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COMPLETE SUMMARY OF THE 2ND YEAR BACHELOR PHARMACY
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WHOLE SUMMARY OF ALL COURSES GIVEN IN THE 2ND YEAR BACHELOR OF PHARMACY AT THE UNIVERSITY OF GRONINGEN
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S4251512 SUMMARY 2nd YEAR PHARMACY S.K. TimmerImportant Lecture Notes Instrumental Analysis (WBFA19001)
Lecture 1: Chemical Separations part I: Chromatography Chapter 23 (dr. P. de Haan)
Chemistry → Derived from Alchemy → Synthetic Chemistry (making molecules)
Scheikunde → the art of separating (separating molecules)
Quantitative Conversion: Pb(s) –(Quant)→ Au(s)
Reagents A and B form a product C and a by-product D (which can be toxic); catalyzed by catalyst X (metal)
Natural synthesis in plants:
- Secondary Metabolites → f.e. Morphine from papaver somniferum, but mixed with 100s of different compounds!
How can a specific compound be isolated from its matrix?
- Separation based on different physical-chemical properties → Preparative-scale separation & Analytical-scale properties
Liquid-Liquid Extraction
On the left a beaker with 2 liquids; water at the bottom and oil (lower density on top of water)
Dissolve red spheres into water and wait; following “Le Chatelier” some of the molecules move toward
Oil phase and some in water phase → it will eventually reach equilibrium
Kd = equilibrium constant; for equation see left.
If this is performed in N-octanol (oil) and water (aqueous)
Log(P) = partition coefficient → can use this to predict if drug is taken up in the body (see bioanalysis)
HIGHER log(P) it has a better permeability over BBB → better uptake in the body
[𝑆]𝑜𝑐𝑡𝑎𝑛𝑜𝑙
𝐿𝑜𝑔(𝑃) = log( [𝑆]𝑤𝑎𝑡𝑒𝑟 )
Hydrophilicity: “LIKES” water → POLAR; H-bridge donor/acceptor; charge; strong dipoles
(Sucrose; Adenosine Triphosphate)
Hydrophobicity: “FEARS” water → NON-POLAR; hydrophobic interactions; van-der-Waals-forces
(Benzene; Hexane; Cholesterol) HIGHER LOG(P)
Drug that is a Weak Acid (pKa around 4):
- In water with Hexane the distribution is equal to neither acid or base
- In HCL the behavior is THE SAME
- If NaOH is the water-phase → the weak acid HA (slightly hydrophobic) reacts with OH
And forms A- ion (conjugate weak base) → more hydrophilic due to charge
Almost all molecules moved to NaOH phase
Neither acid nor base:
- Hexane (organic); Water (aqueous) → some stay in oil phase and some in water
HCL or NaoH it does NOT CHANGE the distribution of those molecules
Weak Base (pKa around 10)
- Weak Base may absorb proton to form HB+ → more ionized and more hydrophilic
- In neutral an equal distribution; in NaOH-solution the situation is the same; in HCL the B molecules are positively charged →
more in the aqueous phase
Compounds partition in two immiscible solvents Equilibrium between aqueous phase + non-aqueous phase
pH of aqueous phase affects weak acids and bases
Paper Chromatography
Separate dyes by affinity for paper
Stationary Phase: Paper Mobile Phase: Water
5 samples applied to bottom of paper; Paper transferred into water;
spots dissolved in water and dragged along to top of paper
Green dot has split into blue and yellow dot so it shows it’s a mixture
Separate compounds on the basis of affinity for the stationary phase!!
Thin-Layer Chromatography (TLC)
Same principle as paper chromatography
Glass slide with silica coating (SiO2)
Stationary Phase: Polar, Silica (SiO2) Mobile Phase: Non-Polar (f.e. hexane)
Separates compounds based on interaction with silica
High retention means it has a LOT of interaction with the stationary phase
Low Retention means that it does NOT have a lot of interaction with the stationary phase → further travel
Column Chromatography
Used to separate compounds in a mixture, e.g. to purify drug molecule from by-product
Preparative-scale separation (sample: grams or kilograms)
Stationary Phase: POLAR, silica (SiO2) Mobile Phase: NON-POLAR (hexane) NORMAL-PHASE CHROMATOGRAPHY
Image (left) → glass column filled with packing (usually silica); filter, outlet and inlet
Samples will have different interactions with stationary phase → more interaction = more retention (slowing down)
Retention is the act of retaining; more interaction with the stationary phase means more retention (sticking)
Silica particles is a POLAR stationary phase, hexane is a NON-POLAR mobile phase
Different types of chromatography:
- Due to Adsorption (due to electrostatic interactions, positive and negative charge)
- Due to Partition (e.g. between 2 liquid phases, one of which is stationary)
- Due to Molecular Recognition (e.g. in affinity chromatography; antibodies)
- Due to Molecular Exclusion/Inclusion (e.g. in size exclusion chromatography)
Analytical-Scale Separations: Sample size <<< 1 mg (and far lower!)
Column-based Separations: Gas Chromatography (GC) & Liquid Chromatography (LC)
Qualitative Analysis: WHICH compounds are present in a sample?
Quantitative Analysis: WHAT is the concentration of that compound?
1
,S4251512 SUMMARY 2nd YEAR PHARMACY S.K. Timmer
The Chromatogram: Y-axis: Detector Response X-axis: Time
Separation inside the column, different compounds at different timepoints due to retention
At some point you inject sample, detector at the end of column detects the response (mostly UV-detector)
4 peaks are seen for CH4, Octane, Nonane and an Unknown sample
- tM: dead time → time required for an non-retained compound to pass from injector to detector
- tR: retention time of a given compound (time between injection and a peak)
- 𝑡′𝑅 = 𝑡𝑟 − 𝑡𝑚 → adjusted retention time of a given compound (retention time minus dead time)
𝑡′𝑟2
- 𝛼= : relative retention between compound 2 and compound 1 (this is related to selectivity)
𝑡′𝑟1
Can tell something about the selectivity of the method
𝑡𝑟 −𝑡𝑚 𝑡𝑖𝑚𝑒𝑠𝑝𝑒𝑛𝑡𝑖𝑛𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦𝑝ℎ𝑎𝑠𝑒
𝑘′ = = → capacity factor
𝑡𝑚 𝑡𝑖𝑚𝑒𝑠𝑝𝑒𝑛𝑡𝑖𝑛𝑚𝑜𝑏𝑖𝑙𝑒𝑝ℎ𝑎𝑠𝑒
→ describes how much this molecule has spent in stationary and mobile phase → refers to partition
𝑛𝑢𝑚𝑏𝑒𝑟𝑜𝑓𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠𝑖𝑛𝑡ℎ𝑒𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦𝑝ℎ𝑎𝑠𝑒 𝑡𝑖𝑚𝑒𝑎𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑𝑠𝑝𝑒𝑛𝑑𝑠𝑖𝑛𝑡ℎ𝑒𝑠𝑡𝑎𝑡𝑢𝑜𝑛𝑎𝑟𝑦𝑝ℎ𝑎𝑠𝑒 𝐶𝑠 𝑉𝑠
Corresponds to = = = 𝑘′
𝑛𝑢𝑚𝑏𝑒𝑟𝑜𝑓𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠𝑖𝑛𝑡ℎ𝑒𝑚𝑜𝑏𝑖𝑙𝑒𝑝ℎ𝑎𝑠𝑒 𝑡𝑖𝑚𝑒𝑎𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑𝑠𝑝𝑒𝑛𝑑𝑠𝑖𝑛𝑡ℎ𝑒𝑚𝑜𝑏𝑖𝑙𝑒𝑝ℎ𝑎𝑠𝑒 𝐶𝑚 𝑉𝑚
CS: Concentration of Compound in the stationary phase VS: Volume of the stationary phase
CM: Concentration of Compound in the mobile phase VM: Volume of the mobile phase
𝑡′𝑟2 𝐾2
K → partition coefficient → relates to the relative retention (alpha): 𝛼= = 𝐾1 → also ratio between the partition coefficients
𝑡′𝑟1
Chromatographic Efficiency – Peak Width: Ideally have a Gaussian Shape → Bell-shaped curve
Width of the bell-curve is described by the standard deviation; the peak height is h
Resolution of 2 peaks: Separation of the 2 peaks
3 sigma → little dimple in the middle so not fully separately distinguishable
Separation of more than 6 sigma. The peaks are mostly separated → full separation of 2 molecules
∆𝑡𝑟 0.589∆𝑡𝑟
𝑅𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 = =
𝑤𝑎𝑣 𝑤1/2𝑎𝑣
Wav is the baseline-width; but difficult to measure so therefore often the half-width is measured (see right equation)
HIGHER resolution is desired
If you have an injection of 1 compound at the left with a very concentrated band;
when moving through the column it BROADENS → due to diffusion
Standard deviation of band: 𝜎 = √2𝐷𝑇
D: Diffusion Coefficient of that compound (m2/s); see table 23-1 DC Harris
T: time (minutes)
Efficiency of Separation
- The exchange of molecules between the stationary and the mobile phase is not infinitely fast. It takes some time for molecules to
travel between the mobile and the stationary phase and back. Each exchange is called a Theoretical Plate, according to
terminology coming from distillation.
- If a column is 500 mm long and a compound undergoes 500 exchanges between mobile and stationary phase, then the column has
500 Theoretical Plates and the Height Equivalent to a Theoretical Plate (HETP) is 1 mm
Standard Deviation of band: 𝜎 = √2𝐷𝑡
Variance: 𝜎 2 = 2𝐷𝑡
𝜎2
Plate Height: 𝐻 = → X = distance travelled at linear velocity μx
𝑥
The smaller the plate height, the narrower the bandwidth
𝐿 𝐿𝑥 𝐿2 16𝐿2 16𝑡𝑟2 𝑡𝑟2
Plate Number: 𝑁 = = = = = =
𝐻 𝜎2 𝜎2 𝑤2 𝑤2 𝜎2
High plate numbers are desired; the easiest way is to elongate the column 𝑅𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛~√𝑁
𝑩
Van Deemter Equation for Plate Height 𝑯 ≈ 𝑨 + + 𝑪 ∗ 𝝁𝒙
𝝁𝒙
In the graph is seen that the A-term is CONSTANT for all flow rates
The B-term starts HIGH and DROPS to low value
The C-term starts at 0 and GOES UP at flow rate
A-term: Multiple Paths
Independent of linear velocity μx
Reduce A by improving column packing and avoiding turbulent flow
Each of the 3 molecules in the pictures follows a different route through the packed column → each has a different retention time
MAKE SURE column is homogeneously packed and there are no cavities inside to make sure most routes are the same length
B-term: Longitudinal Diffusion
Decreases with linear velocity μX
Diffusion distance increases with time → limit analysis time → higher flow rates
Diffusion is slower in more viscous media → so less diffusion
Band becomes broader due to diffusion (which is dependent on time); the FASTER the separation the better
C-term: Mass Transfer
Increases with linear velocity μX → mobile phase moves from left to right
Molecules try to equilibrate with stationary phase but are stuck to it, mobile phase keeps on flowing
When detached from the mobile phase they redissolve in a part of the mobile phase that does not contain any molecules yet
Stationary phase is “lagging” behind on the mobile phase → hop on and hop off the mobile phase
It takes time for a molecule to transfer from mobile to stationary phase
Effect of particle size: Smaller Particles have a larger interface area → faster mass transfer
Decreasing Plate Heigth; BUT more pressure needed
Fronting: Overloading (too high concentration of analyte) leads to assymetric peaks due to early elution
Tailing: Stronger retention of some molecules leads to later elution
Often interactions with bare Si-OH groups on Si-C18 columns
o Chromatography based on partitioning of molecules between a “non-moving” stationary phase and mobile phase (K)
o The number of partitioning events (equilibrations) is called Number of Theoretical Plates (N)
o Resolution between molecules by chromatography is due to selectivity (K) and efficiency (N)
o Band Spreading (sigma squared) limits resolution
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,S4251512 SUMMARY 2nd YEAR PHARMACY S.K. Timmer
Lecture 2: Chemical Separations part II: High Pressure Liquid Chromatography (dr. P. de Haan)
Chapter 25 & 26 (up till 26-5)
Chromatography separates compounds with retention
Partition over stationary phase/mobile phase determines retention
Liquid Chromatography: Solid Stationary phase in Column, Liquid Eluent
- Normal-Phase Liquid Chromatography: Polar Stationary Phase (e.g. SiO2); Non-Polar Mobile Phase (e.g. hexane)
- Reversed-Phase Liquid Chromatography: Non-Polar Stationary Phase (e.g. Si-C18); Polar Mobile Phase (aq buffer + add)
In columns (glassware) or analytical instrumentation (this lecture)
2 solvent bottles on the top; through the degasser and goes on to the pumps; injection valve
Through the filter and on to the columns where the separation happens
After the column it goes to the detector (communicates with computer) and flows to waste’
ELUENT goes into the HPLC-system
Column -> Separation happens here
LOW plate height and HIGH plate number → better separation efficiency
Guard Column removes most stuff you don’t want in the column (dust e.g.)
The main column is filled with small particles and there is a sieve at the bottom
𝑩
𝑯 ≈ 𝑨 + + 𝑪 ∗ 𝝁𝒙
𝝁𝒙
Plate height depends on the A, B and C factor in the van Deemter equation! Smaller particles means there is a lower A-term
𝑭𝜼𝑳
𝑷=𝒇 → F (volumetric flow rate); n = viscosity; L = column length; r = column diameter; dp = particle diameter; f = factor
𝝅𝒓𝟐 𝒅𝟐𝒑
The required pressure increases with the particle diameter squared
Reversed-Phase LC (RP-LC): Stationary phase: non-polar → silica with immobilized octadecyl (C18 chains)
Mobile phase: polar → aqueous buffers (water with acetate buffer)
OH-groups are the silanol-groups to which a different compound with a long hydrophobic tail attached
So hydrophobic tails everywhere on the particle → the whole surface covered with hydrophobic tails → “non-polar)
Eluent:
Elution process → the process of washing things off the column
Eluent is the mobile phase
Blue triangles are on the stationary phase and the solvent goes by this area and pick up some of the solutes
RP-LC: LESS POLAR solvents elute more strongly (less polar solvents mixed in with aqueous buffers)
This is done to speed the elution process → these are called modifiers (acetonitrile, methanol)
Lower retention and faster elution
Eluent composition e.g.: A: aqueous buffer B: Acetonitrile (weakens interaction stationary phase)
On the right it is seen that from 60 to 70% B (more acetonitrile, less water) and up to 90% B and 10% water
The elution speeds up; separated on a standard hydrophobic Silica-C18-column
In the chromatograms on the right they have used fixed concentrations of the modifier → isocratic elution
Isocratic elution (same strength) → constant % vol/vol
Gradient Elution: Same compounds, %B increased gradually in this separation
Start with low concentration of modifier, and it gradually increased! → FASTER ELUTION of strongly retained species
Gradient elution takes less time → MORE SENSITIVITY (higher and less broad peaks)
Pumps: High pressures up to 400 bars (normally 80-100 bars)
Delivers constant flow of eluent or eluent mixture to column
Two pistons move back and forward and pump liquid through the outlet valve (1 in = 1 out)
Injection Valve: 6 ports, load sample in syringe, and goes through valve into sample loop with fixed volume
Make sure to inject same volume; inject in sample loop and overfill into waste
When knob in the middle is rotated, the sample loop is connected to the pump and transfers everything inside loop into column
Detector: Most common: UV-detector (UV/Vis spectrophotometer with flow cell)
The eluate comes from the column, goes through a Z-shaped channel
The light goes through the pathlength and reaches the detector (see spectrometry lecture)
The concentration of a compound is directly proportional to its absorption in the flow cell (Lambert-Beer)
Alternatives: Mass Spectrometry, Fluorescence, Electrochemical detection etc.’
Data Interpretation
According to Lambert-Beer the absorbance is proportional to the concentration of the analyte
Integrate peak to obtain the AUC → peak area
We need analyte concentration calibration curves to obtain quantitative information
Calibration Curve: Make 6 different concentrations of analytes and analyze these six samples
Plot the Detector Signal against the Concentration of the Analyte (C-analyte)
Linear Regression is used to find the correlation, C-unknown is calculated from the linear regression
In RP-HPLC hydrophobic molecules have the MOST RETENTION and ELUATE as last!!
Affinity Chromatography: SELECTIVELY BINDS ONE SPECIES (not the others)
First load the sample → wash away all the others → use different buffer to elute all others that is trapped → 1 peak of materials
Ion-Exchange Chromatography:
E.g. Negatively charged stationary phase will retain negatively charged ions on its surface
If you have a strongly charged stationary phase, it can be eluted with high-ionic-strength buffers
First inject the sample and wash away everything and wash away everything that’s not bound
Slowly increase the concentration of NaCl to elute the different fractions from the column based on retention (charge)
In the end a high salt wash to wash away everything that is still present in the column
RP-HPLC with ion pairing:
Silica particles with hydrophobic (OH) tails; soap like molecules (surfactants) charges interact with positive charges and keep them at the
stationary phase for some times. This will cause the positively charged species to have a higher retention
Anionic surfactant (hydrophilic head; hydrophobic tail) present in stationary phase
Stronger retention of cations, mixed with hydrophobic retention
3
, S4251512 SUMMARY 2nd YEAR PHARMACY S.K. Timmer
Lecture 3: Chemical Separations Part III: Gas Chromatography Chapter 24 (dr. P de Haan)
Chromatography separates compounds with retention, so compounds have more or less retention inside a column
The retention is caused by partition over a stationary and mobile phase
Liquid Chromatography: Stationary Phase: Solid Mobile Phase: Liquid (“eluent”)
Gas Chromatography:
Stationary Phase: Liquid (film) or Solid (film or fully packed)
Mobile Phase: Gas
Circular column, in blue is a thin film of the stationary phase that is coated on the walls
Analytes move through the column in the gas phase → “Boiling point is highly relevant” → e.g. not for proteins (no boiling point)
GC can not be done in a glass column, only with instrumentation.
Gas Chromatograph:
Injector oven has a septum (slice of rubber which needle can go through) → mobile phase (carrier gas) flows through very long column in a
column oven → afterwards goes to detector, which detects molecules that may/may not be inside the column.
Column sits inside a column oven which is temperature controlled → VERY important for separation of the molecules
Columns:
WCOT (Wall-coated open tubular column) long open capillary open on inside, and thin film of liquid on the wall
SCOT (Support-Coated Open Tubular Column) → Solid Support with stationary Liquid Phase
PLOT (Porous-layer open Tubular Column) → Stationary Solid-Phase Particles
30 m long SiO2 capillary with an inner diameter of 0,1-0,53 mm
Stationary Phase Material:
Dependent on the separation that you want to do
Can either be polar or non-polar, depending on application
“Like-dissolves-Like” → more retention of polar compounds use more polar stationary phase
More retention of non-polar compounds, use more non-polar stationary phase
Non-Polar: (Diphenyl)x(Dimethyl)1-xpolysiloxane Polar: Carbowax (PEG = polyethylene glycol)
Mobile Phase: Carrier Gas
Inert Gases: Helium (He) → MOST COMMON; Nitrogen (N2) & Hydrogen (H2)
There is no pump in a Gas Chromatograph since you use pressurized gas source (big tank)
Sample Injection: slightly different than in LC, different forms of sample injections
Compounds used need to be volatile enough to evaporate, otherwise condensation which is not good → QUICK EVAPoration
Split Injection: High flow of carrier gas (102 ml/min) in which the sample is mixed, injector oven at high temperature so all
molecules evaporate quickly. Mix with carrier gas and tiny fraction of carrier gas is injected onto the
column. Most of carrier gas mixed with sample goes to waste. Quick evaporation and “diluted” in carrier
gas. This works best for higher concentration in the sample
Splitless Injection: Lower flow of carrier gas into the injector, syringe is loaded with sample and injected through septum.
Evaporates and most of sample injected onto the column. Sample is given some time to evaporate inside the
hot injector before it goes into the column. More applicable for traces of molecules inside a sample
On-Column Injection: A direct injection of the sample onto the column, inject the needle of the syringe directly into the column.
Make sure that everything is volatile, otherwise it settles in the column. The injector NOT at a high
temperature, so first evaporation needed before moving into the column
Headspace-Gas-Chromatography:
In this case you load a sample (volatile) into a glass vial, which is closed with a cap and a septum
Gas Phase above this sample is called the “Headspace”, if you heat this sample just before injection
Yellow molecules will evaporate and will be in gas phase above the sample, autosampler takes little sample of “headspace”
This gets injected onto the column (with or without split)
→ usually done to avoid matrix effect & other purification steps elimination. Used to analyze dirty or complex samples
Detector: (NOT EXAM MATERIAL)
Ones the compound have made their way through the 30 m column it reaches the detector
Most common detector: Flame Ionization Detector
UV-detector is not possible in GC so therefore the FID has a constantly burning H2-flame
Sample that are coming from the column are mixed with hydrogens and burned there.
Any organic materials, the carbons will react with the hydrogen and the oxygen to form CHO+-ions
These will conduct a small current in the flame → this is measured
Typical Results → Chromatogram
Retention time is plotted on the x-axis and the Detector Response is plotted on the Y-axis’
4 peaks observed: Methane (CH4), Octane, Unknown Sample and Nonane (C9H20)
Methane is a gas, Octane is a liquid and Nonane a liquid with their respective boiling points in the figure
The same terminology used as for LC: Tr = retention time, Tm = dead time; Tr’ = adjusted retention time
Also the same equations hold as for LC
Resolution Improves with Column length!
Resolution is proportional of the square root of the plate number (N); and the N is calculated by L/H
Easiest way to make the N higher is to make a longer column
Highest plate number with low plate height is desired
Column Temperature (regulated with column oven) → important for separation (boiling point)
Isothermal Elution → during 1 run the column is kept at the same temperature (recall isocratic)
Last peaks very broad and asymmetric so not good
Programmed-temperature Elution → the temperature was gradually increased to 250 °C so a very nice separation with sharper peaks!
Leads to better resolution and faster analysis time
Advantages GC:
Fast Analysis, High Resolution due to high efficiency, Sensitivity (ppb level); High quantitative accuracy, Well established
Disadvantages GC:
Limited to volatile substances, not suitable for thermally labile substances & preparative purposes, Most detectors are destructive
4
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