Chromatography PDF

Introduction to Chromatography  Chromatography: (Greek = chroma “color” and graphein “writing” ) Tswett named this new technique chromatography based on the fact that it separated the components of a solution by color. Common Types of Chromatography Tswett’s technique is based o...

Introduction to Chromatography  Chromatography: (Greek = chroma “color” and graphein “writing” ) Tswett named this new technique chromatography based on the fact that it separated the components of a solution by color. Common Types of Chromatography Tswett’s technique is based on Liquid Chromatography. There are now several common chromatographic methods. These include: Paper Chromatography (PC) Thin Layer Chromatography (TLC) Liquid Chromatography (LC) High Pressure Liquid Chromatography (HPLC) Ion Chromatography (IC) Gas Chromatography (GC)  A Quick Historical Perspective 1901 Mikhail Tswett invented chromatography during his research on plant pigments. 1903 Adsorption chromatography 1952 Gas chromatography (GLC) 1968 High performance liquid chromatography (HPLC) 4  According to the nature of the mobile phase, chromatographic techniques can be classified into three classes: 1. Liquid chromatography (LC) 2. Gas chromatography (GC) 3. Supercritical fluid chromatography (SFC) Other classifications are also available  Column chromatography where chromatographic separations take place inside a column.  Planar chromatography, where the stationary phase is supported on a planar flat plate, are also used. 5  General Description of Chromatography In a chromatographic separation of any type, different components of a sample are transported in a mobile phase (a gas, a liquid, or a supercritical fluid). The mobile phase (also called eluent) penetrates or passes through a solid or immiscible stationary phase. Solutes (eluates) in the sample usually have differential partitioning or interactions with the mobile and stationary phases. Since the stationary phase is the fixed one then those solutes which have stronger interactions with the stationary phase will tend to move slower (have higher retention times) than others which have lower or no interactions with the stationary phase will tend to move 6 faster. Therefore, chromatographic separations are a consequence of differential migration of solutes. It should be remembered that maximum interactions between a solute and a stationary phase take place when both have similar characteristics, for example in terms of polarity. However, when their properties are so different, a solute will not tend to stay and interact with the stationary phase and will thus prefer to stay in the mobile phase and move faster; a polar solvent and 7 a nonpolar stationary phase is a good example.  Elution Chromatography The term elution refers to the actual process of separation. A small volume of the sample is first introduced at the top of the chromatographic column. Elution involves passing a mobile phase inside the column whereby solutes are carried down the stream but on a differential scale due to interactions with the stationary phase. As the mobile phase continues to flow, solutes continue to move downward the column. Distances between solute bands become greater with time and as solutes start to leave the column they are 8 sequentially detected. 9  Fig. Chromatogram and Related Terms 10  Chromatograms The plot of detector signal (absorbance, fluorescence, refractive index, etc..) versus retention time of solutes in a chromatographic column is referred to as a chromatogram. The areas under the peaks in a chromatogram are usually related to solute concentration and are thus very helpful for quantitative analysis. The retention time of a solute is a characteristic property of the solute which reflects its degree of interaction with both stationary and mobile phases. Retention times serve qualitative analysis parameters to 11 identify solutes by comparison with standards.  Introduction to Gas Chromatography  Gas chromatography (GC) is a technique used for separation of volatile substances, or substances that can be made volatile, from one another in a gaseous mixture at high temperatures. A sample containing the materials to be separated is injected into the GC. A mobile phase (carrier gas) moves through a column that contains a wall coated or granular solid coated stationary phase. As the carrier gas flows through the column, the components of the sample come in contact with the stationary phase. The different components of the sample have different affinities for the stationary phase, which results in differential migration of solutes, thus leading to separation 13  GC can be used for both qualitative and quantitative analysis. Comparison of retention times (Rt) can be used to identify materials in the sample by comparing Rts of peaks in a sample to Rts for standards. Quantitative analysis is accomplished by measurement of either peak height or peak area. 14  Gas - Solid Chromatography (GSC) The stationary phase, in this case, is a solid like silica or alumina. It is the affinity of solutes towards adsorption onto the stationary phase which determines, in part, the retention time. The mobile phase is, of course, a suitable carrier gas. This gas chromatographic technique is most useful for the separation and analysis of gases like CH4, CO2, CO,... etc. The use of GSC in practice is considered marginal when compared to gas liquid chromatography. 15  Gas - Liquid Chromatography (GLC) The stationary phase is a liquid with very low volatility while the mobile phase is a suitable carrier gas. GLC is the most widely used technique for separation of volatile species. The presence of a wide variety of stationary phases with contrasting selectivities and easy column preparation add to the assets of GLC or simply GC. 16  HPLC GC Mobile phase  changes  constant Temperature  constant  Increasing from the mobile  from the mobile Compounds partition phase based on phase based on solubility. volatility.  is generally  is generally Elution time or volume temperature dependent dependent  GC Function Separation of volatile organic compounds Volatile – when heated, VOCs undergo a phase transition into intact gas-phase species Separation occurs as a result of unique equilibria established between the solutes and the stationary phase (the GC column) An inert carrier gas carries the solutes through the column  Gas Chromatography  Good for volatile samples (up to about 250 oC)  0.1-1.0 microliter of liquid or 1-10 ml vapor  Can detect 99.9%) 2. Inert so that no reaction with stationary phase or instrumental components can take place, especially at high temperatures. 3. A higher density (larger viscosity) carrier gas is preferred. 4. Compatible with the detector since some detectors require the use of a specific carrier gas. 5. A cheap and available carrier gas is an advantage. 35  Injector A GC syringe penetrates a septum to inject sample into the vaporization camber Instant vaporization of the sample, 280 C Carrier gas transports the sample into the head of the column Purge valve controls the fraction of sample that enters the column  Splitless (100:90) vs. Split (100:1) Syringe Syringe Injector Injector He He Purge valve closed Purge valve open GC column GC column  Split/Splitless Injector Splitless Injection, (where the split vent is closed) attempts to transfer all of the sample to the column and is used for trace analysis. Split Mode, only a small portion (maybe 1-10%) of the sample moves into the column, and the rest is sent to waste. This is used when the analytes are in high concentration and would overload the column. Sample is injected through the septum with a syringe. The oven Inside here Column Question: The injection port is at a HIGH temperature 250oC is common. What happens when the hot, volatized sample hits a “cold” (50oC) GC column?  The volatile compounds condense at the front end of the column. Raising the temperature of the column allows for the separation of the compounds as they boil away at different temperatures. If two compounds have the same volatility, the compound with the least affinity for the stationary phase will volatilize first. Gas chromatography separates molecules mostly based on: 1. Volatility (Oven) 2. Polarity (Column)  Polar vs. nonpolar Separation is based on the vapor pressure and polarity of the components. Within a homologous series (alkanes, alcohol, olefins, fatty acids) retention time increases with chain length (or molecular weight) Polar columns retain polar compounds to a greater extent than non-polar – C18 saturated vs. C18 saturated methyl ester Oven Temperature Program Increase Temp. Linearly or Stepwise Time  Oven Programmable Isothermal- run at one constant temperature Temperature programming - Start at low T and gradually ramp to higher T – More constant peak width – Better sensitivity for components that are retained longer – Much better chromatographic resolution – Peak refocusing at head of column  Typical Temperature Program 220C 160C 50C 0 60 Time (min)  Isothermal at 45° Isothermal at 145° Programmed 30 to 180° 47 48 49  Columns GC column nomenclature can be confusing. Carbowax, DB-1, DB-5, PDMS ….. Columns are often referred to by their polarity, like most things with chromatography. The most non-polar stationary phase is polydimethyl siloxane (PDMS). Polarity of a column is increased by adding phenyl groups to PDMS (1% = DB-1; 5% = DB-5). For more polar analytes, polyethylene glycol (carbowax) is used as the stationary phase  Detectors Flame Ionization Detectors (FID) Electron Capture Detectors (ECD) Electron impact/chemical ionization (EI/CI) Mass spectrometry  FIDs Effluent exits column and enters an air/hydrogen flame The gas-phase solute is pyrolyzed to form electrons and ions All carbon species are reduced to CH2+ ions These ions collected at an electrode held above the flame The current reaching the electrode is amplified to give the signal  FID A general detector for organic compounds Very sensitive (10-13 g/s) Linear response (107) Rugged Disadvantage: specificity  ECD Ultra-sensitive detection of halogen containing species Pesticide analysis Other detectors besides MS – IR – AE Mass Spectrometry  What kind of info can mass spec give you? Molecular weight Elemental composition (low MW with high resolution instrument) Structural info (hard ionization or CID)  How does it work? Gas-phase ions are separated according to mass/charge ratio and sequentially detected  Parts of a Mass Spec Sample introduction Source (ion formation) Mass analyzer (ion sep.) - high vac Detector (electron multiplier tube)  Sample Introduction/Sources Volatiles Probe/electron impact (EI),Chemical ionization (CI) GC/EI,CI Involatiles Direct infusion/electrospray (ESI) HPLC/ESI Matrix Assisted Laser Adsorption (MALDI) Elemental mass spec Inductively coupled plasma (ICP) Secondary Ion Mass Spectrometry (SIMS) – surfaces  EI, CI EI (hard ionization) – Gas-phase molecules enter source through heated probe or GC column – 70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions – EI spectra can be matched to library stds CI (soft ionization) – Higher pressure of methane leaked into the source (mtorr) – Reagent ions transfer proton to analyte  EI process M + e- M+* f1 f2 f4 f3 This is a remarkably reproducible process. M will fragment in the same pattern every time using a 70 eV electron beam Ion Chromatogram of Safflower Oil  CI/ ion-molecule reaction 2CH4 + e-  CH5+ and C2H5+ CH5+ + M  MH+ + CH4 The excess energy in MH+ is the difference in proton affinities between methane and M, usually not enough to give extensive fragmentation EI spectrum of phenyl acetate  Mass Analyzers Low resolution – Quadrupole – Ion trap High resolution – TOF time of flight – Sector instruments (magnet) Ultra high resolution – ICR ion cyclotron resonance  Resolution R = m/z/Dm/z Unit resolution for quad and trap TOF up to 15000 FT-ICR over 30000 – MALDI, Resolve 13C isotope for a protein that weighs 30000 – Resolve charge states 29 and 30 for a protein that weighs 30000  High vs low Res ESI Q-TOF, ICR – complete separation of the isotope peaks of a +3 charge state peptide – Ion abundances are predictable – Interferences can be recognized and sometimes eliminated Ion trap, Quad – Unit resolution  594.3 594.7 MVVTLIHPIAMDDGLR Q-TOF C78H135N21O22S2+3 595.0 601.3 595.3 601.7 601.0 602.0 m/z 901.4 100 891.7 95 90 LCQ 85 891.2 80 902.3 R = 0.88 75 70 65 60 892.6 55 50 45 40 900.6 35 30 25 20 15 10 5 0 m/z Quadrupole Mass Ion Filter Ion Trap Time of Flight -TOF Where: mi = mass of analyte ion zi = charge on analyte ion E = extraction field ti = time-of-flight of ion ls = length of the source ld = length of the field-free drift region e = electronic charge (1.6022x10-19 C)  TOF with reflectron http://www.rmjordan.com/tt1.html Sector instruments http://www.chem.harvard.edu/mass/tutorials/magnetmovie.ht ml  FT-ICRMS http://www.colorado.edu/chemistry/chem 5181/MS_FT-ICR_Huffman_Abraham.pdf  Mass accuracy Mass Error = (5 ppm)(201.1001)/106 =  0.0010 amu 201.0991 to 201.1011 (only 1 possibility) Sector instruments, TOF mass analyzers How many possibilities with MA = 50 ppm? with 100 ppm?  Exact Mass Determination Need Mass Spectrometer with a high mass accuracy – 5 ppm (sector or TOF) C9H15NO4, FM 201.1001 (mono-isotopic) Mass accuracy = {(Mass Error)/FM}*106 Mass Error = (5 ppm)(201.1001)/106 =  0.0010 amu  COLUMNS Compound Polarity Max Column ID Manufacturer Temp oC Poly(methyl siloxane) Low 300-350 HP-1 Agilent AT-1 Alltech DB-1, SE-30 J&W OV-1 Ohio Valley ZB-1 Phenomenex RTx-1 Restek BP-1 SGE SPB-1 Supelco CP-Sil 5 CB Varian 95% Dimethyl, Low 300 HP-5 Agilent AT-5, EC-5 Alltech 5%phenyl DB-5, SE-54 J&W Poly(methyl siloxane) OV-5 Ohio Valley ZB-5 Phenomenex RTx-5 Restek BP-5 SGE SPB-5,MDN-5 Supelco CP-Sil 8 CB Varian Polyethylene glycol Medium 250 HP-20M Agilent AT-Wax Alltech DB-Wax J&W Carbowax 20M Ohio Valley ZB-Wax Phenomenex Stabilwax Restek BP20 SGE Supelcowax 10 Supelco CP-Wax 52 CB Varian About a dozen different types in common usage.  GC Applications Analysis of foods is concerned with the assay of lipids, proteins, carbohydrates, preservatives, flavors, colorants, vitamins, steroids, drugs, and pesticide residues. Most of the components are non-volatile (thus the use of HPLC) but with modification, GC can be effectively used. Derivatization of lipids and fatty acid to their methyl esters Proteins are acid hydrolyed followed by esterification (N-propyl esters) Carbohydrates derivatized by silylation to produce a volatile compound  Quantitative Analysis  GC is an excellent quantitative technique where peak height or area is proportional to analyte concentration.  Thus, the GC can be calibrated with several standards and a calibration curve is obtained, then the concentration of the unknown analyte can be determined using the peak area or height.  The detector response factor for each analyte should be considered for accurate quantitative analysis. 83  GC  Are widely used as criteria for establishing the purity of organic compounds.  Contaminants, if present, are revealed by the appearance of additional peaks.  Qualitative Analysis is usually done by comparison with Rts of standards, which are very reproducible in GC, provided good injection practices are followed. Injection should be done with a suitable Hamilton type syringe through the heated septum injector till all needle disappears, then the needle is drawn back as steadily and fast as possible. This is important for reproducible attainment of retention times. 84