Chromatography is widely used by forensic teams to analyse blood and urine samples for drugs, for paint analysis and testing for the presence of explosives. Most chromatography uses modern instrumentation and involves placing the sample to be analysed on a support (paper or silica) and transporting it along a mobile phase. The mobile phase can be a liquid (liquid chromatography) or a gas (gas chromatography). ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect blood or alcohol levels in a patient’s blood stream Law Enforcement – to compare a sample found at a crime scene to samples from suspects Environmental Agency – determine the level of pollutants in the water supply Manufacturing Plant – to purify a chemical needed to make a product
Most chromatography uses modern instrumentation and involves placing the sample to be analysed on a support (paper or silica) and transporting it along a mobile phase. The mobile phase can be a liquid (liquid chromatography) or a gas (gas chromatography).
separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase) _________________________________________ The principle is that the inks are water soluble and travel up the paper. Each marker is composed of more than one dye so each dye will separate as it moves up the paper with the water. The inks used in the dye are soluble in water and so will travel up the paper. The sample refers to what you want to test which in this case are colour markers. The standard refers to the reference material you will compare the sample to. In this case it is Goodalls food dye. Each food dye contains E numbers. The stationery phase is the support you apply your sample to – in this case chromatography paper. You spot each sample on the paper and allow it to dry. The mobile phase is water and the paper is placed in the water. Since each E number is a different chemical with its own characteristics and properties it will behave differently when you run it on the chromatography paper.
First you spot the food dye. Check on the bottle and confirm which E numbers are contained in the food dye. Make a note of it in your workbook. Then you spot your marker. Make sure you note down which spot is which because once they move along the paper you won’t be able to recognise them. The key point is that you don’t just visually compare the two. You must calculate a value for each spot and compare them so you can conclusively say whether the E numbers in the food dye are the same as the ones in the marker.
The Rf value is a measure of how far each spot has moved relative to the solvent front. Each dye will have its own Rf value so you can compare Rf values and confirm whether food dyes were used in making these markers.
Direct Inj - 8141, 625, 551.1, 604 P & T - 502.2
Isothermal - Keep oven at one temp thru run. Not very useful. Possibly useful for series of very similar compounds differing by boiling points such as alcohols ( MeOH, EtOH, n-PrOH, i-PrOH, BuOH, i-BuOH). BP 64.6 78.3 97.2 82.4 117.6 99.5 Gradient - temp profile: 40 deg hold for 10 min then 10deg/min to 240 deg and hold there for 20 min. Advantages: 1- resolution and 2- analysis time.
Packed - As suggested by the term, it is filled with a coated inert solid support such as fire brick, alumina, and graphite with a specific mesh size. The coatings are called phases and for best results are chemically bonded to the support. Chemical bonding provides for longer column life and less bleeding (major source of background noise) contributing to lower sensitivity. Column dimensions 1/8” - 1/4” ID x up to about 6’ using glass or stainless steel. Advantages - higher capacity (higher conc). Disadvantages: low resolution and low S/N. Capillary - Here the phase (film) is coated on the inside diameter of the capillary wall with film thickness range of 0.1 to 5μ where the ticker film provides for better resolution but also allows for more bleed. Typical dimensions .25mm - .53mm ID x up to 60m made of fused silica coated with polyamide. Advantages: high resolution and better S/N. Disadvantages: low capacity and cost.
Non - Polar : Equal distribution of electrons over the entire molecule. Look at the structure of fluorene. Polar: Non-equal distribution of electrons in a molecule causing one size of the molecule to be more positive or negative thus creating poles of charges. Look at 2,4,5-T (2,4,5-trichlorophenoxyacetic acid).
Here are some of the commonly used phases. They range in polarity from non-polar such as low polarity DMS to the higher polarity of mixtures with DPS with any combination available. There are specialty phases with very high polarity such as cyanopropylphenyl siloxane or trifluoropropyl methyl siloxane for fluorinated compounds. Some of these columns are used for separation confirmation such as the cyanopropylphenyl siloxane column for method 551.1.
MS - Mass Spectral - EI Electron impact - used for absolute confirmation. Molecules are ionized and their mass to charge ratio is plotted against its abundance. The resulting spectrum is unique to each molecule and can be looked up in a standard library and the % fit is noted. CI- chemical ionization is used for research and for structure elucidation. FID - Air/Hyd. Flame combusts the compound and the conductivity due to the ionization of the resulting carbon is determined and is presented as a signal. Very large range. NPD - Similar to FID except the combusted compound is passed over a heated bead of rubidium which provides for specificity in determining N and P where P is 500x more sensitive than N. NPD-P limited range, NPD-N broader range. FPD - Similar to FID but detector is light-tight where a PMT/filter assembly collects signal from emission for P at 393nm and S at 529nm. Haal/ELCD - furnace at about 900°C produces ionized acidic gases such as HCl or HF which is dissolved into a deionized solvent to produce conductivity proportional to the mass of the halogen in the org. compound.
TCD -Not normally used. Low sensitivity but good range. Change in resistance due to cooling effect of effluent over the resistance wire. ECD - limited range but very sensitive mainly to Cl org samples. The sample passes thru a Ni-63 foil where the Ni-63 gives off a constant amount of β particles and the Cl captures these electrons and the resulting loss produces a signal. PID - sample passes thru chamber it is constantly bombarded with high energy, 10.2 eV, where resulting ionization is produced and the ion current collected.
Note peaks 15, 16 17 & 18 on the DB-5 column and note the same peaks on the DB-1701 column. This shows the need for confirmatory columns (columns with different phases) so that separation of the compounds can be verified.
Note peaks 1 and 2 using the different columns. The separation is achievable because the compounds are different. Also note peaks 4 and 5 and 11 and 12. Even using different columns, positional isomers, o-, m- and p- cresol are difficult to separate.
Prepared By : Yahia Mohamed Reda Benha university – faculty of science Chemistry department
What is Chromatography?Derived from the Greek word Chroma meaning colour,chromatography provides a way to identify unknown compoundsand separate mixtures
What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. • Analyze Separate • Identify • PurifyMixture Components • Quantify
Applications of Chromatography Forensics Research Pharmaceutical industry
Types of Chromatography… Thin layerPaper HPLC Gas Column
•What Are The Branches of Chromatography According To DistributionBetween The Mobil & Stationary Phases ??
1- Adsorption Chromatography It Depends On The Ability Of Different Solutes To be Adsorbed On The Surface Of The Stationary Phase At Different Strengths It Can Be Found In GLC & LSC
2-Partition Chromatography It Depends On The Difference in Solubility Of Different Solutes In The Stationary Liquid Phase The Rate Of Separation Depends On The Equilibrium Of The Solute Between The Mobil Phase “Liquid “ & Stationary Phase ”liquid”
3 - Ion Exchange Chromatography In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.
4 - Molecular Exclusion Chromatography Also known as gel permeation or gel filtration, this type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.
5 - Affinity Chromatography This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.
•What Is The Main Types OfChromatography According ToThe Kind Of Mobil & Stationary Phases?
•What Are The Techniquesaccording To Chromatography Red Shapes?
1- Column Chromatography Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample
2- Planar chromatography Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate ( thin layer chromatography). Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retention factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.
•Compare Between Paper & Thin Layer Chromatography ?
1 – Paper Chromatography•separates dried liquid samples with a liquidsolvent (mobile phase) and a paper strip (stationaryphase)
Principles of Paper Chromatography Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.
Illustration of Chromatography Stationary Phase Separation Mobile Phase Mixture Components Components Affinity to Stationary Phase Affinity to Mobile Phase Blue ---------------- Insoluble in Mobile Phase Black Red Yellow
Paper Chromatography Experiment What Color is that Sharpie?
Overview of the ExperimentPurpose: To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in Sharpie Pens with paper chromatography.Time Required: Prep. time: 10 minutes Experiment time: 45 minutesCosts: Less than $10
Materials List 6 beakers or jars 6 covers or lids Distilled H2O Isopropanol Graduated cylinder 6 strips of filter paper Different colors of Sharpie pens Pencil Ruler Scissors Tape
Preparing the Isopropanol Solutions• Prepare 15 ml of the following isopropanol solutions inappropriately labeled beakers: - 0%, 5%, 10%, 20%, 50%, and 100%
Preparing the Chromatography Strips Cut 6 strips of filter paper Draw a line 1 cm above the bottom edge of the strip with the pencil Label each strip with its corresponding solution Place a spot from each pen on your starting line
Developing the Chromatograms Place the strips in the beakers Make sure the solution does not come above your start line Keep the beakers covered Let strips develop until the ascending solution front is about 2 cm from the top of the strip Remove the strips and let them dry
Observing the Chromatograms0% 20% 50% 70% 100% Concentration of Isopropanol
Black Dye1. Dyes separated – purple and black2. Not soluble in low concentrations of isopropanol3. Partially soluble in concentrations of isopropanol >20% 0% 20% 50% 70% 100% Concentration of Isopropanol
Blue Dye1. Dye separated – blue2. Not very soluble in low concentrations of isopropanol3. Completely soluble in high concentrations of isopropanol 0% 20% 50% 70% 100% Concentration of Isopropanol
Green Dye1. Dye separated – blue and yellow2. Blue – Soluble in concentrations of isopropanol >20%3. Yellow – Soluble in concentrations of isopropanol >0% 0% 20% 50% 70% 100% Concentration of Isopropanol
Red Dye1. Dyes separated – red and yellow2. Yellow –soluble in low concentrations of isopropanol and less soluble in high concentrations of isopropanol3. Red – slightly soluble in low concentrations of isopropanol, and more soluble in concentrations of isopropanol >20% 0% 20% 50% 70% 100% Concentration of Isopropanol
Alternative Experiments Test different samples: Other markers, pens, highlighters Flower pigments Food Colors Test different solvents: Other alcohols: methanol, ethanol, propanol, butanol Test different papers: Coffee filters Paper towels Cardstock Typing paper
2 - Thin Layer Chromatography Sample – marker Standard – food dyes Stationary phase – chromatography paper Mobile phase - water
Structures of E numbers….. E122 pink E110 yellow E124 red E133 blue
So what will happen? Each dye will travel up the paper at different speeds The speed depends on the solubility of the dye in water and its interaction with the paper The dyes are all different molecules with different characteristics
Analysis Calculation of resultsYou must now calculate an Rf value for each spot.Rf = Distance from the start to the middle of a spot Distance from start to finish point of the water
Conclusions – writing up One of the key elements of all scientific experiments is to write up your results At the end of this experiment we would like each person to conclude from the Rf values as to which E numbers are contained in the markers
Gas Chromatography•Separates vaporized samples with a carrier gas (mobilephase) and a column composed of a liquid or of solid beads(stationary phase)
THE CHROMATOGRAPHIC PROCESS - PARTITIONING (gas or liquid) MOBILE PHASE SampleSample out in STATIONARY PHASE (solid or heavy liquid coated onto a solid or support system)
Properties of Selected Gas Chromatography Detectors Approximate Limit Approximate Type Comments of Detection (gs-1) Linear RangeThermal conductivity Universal detector -measures 10-5-10-6 103-104(TCD) changes in heat conduction Universal detector -measures ionFlame ionization (FID) 10-12 106-107 currents from pyrolysis Selective detector for compoundsElectron capture (EC or 10 -14 10 -10 2 -3 containing atoms with highECD) electron affinitiesFlame photometric Selective detector for compounds 10-13 102(FPD) containing S,P Selective for N,P containingNitrogen-phosphorus 10-8-10-14 105-107 compounds Universal (some selectivity due toPhotoionisation (PID) 10-8-10-12 105 identity of gas in lamp) Specific detector for compoundsHall Detector 10-11 105 which contain halogen, S, or N variable,Mass spectrometer (MS) 10-12 depends on MS UniversalFourier-transform 10-10 102 Polar moleculesinfrared (FTIR)
1- Mass Spectrometry Molecular weight can be obtained from a very small sample. It does not involve the absorption or emission of light. A beam of high-energy electrons breaks the molecule apart. The masses of the fragments and their relative abundance reveal information about the structure of the molecule.
Electron Impact Ionization A high-energy electron can dislodge an electron from a bond, creating a radical cation (a positive ion with an unpaired e-). H H H C C H H H H H H H e- + H C C H H C C+ H H H H H H H H C+ C H H H
Separation of Ions Only the cations are deflected by the magnetic field. Amount of deflection depends on m/z. The detector signal is proportional to the number of ions hitting it. By varying the magnetic field, ions of all masses are collected and counted.
The Mass SpectrumMasses are graphed or tabulated according to their relative abundance.
The GC-MSA mixture of compounds is separated by gas chromatography, then identified by mass spectrometry.
High Resolution MS Masses measured to 1 part in 20,000. A molecule with mass of 44 could be C3H8, C2H4O, CO2, or CN2H4. If a more exact mass is 44.029, pick the correct structure from the table: C3H8 C2H4O CO2 CN2H4 44.06260 44.02620 43.98983 44.03740
Molecules with Heteroatoms Isotopes: present in their usual abundance. Hydrocarbons contain 1.1% C-13, so there will be a small M+1 peak. If Br is present, M+2 is equal to M+. If Cl is present, M+2 is one-third of M+. If iodine is present, peak at 127, large gap. If N is present, M+ will be an odd number. If S is present, M+2 will be 4% of M+.
Mass Spectra of AlkanesMore stable carbocations will be more abundant.
Mass Spectra of AlkenesResonance-stabilized cations favored.
Mass Spectra of Alcohols Alcohols usually lose a water molecule. M+ may not be visible. =>
2- Flame Ionisation Detector (FID)Destruction of combustible sample in flame produces measurable current •Elute is burnt in a mixture of H2 and air •Electrical conductivity of a gas is directly proportional to the concentration of charged particles within the gas •It responds to most hydrocarbons Compounds that give no response include: air, water, inert gases, CO, CO2, CS2, NO, SO2 and H2S • Very sensitive detector with a wide linear range • Excellent detector for quantitative trace analysis
Thermal Conductivity cell (TCD) (most common in the past)•Thermal conductivity measures the ability of a substance to transport heat from a hot region to a cold region•Differences in the thermal conductivity of gasses are based on the mobility•The smaller the molecule the higher the mobility and thermal conductivity•Helium is the carrier gas commonly used•Simple and universal•Respond to all analytes•104 linear response range•Not sensitive to detect minute quantities•Mostly used with tubular columns with >0.053 mm in diameter or packed columns•Sensitivity increases with: Increasing filament current Decreasing flow rate Lower detector block temperature
Other detectors Nitrogen-phosphorus detectorModified Flame Ionization detector. Especially sensitive to N and P, 10 4-106 greater response than to carbon. Important for the analysis of drugs,herbicides and pesticides Sulfur chemiluminescence detectorSulfur is oxidized to SO during ionization and converted to blue lightemitting SO2 by reaction with ozone. Intensity of emission is proportional tothe mass of S eluted. The response to Sulfur is 107 greater than to carbonA nitrogen chemiluminescence detector works in analogousmanner, combustion of eluent converts to NO which reacts with O 3 to forma chemiluminescent product. Response to N is 107 greater than to carbon Atomic emission detectorDetector can be set to observe almost any element in each analyte asit emerges through the column.
Desirable characteristics of detectors•High sensitivity•Negligible baseline noise•Large linear response range (analyte concentration range overwhich detector response is proportional to concentration)•Insensitive to temperature changes and solvent composition•Universality or predictable specificity•Low dead volume•No sample destruction•Stability over time•Reliable•Inexpensive to produce and continuous operation•Capable of providing information on solute identity
Characteristics of Selected Liquid Chromatography Detectors Approximate Limit Approximate Type Comments of Detection Linear RangeUltraviolet and visible Specific for light-absorbing 10-11 g 104absorption compounds Universal detector -measuresDifferential Refractive 10 -10 -9 -10 g 103 changes in refractive index.Index Cannot be used with gradientsElectrochemical Specific detector. Compound 10-10-10-11 g 105Amperometric must be electroactiveElectrochemical 10-8 g/mL 105 Specific detector, but for all ionsConductometric Specific detector. CompoundFluorescence 10-14 g 105 must be fluorescent Universal detector. Also can beMass Spectrometry 10 -10-7 -9 105 used to identify analytes with great certainty Used to determine MW’s ofSolution Light polymers as they elute. 10-6 g/mL 105Scattering Concentration usually far above limits of detectionEvaporative Light Universal except for volatile 10-9 g 106Scattering analytes. Not a linear response
HPLC Detectors respond to:Solute property not Bulk property that Directexhibited by MP changes with solute eluted solute detection UV/VIS (0.1-1 ng) •Fixed Refractive Mass •Variable (VWD) Index Spectrometry •Photodiode array (DAD) (100-1000 ng) (1-1000 pg) FTIR (1mg) Conductivity ELSD (500-1000 ng) (100-1000 pg) Electrochemical (10-1000 pg) •Amperometric •Coulobmetric Fluorescence (1-10 pg) Requires fluorophore
UV-vis Light Absorption Detectors•Most common HPLC detector•Many solutes absorb ultraviolet (UV) light•Most employ the most intense 254 nm(emission of a mercury lamp) Light•Modern HPLC instruments have the source Detectorcapability to choose the appropriate wavelength for a given analyte•Use of photodiode arrays which can recordthe entire UV region at once in a fraction of asecond (spectrum of each solute as it iseluted)•Full scale absorbance range 0.0005-3 absorbance units•Linear range 5 orders of magnitude of solute concentration•Good for gradient elution
Refractive Index (RI) Detector• Light passes through the celland is directed to the photocellby the deflection plate. Whensolute with a different RI entersthe cell, the beam is deflectedand the photocell outputchanges.• Respond almost to every solute but sensitivity is lowered by a factor of 1000 compared to a UV detector• Sensitive to changes in pressure and temperature• Unsuitable for gradient elution due to problem of matching sample and reference while the composition is changing• Its primary appeal is due to a nearly universal response to all solute including those with little UV absorption
Electrochemical Detector •Respond to analytes that can be oxidized or reduced •Potential is maintained at a selected value with respect to Ag/AgCl reference electrode and current is measured between the working and counter electrodes.• Current is proportional to the concentration ofsolute over 6 orders of magnitude• Oxygen free aqueous or polar solvent containingdissolved electrolyte are required.•Very sensitive to fluctuations of temperature and the flow rate
Electrochemical Detector Pulsed electrochemical detection of alcohols in an ion exchange column with 0.05M HClO4Peaks: 1, glycerol; 2,ethylene glycol; 3, propylene glycol 4, methanol; 5,ethanol; 6, 2-propanol; 7, 1-propanol; 8, 2-butanol; 9, 2-methyl-1-butane; 10,1-butanol; 11, 3-methyl-1-butanol; 12, 1-pentanol; 13, cyclohexanol; 14,diethyleneglycol.
Evaporative Light-Scattering Detector (ELSD)•Responds to any solute which is less volatile than the MP•Response is related to mass of material (large peak = more material in contrast to UV detectors peak intensity is related to how well the solute absorbs UV)•Response is non linear, so polynomials are used toconstruct calibration curve•Compatible with gradient elution. No peaks associated with the solvent front, so less interference fromearly eluting peaks
ELSD vs UV• Obtain a more accurate representation ofsample mass than UV• See what may be missing from the UVchromatogram• Detects compounds without chromophores
Conclusion: Is ELSD the Ideal Detector?Advantages Disadvantages•Universal: Detect any • Destructivecompound less volatile than theMP • Compatible with isocratic and gradient elution but NOT•Very Sensitive: Typical buffers/salts.detection limits 0.1-1 ng.•The response is proportionalto the analyte concentration,and not affected by solventproperties•Reliable and easy to use
Important Definitions K partition coefficient :- defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase. retention time (tR ) : The time between sample injection and an analyte peak reaching a detector at the end of the column
Retention factor, k : is often used to describe the migration rate of an analyte on a column. You may also find it called the capacity factor. The retention factor for analyte A is defined as; kA = (t R - tM) / tM Selectivity factor, a : which describes the separation of two species (A and B) on the column; a = k B / k AWhen calculating the selectivity factor, species A elutes faster than species B. The selectivity factor is always greater than one.
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