Sample Preparation and Validation Flashcards
Aim and Objectives
* Aim
o To give an overview of the main sample preparation techniques in analytical chemistry
Principle of methodology
Advantages and disadvantages
*** Objectives **
o Understand the importance of sample preparation
o Understand the molecular properties that aid with separation
o Critically assess the advantages and disadvantages of some of the available separation techniques
Additional resources
- Royal society of chemistry website
- CHROMacademy website
- Textbooks
o Quantitative chemical analysis
o Analytical chemistry
o Fundamentals of analytical chemistry
What are some considerations for sample preparation?
- Sample matrix (solid/ liquid/ gas)
- Pre-treatment requirements (e.g., dilution, filtration etc.)
- Is extraction needed?
o Concentration of analyte
o Interferents
o Compatibility with analytical techniques available - Molecular properties of analyte (e.g., BP, acidic/ basic etc.)
- Chemical equilibrium (temperature, pH, solubility, solvent)
- Do analytes require chemical alteration for analysis? (e.g., derivatisation, or cleavage from other compounds)
What are some basic and advance techniques for sample preparation?
For the basic techniques for sample preparation what are the advantages and disadvantages?
For the advance techniques for sample preparation what are the advantages and disadvantages?
What are the different sample types?
- Solid (e.g., tablet)
- Liquid (e.g., biological fluid)
- Gas (e.g., environment pollution)
Forensic toxicology
- Blood
- Urine
- Stomach contents
- Vitreous humour
- Liver, lung, brain, muscle tissue
- Alternative matrices (e.g., hair, saliva)
What are some chemical properties consider during sample preparation?
- Acid/ Base (pKa)
- Size
- Polarity
pKas of inorganic and oxo-acids
pKas of nitrogen acids
For the Henderson-Hassebalch equation answer the following:
What is pH and how is it measured?
How do we calculate pH?
The equation?
What things are important for pH of a solution?
- Important for analytes
- Acid dissociation constant (pKa)
- E.g., for acidic analytes, the effect of **[A-]/ [HA] **on the pH of analytes is as follows
What are amphoteric compounds?
- Both proton donors and proton acceptors (AA are good examples of amphoteric compounds)
- At a certain pH this AA can exist as a **zwitter ion **
- Where only one weakly acid and one weakly basic function
o **Zwitterionic, net charge of 0 (isoelectric point, pI) **
o Ionisation of basic group has ended but the acidic hasn’t begun
o Halfway point between pKa values (unionised compound)
Amphoteric compounds with example: **alanine **
What pH will morphine be unionised?
What is molecular hydrophobicity and what is the equation to calculate it?
- partition coefficient (LogP octanol/ water)
- ratio of unionised drug distributed between organic and aqueous phase at equilibrium
- hydrophobicity** only holds true for unionised compounds**
What is the distribution coefficient and the equation for it?
- Considers the hydrophobicity of a molecule as well as the proportion ionised at a particular pH
- **LogD (octanol/pH buffer) **
*** Two different equations for acids and bases ** - The greater the solubility of a substance, the higher its partition coefficient, and the higher the partition coefficient, the higher the permeability of the membrane to that substance
Example) Amitriptyline has a LogP=4.92 and a pKa of 9.4. What is LogD at pH7?
What does sample pre-treatment depend on?
**Sample pre-treatment **
- Dependent on analyte, sample matrix, and nature of retention chemistry; involves pH adjustment, centrifugation, filtration, dilution, buffer addition etc,.
o Protein/ protein bound - Organic solvent displacement (e.g., acetonitrile crash)
- pH shift
- sonication
- o conjugated analytes?- especially when dealing with urine
- Glucuronide (a monosaccharide with free carboxylic acid grp)
- Sulphate
What are the types of hydrolysis and the advantages and disadvantages of each?
*** Acid or alkaline **
o Adjust the pH of a sample with a strong mineral acid (pH) or alkali (pH 14)
Advantage: rapid (e.g., 10-30 min at 100˚c)
Disadvantage: can generate by-products analyte (introduction of double bond) or destroy analyte altogether
*** Enzymatic **
o Adjust pH of sample to optimum pH of enzyme and add glucuronidase (and/ or sulphatase)
Advantage: much fewer artefacts
** Disadvantage: ** slower than acid hydrolysis. Not 100% efficient (not all conjugates cleaved)
*** Solvolysis **
o Similar to protein precipitation. Effective for deconjugation of sulphates not effectively cleaved by enzymes
Advantage: can achieve deconjugation
Disadvantage: slow and conditions must be optimised
Shows typical drug excreted as a glucuronide
What is liquid-liquid extraction (LLE) and tell me about it
- Solvent extraction or partitioning
- Partition of a compound between two immiscible phases
* Partition coefficient KD (looks at solubility of analyte in organic phase over the solubility of analyte in aqueous phase) - The larger the KD value the better the separation. However, the partition coefficient is only useful if the analyte remains aqueous in solution
What are the considerations for LLE?
o Organic solvent type and volume
o Miscibility
o Neutral, weakly acidic and weakly basic organic analytes
o pKa of analyte and pH of solution
o LogP
o [salt] or [buffer]
o Temperature (and pressure)
o Agitation (shake to move analyte from one phase to another)
o Time (of extraction)
How is the polarity of organic solvents known?
- Dielectric constant is a measure of a substances ability to insulate charges from each other
- Measure of solvent polarity (higher the polarity the greater the ability to stabilise charges)
- Dielectric constant of some solvents
o Water= 80.1
o Methanol= 32.7
o 1-chlorobutant=7.4
o Ethyl acetate= 6.0
o Diethyl ether= 4.3
o Toluene= 2.4
o Hexane= 1.9
Where no secondary reactions occur the efficiency of extraction is only dependent on what?
- Where no secondary reactions occur the efficiency of extraction only dependent on the solutes partitioning between two phases
What does the distribution ratio not depend on?
- The distribution ratio does not depend on the composition of the aqueous phase or the organic phase. A change in the pH of the aqueous phase, for example, will not affect the solutes extraction efficiency when kD and D have the same value
LLE involving acid-base equilibria
What are the pH effects on extractions, in particular with a weak acid?
What is solid-liquid extraction (SLE)?
- Solid-liquid extraction is similar to liquid-liquid extraction, except that the solute is dispersed in a solid matrix rather than in a carrier liquid
- Removal of analyte from a solid by interacting with a solvent that its dissolved in
How could SLE be improved?
o Grind sample to fine particles (increase SA)
o Increase temperature (and pressure if unstable)
o Adjust pH of the extraction solution only
o Add low salt concentration- “salting in”
o Use high solubility solvent (polarity match)
o Check compatibility with analytical instrument
Name a type of SLE tell me about it
Soxhlet extraction- a type of SLE
* Invented in 1879 by Franz von Soxhlet
* Originally designed for the extraction of a lipid from a solid material
* Used when the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent
* Allows for unmonitored and unmanaged operation while efficiently recycling a small amount of solvent to dissolve a larger amount of material
* Only small amount of solvent needed as is recycled throughout
Tell me the following about supercritical fluids:
* What they are
* How they are produced
* their gaseous and liquid property
* pros and cons
* uses
- Supercritical fluids are highly compressed gases that have the combined properties of gases and liquids of vital importance for extraction and processing
- Produced by heating a gas above its critical temperature or compressing a liquid above its critical pressure
- Gaseous property of being able to penetrate anything
- Liquid property of being able to dissolve materials into their components
- Advantages: selectivity and speed
- Disadvantage: requires a high pressure
- Uses (e.g.,): extraction of metal ions from aqueous solutions and solid and liquid matrices, decaffeination of tea and coffee, separation of lecithin from oil
What is pressurised liquid extraction?
- Combines elevated temperature and pressure with liquid solvents to achieve fast and efficient extraction of the analytes from the solid matrix
- Can reduce solvent consumption (compared to traditional solvent extraction methods)
- Can increase sample throughput both rapid and automated
- Increase analyte capacity of high temperature solvent
- Higher analyte stability due to lower temperatures and higher pressures
SLE comparative performances
What is solid phase extraction (SPE)
- Sample preparation technique that is used to extract dissolved solutes in the liquid (or gas) phase onto a solid sorbent
- Exhaustive (fully comprehensive) analytical technique
- Designed to minimise matrix and concentrate analyte of interest
- Image shows different chromatography techniques which can be used
o Ion-exchange chromatography
o Partition chromatography
o Adsorption chromatography
o Size-exclusion chromatography
SPE cartridge
Multiplexing SPE
SPE is a 5 step process. What is each step and tell me a bit about each
**1. Conditioning ** (pre-treat the sample e.g., dilution or adjustment of pH)
a. Solvent passed through SPE material
b. Allows interaction between solvent and analytes
**2. Conditioning ** (condition the cartridge and run water or solvent through it)
a. Equilibration occurs
b. Sorbent treated with solution of similar polarity and pH of matrix
c. Maximises the retention of analyte when applied to matrix later
**3. Loading sample **
a. Sample onto cartridge and bind with sorbent
b. Don’t want step to be too fast otherwise analyte doesn’t have enough time to interact with sorbent
**4. Wash **
a. Use solvents which don’t interact with out sorbent
b. Can use those that interact with others in order to remove interference
**5. Elution **
a. Focus on removing analyte of interest with solvent that overcomes chemistry which overcomes primary and secondary retention interaction between analyte and sorbent of interest
Whats are the pros and cons to SPE?
*** Advantages **
o Great selectivity
Wide range of chemistries available
o Wide variety of sample matrices
o High recovery and good reproducibility
o Amenable to automation
o Low solvent volumes
*** Disadvantages **
o Method development
o More steps (method development)
o Cost per sample?
What are some SPE strategies?
1. Bind and elute
a. Bind analyte of interest and elute interference
**2. Interference removal strategy **
a. Bind interferent and elute analyte
**3. Fraction strategy **
a. Retain and sequentially elute different classes of compounds
What are some different sorbent chemistries in SPE?
- non-polar
- polar
- ion exchange
- mixed mode
Tell me the following about non-polar sorbent chemistries:
* phase type
* reaction mechanism
* Analyte characteristics
* how elution is achieved
Sorbent chemistries- non-polar
* Reversed phase SPE
- Reaction mechanism: non-polar or hydrophobic
- Analyte characteristics-exhibiting non-polar functionalities (most organic analytes, alkyl, aromatic, alicyclic functional groups)
- Elution is achieved by disrupting sorbent analyte hydrophobic interruption with a solvent with strong interaction i.e., adequate non-polar characteristics (e.g., methanol, acetonitrile, dichloromethane, or buffer mixes)
Tell me the following about polar sorbent chemistries:
* phase type
* reaction mechanism
* Analyte characteristics
* how elution is achieved
**Sorbent chemistries- Polar **
* Normal phase SPE
- Reaction mechanisms: polar or hydrophilic
- Analyte characteristics- exhibiting polar functionalities (hydroxyl groups, carbonyls, amines, double bonds, hetero ions (O, N, S, P))
- Elution is achieved by disrupting sorbent analyte hydrophilic interruption with a solvent with stronger interaction i,.e., adequate polar characteristics (e.g., methanol, acetonitrile, isopronanol, or buffer mixes)
Tell me the following about ion exchange sorbent chemistries:
* phase type
* reaction mechanism
* Analyte characteristics
* how elution is achieved
**Sorbent chemistries- ion exchange **
* Electrostatic attraction of charged functional groups on the sample to oppositely charged functional groups on sorbent
- Used for compounds that are charged in solution
- Reaction mechanism: anionic (-) or cationic (+)
- Analyte characteristics: anionic for basic compounds (amine groups) and cationic for acidic compounds (carboxylic acids, sulfonic acid)
- Elution is achieved by pH modification to neutralise compound and/ or sorbent functional groups. It is also possible to increase salt concentration or use a more selective counter ion to compete for ion-exchange binding sites
Tell me the following about mixed mode sorbent chemistries:
* phase type
* reaction mechanism
* Analyte characteristics
* how elution is achieved
Sorbent chemistries- mixed mode
* Rely on two or more retention mechanisms to simultaneously extract a broad range of compounds from a single biological sample
- Slight changes to eluant conditions bring about selective elution with highly reproducible recovery of trace levels of analytes that are free of contamination from the sample matrix
- Used for compounds that are charged in solution
- Reaction mechanism: combination e.g., hydrophilic, and hydrophobic, or ionic and hydrophobic
The solution/ analyte is dissolved in…
Tell me what validation is and why it is important
- What is quality?
- Validation is the process of providing documented evidence to demonstrate that a process or procedure will consistently produce a product
- Proves that performance characteristics of the method meet the requirements for the intended analytical applications
o Analytical procedure includes all the steps in analysis (includes sample preparation, analytical analysis, interpretation of results)
What are the parameters to validation?
- Accuracy
- Precision
- Specificity
- Recovery
- Limit of detection (quantitative, influence by instrumentation and matrix interference)
- Limit of quantification
- Linearity (quantitative)
- Range (quantitative, calibration curve)
- Robustness
Tell me about validation recovery:
* why it is important
* what is measures
* how it is expressed
