Module E: Chemicals in liquids and gases Flashcards
What is chemical sensing? What are they composed of?
Transforming chemical information, ranging from concentration to total composition analysis, into an analytically useful signal
Recognition element, transduction element, signal processor capable of continuously and reversibly reporting a chemical concentration.
What is chromatography?
Process used to separate compounds into their individual components (separate compounds from one another).
Solution is passed through column that retains some compounds longer than others.
- Involves flowing an analyte (liquid or gaseous) into a CAPILLARY OR COLUMN
- The column contains the stationary phase (solid or adsorbed liquid)
Based on the idea that different molecules interact differently with the analyte, affecting their RETENTION TIME.
CHROMATOGRAPHY: What is the stationary phase?
Solid or adsorbed liquid phase, fixed inside the column. It is a matrix packed inside the column or a coating on the walls of the capillary.
Adsorbed vs Absorbed?
Adsorbed: gathering on surface
Absorbed: soaked up entirely by component
CHROMATOGRAPHY: What is a mobile phase?
Liquid or gas moving through the column and containing the analyte
CHROMATOGRAPHY: What is elution, eluent, eluate?
Elution: process of passing the mobile phase through the column.
Eluent: (analyte), fluid entering the column
Eluate: fluid exiting the column.
What does liquid/solid chromatography mean? gas/solid? gas/liquid? liquid/liquid?
Liquid phase: mobile phase is a liquid
Gas phase: mobile phase is a gas
Liquid/solid: mobile phase is liquid, stationary phase is solid
Liquid/Liquid: mobile phase is liquid, s phase is liquid
What is adsorption chromatography? BRIEF
Solute is ADsorbed on the surface of the stationary phase. Separation is based on adsorption ability (based on polarity).
What is partition CHROMATOGRAPHY? BRIEF
Separation occurs based on partitioning of a component between stationary and mobile phases.
What is Ion-Exchange C? BRIEF
Anion and cation exchange:
Anion-exchange - only compounds containing anions can be attracted to it. mobile anions are covalently attached to this stationary phase.
Affinity to the ions in the resin will affect retention time.
What is size-exclusion c? BRIEF
Size of molecules affect passing through stationary phase. Some molecules are excluded from the resin.
What is Affinity C? BRIEF
Only one kind of molecule in a complex mixture becomes covalently bound to the stationary phase
- all the other molecules pas directly through.
What affects chromatography performance?
- Nature of the analyte
- nature and amount of the stationary phase / mobile phase
- temperature of the column (solubility)
- speed of the mobile phase
- column length (bigger = increase in retention time)
- polarity of the two phases
- interactions between the analyte and the stationary phase
PERFORMANCE PARAMETERS - C What is the capacity factor (k’)?
Indicates the speed of passage of the analyte through the column.
k’ = tr-t0/ t0
= The lower the k’ value, the faster the elution
tr: retention time of the compound
t0: unretained compound
Retention time is inversely proportional to the speed of the analyte passing through the column!
PERFORMANCE PARAMETERS - C: What performance parameter is used for partition chromatography only? How is it calculated?
Distribution coefficient:
We look at equilibrium between the concentration of analyte in the mobile phase and concentration in the stationary phase.
[For elution time to be quicker, we want a higher concentration in the mobile phase]
[For separation to occur, the distribution coefficient must be different]
Cm (concentration mobile: tendency to stay in mobile phase)
Cs (concentration stationary: tendency to stay in the stationary phase)
Kd = Cs / Cm
PERFORMANCE PARAMETERS - C: What is the selectivity factor (alpha)?
The selectivity factor indicates the RELATIVE retention of two compounds. It uses k’ from capacity factor.
We look at peaks “n” and “n+a”
alpha (n+a,n) = k’n+a / k’n
Numerator: greater retention time
Denominator: smaller retention time
This is always a comparison of two compounds, meaning an alpha of 1 means that the compounds are not separated.
PERFORMANCE PARAMETERS - C: What are the number of theoretical plates?
We compare a distillation column to a chromatography column.
in distillation column, liquid-vapor equilibrium can form at every plate in the column, and the more plates, the better the separation.
Theoretical plates represents an imaginary number of plates for a chromatography column - it measures the efficiency of the column
= (tells us about how narrow peaks can be and how well retained compound is within column).
Nth = 5.54 (tr / w1/2)^2
How is height of the theoretical plate linked to efficiency of separation?
The smaller the plate height, the narrower the peaks and the more efficient the separation
HETP = length column / number of theoretical plates
What is velocity of the mobile phase?
Based on the retention time of the REFERENCE UNRETAINED COMPOUND:
u = L / t0
where L: length of the column`
What is peak broadening? What are the 3 situations where this occurs?
PEAK BROADENING: RESULTS FROM DIFFUSION OF PARTICLES IN STATIONARY PHASE
As compounds pass through the column, they diffuse and cause peaks to broaden
- Diffusion due to particles in the column
Large particles: many different paths; broad peak
Small particles: flow better distributed, small peaks. bigger surface area, more opportunities for interaction, higher resolution - Diffusion of molecules in the mobile phase (longitudinal diffusion)
The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges - Diffusion due to interactions with the stationary phase
Occurs when retention time is higher; particles have time to broaden by interaction with the column during slow flow
PERFORMANCE PARAMETERS - C: Resolution. What is it and what are the two ways it can be calculated?
Resolution is how distinguishable peaks are, calculation involves using retention time and width of the band (between two consecutive peaks).
- It can use baseline width of width at half max
- It can use number of plates, selectivity factor (relative retention of TWO compounds) and capacity factor (retention of compound wrt t0)
What happens if resolution is:
a) R <1.25
b) 1.251.5
a) no separation at baseline
b) separation depends on symmetry of peaks
c) complete separation at the baseline
Essentially, we aim for an R bigger than 1.5
What are techniques to improve resolution?
Increase the number of theoretical plates (increases retention time, decreases peak width), increase selectivity factor (alpha) by increasing capacity factor (k’).
Ion-Exchange C: What are the two ways to change elution time?
Changing pH or changing ionic strength [ still needs to fall within stability range with sufficient charge to perform the experiment]
Weak anions/ cations operate over small pH ranges (depend on the pKa of the resin). Strong anions and cations operate over entire pH range.
CHROMATOGRAPHY: Ion-Exchange: What is gradient elution? Step elution?
Gradient elution: gradually increasing pH or ionic strength
Step elution: peaks eluding together (fractions are not small enough to separate) eg peak 1 = compounds 1+2
CHROMATOGRAPHY: Ion-Exchange - What is the relationship between isoelectric point and pH?
Each protein has an isoelectric point (pI), the pH at which the overall number of negative and positive charges is zero.
We need to have sufficient charge to perform experiment
Anion exchange resin: binds negatively charged protein, pH needs to be slightly above pI
Cation exchange resin: binds positively charged protein, pH needs to be slightly below pI.
If protein is basic (negatively charged), it’s photoelectric point will need to be below pH 7
If protein is acidic (+ charged), it’s photoelectric point will need to be above pH 7
PERFORMANCE PARAMETER: Retention time [what about retention volume]?
Retention volume is dependent on retention time. It is based on flow rate of the mobile phase and retention time of the analyte.
Vr (retention volume) = tr (retention time) x F (flow rate)
Size-exclusion c: What are the exclusion and penetration limits?
Retention time is inversely proportional to particle size.
[the log of the molecular weight is proportional to the elution volume]
Exclusion limit: molecules larger than the pore size pass straight through [Elution volume is smaller for a higher molecular weight]
Penetration limit: Molecules below a certain molecular weight penetrate all pores and elute at the same position [Elution volume is higher for a smaller molecular weight]
[columns must be selected according to the range of molecular weights in the mixture]
ADSORPTION C: High performance liquid C (HPLC)
Mobile phase is a pressurized liquid. Technique is used for liquids or compounds with low volatility.
Interaction between mobile and stationary phases is based on polarity (adsorption)
If stationary phase is polar, mobile phase is non-polar (to pass directly through)
if stationary phase is non-polar, mobile phase is polar (to pass directly through)
= Separated from each other due to their different degrees of interaction with the absorbent particles.
ADSORPTION C: Gas C (GC)
Mobile phase is an inert carrier gas (helium, argon, nitrogen)
Separation is based on polarity and volatility.
Stationary phase is solid or liquid, typically polar (meaning non-polar compounds will elute first and more volatile compounds will elute first (they stay in gas phase longer)
Suitable for volatile compounds, molecules that are INSENSITIVE to thermal decomposition (given the heating of the sample).
What detectors are used for chromatography?
Two techniques: destructive and non-destructive
Non-D: UV-visible absorbance, fluorescence, conductivity, thermal conductivity
Destructive: mass spectroscopy, flame ionization
What is quantitative analysis and how do we perform it?
= Quantifying the amount of analyte in a given sample
We need:
- An appropriate detector or a method of analyzing fractions collected from a column
- A sufficiently large concentration (above the detection limit of the detector or instrument)
- Standard samples with known concentrations (internal standards)
Why must we use an internal standard for quantitative analysis?
necessary to quantify unknown peaks, always run with sample of interest.
internal standards are of known concentration
This shows a RESPONSE FACTOR (RF) = the ratio between the concentration of a compound being analyzed and the response of the detector to that compound.
What are the necessary steps to running a chromatography experiment?
- Choose an appropriate type of stationary phase
- Choose a detector
- Determine the detection limit of the detector and plan the analyte injection, and the flow rate of the mobile phase
- CALIBRATE the instrument with standard solutions of known concentrations
- calibration to determine retention times and response factor - Run the UNKNOWN samples, along with internal standards
- Identify the peaks
- Determine the concentration of the analyte
DETECTORS FOR C: Mass spectrometry. What are the steps?
Most powerful detector for chromatography (mainly for gas)
Separates compounds based on their mass to charge ratio.
- Sample is vaporized through heating OR is already in the gas phase
- Molecules are ionized to produce a molecular cation (through bombarding with high energy electrons)
- Cations are accelerated in an electric field (towards negative electrode)
- Accelerated cations are deflected in a magnetic field
= molecules with LARGER MOLECULAR WEIGHT (heavier cations) MOVE SLOWER, and are DEFLECTED LESS
= molecules with SMALLER MOLECULAR WEIGHT (lighter cations) MOVE FASTER, and are DEFLECTED MORE.
= molecules with SAME MASS but different charge mean that higher charge will lead to bigger deflection - deflected cations are detected
- quantity of molecules (cations)
- mass to charge ratio = molecular weight of compount or molecule identity
DETECTORS FOR C: Mass spectrometry. What does mass spectrum illustrate?
(y) Relative abundance of a molecule vs (x) its mass/ charge ratio
y = 3 and y=1 —-> number of isotopes of 35cl vs 37 cl.
DETECTORS FOR C: Mass spectrometry. What kind of chromatography is used in combination with MS?
Gas C (separate, quantify and identify unknown chemical species)
What is titration? BRIEF
Technique used to DETERMINE THE CONCENTRATION of a dissolved substance through measuring the effect of a reaction with ANOTHER substance.
How?
Solution of known concentration is added to a solution of unknown concentration until the reaction is complete.
A SENSOR (generally chemical indicator, pH meter or voltmeter) is used to signal the end of the reaction
What is a titrant?
Solution of known concentration that reacts with the analyte
What is the analyte?
Solution of interest with an unknown concentration
What is the indicator?
A substance, added to the analyte, that changes the color as the reaction between the titrant and the analyte proceeds (an indicator = a chemical sensor).
What is the equivalence point?
Volume of a titrant for which there is a stoichiometric mixture of analyte and titrant
What is the endpoint?
Volume of titrant for which the indicator changes color (an indication of the equivalence point)
What must be known for a titration reaction?
- Identity of the chemical species
- Titrant concentration
- Phase (LIQUID OR GAS ONLY)
= this allows us to determine the concentration of the analyte
What is molarity vs normality and why must it be considered for a titration reaction?
Molarity: moles/ L
Normality: number of equivalents/ L
Equivalents correspond to the number of moles of reactive groups in a molecules.
ex: 2M h2so4 = 4N (2 *2)
How are derivatives used in titration curves?
First derivative can be used to identify the equivalence point (given that the slope is the highest)
Second derivative = 0 at equivalence point
Besides the identity of the species and the concentration of the titrant, what are the other requirements for a titration reaction?
- The stoichiometry of the reaction must be known to quantify the analyte
- The reaction between the analyte and the the titrant must be rapid (faster than the rate of addition of titrant)
- A method must be available to determine the equivalence point (or endpoint).
What are the four main types of titration?
- Acid-base
- Complexometric
- Redox
- Precipitation
- Acid-base titration: How does it work?
Acidic or basic titrant reacts with a known analyte that is acidic or basic
At the equivalence point, the molar concentration of the acid* the volume of the acid = the molar concentration of the base* the volume of the base
For strong acids and bases, the equivalence point is always at pH = 7
1.Acid-base titration: Why are strong acids and strong bases preferable?
Strong acids and bases dissociate completely in aqueous solution, whereas weak acids and bases do not dissociate completely.
1.Acid base titration: What sensors are used?
- Colorimetric indicator
ex: bromothymol blue
An indicator must change color at the predicted equivalence point - indicators have different ranges of pH that must be taken into account
- pH meter
pH meter is more accurate because it can be CALIBRATED
= they both work by sensing the concentration of protons in the solution
- Complexometric titration. How does it work?
The titrant is EDTA, referred to as a ligand, and is used to determine the concentration of METAL IONS.
The titrant forms strong complexes with the given metal ions (regardless of their charge).
When all metal ions are bound to EDTA, reaction is complete and indicator changes colour.
MedtaVedta = MmetalionVmetalion
Step 1: Add indicator to the metal ion solution (make sure analyte is at appropriate pH)
Step 2: Add EDTA to displace the indicator
y axis is the pMetal
- Complexometric titration: What sensors are used?
Metallochromic indicators are used: they change colour when bound to metal ions [only valid for given pH region: for too high or too low pH, metal ions start to precipitate unless other ligand stabilizes ion in solution]
- Redox titration: How does it work?
Titrant is an oxidizing or reducing agent: oxidizing - receives electrons, reducing - gives electrons
The titrant participates in a redox reaction with the analyte
- Redox titration: What are the sensors?
Voltmeter or colorimetric indicator:
Colorimetric indicator is sometimes the oxidized or reduced species itself that changes colour when it binds to the oxidized or reduce species
y axis = voltage or potential (if voltmeter is used)
- Precipitation Titration: How does it work?
The titrant and the analyte react to form an insoluble precipitate.
Well known precipitation: titration using Mohr’s method for quantification of chlorides (Cl-)
- Precipitation Titration: Mohr’s method
The titrant dissolves into cation and anion; cation reacts with chloride in seawater; moles cl- = moles cation
Indicator: K2CrO4 - when cation is slightly in excess a precipitate is formed.
What are examples of random errors in titration ?
Random error = uncertainty - cannot eliminate but must account for:
- volume reading (meniscus)
- misjudging the indicator endpoint
- errors in titrant concentration (volume or mass)
What are examples of systematic errors in titration?
- Believing endpoint is at a darker or lighter colour than we think
- Reaction does not go to completion
- Assumption that we have a strong acid or base but that it is weak
- Miscalibration of the voltmeter or ph-meter
- *TEMPERATURE OR PH DEPENDENCE OF INDICATORS
What are some other limitations to titration?
- Large volumes are required
- It is difficult to use indicators for coloured or analyte solutions
- Stoichiometry and identity of analyte and titrant must be known
How could one improve titration accuracy?
- Use a ph meter instead of indicator
- Use a more accurate burette
[Class A and B burettes - tolerance is larger for class B, tolerance increases with volume] - Minimize the volume added
How could one improve titration precision?
- use small drops of a dilute solution
- always identify the correct endpoint for the same colour value
How could one improve titration sensitivity?
- Use a well calibrated instrument that is more sensitive than the human eye.
SPECTROSCOPY: What is it?
Study of how matter interacts with light or how matter emits electromagnetic radiation TO extract molecular information from that data
SPECTROSCOPY: How do molecules react under given conditions?
- Molecules can absorb light and enter an excited state [absorption spectra]
- Molecules can emit a spectrum of radiation by returning to their normal state [emission spectra]
[each peak in the spectrum corresponds to a specific atoms of a chemical group]
SPECTROSCOPY: What is the relationship between wavelength and energy?
The smaller the wavelength, the greater the energy.
Different wavelengths of light can cause;
- molecular motion (vibration, stretching…)
- electronic transition to a higher energy level
Not all wavelengths are visible to the human eye
SPECTROSCOPY: What is transmittance, absorbance?
Transmittance is a measure of the amount of light that passes through a given substance
T = current entering/ current exiting
Absorbance is a measure of the amount of light that is absorbed by the substance
A = -log(T)
Absorbance and transmittance go hand in hand. 100% transmittance = 0% absorbance
** 10* transmittance is NOT 90% absorbance**
SPECTROSCOPY: What is transmittance, absorbance?
Transmittance is a measure of the amount of light that passes through a given substance
T = current entering/ current exiting
Absorbance is a measure of the amount of light that is absorbed by the substance
A = -log(T)
Absorbance and transmittance go hand in hand. 100% transmittance = 0% absorbance
** 10* transmittance is NOT 90% absorbance**
SPECTROSCOPY: What is the Beer-Lambert Law?
Measure of the absorbance at a given wavelength can be found IF:
molar absorptivity, concentration and path length for the substance are known.
For a mixture, same thing applies given that they all absorb at the same wavelength
SPECTROSCOPY: How can we calibrate experiment?
By producing a calibration curve for absorbance or transmittance wrt to concentration:
Absorbance will have linear relationship and transmittance will have an exponential relationship
SPECTROSCOPY: How can we calibrate experiment?
By producing a calibration curve for absorbance or transmittance wrt to concentration:
Absorbance will have linear relationship and transmittance will have an exponential relationship
SPECTROSCOPY: How can we calibrate experiment?
By producing a calibration curve for absorbance or transmittance wrt to concentration:
Absorbance will have linear relationship and transmittance will have an exponential relationship
SPECTROSCOPY: How can we calibrate experiment?
By producing a calibration curve for absorbance or transmittance wrt to concentration:
Absorbance will have linear relationship and transmittance will have an exponential relationship
SPECTROSCOPY: How can we calibrate experiment?
By producing a calibration curve for absorbance or transmittance wrt to concentration:
Absorbance will have linear relationship and transmittance will have an exponential relationship
UV-Vis Spectroscopy: What is the spectral range?
UV: 100 to 380 nm
Visible: 380 to 800 nm
UV-Vis Spectroscopy: what causes absorption
Absorption is due to chromophores (absorb light, responsible for colour)
Auxochromes (modify the absorption of chromophores)
Maximum wavelengths for compounds is known, which can help narrow down groups.
Chromophore is that part of the molecule which when exposed to visible light will absorb and reflect a certain color
Auxochrome is a group of atoms which will impart a particular color when attached to a chromophore
UV-Vis Spectroscopy: what are limitations and requirements?
- Absorbance: 0.1 to 1
- Solution must be uniform
- Must have standard solutions available for calibration OR must know the molar absorptivity (given that A VS C is dependant on epsilon and path length)
- Blank solution required
Energy can produce electronic transitions in addition to movement
Infrared spectroscopy: What is the spectral range?
2500 nm to 16 000 nm
wavelength too high to produce electronic transitions
Infrared spectroscopy: What are the uses?
- identifying chemical groups
- determining surface composition of sample
- detector for chromatography
FTIR: What is it?
transforms input from IR source to output:
- intensity vs frequency
- % transmission vs wavenumber (instead of absorbance)
wavenumber is 1/wavelength
It is made up of a pellet containing the sample of interest
Infrared spectroscopy; IR vibration frequency - what does it depend on?
vibration frequency can be found for molecules subjected to IR spectroscopy:
It depends on
- mass of the atoms
- strength of the bond (spring constant)
- environment
Infrared spectroscopy: What happens during stretching, deformation?
stretching of bonds results in high frequency and is possible for low reduced masses
deformation of bonds results in low frequency and is possible for molecules with more atoms
SPECTROSCOPY: Advantages vs limitations
+:
- non destructive (as opposed to titration)
- provides qualitative and quantitative information
- various material forms can be used
- :
- difficult interpretation of data for mixtures
