Exam 3 Flashcards
What is chromatography
*general term applied to wide variety of separation techniques based on patitioning of sample (solute) between moving (mobile) phase and a fixed (stantionary) phase
*relative interaction of the solute with these two phases is described by the partition coefficient
what is the mobile phase (ex.)
gas (GC), liquid (LC), or supercritical fluid (SFC)
what is the stationary phase
liquid or solid
what determined the elution time
interaction of the solute with the stationary phase
Thin-Layer Chromotography
*separation based on polarities
*TLC plates with thin layer of stationary phase
*sorbents used: silica gel, alumina, diatomaceous earth, cellulose
*TLC put into solvent (mobile phase)
*subst. with polar prop. will interact strongly with stationary phase (move slower)
*subst. with nonpolar prop. will interact strongly with mobile phase (move up & faster)
How can separation occur in TLC
*adsorptive
*partition (normal/reversed phase)
*ion exchange
*size-exclusion
Advantages & applications to TLC
ADVANTAGES:
*better resolution, faster, more reproducible, easier to handle
APPLICATIONS:
*screen corn/peanuts for mycotoxins before processing, lipids, carbs, vitamins, amino acids, pigments
Visualization/ Quantitation of TLC
VISUALIZATION:
*colorimetric, fluorescence, UV light
QUANTITATIVE:
*scraping off the zone, eluting the compound and then analyze the resulting solution
Column Chromotography (basics)
*separation to isolate/separate solutions
*mobile phase = liquid
*stationary phase = solid or liquid supported by inert solid
*stationary phase is packed in column and equilibrated with mobile phase
*mobile phase moves through column by a pump
*components will separate as they move down column
What are the essential components of a column chromotagraphy system
*magnetic stirrer
*peristatic pump
*column
*spectrophotometer
*fraction collector
Adsorption
*solute is adsorbed on surface of stationary phase (silica, alumina)
*elution order of solutes depends on relative polarities (polar = last, nonpolar = first)
*Van der waals, electrostatic, H bonding, hydrophobic interactions
*uses: separates aromatic or aliphatic nonpolar compounds, base primarily on type of # of functional groups present
External Standards
standard solutions are chromatographed and peak area (or height or mass) are plotted vs. concentration on standard curve
Internal Standards
*amount of each component in sample is determined by comparing area (or height or mass) of component peak to that of the internal standard peak
*need to run set of standards at varying concentrations, with each solution containing constant amount of internal standard
Partition Chromatography
*liquid-liquid chromatography
*hydrophobic remains in organic phase
*hydrophilic remains in aqueous phase
*column with particle of solid support coated with liquid stationary phase
*pour mixture into column and mixture dissolves into stationary phase
*mobile phase will be poured in and one comp. will interact strongly with mobile while other interacts with stationary phase
*collected as fractions
Normal Partition Chromatography
*polar stationary phase vs. nonpolar mobile phase (nonpolar solutes elute first)
*common stationary phases: silica, organic moieties w/ cyano and amino funct. groups
*uses: separate and quantitate polar hydrophilic substances, ex. carbohydrates, water-soluble plant pigments
Reversed Partition Chromatography
*nonpolar stationary phase vs. polar mobile phase
*polar solutes first to elute
*common stationary phase: alkyl hydrocarbons
*uses: separate and quantitate lipophilic compounds ex. lipids and fat soluble pigments, separate and quantitate polyphenols
Ion-Exchange Chromatography
*solid-liquid chromatogrpahy
*based on attraction between opposing charges
*stationary phase contains fixed funcitonal groups with either positive or negative charge
*cation exchnager (has neg. charge)
*anion exchanger (has pos charge)
*elution: change mobile-phase pH or inc ionic strength so solute no longer binds
*uses: amino acids, sugars (HPAEC), alkaloids, proteins, drugs, fattya cids, acids of fruit
Size-Exclusion Chromatography
*solid-liquid chromatography
*molecules are separated based on size
*stationary phase is porous
*molecules too large to enter the pores travel with the mobile phase in the interstitial phase at elute first
*solutes of low molecular weight get slowed down by entering the pores
Affinity Chromatography
*solid-liquid chromatography
*isolates biomolecule from sample
*separation based on reversible interaction between a solute and an immobilized ligand (antibodies, enzyme inhbitiors, lectins)
*pour mobile phase into column w/stationary phase
*biomolecule will attach to ligand
*everything else will be washed away
*then another solvent will be poured in that competes with biomolecule to bind to ligand
*biomolecule will be separated/isolated
*uses: separate and purify enzymes or enzyme inhibitors, separate and purify glycoproteins
what is special about HPLC
*it can separate parts of a compound, tell you how much of each compound is in a mixture, and help identify what each compound is
what are the components of an HPLC system
*eluent reservoir
*proportioning valve
*pump
*injector
*column
*detector
*computer/data system
How do piston pumps function in one-way flow
*they are reciprocating, piston-type pumps
*pump will reciprocate and will open up the suction valve and allow eluent to flow into pump
*pump will come back which will cause the suction valve to close but the delivery valve to open up allowing the eluent to flow out
*eluent will flow into column
How is a gradient formed?
*gradient elution system is used to vary the mobile phase concentration during the run (mixes from two or more reservoirs)
*the multi-valve is what varies mobile phase conc.
*the valves are open or closed to a certian degree by an electrical current and low-pressure will mix them
*the flow of eluent draws out each eluent proportionally; they then mix into one system and enter the high pressure pump
How does the injector work (HPLC)
*places sample into flowing mobile phase, so it can enter the column
*valve injectors that separate sample introduction from high-pressure eluent system (after the pump)
Which detectors are common in HPLC
*fluorescence
*refractive index (RI)
*UV-Vis absorption
*electrochemical
HPLC Detector (RI)
*detects solutes by monitoring the refractive index of the column eluent relative to a reference cell containing air, mobile phase, or a transparent material with a specified RI
HPLC Detector (UV-VIS)
*measures the intensity of light that passes through a sample
*measures how much light a sample absorbs
What are applications of HPLC
*normal phase; analysis rice bran oil
*reversed phase; analysis of vitamin B6 compounds
*anion exchange; analysis of isoamylase-treated waxy corn starch
*size exclusion; analysis of tomato cell wall pectin from hot/cold break tomato prep
Why would someone use a fraction collector (HPLC)
it allows for the isolation of single peaks to obtain enough of chemical to be used as reference standard
How does GC work
*used to separate volatile compounds
*components: mobile/stationary phase, molecular sieve, injector, column (tight coil), detector (FID)
*stationary phase packed into inner wall of column
*molecular sieve separates unwanted hydrocarbons, water vapor, O from mobile phase
*sample is injected into the column
*sample is transported through column by flow of an inert-gas mobile phase (often Helium)
*less volatile comp interact with stationary phase and more volatile interact with mobile phase (helium, nitrogen)
*the column is in an oven to control temp; ex. a gradient of the oven temp
*volatiles are separated based on properties of: boiling point, molecular size, polarity
What characteristics of a sample are necessary for GC
thermally stable volatile substances
GC Applications
*carbohydrate analysis
*lipid analysis
*analysis of contaminants, residues, and constituents of concern
What should the gas supply system have
a gas line with traps to remove moisture and contaminants
injection port- hardware GC
*functions: sample introduction, sample vaporization, possibility for sample dilution and splitting
*contains soft septum with gas-tight seal that can be penetrated by syringe needle
*samples introduced either by manual syringe technique or automated sampling system
Why is there an oven GC
*oven controls the temperature of the column
*higher temp of oven causes sample to elute faster, but at cost of resolution
*capillary column can be heated directly with insulated heating wire based on low thermal mass technology (makes total heating/cooling cycle much shorter)
Capillary Columns GC
*hollow infused silica glass
*5-100m long; very thin walls
Preparation for GC- SPME
*Solid-Phase Microextraction
*stationary phase is bound onto fine fused silica filament or metal filament (fiber)
*fiber immersed in sample, or headspace above sample
*at end of extraction time, fiber is removed from sample and forced through septum of a GC
*adsorbed volatiles are thermally desrobed from fiber
Preparation for GC- Headspace
*direct injection of headspace vapors above a food product is a simple method to isolate volatile compounds
*direct; headspace of sample taken using gas-tight syringe
*dynamic: involves passing large volume of headspace vapors through an adsorbent trap
Why are some samples subjected to a Derivatization
*they are thermally unstable
*are too low in volatility (sugars, amino acids)
*yield poor chromatography separation due to polarity (phenols, acids)
What happens upon injection with either a syringe or a SPE device?
it is vaporized to be able to pass through the column for separation
Detector GC (FID)
*samples flow from coulmn and an igniter will ignite hydrogen and oxygen to make flame
*sample will flow into flame and be ionized
*there are electrodes that measure the current
*this signal is then detected
Ionization, separation (resolution), detection
*Ionization: occurs in ion source by electron impact, matrix-assisted-laser-desorption
*Separation: charged molecular ion and/or fragments are separated according to their m/z (occurs in mass analyzer), higher mass = deflected more, lower mass = deflected less
*separated, charged fragments are then monitored by detector
Types of ionization systems
*electron impact (EI) ionization process
*electrospray ionization (ESI-MS)
*matrix assisted laser desorption ionization (MALDI)
Electron impact (EI) ionization process
*compound is exposed to beam of electrons emitted from filament
*when a direct current is applied to the filament, it heats and emits electrons that move across ion chamber toward pos electrode
*e- will pass through region and extract e- from sample molecules
*this forms an ionized molecule
*ionized molecule contains high enough energy to fragments into even smaller fragments
*then moves through electric plates
Matrix assisted laser desorption ionization (MALDI)
*sample is dissolved in a matrix and ionized using UV laser (soft ionization)
*good for ionization of large biopolymers and other fragile molecules
Types of resolution systems
*time of flight
*magnetic sector
*quadrupole mass analyzers
Time of flight
*separates ions according to time required to reach detector
*ions are pulsed from source with same kinetic energy, so ions of different m/z ratios acquire different velocities, which determines time to reach detector
*useful for analysis of biopolymers and large molecules
*enables high resolution accurate mass applications
Quadrupole mass analyzers
*dervied from “fourfold” and “pole” to describe the four rods used to generate two equal, but out-of-phase electric potentials: one is alternating current (AC) freq, second is direct current (DC) freq
*creates an oscillating electric field between two of the opposite rods
*it filters ions that achieve stability from those that do not
Types of computer calculations for mass detection
*time of flight
*magnet current
*quadrupole voltage
What can we learn from the GC-MS of fatty acids
*fatty acids are not volatile in native form, so they must be derivatized
*fatty acids must be methylated on the carboxyl group = fatty acid methyl esters, this eliminated the polar nature of the group
How does one get carbohydrates linkage data from GC-MS
*begins with methylation of all free OH groups in the intact polysaccharide
*hydrolysis of polymer
*sample is then reduced and acetylated; the methyl ethers are stable to the acetylation conditions
*derivatives are analyzed by GC-MS
