FOOD ANALYSIS: Principles of Chromatography
B. Pam Ismail [email protected] FScN 146 612 625 0147
FOOD ANALYSIS: Principles of Chromatography
The following intermolecular interactions are responsible for adsorption chromatography A. Electrostatic forces B. Hydrogen bonds C. Hydrophobic interactions D. A and B E. B and C F. A and C G. All of the above H. None of the above
1 FOOD ANALYSIS: Principles of Chromatography
In reverse-phase chromatography: A. Polar compounds elute first B. Non-polar compounds elute first C. The mobile phase is polar liquid D. A and B E. B and C F. A and C G. All of the above H. None of the above
FOOD ANALYSIS: Principles of Chromatography
Historical Perspective
David Day ---American Geologist o Crude petroleum---fuller earth
Mikhail Tsvet ---- Russian Botanist o Leaf pigments --- column packed with chalk
o Named the phenomenon chromatography
o Credited for its discovery
1940’s: partition chromatography and paper chromatography
1960’s: Gas chromatography evolved because of petroleum industry followed by liquid chromatography and supercritical fluid chromatography (SFC)
2 FOOD ANALYSIS: Principles of Chromatography
Uses?
FOOD ANALYSIS: Principles of Chromatography
Extraction: Transfer of a solute from one liquid phase to another
Actual means of analysis
Preliminary sample clean up
Concentration of the component of interest
3 FOOD ANALYSIS: Principles of Chromatography
Extraction: At equilibrium: - Batch extraction
– Partition coefficient (K) http://carlwillis.files.wor dpress.com/2009/09/pur ex_4.jpg is constant
– Continuous extraction K = Conc. of solute in phase 1 Conc. of solute in phase 2
– Countercurrent extraction Solute A is more soluble in (basis for partition phase 1 chromatography) Solute B is more soluble in http://195.134.76.37/applets/AppletCraig/Appl_Craig2. html phase 2
FOOD ANALYSIS: Principles of Chromatography
Extraction: – Countercurrent extraction (basis for partition chromatography)
http://195.134.76.37/applets/appletcraig/Images/Ani mation2.gif
4 FOOD ANALYSIS: Principles of Chromatography
Chromatography Terminology
Solute
Stationary phase (liquid or solid)
Mobile phase (eluting solvent/carrier gas/supercritical fluid)
Series of equilibrations
Eluent (noun), elute (verb)
Chromatographic peak
FOOD ANALYSIS: Principles of Chromatography
Chromatography
A separation technique used in food analysis
Based on the partition or distribution of solute between a stationary phase and a mobile phase
K = Conc. of solute in stationary phase Conc. of solute in mobile phase
5 FOOD ANALYSIS: Principles of Chromatography
Chromatography is a separation technique used in food analysis Chromatography
Gas Supercritical fluid Liquid Chromatography Chromatography Chromatography
Paper Thin Layer Gas/Liquid Chromatography Chromatography
(Liquid/Liquid) (Liquid/Liquid)
Column Chromatography
(Liquid/Liquid) (Liquid/Solid)
FOOD ANALYSIS: Principles of Chromatography
Chromatography is a separation technique used in food analysis
Chromatography
Column Liquid Chromatography
Partition Adsorption Size-Exclusion (Liquid/Liquid) (Liquid/Solid)
Reversed Phase Normal Phase
Hydrophobic Ion-Exchange Affinity Interaction
6 FOOD ANALYSIS: Principles of Chromatography
Characteristics of Different Chromatographic Methods
Method Mobile/Stationary Phase Retention Varies with
Gas-liquid chromatography Gas/liquid Molecular size/polarity Gas-solid chromatography Gas/solid Molecular size/polarity
Supercritical fluid chromato- Supercritical fluid/solid Molecular size/polarity graphy
Reversed-phase chromato- Polar liquid/nonpolar liquid Molecular size/polarity graphy or solid
Normal-phase chromato- Less polar liquid/more polar Molecular size/polarity graphy liquid or solid
Ion-exchange chromato- Polar liquid/ionic solid Molecular charge graphy
Size-exclusion chromatography Liquid/solid Molecular size
Hydrophobic-interaction Polar liquid/nonpolar liquid Molecular size/polarity chromatography or solid Table 12-1
Affinity chromatography Water/binding sites Specific structure
FOOD ANALYSIS: Principles of Chromatography
Chromatography is a separation technique used in food analysis Chromatography
Gas Chromatography
Gas/Liquid
Separation of thermally stable volatiles
Controlled temperature gradient
Volatiles are separated based on molecular size, polarity and boiling point
7 FOOD ANALYSIS: Principles of Chromatography
Recap Questions Define chromatography?
What is the difference between stationary phase and mobile phase, what is the function of each?
List three types of chromatography
What is the form of mobile phase in GC and what is the form of the stationary phase?
Same question for partition and adsorption chromatograph
What is partition coefficient?
What are some applications?
FOOD ANALYSIS: Principles of Chromatography
Liquid Chromatography
Paper chromatography
Paper (cellulose) serves as a support for the liquid stationary phase
The stationary phase is usually water
Mobile phase is a solvent immiscible in water
8 FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography
Paper chromatography If sample mixture is colorless (amino acid mixture)
Visualized by autoradiography
FOOD ANALYSIS: Principles of Chromatography
Liquid Chromatography
Paper chromatography
2-D techniques for complex mixtures
9 FOOD ANALYSIS: Principles of Chromatography
Liquid Chromatography
Paper chromatography Components of a mixture is characterized by their relative mobility
o Rf = Distance moved by component Distance moved by solvent
o Rf values are supposedly constant
Can be affected by stationary phase thickness, humidity, developing distance, and temperature
FOOD ANALYSIS: Principles of Chromatography
Liquid Chromatography
Paper chromatography
Can also be reversed-phase
o Support impregnated with a non-polar solvent
o Developed with polar solvents or water
The stationary phase can be ion exchange paper
o OH groups on the cellulose derivatized with acid or base moieties
10 FOOD ANALYSIS: Principles of Chromatography
In reversed-phase paper chromatography
A. The most polar travels the most
B. The least polar travels the most
C. Rf value of the polar compound will be higher than the less polar one
D. A and C
E. B and C
FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography
Thin layer chromatography (TLC)
Similar principle as paper chromatography
Developed to replace paper chromatography
o Better resolution
Smaller more uniform particle size
o Faster,
o Better reproducibility
11 FOOD ANALYSIS: Principles of Chromatography
Liquid Chromatography
Thin layer chromatography (TLC)
Thin layer (250 μm thick) of sorbent or stationary phase bound to inert support (glass, plastic, or aluminum foil)
Sorbent used: silica, alumina, or cellulose
Can be applied as adsorptive, normal phase or reversed- phase partition, ion exchange chromatography
FOOD ANALYSIS: Principles of Chromatography Liquid Chromatography Thin layer chromatography (TLC)
Different techniques can be applied to detect or visualize the components
o Colorimetrically (ninhydrin, sulfuric acid, iodine vapor, dichloroflurescein)
o Measure of absorbed or emitted radiation (fluorescence)
o Measure of radioactivity of radioactive labeled compounds
Quantitative evaluation can be carried out by means of
o Densitometer
o Scraping off the zone, eluting the compound and then analyze the resulting solution
12 FOOD ANALYSIS: Principles of Chromatography
Column liquid chromatography
General procedure
Stationary vs. mobile phase Column dimensions Packed dry or wet (making a slurry) Isocratic vs. gradient mobile phase
FOOD ANALYSIS: Principles of Chromatography
Chromatography is a separation technique used in food analysis Chromatography
Supercritical fluid Chromatography
Mobile phase commonly used liquid CO2, can sometimes be combined with methanol
High diffusivity and low viscosity – Stationary phase similar to that of better resolution and less time HPLC
Used for separation of non-polar Equipment similar to HPLC, and molecules, and non-volatile detectors used are similar to those compounds that are thermally used for GC labile (instead of GC)
13 Physicochemical principle of chromatography separation
FOOD ANALYSIS: Principles of Chromatography
Adsorption (solid/liquid) chromatography:
Stationary phase: (adsorbent) finely divided solid; permit differential interaction with components to be separated
Intermolecular forces: Van der Waals forces Electrostatic forces Hydrogen bonding Hydrophobic interactions
Adsorption coefficient is not a constant
14 FOOD ANALYSIS: Principles of Chromatography
Adsorption (solid/liquid) chromatography:
Stationary phase:
o Silica: (slightly acidic) (Silica bounded to C8 or C18)
o Alumina: (slightly basic)
o Charcoal: (non-polar)
The elution order of solutes depend on their relative polarities
Solute and solvent (mobile phase) compete for active sites on the stationary phase
Changing the strength of the mobile phase will alter solute – stationary phase interactions
FOOD ANALYSIS: Principles of Chromatography
Recap Questions
What is supercritical fluid chromatography?
What is reverse phase chromatography?
In reverse phase paper chromatography what type of compound travels the most with the mobile phase?
What does TLC stand for?
How does TLC differ from paper chromatography?
What is the difference between isocratic and gradient elution?
What does increasing the strength of the mobile phase mean?
15 FOOD ANALYSIS: Principles of Chromatography
Partition (liquid/liquid) chromatography:
Solutes partition between two liquid phases
Elution of solutes depends on changing the nature of the phases o Combining solvents o Changing the pH
When the most polar phase is held stationary - - normal phase chromatography (polar hydrophilic substances)
When the least polar phase is held stationary – reversed-phase chromatography (non-polar hydrophobic substances)
FOOD ANALYSIS: Principles of Chromatography
Partition (liquid/liquid) Chromatography:
Coated supports: Liquid coating on a solid matrix
o Silica gel (hydrated silica)
o Cellulose
o glass beads
Bonded supports: liquid stationary phase covalently bonded to a support via a chemical reaction
Bonded support is very common in reversed-phase HPLC (Silica bounded to C8 or C18)
16 FOOD ANALYSIS: Principles of Chromatography Hydrophobic interaction chromatography (solid/liquid)
Interaction of solute with stationary phase is hydrophobic in nature (weak)
Biomolecules adsorb to a weak hydrophobic surface at high salt conc.
Stationary phase:
hydrophilic support bonded to hydrophobic ligands (e.g., butyl- Sepharose®)
Mobile phase:
Start with buffered 1 M ammonium sulfate, then decrease salt to 0 M
FOOD ANALYSIS: Principles of Chromatography Hydrophobic interaction chromatography (solid/liquid)
https://technologyinscience.blogspot.com Separation/purification of proteins
17 FOOD ANALYSIS: Principles of Chromatography
In hydrophobic interaction chromatography, interactions between solute and stationary phase are mostly:
A. Hydrophobic B. Electrostatic C. Hydrogen bonding D. Covalent bonding
FOOD ANALYSIS: Principles of Chromatography Ion-exchange chromatography (solid/liquid)
Interaction of solute with stationary phase is electrostatic in nature
Stationary phase: Cross-linked polyelectrolytes
Highly cross-linked (small pore size, more capacity and selectivity; usually used for small molecules)
Lightly cross linked (large pore size, used to separate large molecules – proteins and nucleic acids)
18 FOOD ANALYSIS: Principles of Chromatography Ion-exchange chromatography (solid/liquid)
Polystyrene (polymeric resins)
FOOD ANALYSIS: Principles of Chromatography Ion-exchange chromatography (solid/liquid)
Polysaccharide- based
19 FOOD ANALYSIS: Principles of Chromatography
Ion-exchange chromatography (solid/liquid)
Selectivity of an exchanger depends on
o Characteristics of the ion: ionic valence, radius, and concentration
o Nature of the exchanger Strongly acidic (negatively charged at any pH above 2) Strongly basic (positively charged at any pH below 10) Weak cation-exchanger (exchange capacity varies a lot between pH 4-10) Weak anion-exchanger (de-protonated in moderately basic conditions)
o The composition and pH of the mobile phase
FOOD ANALYSIS: Principles of Chromatography
In ion-exchange chromatography, interactions between solute and stationary phase are mostly
A- Hydrophobic B- Electrostatic C- Hydrogen bonding D- Covalent bonding
20 FOOD ANALYSIS: Principles of Chromatography
Affinity chromatography (solid/liquid)
Separation based on reversible interaction between a solute and an immobilized ligand: o Antibodies (specific) o Enzyme inhibitors (specific) o Lectins (general)
Stationary phase o High surface area material o Porous o Does not absorb anything o Agarose, cellulose, dextrans, polyacrylamide, glass
FOOD ANALYSIS: Principles of Chromatography
Affinity chromatography (solid/liquid)
Elution methods
o Nonspecific: changing pH, ionic strength or temperature
o Bio-specific: used for general ligand binding – add excess ligand to the eluent
21 FOOD ANALYSIS: Principles of Chromatography
In affinity chromatography, interactions between solute and stationary phase are mostly
A- Hydrophobic B- Electrostatic C- Hydrogen bonding D- Van der Waals
FOOD ANALYSIS: Principles of Chromatography
Size-exclusion chromatography (solid/liquid)
Molecules are separated based on size
Large molecules travel with the mobile phase in the interstitial phase -- void
volume (Vo)
Solute of low molecular weight get slowed down
22 FOOD ANALYSIS: Principles of Chromatography
Size-exclusion chromatography (solid/liquid)
Molecules are separated based on size
Large molecules travel with the mobile phase in the interstitial phase -- void
volume (Vo)
Solute of low molecular weight get slowed down
http://slohs.slcusd.org/pages/teachers/rhamley/ Biology/Basis%20of%20Life/gelfiltr.gif
FOOD ANALYSIS: Principles of Chromatography
Size-exclusion chromatography (solid/liquid)
Solute with very low molecular weight have access to all the available pore
volume and get eluted in Vt ; Vt = Vo + Vi
Elution volume Ve is the volume required to elute a certain solute
23 FOOD ANALYSIS: Principles of Chromatography
Size-exclusion chromatography (solid/liquid) Available partition coefficient -- define solute behavior
Kav= (Ve-Vo)/(Vt-Vo)
FOOD ANALYSIS: Principles of Chromatography
In size-exclusion chromatography, interactions between solute and stationary phase are mostly
A- Hydrophobic B- Electrostatic C- Hydrogen bonding D- Covalent bonding
24 Separation Mode Stationary Phase Mobile Phase Increasing Mobile Phase Strength Compounds eluting Type of Interactions first/ eluting last between Solutes and Stationary Phase Normal Phase (can Polar sorbent (e.g. Non-polar Decreasing concentration of organic solvent (i.e. increasing Least polar/most H-bonding be in the form of silca, alumina, water) solvent (e.g. polarity, making the mobile phase more like the stationary phase) polar mostly adsorption or aqueous partition methanol, chromatography) acetonitrile) Reversed-Phase Non-polar sorbent Polar solvent Increasing concentration of organic solvent (i.e. decreasing Most polar/least H-bonding; Van (can be in the form (e.g. bonded silica, C8 (e.g. water) polarity, making the mobile phase more like the stationary phase) polar der Walls, of adsorption or or C18) hydrophobic partition interactions chromatography) Hydrophobic Non-Polar sorbent Salt solution/ Decreasing concentration of salt (result in reduced interaction of Least Hydrophobic Interaction (e.g. butyl-sepharose buffer (e.g. 1 M solute with the sorbent) hydrophobic interactions and phenyl-sepharose) ammonium surface/most sulfate; hydrophobic phosphate surface buffer) Cation Exchange Negatively charged Buffers of Increasing the pH (in case of weak cation exchanger) or Solutes with the Electrostatic functional groups (e.g. specific pH and increasing salt concentration (e.g. increasing the pH will result in least positive - - RSO3 , RCO2 ) ionic strength deprotonation, i.e. loss of positive charge of the solute so it no charge longer interacts with the functional group of the stationary density/most phase, and increasing the salt concentration will provide counter positive charge ions that will displaces the solute on the functional groups of the density stationary phase) Positively charged Buffers of Decreasing the pH (in case of weak anion exchanger) or Solutes with the Electrostatic Anion Exchange functional groups (e.g. specific pH and increasing salt concentration (e.g. decreasing the pH will result least negative RNR'3+, R-NHR'2+) ionic strength in protonation, i.e. loss of negative charge of the solute so it no charge longer interacts with the functional group of the stationary density/most phase, and increasing the salt concentration will provide counter negative charge ions that will displaces the solute on the functional groups of the density stationary phase) Highly specific ligand Buffer Changing the pH, ionic strength, or adding a ligand similar to the Solutes with least H-bonding; Van Affinity bound to inert surface bound ligand of the stationary phase affinity to bound der Walls, (e.g. antibodies, ligands/most hydrophobic enzyme inhibitors, affinity to bound interactions, lectins) electrostatic Size Exclusion Porous inert material Mostly water or Not applicable Largest in None (e.g. Sephadex, a buffer size/smallest in crosslinked dextran) Table 12-3 size
FOOD ANALYSIS: Principles of Chromatography
Recap Questions
Name examples of adsorption chromatography?
What molecular interactions between solute and stationary phase are involved in size exclusion? Ion exchange? HIC? Affinity chromatography?
What is the difference between cation and anion exchange chromatography?
How do you increase the strength of the mobile phase in reverse phase chromatography, HIC, ion exchange, and affinity chromatography?
What is the order of elution in HIC? Ion exchange? Size exclusion?
25 FOOD ANALYSIS: Principles of Chromatography
Analysis of Chromatographic Peaks
Developing a separation
o What is known about the sample
o How many components need to be resolved
o What degree of resolution is needed
o Is qualitative or quantitative information is needed
FOOD ANALYSIS: Principles of Chromatography
Analysis of Chromatographic Peaks
Developing a separation
o Choice of chromatographic separation mode
o Choice of stationary and mobile phase
o Elution mode Isocratic Gradient Flow rate Temperature
26 FOOD ANALYSIS: Principles of Chromatography
Choosing a Chromatographic Separation Mode
(Fig. 12-10)
FOOD ANALYSIS: Principles of Chromatography
Analysis of Chromatographic Peaks
Developing a separation
o Choice of chromatographic separation mode
o Choice of stationary and mobile phase
o Elution mode Isocratic Gradient Flow rate Temperature
27 FOOD ANALYSIS: Principles of Chromatography
Chromatographic Resolution
Retention volume (VR)
Retention time (tR)
Compare VR or tR to a known compound
Two compounds can co-elute – further separation using another technique
Adjusted t’R
Identification of peaks - - Separation factor (affected by temperature, stationary and mobile phase)
FOOD ANALYSIS: Principles of Chromatography
Separation and resolution
Resolution of two peaks from each other
Rs = 2 Δt / w2 + w1
Resolution is a function of efficiency, selectivity and capacity
1 1 R = N s 4 1 a b c
28 FOOD ANALYSIS: Principles of Chromatography
Recap Questions
What is peak resolution?
What is a solvent front peak?
What is the difference between retention volume and retention time?
What is the relationship of peak width to resolution?
What is the first step in developing a separation between solutes of interest in your sample?
FOOD ANALYSIS: Principles of Chromatography
Separation and resolution Varies with type of solute Efficiency
Number of theoretical plates Proportional to length of the column
2 2 2 tR tR tR N= 16 5.5 1/ 2
29 FOOD ANALYSIS: Principles of Chromatography
Separation and resolution
Efficiency
o HETP
HETP = L/N
Van Deemeter equation
Flow rate effect Temperature effect
FOOD ANALYSIS: Principles of Chromatography
This image cannot currently be displayed. Separation and resolution
Selectivity
-Nature of the stationary phase -Nature of the mobile phase
Good selectivity can be more important than high efficiency; resolution is directly related to selectivity but is quadratically related to efficiency 1 1 Rs= N 4 1
30 FOOD ANALYSIS: Principles of Chromatography Separation and resolution A A
Capacity
o Column losses capacity resulting in less time on Loss of column functional groups Capacity factor B A K’ = tR –t0
t0
FOOD ANALYSIS: Principles of Chromatography
Qualitative Analysis
Peak retention time and standards Spiking of a sample Photo diode array detector Detection using multiple detectors Peak ratio at two different wave lengths Separation using another Chromatography mode Mass Spectrometry
31 FOOD ANALYSIS: Principles of Chromatography
Quantitative analysis This image cannot currently be displayed.
Peak height vs. peak area
Area determination
Strip Chart Recorder Data Analysis Software
FOOD ANALYSIS: Principles of Chromatography
This image cannot currently be displayed. Quantitative analysis
External vs. internal standard
32 FOOD ANALYSIS: Chromatographic Techniques
Laboratory- HPLC/GC
Groups A, B, and D will be doing the HPLC lab, Groups C, E, and F will be doing the GC lab (Please come on time) HPLC Analysis: Quantification of Caffeine in several beverages, using external standard calibration curve
GC Analysis: Qualitative and semi-quantitative profiling of fatty acid content in several oils, using an external standard mix
Read Chapters 12, 13 and 14 and Lab addendums (found on Canvas) Complete pre-lab quiz (HPLC or GC, found on Canvas) and hand to the TA Monday or Wednesday morning Bring calculators and laptops, excel data files will be e-mailed to you
FOOD ANALYSIS: Chromatographic Techniques
Laboratory- HPLC/GC
HPLC intro you-tube http://www.youtube.com/watch?v=I-CdTU5X4HA
Determination of caffiene content in different beverages using reverse phase chromatography
GC intro you-tube https://www.youtube.com/watch?v=iX25exzwKhI
Saponification of oil and derivatization of fatty acids into FAME (thermally stable and volatile). Running on GC with FID
33