploited for detection by amperometric, polaroraphic or coulometric methods (Fi 28-12). In the thin-layer flow cell for amperometric detection, the worin electrode is part of a channel wall (Fi 28-13). The reference and counter electrodes are located downstream. Detectors with dual worin electrodes are also available. Mass spectrometric detector A major problem in interfacin LC with MS is the enormous mismatch between the relatively lare solvent volume from the former and the vacuum requirement of the latter. For microbore columns with µl/min flow, the method of split flow can be used. In the thermospray method (for flow up to 2 ml/min), the liquid is vaporized as it passes throuh a heated SS capillary tube to form an aerosol jet of solvent and analyte molecules, which are ionized throuh a chare-exchane mechanism with a salt (e.. NH 4 OAc) present in the mobile phase. However, the method is applicable only to polar analytes and mobile phase and can only provide MW data (no framentation). In the electrospray method, the liquid is pumped throuh an SS capillary needle maintained at a hih potential (few V) with respect to a cylindrical electrode surroundin the needle (R6 Fi 25-30, Fi 25-31). A chared spray is resulted and desolvation, solvent evaporation, chare attachment to the analyte molecules occur. Comparison of various HLC detectors is iven in Table 28-1 or R5 Table 6.2. Stationary phase Amon various LC modes (LSC, TLC, IEC, SEC), partition chromatoraphy has become the most widely used one. Liquid-liquid chromatoraphy: the liquid stationary phase, which is physiosorbed on the column pacin, may bleed away. Bonded-phase chromatoraphy: the liquid stationary phase which is chemically bonded to the silicabased column pacin is the predominant form. The bonded phase are siloxanes formed by reaction of the silanol (Si-OH, 8µ/m 2 ) with an oranochlorosilane (Fi in 28D-1). Surface coverae by silanization is limited to 4 µmol/m 2 (i.e. 50%) or less. The unreacted silanol roups are capped by reactin with less sterically hindered chlorotrimethylsilane to avoid tailin problems, especially for basic solutes. 2 types of stationary phases: normal-phase: hihly polar (e.. triethylenelycol), and a non-polar M (e.. n-hexane) is used to elute polar solutes. Reversed-phase (75% usae): non-polar (e.. C18), and a polar M (e.. water, MeOH or MeCN) is used to elute less polar solutes. In reversed-phase chromatoraphy, the most polar component is eluted first because it is more soluble in the polar M and least soluble in the non-polar S (Fi and Fi a & b). Increasin M polarity increases the elution times of all components (e.. water > 50% MeOH/50% water > 80% MeOH/20% water). The ph of water should not be reater than 7.5 to avoid base hydrolysis of siloxane S. Chem 316/. Li/HLC/.3

4 Reverse reasonin applies to normal-phase chromatoraphy (Fi and R1* Fi ). Loner chain lenth in the reversed-phase S will provide reater solute retention and larer sample capacity (Fi 28-15). Mobile phase Unlie GC, separations are hihly affected by the composition (or polarity) of the M. A rule of thumb is to select a S with polarity that is rouhly matched with that of the solutes; a M of considerably different polarity is then used for elution. Gradient Elution Isocratic elution is first carried out to determine the appropriate radient elution for optimizin separations (R6 Fi & 25.11). R6: DC Harris Quantitative Chemical Analysis 5 th ed, 1999 olarity index, This parameter is based on solubility measurement for the substance in 3 solvents (Table 28-2): Dioxane (low dipole proton acceptor) Nitromethane (hih dipole proton acceptor), Ethyl alcohol (hih dipole proton donor) = lo( K ) + lo( K ) + lo( K ) e d n For a binary solvent mixture, AB = φ A A + φ B B φ is volume fraction Chane in M composition will affect 2-unit difference in results in 10-fold increase in (E and Fi a & b). 2 ( 2 1 )/ 2 2 ( 1 2 )/ 2 Re versed phase = 10 Normal phase = In many cases, adjustin is all that is needed to produce a satisfactory separation. If not, adjustment of α is necessary (Fi c-e). More details are available in R3. Moreover, a systematic way to achieve this has been demonstrated usin an optimization procedure usin 4 solvents. Derivative formation 1. To reduce polarity of the analyte so that partition, rather than adsorption or ion-exchane columns are used. 2. To increase the detector response and thus sensitivity of all or some analytes (Fi 28-18) Other partition chromatoraphy techniques: chiral chrom (chirality in S), ion-pair chrom (counter ions in M) Other LC modes: Adsorption chrom or LSC, IEC or IC, SEC and TLC. Different modes of LC used accordin to the MW and polarity of the solutes (Fi 28-1 or equiv) Exercise 28-1 a-c, 28-2 to 4, 28-6 a-j, 28-8, 28-10, to 13, a-b, to 19. Chem 316/. Li/HLC/.4

5 Chem 316/. Li/HLC/.5

High Performance Liquid Chromatography

Updated: 3 November 2014 Print version High Performance Liquid Chromatography David Reckhow CEE 772 #18 1 HPLC System David Reckhow CEE 772 #18 2 Instrument Basics PUMP INJECTION POINT DETECTOR COLUMN