Sampling Flashcards
Does sampling come before or after analysis in the pharmaceutical process?
Sampling of a substance and the sample preparation needed for it, comes before analysis- it is preparing a substance for analysis.
Why do we need to do sample preparation before analysis?
> To remove interferents (toxins impurities) from solution, that could possibly hinder the results.
Actually extracting the analyte for a more accurate and precise reading.
Pre-concentrating the specimen; if we were to go into the analysis with the raw sample, there may not return any reading. This may be because the analyte is a trace amount in the substance, or it has a low concentration. Pre-concentrating ensures we get a reading back.
Converting the analyte to a measurable form. In its original form, the analyte may not be directly measurable with any analytical instruments, so much that it is unable to produce back a signal or property (colour, absorbance or electrical current).
Soldi samples into solution?
What things in the real world made be sampled to be analysed?
> Biological fluids such as plasma, saliva or urine.
Environmental samples like water, air or soil.
Food products like meat. grain and seafood.
Pharmaceuticals and nutraceuticals.
Name the main stages to go through in order to analyse something, and the method within each stage.
Sampling - Collecting the sample, storing the sample.
Sample clean up - Extracting, concentrating and isolating the sample.
Analysis - Identification and quantification.
What is a bulk sample?
A large sample of mineralised rock, normally hundreds of tonnes heavy.
What is a gross sample?
The sample which consists of many portions. Can be impure or pure. May be heterogeneous or homogeneous.
What is a laboratory sample?
When a gross sample is reduced in size to obtain a laboratory sample which has mass of only a several grams.
What is an analysis sample?
When a sample of a few grams is reduced to milligrams to be taken to be analysed.
What is a heterogeneous sample?
A sample that contains particles of different shapes and sizes. The composition of one sample can differ from that of another sample.
What is a homogenous sample?
A homogeneous sample is uniform and the composition is consistent throughout different samples, regardless of where you sample them.
What questions need to be asked through the process of sampling and separation?
What form does the sample need to be in- solid, liquid or a gas?
Is the sample organic or inorganic?
Is the sample soluble in polar or non-polar substances? Separation by solubility?
Is chemical separation or masking of interferents needed?
Do we need to concentrate the sample to obtain a better and clearer reading during analysis?
Do we need to adjust solution conditions (like pH, or adding any reagents)?
What are some main methods or processes of chemical separation?
Distillation.
Precipitation.
Solvent extraction.
Solid phase extraction.
Chromatography.
Electrophoresis.
Describe the main apparatus and set up during distillation.
Firstly, there is a round bottom flask sitting on top of a mantle.
At the top of the round bottom flask, there is a 3-way adaptor; one tube is connected to the round bottom flask, one goes up and is open ended, and the last tube is connected to a Liebig condenser that lies perpendicular to the round bottom flask.
The Liebig condenser is perpendicular, but is slightly at a slant downwards.
This Liebig condenser is connected to a receiving flask via another adapter.
The round bottom flask and receiving flask is held upright via a standing clamp (there may also be one needed for the Liebig condenser).
The Liebig condenser has two ports for water to enter and leave. The port at the bottom is for water to enter, whilst the port at the top is for water to leave. (Done by connecting tubes to the ports from the sink).
How is simple distillation and steam distillation different?
In steam distillation, steam or boiling water is introduced into the round bottom flask that contains the mixture to be distilled.
In simple distillation, the round bottom flask contains the mixture to be distilled and nothing else.
Why would we use steam distillation over simple distillation?
When there is a mixture that contains components with high boiling points, continuously heating the mixture at high temperatures can cause temperature-sensitive components to be damaged during distillation. Not to mention that continuously heating the mixture results in high energy usage and is cost inefficient.
Introducing steam into the mixture to be distilled lowers the boiling point of the compounds. Steam has a relatively lower boiling point and when it mixes with the distillation mixture, it also reduced the boiling point of the target compound significantly.
Now, the energy needed to evaporate this compound is lower, and no damage will be sustained from the compound.
Where is steam distillation commonly used?
Extracting essential oil from flowers.
Some solutes completely dissolve in an aqueous (polar) layer, and some solutes completely dissolves in an organic (non-polar) layer. On the other hand, most solutes may be partially soluble in the aqueous phase AND partially soluble in the organic phase. How can we calculate the ratio of concentration of solute A in each phase (can be before or after extraction)?
The ratio of concentration of solute A in each phase is called a Partition ratio.
It can be calculated by:
Kd = [A organic phase] / [A acquiesce phase]
How does the partition rate (Kd) of solute A vary with different solvents, or different quantities of A (different quantities / volume of A for the same solvent)?
In every case, the Kd for solute A changes if the solvent changes.
However, if we have solute A and the same solvent every time, the partition ratio should be consistent. The partition ratio should still be constant even if the quantity of A changes.
Give an example of a solute that may be present between two immiscible phases (between the organic and acquiesce layer).
A weak acid.
A weak acid partially dissociates: HA > A- + H+. The H+ and A- ions will be found in the acquiesce layer, whilst the HA will be found in the organic layer (like dissolves like).
What are the limitations of the Partition ratio, Kd?
If we are looking at any species that ionise in an acquiesce layer (like an acid), the Kd ratio does not give an accurate representation of the concentration of solute in each phase.
This is because in the acquiesce phase we will have H+ and A-, while in the organic phase we have HA.
The Kd ratio only allows us to take in account of one species in each phase, so we can have:
Kd = [HA] / [H+] or
Kd= [HA] / [A-]
Any of these equations is not an accurate representation of concentrations of the weak acid in the acquiesce phase.
In situations like this, we use the distribution ratio, D.
What is the distribution ratio, D?
The distribution ratio is an equilibrium constant that shows the ratio of the concentrations of ALL species of the solute in each phase.
So the distribution ratio of a weak acid would be:
D= [HA] organic / ([HA] acquiesce + [A-] acquiesce)
Why is the distribution ratio D constant, even when the volume ratio of the two phases change, while the fraction of solute extracted is dependent on the volume ratio?
The distribution ratio D is a instant independent of the volume ratio. If a larger volume of organic solvent is used, the volume ratio of the two phases changes, but more solute dissolves in the organic layer in order to keep the concentration ratio constant and to satisfy distribution ratio D.
However, if the solute were to be extracted from the organic phase (which has just grown bigger in volume), more fraction of the solute will be extracted. This is because, as said above, with a larger volume of the organic layer, more solute dissolves in this layer to maintain distribution ratio. If there is more solutes in the organic layer, there is a chance to extract more solutes, and hence the fraction of solutes extracted increases while the distribution constant, D, stays constant.
How can we calculate the fraction of solute extracted?
Fraction of solute extracted=
(millimoles of solute in the organic layer) / (total number of millimoles of solutes)
Solvent extraction is a generalised term used for liquid-liquid extraction, solid-liquid extraction and supercritical fluid extraction.
What does solvent extraction mean?
Solvent extraction is essentially the process in which compounds are separated depending on their relative solubilities. So, it involves using a solvent that one solute in the mixture (can be a liquid-liquid mixture or solid-liquid mixture) is soluble in, but the other is not.
Solvent extraction is simply a technique used in multiple processes like liquid-liquid extraction and solid-liquid extraction.
Define solubility.
The upper limit of solute that can be dissolved in a given amount of solvent at equilibrium.
What is the ‘like-dissolves-like’ rule?
Polar solutes tends to dissolve in polar solvents and non-polar solutes tends to dissolve in non-polar solvents.
How is normal solvent extraction different from accelerated solvent extraction?
Normal solvent extraction is separation of a compound from a liquid or solid at room temperature and pressure. Extraction takes place over a duration of hours to days.
On the other hand, accelerated solvent extraction is performed at higher temperatures or pressures, to speed up the process (it improves the solvent’s abilities to extract compounds in a shorter time period). Extraction takes place within a few minutes.
When may accelerated solvent extraction be used?
ASE may be used when there is the extraction of a compounds from complex matrices in a solid.
ASE can also just be used for a faster, more efficient extraction than normal solvent extraction (as normal solvent extractions can take from hours to days).
Describe the main principle of a Soxhlet extraction, and it’s set up.
A Soxhlet extraction is a solid-liquid extraction (in which some liquid is extracted from a mixture, to leave a solid).
The set up is basically a round bottom flask that sits on a mantle (heat source). In the round bottom flask is the solvent that is used to dissolve ONLY the target molecule in the solid mixture. On top of the round bottom flask is a solvent extractor, held upright using a standing clamp.
It is similar to a Liebig condenser in the fact that cool water is fed into the cooling jacket of the Soxhlet extractor to condense any gas that has moved up. The top of the Soxhlet extractor is also open ended like a Liebig condenser. Of course, there will be ports in the Soxhlet extractor to feed cool water in and out from the sink.
The difference between a Soxhlet extractor and Liebig condenser is that a Soxhlet extractor is wide enough around the middle to contain a thimble for a sample (another small container). The thimble contains the solid-liquid mixture.
Describe how the Soxhlet extraction process works.
The solvent in the round bottom flask is heated by the mantle, until it evaporates and travels up the Soxhlet extractor, going through the sidearms.
When the solvent has reached the top, it reaches the condenser of the Soxhlet extractor where cool water is fed in. The cool water causes the solvent vapours to condense into a liquid, which results in it falling into the thimble containing the solid mixture.
When the solvent mixes with the solid-liquid mixture, it causes the target compound to dissolve in the solvent. The solvent then drops back into the round bottom flask, so is ‘recycled’.
The solvent is continuously run through the extractor as the same amount of solvent is conserved, as it is essentially ‘recycled’ after it runs through the Soxhlet extractor each time.
Overtime, all the soluble components will be extracted into the round bottom flask. The extracted component and the solvent can then be separated by rotary evaporation.
Accelerated Solvent extraction can be said to be a modernised version of the Soxhlet extraction. How do they differ?
For extraction, the Soxhlet technique relies on repeated solvent contact with the sample to provide a sufficient extraction (continuous solvent contact brought about via continuous heating, evaporation, condensation of the solvent in the Soxhlet extractor). This process can vary from hours to days.
On the other hand, ASE sues increased temperature and pressure to speed up the Soxhlet extraction process. So, the solvent is heated above their boiling point, at a higher pressure. The pressure prevents the solvent boiling off due to the high temperatures.
How is an accelerated solvent extraction better than a Soxhlet extraction?
With a Soxhlet extraction:
> Much smaller volume of solvent is used.
> Extraction takes less time.
> Extractions are automated.
> Multiple samples can be managed at the same time.
Where is solid-liquid extraction typically used?
Typically used for analysing tablets, capsules and plant materials.
Is a Soxhlet extraction used for extraction from a solid mixture, or extraction from a solid-liquid mixture?
The sample in the Soxhlet extractor is a solid mixture, but it is said to be a solid-liquid extraction once the solvent passes through the sample.
Ethyl acetate, diethyl ether and dichloroethane are good examples of efficient organic solvents that are used in extraction. Why are they efficient for forming the organic layer during liquid-liquid extraction? Out of the three stated above, why would we prefer to use one over the other?
These solvents are good for forming an organic layer because they are immiscible with water; this means there is a good and distinct separation between the organic phase (so the organic solvent) and the aqueous phase (the water).
One would prefer to use one over the other, because some of these (diethyl ether and Ethyl acetate) are highly flammable. Dichloromethane is less flammable than the other two so may be used if the experiment is conducted in an area containing naked flames.
Compared to Ethyl acetate, diethyl ether is more volatile, so would evaporate more easily. This would be dangerous as diethyl ether is toxic, especially when breathed in.
What is liquid-liquid extraction commonly use for?
> Used for extracting substances from biological fluids, like blood and urine.
used in pharmaceutical products like ointments.
What apparatus is needed for a liquid-liquid extraction?
Separating funnel
How would you carry out a liquid-liquid extraction using a separating funnel?
Prior to the extraction, the sample may have been a solid that was dissolved in the organic layer. If the mixture was a liquid, the organic layer may have just been added to the liquid mixture. If both compounds in the mixture (liquid or solid) have some sort of solubility in the organic layer added, an additional reagent may be added to the mixture to make one compound increasingly more soluble (a common example is an acid-base reaction if one of the compounds in the mixture is a acid or base). This is not necessary if one compound is definitely soluble in the organic layer, and the other compound in the mixture is not.
So, the mixture would initially be put in the separating funnel, and then the organic layer would then be added. This can be added by making sure the tap is closer, removing the stopper at the top and using a glass funnel to add the organic solvent.
After the organic solvent is added, place the stopper on top and invert the separating funnel and start swirling and shaking the separating funnel to ensure the contents are properly mixed.
As one of the compounds are dissolving in the organic layer, a reaction would have had to occur, which could release a gas. So from time to time, after shaking, open the tap at the top to release some gas that is produced. This is to prevent the build of pressure in the funnel that could possible cause the funnel to shatter. After this, close the tap and continue swirling.
Repeat this process for around 5 minutes, and when you invert the funnel back around, wait for the two layers (organic and aqueous) to form.
Drain the bottom layer (typically the aqueous layer) into a flask by opening the tap.
After extraction and draining the aqueous layer in a separating funnel during liquid-liquid extraction, why do we add additional water to the funnel after and then drain again?
Adding water after extracting the aqueous layer, helps to pick up any leftover aqueous solution (that lies within the organic layer) that remains in the separating funnel.
We add water, invert and shake the funnel, and drain the aqueous layer.
This ensures an efficient extraction.
What does a continuous liquid-liquid extraction look like?
Like a Soxhlet extraction (solid liquid extraction), there is a round bottom flask that sits on a mantle. Inside the round bottom flask is the organic solvent that evaporates, and travels up an individual tube to the condenser. The condenser causes the solvent vapors to condense and fall into a flask containing the mixture. This flask is independent and separate to the tube mentioned before (tube between round bottom flask and condenser).
The solvent vapour have become a liquid, and when it drops into the flask containing the mixture. The additional of the organic solvent allows to immiscible layers to form- an organic and an aqueous layer.
The flask has a tube to connect the flask to the round bottom flask (or the tube of the round bottom flask). Through this tube, the organic compound dissolved in the organic solvent both travels and is collected in the round bottom flask. It is continuous because once the solvent (and soluble organic compound) is collected, it is then heated once again to evaporate, condense and fall into the flask.
The position of the tube in the flask is dependent on whether the organic layer is more or less dense than the aqueous layer. In most cases, the organic layer is less dense than the aqueous layer, so the tube is found further up the top of the flask.
What are some advantages of a continuous extraction?
> Only small volumes of solvents are used (as it is continuously circled through the system).
A high percentage of solute can be extracted.
Extraction can be left unattended for long periods of time (as it an automatic process with the element of heat).
Can extract solids as well as liquids.
Why must the solute extracted from the mixture be thermally stable and less volatile than the extracting solvent in a continuous solvent?
So the solute extracted does not continuously run the system again- this way traces of the solute may still remain in the mixture after extraction, making the process less efficient.
How is SLE different to LLE?
SLE is more difficult for extraction from a solid sample.
Often have to do multiple extractions from SLE.
Can use solvents that are miscible with water, as long as major matrix constituents are not dissolved by solvents.
LLE is more easy to extract from a liquid sample.
LLE can possibly be done within a single extraction. Immiscible solvents must be used.
What is the difference between solid-liquid extraction and liquid-liquid extraction?
In solid-liquid extraction, the initial phase in the state of a solid mixture. A liquid solvent is added to the solid mixture to dissolve the desired compound into the liquid phase. The mixture may be left to sit for some while, shaken or agitation, and after the solid can be separated from the liquid phase via filtration, centrifugation and decantation. So its key principle is dissolving the solid in a liquid solvent, then extracting the solid from it afterwards.
In liquid-liquid extraction, the initial phase is in the state of a liquid mixture. A particular solvent is added (that is immiscible in water) to create two layers and dissolve one of the compounds in the liquid mixture. So the key principle is adding a solvent to create soluble partitions between two immiscible solvents.
What is the principles of solid phase extraction?
After a solid is dissolved in a solvent (can be water miscible , or does not have to be water miscible), it is running through solid phase extraction.
The mobile phase in this extraction is actually the sample that had just been dissolved, and this moves down a cartilage device (like a syringe) through the solid phase.
The solid phase can also be referred to as the stationary phase, and this phase contains an absorbent (an adsorbent is a solid that absorbs gases or dissolved substances) such as silica, polymer and more. The adsorbent forms certain interaction with targeted analytes in solution, so these targeted analytes are retained in the column, while other substances pass through.
What are the four stages of solid phase extraction?
Conditioning, sample addition, washing and elution.
What is the conditioning phase of SPE?
Conditioning phase is the process of rinsing the SPE cartridge with a solvent mixture that is similar to the matrix of the sample to be extracted.
The nature of the solid phase before conditioning is in a collapsed state, which makes it unable to form necessary bonds with analyte- after conditioning, these interactions are then possible. So, conditioning basically readies the solid phase for when the sample is added (to make sure it effectively retains target analytes).
What is the sample addition and washing phases of SPE?
Sample addition is the process of adding the analyte to the SPE cartridge, then allowing the target molecule to form certain bonds with the solid phase, depending on the target molecules properties. So, this is the retention of the target molecules, while all other molecules are filtered out.
Washing in SPE is about washing the cartridge with a solvent (like a weak solvent or buffer) to get rid of any leftover interferents that did not filter out before. Unwanted interfering substances includes salts, proteins and other contaminants.
What is the elution stage of SPE?
The target analytes that have retained to the solid phase are now elution (washed out with a solvent) to be extracted (similar to the washing phase, except these analytes are not interferents, but the compounds we want to analyse).
Elution is done by adding a strong solvent that will interrupt any interactions between the analytes and solid phase. This elution solvent is important because the right one needs to be chosen. This is because of its strong nature, the elution solvent can still pick up any interferents that remained even after the washing stage.
Where may SPE be used?
Sample preparation of biological fluids such as plasma, serum and urine.
A mixture of liquids can also be placed through an SPE cartridge for extraction. Why may we want to use SPE over LLE?
> Solvents in LLE is limited to ones that are water immiscible (to form two clear layers).
Emulsions can often form.
Relatively large solvents needs to be used.
Multiple extractions may need to occur to increase efficiency and yield of extracted material.
What is the solid phase of SPE?
The solid phase sits near the bottom of the SPE cartridge and basically just holds adsorbents. Adsorbents are solids that adorbs (by forming bonds) gases and dissolved substances.
Does the type of adsorbent change with different samples during SPE extractions?
The adsorbent does change with different samples. In order to effectively retain the analyte, the right bonds needs to be formed between solid phase and analyte, and in order for the right bond to be formed, they need to have similar properties (like the like-dissolves-like rule when dissolving substances).
For example, you would not use an alkyl adsorbent to retain an analyte like propanol- the right bond would not be formed that would be strong enough to retain propanol; at best we would get London forces formed, which is weak. For propanol, we need an adsorbent that is equally polar, or maybe an adsorbent which propanol can form hydrogen bonds with.
Note that the frame work or base of any adsorbent is mostly the same- an oxygen on the walls of the SPE cartridge, that is bonded to a silicon atom. The silicon atom is bonded to two other silicon atoms that runs along the wall of the cartridge. The last bond of silicon is connected to a substituent which can change to change the properties of the adsorbent.
What type of analyte would an adsorbent like silica retain?
A silica adsorbent involves 3 oxygens bonded to one silicon atom. These oxygens also form a bond with the wall of the SPE cartridge. The last bond of silicon atom goes to a hydroxyl group, which made silica a very polar group.
Hence, the analyte it retains will most likely be very polar, where they can form permanent dipoles between one another, or hydrogen bonds.
What are the types of column packaging in SPE?
Dispersive- alkyl groups
Pi Pi interactions- phenyl
Electrostatic/dipole- Cyano
H bonding- Silica
What are primary interactions between the analyte and solid phase in SPE?
Hydrophobic and hydrophilic interactions.
As said before, different samples require different adsorbents, which is also why there is a subset of different SPE extractions. Bearing this in mind, what is the difference between normal phase extraction and reversed phase extraction?
Normal phase extraction uses a polar adsorbent because the analyte to be extracted is also polar. Polar adsorbents includes silica and alumina.
On the other hand, if the analyte to be extracted in non-polar, for more effective interactions and bonds, it would make sense to pick a non-polar adsorbent. Such adsorbents includes C18, C8 (different sized alkyl groups attached to the silicon atom). Most interactions here are hydrophobic. If what keeps the analyte to the solid phase is hydrophobic interactions due to their non-polar nature, it would make sense than polar substances are washed out or filtered out first. However, these types of non-polar adsorbents are not really selective.
So the main difference between normal and reversed phase extraction is that normal uses a polar adsorbent and reversed uses a non-polar adsorbent. This is reliant on what type of analyte is being extracted.
What is ion exchange SPE?
Ion exchange SPE uses a charged or partially charged adsorbent (like ions) to retain charged analytes depending on their ionisation states, via electrostatic attractions.
What are functionalist adsorbents?
Adsorbents with specific functional groups to extract specific classes of compounds, via conducting specific reactions in the SPE cartridge.
Different phases (normal, reversed, ion exchange) can be added together in different ways, like mixed mode, layered and stacked phases (for a higher efficiency of extraction). How do they differ from one another?
Mixed mode- 2 different phases are simply mixed together in the cartridge.
Layered- 2 different phases are stacked on top of one another in the cartridge.
Stacked phases- 2 different phases are conducted in different cartridges, but these cartridges are stacked on top of each other.
What are the key benefits of SPE?
> Washing steps removes interferents.
Removing interferents gives an increased sensitivity when conducting masses spectrometry.
We can divide the sample up into different fractions (water soluble, soluble in methanol).
Concentrating analyte in sample for analysis to get back a clear and loud reading.
Why may we want to concentrate or isolate proteins in biological samples?
Concentrate and isolate proteins found in the plasma and the blood. If there is an unwanted function or no function at all, we can determine whether it is because there had been a mutation in DNA, by looking at protein structure.
Concentrating and isolating proteins from urine. Proteins should not be found in the urine, and by isolating the proteins found in the urine, we can determine what the problem may be (kidney disease, urine infection).
To analyse proteins, we may want to precipitate them. Ways of precipitating them is by using their chemical properties- changes in pH, temperature, introduction is more salts, heavy metals salts.. etc. Why may these not be the best way to precipitate protein?
While the factors stated may help with precipitating a protein, these factors can also denature a protein. Precipitating a protein would be meaningless if we cannot analyse it because it has denatured.
What is a common way of extracting proteins from biological samples?
For a sample like plasma, we first add a solvent to the solution. This solution can be acetonitrile (quite polar). The idea is the protein cannot interact with the acetonitrile, so precipitates out to form a solid, or a semi-mass of solid.
Centrifuging is next, and is basically separating components based on their size, shape and density, by spinning the solution at high speeds. In this case, the precipitated proteins will be at the bottom due to its higher density and the supernatant (liquid lying at the top after centrifuging) would be the plasma. The plasma would then go on to be further analysed.
What is green extraction?
Green extraction is the design of extraction processes, done in a way to reduce energy consumption, using alternative solvents (that is less damaging to the environment, less toxic and more safe), renewable natural products, while ensuring the whole process is high quality and safe.
