1	HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AMANDA CAMERON Forensic Chemist Georgia Bureau of Investigation Atlanta, Georgia
2	Chromatography chro·ma ·tog ·ra ·phy: n. Any of various techniques for the separation of complex mixtures that rely on the differential affinities of substances for a gas or liquid mobile medium and for a stationary absorbing medium through which they pass, such as paper, gelatin, or magnesia. -American Heritage Dictionary of the English Language. 4th ed. 2000. From the Greek words “chromatos” meaning “color,” and “graphy” meaning “writing”
3	History of Chromatography 1903: Russian botanist, Mikhail S. Tswett experimented with separation of leaf pigments from plants using solvents Using an open glass tube packed with calcium carbonate and alumina, he poured the solvent extract of leaves into the column Then, pure solvent was poured through the column, washing the extract down the column through gravity Tswett related the resulting different colored bands to different compounds present in the original leaf sample
4	"The components of the pigment..." The components of the pigment were separated based on their individual attraction to the column packing (stationary phase) Components that were more attracted to the stationary phase remained in the column longer while components that were more attracted to the solvent (mobile phase) moved through the column faster
5	Common Types of Chromatography
6	Thin Layer Chromatography Preliminary technique for qualitative analysisàR_f “Normal Phase” = relatively POLAR stationary phase, relatively NONPOLAR mobile phase Stationary phase is usually a silica gel coated on glass or plastic plate Mobile phase is solvent or combination of solvents Useful when target analyte is fairly polar
7	Basic Process for TLC Perform extraction to get compound in solution “Spot” sample solution, negative control and any standards at origin of plate, allow solvents to evaporate To separate, place plate in closed chamber contacting the bottom edge of the plate with the mobile phase. Mobile advances up the plate through capillary action Once solvent has reached top of plate, plate is removed and the results are visualized using an appropriate technique. Examples of visualization methods: Sulfuric acid/heat: destructive, leaves charred blots behind Ceric stain: destructive, leaves a dark blue blot behind for polar compounds Iodine: semi-destructive, iodine absorbs onto the spots, not permanent UV light: non-destructive, long wavelength (background green, spots dark), short wavelength (plate dark, compounds glow)
8	TLC process in pictures
9	High Performance Liquid Chromatography
10	Example of Specific System: The Agilent 1100 HPLC
11	Solvent Compartment Holds solvent bottles Can use up to 4 solvents SOLVENT CHOICE! Useful to employ solvent filters to prevent particulate or impurities from entering system
12	Vacuum Degasser Pump draws solvent from solvent bottles through degasser Vacuum chamber draws gases dissolved in solvents out into chamber through semi-permeable membrane of tubing Bubbles can cause problems in pressure and flow rate, chromatography/retention time, as well as problems in the detector
13	Pump Used to draw solvent from bottles and push solvents continuously through system Quaternary Good for use in a wide range of research and routine applications Either isocratic or gradient analysis Flow rates up to 10 mL/min Pressure range up to 400 bar at max of 5 mL/min Binary High pressure mixing Extremely reproducible gradients at low flow rates Flow rates as low as 50 μL/min, pressures as high as 600 bar in SL system
14	Agilent: Operating Principle of the Dual Piston Pump
15	Isocratic vs Gradient Elution Isocratic elution Utilizes only one solvent “Single solvent” can mean combination of solvents, but in a constant ratio throughout analysis Depending on target analyte(s), single solvent may not adequately separate components Gradient elution Utilizes a combination of solvents in varying ratios throughout the analysis Mobile phase can range from more polar to less polar (or vice versa) to ensure adequate separation in a useful amount of time Disadvantage: column must re-equilibrate after each run (post-run)
16	Isocratic vs Gradient Elution
17	Automated Liquid Sampler (ALS) Arm retrieves sample vial from tray position and carries it to the needle-seat. The needle pierces the septum of the vial cap and draws up some of the sample before the arm picks up the vial and returns it to its original tray position. The needle then injects the sample into the solvent flow.
18	Sample Introduction
19	Column Compartment Depending on the manufacturer, the column is positioned in a thermostatted chamber and connected to the injection valve and detector through a series of capillary tubing. Compounds exit the column at a retention time dependent on system parameters Samples continue through the system to the detector
20	Column Choice Several column options are available from several manufacturers Consider samples to be analyzed Consider goals of separation Physical features of column: Particle size --Surface area Column length --Pore size Internal diameter --Carbon load Bonding type --Particle shape www.alltechweb.com (http://www.discoverysciences.com/Productinfo/Technical/posters/MethodDevelopment.ppt#1)
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22	Choose bonded phase: (Porous bonded silica columns) C18 (Octadecylsilane)—very nonpolar; retention based on London interactions with hydrophobic compounds Phenyl—nonpolar; retention is a mixed mechanism of hydrophobic and π-π interactions Cyanopropyl—intermediate polarity; retention is a mix of hydophobic, dipole-dipole and π-π interactions
23	Other column types Specialty silicas Sulfono --Fluoro Diols --Miscellaneous Amino Polymers: PS-DVB, polyvinyl alcohol Zirconium oxide
24	Choose Physical Characteristics Length: Longer columns = higher resolution but longer run times Shorter columns = short run time, low back pressue, less solvent usage and waste Diameter: Narrow columns = higher detector sensitivity, higher pressure required, less sample can be injected while maintaining good peak shape Wide columns = generally used for preparative HPLC
25	Physical Characteristics (cont’d) Particle Characteristics Shape Spherical ▪Irregular Size Pore size Surface Area Bonding type Monomeric ▪Polymeric Carbon Load All factors have an effect on the number of theoretical plates, and therefore the efficiency of the column
26	Van Deemter Equation Takes into account all factors that contribute to band-broadening H = plate height λ = particle shape (with regard to the packing) d_p = particle diameter G, ω, and R are constants D_m = diffusion coefficient of the mobile phase d_c = capillary diameter d_f = film thickness D_s = diffusion coefficient of the stationary phase.
27	"A = Eddy-diffusion" A = Eddy-diffusion B = Longitudinal diffusion C = Resistance to mass transfer u = Flow rate
28	Even simpler… Plate Height can be calculated using the equation Where N = number of plates and L = length of the column
29	Calculating N
30	Example of Jasco X-LC^TM Column
31	Detector Options Optical Detectors: Fixed wavelength Fluorescence Refractive Index Diode Array Detector Electrochemical Infra-red Mass Spectrometry
32	Review of Ultraviolet Spectroscopy
33	Example of phenethylamine UVs
34	Chromophore: A chemical group capable of selective light absorption resulting in the coloration of certain organic compounds.
35	"Absorbance = -log T..." Absorbance = -log T = -log(I / I0) = εbc where: ε = molar absorptivity b = length of sample cell (cm) c = molar concentration of solute T = I/I₀ = Intensity of light after passing through cell/Intensity incident light
36	Transmittance v. Absorbance Transmittance is measured as a percentage while absorbance becomes arbitrary > 1.0
37	Fixed Wavelength Detector Contain low-pressure mercury lamps with an intense emission line at 254 nm Other types of lamps are available Some have optional additional wavelength settings Many organic compounds absorb at 254 nm Best for situations when wavelength is rarely varied
38	Fluorescence Detector Solute is excited with UV radiation and emits radiation at a longer wavelength Can offer great selectivity since excitation and emission wavelengths, as well as retention times can be used to identify drugs Solvent choice is important: Mobile phase must neither fluoresce nor absorb at chosen wavelengths pH: some drugs only fluoresce in certain ionic forms
39	Refractive Index Detector Refractive Index of a medium is a measure for how much the speed of light is reduced inside the medium Changes in refractive index (positive or negative) that arise from the presence of a compound in the eluent are recorded Universal detector, but lease sensitive Factors must be controlled during separation: Temperature Eluent composition Pressure
40	Diode Array Detector (DAD) Sample travels from column through flow-cell where polychromatic light is focused on the flow cell and is dispersed onto a chip containing light sensitive diodes arranged side-by-side Each diode registers a well-defined fraction of the UV-spectrum so it can measure all wavelengths at the same time
41	Electrochemical Detector Measure the gain or loss of electrons from migrating samples as they pass between electrodes at a given difference in electrical potential Eluents must be electrically conductive (addition of inert electrolytes) Most easily used in the oxidative mode (use in reductive mode requires the removal of dissolved oxygen from the eluent)
42	Infra-red Detector Non-destructive technique Scans compounds in the IR region and measures the stretching and bending vibrations of chemical bonds Advantage of 3 pieces of data from 1 sample/extraction: retention time, UV, IR
43	Mass Spectrometer (LC/MS) Presents unique challenge because liquid phase must be converted to gas phase for normal mass spectral analysis Different methods of atmospheric pressure ionization: Electrospray ionization (ESI) - voltage applied to the solvent - as solvent evaporates electrical field at the surface of the drop increases due to the decreasing radius of the droplet Atmospheric pressure chemical ionization (APCI) - heat evaporates the solvent and sample charged via charged probe. API: need volatile mobile-phase modifiers in the chromatographic separation
44	Fraction Collection Additional component that allows for collection of individual components from a multi-component sample Can be set to collect certain peak from multiple injections of sample in one vial
45	Applications of HPLC Pharmaceutical (drug development, research, QA) Clinical (drug monitoring, drugs of abuse, disease markers) Environmental analysis Food & beverage Polymers & plastics Forensics!!
46	Qualitative Analysis GBI: (primarily) Agilent 1100 HPLC, Zorbax XDB C18 4.6 x 150 mm, 5 μm column, DAD detector
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48	Sample shots
49	Mixed Standard: Nicotinamide, Ephedrine, Amphetamine, Methamphetamine, Phentermine
50	Final negative control
51	Potential Causes of Carryover Mobile Phase pH Injection Valve Metering Device Needle and Needle Seat Column Compartment Capillary/Peek Tubing and fittings Article on carryover: http://www.forumsci.co.il/HPLC/Carryover_problems.pdf
52	Quantitative Analysis HPLC is well suited to quantitative analysis Reproducibility Metered injection Stable solvents Temperature control Peak resolution Relatively short analysis time Ease of sample prep Ease of calculation Ease of explanation
53	Calibration Methods Standard Addition Internal Standard External Standard
54	Standard Addition Useful when sample is in a complex matrix Multiple aliquots of unknown sample are prepared. One sample is analyzed straight for the target Other samples are spiked with precise but varying amounts of the target Calibration curve of response vs. concentration is produced to directly determine the unknown Disadvantages: Difficult to explain --Time-consuming preparation Requires greater volume of sample Associated uncertainty is large
55	Internal Standard Tends to yield the most accurate and precise results A known amount of a component that is chemically similar and has a retention time close to the target analyte, but that is not present in the sample, is added to the target and a standard solution Ratios of peak height or area of the target to the internal standard gives quantitation data
56	"Conc_unk=Area_int." Conc_unk=Area_{int. std.in known} x Area_unknown x Conc_known Area_{int. std.in unknown} Area_known
57	External Standard GBI Reference standards of varying amounts encompassing the expected range of sample concentration are used to generate a calibration curve Simplest quantitation technique Easy sample prep Easy to plot and explain Requires consistency in analysis (consistent, reliable injections) Requires additional QA/QC procedures
58	Building the Curve….
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60	Chemstation Software
61	Excel
62	Additional QA/QC Once calibration curve is established, it is considered valid until something invalidates it **Need additional QA/QC measures to ensure instrument is still working properly and to ensure that quantitation procedure is followed properly
63	Calibration Check Standard (CCS) Analyzed with a negative control (ethanol) at the beginning and end of each sequence Also analyzed before and after each scientist to bracket individual data Considered valid if within 5% of known concentration
64	Scientist Control Standard (SCS) Standard of known purity Quantitatively weighed and analyzed along with samples Calculate the % recovery of the SCS for each scientist %Purity = conc (mg/mL) * Vol (mL) * 100 weight (mg) %Recovery = . % Purity . *100 Actual Purity
65	Measurement Uncertainty GBI reports at a 95% confidence interval (corresponds to 1.96 σ) For sample/dup, meas. unc. for process = 4.25% For 5 bag quant: Meas Unc =( T_N-1 * SD)/ √N where N = number of pkgs tested, T_N-1 = T value at 95% confidence and N-1 degrees of freedom, SD = Standard deviation of samples
66	Example of Quant Spreadsheet
67	Conclusion: Advantages of High Performance Liquid Chromatography Convenient separation of wide range of sample types Excellent resolving power Speed of analysis Low detection levels for weak samples
68	Questions