Chromatography And Analysis Flashcards
Summary of Phase I Metabolism
- Almost any chemical transformation can be catalyzed by enzyme systems, mainly in the liver
- These systems developed primarily to process endogenous compounds and dietary xenobiotics
- Many xenobiotics are substrates for a number of different Phase I reactions, e.g. diazepam
- Phase I reactions can be used to activate or alter the activity of drugs, but are primarily employed to prepare xenobiotics for Phase II processes
Cytochrome P450 Oxidations
O-dealkylation
Codeine –> (CYP2D6) Morphine
H3CO –> HO
Summary of Phase II Metabolism
- Reactions are generally with Phase I products
- Common requirement for an energy rich or “activated” intermediate
- Products are generally more water soluble and are ready for excretion
- There are many complementary, sequential and competing pathways
- Together with phase I metabolism, this is a coupled interactive system interfacing with endogenous metabolic pathways
Glucuronidation
ROH - not excreted in large amounts
UDP-glucuronosyl transferase - not water soluble
Glucuronide conjugate (B) - water soluble, excreted in large quantities
Glucuronide will be hydrolysed by enzymes
by increasing stationary phase
retention can be increased
Chromatography
- A technique used to analyse and separate a mixture of compounds into individual components
- Stationary phase versus mobile phase
- Traditional view is that separation is achieved by the distribution of molecules between a stationary phase and a mobile phase
- Many different chromatography experiments
- Chromatography can be linked with other methods of analysis (hyphenated techniques)
Chromatographies
- Adsorption chromatography, e.g. TLC, column
- Stationary phase is solid, e.g. silica, alumina
- Partition chromatography, e.g. HPLC
- Both stationary and mobile phase are ‘liquids’
- Ion-exchange chromatography
- Stationary phase is an ion-exchange resin
- Gel permeation chromatography
- Size-exclusion chromatography
- Affinity chromatography
- Ligand immobilised on solid, stationary phase
Thin Layer Chromatography (TLC)
qualitative technique
Rf of compound 1 = X1/Xs
• Silica stationary phase: non-polar compounds eluted first
• Non-polar versus polar solvents, reverse phase TLC
• Visualisation using UV, iodine, sulfuric acid, molybdate
what phases used mostly in TLC
polar mobile phases
‘Lab-Scale’ Column Chromatography
- qualitative technique
• Sometimes viewed as a black art
• Typically the separation of organic compounds on an inert stationary phase, e.g. silica or alumina
• Column packing method can affect results
• Gravity columns driven by the solvent head
• Flash column chromatography is driven by the application of pressure to the solvent head
• Must do TLC first before starting column and carry out TLC throughout to detect eluted compounds
• Excellent technique but preparative, not analytical
Analytical Techniques
- Used for the quantitative analysis of components of a (complex) mixture e.g. a pharmaceutical formulation
- Analytes are typically separated based on differing affinities for the stationary and mobile phases
- Mobile phase can be a gas e.g. gas chromatography (GC) or a liquid e.g. high performance liquid chromatography (HPLC)
- HPLC is widely used in pharmaceutical analysis
Retention factor k
- measures how long the material is retained in the column
- retention factor is affected by the mobile stationary phase
• Independent of column length or flow rate
• Need to measure column dead time to - using dead time you can get your retention factor
Separation Factor a
- Identifies when peaks elute relative to one another
- Ratio of the retention factors (k2 > k1)
- Separation factor >1 to achieve separation
- Governed primarily by the stationary phase selection
a =
k2 / k1 = tr,2 - to/ tr,1 - to
Column Efficiency (Plate Number) N
• Represents narrowness of the peak
• Columns with large values of N give narrower peaks
-the narrower the peak, the better the separation
-the larger the number of plates you have, the higher the amount of separation
-the greater the depth of stationary phase , the better the separation
-the more densely the column in HPLC is packed, the greater the efficiency
what is plate number and equation
each layer is called a plate
N = 5.54 (tr/w0.5)2
Asymmetry Factor As
- In practice, peak shapes are not gaussian and have ‘tails’
* As 0.9 → 1.2 acceptable; As >1 tailing, As <1 fronting
When does tailing increase
tailing increases when the column becomes worn out, the more tailing you get the more likely the peaks are to overlap
Resolution R
- Ideal valley between peaks should return to the baseline
- R is a quantitative measure of separation
R = (square root)N / 4 x k/k + 1 x a-1/a
don’t need to remember the formulae, just understand it
• To achieve resolution:
- Peaks should be retained on the column (k > 0) - need to have a decent retention time
- Peaks have to be separated from one another ( > 1) - need a good value of alpha
- The column must develop some min value of N - the more efficient the column, the greater the resolution. the older the column, the older the mobile phase
Varying Conditions
resolution affected by the efficiency of the column and mobile phase
- initial
- vary k’ - if retention time is increased
- increase N - brand new column, more narrow peaks
- increase a - combination of a good efficient column and mobile phase
Gas Chromatography (GC)
- turns material into gas
-used to detect volatile agents but not small molecules
• Sample is injected on to column (i.d. 0.1–0.5 mm 60 m)
• The column is heated to release the volatile components
• The mixture is separated on the column and various methods used to detect each component
