Blog

• #hplc_method_development
• Controlling selectivity-Additional factors-2.0 - temperature
  

  In the earliest posts we saw the how solvent concentration and type are powerful tools for controlling separation and also discussed these variables alone may not be sufficient for achieving the desired sample resolution. In such cases chromatographers may seek to employ other parameters to adjust selectivity. In the last post the discussion was focused on the influence of pH on selectivity. In this blog the focus will be on the effect of temperature on selectivity.
• Controlling selectivity-Additional factors-1.0 - pH
  

  Although varying solvent concentration and type are powerful tools for controlling separation, they may not be sufficient for achieving the desired sample resolution. In such cases the chromatographers may use other parameters to adjust selectivity viz. pH; temp.; column selectivity - C8/C18/PH/CN/NH2. In this blog the focus will be on pH mobile-phase.
• Controlling selectivity-Solvent role-2.0
  

  In this post the focus is on the second approach, blending of solvents, to arrive at a good separation of difficult-to-separate peak pairs in HPLC method development.
• Controlling selectivity-Solvent role-1.0
  

  The fundamental resolution equation in particular will make it easier to assess how different selectivity-controlling variables will affect a method's performance. An overview of the variables that control chromatographic separation is depicted in the image below. In the current post the focus will be on the role of solvent in controlling chromatographic separation in particular retention factor and selectivity during method development process.
• Measuring quality of chromatogram- Tools 3.0
  

  The chromatogram observations like retention factor, peak asymmetry, peak shape, column plate number, and related aspects are important, but the main purpose of chromatography is to achieve a separation that is reasonable. To ensure whether the separation is satisfactory one need to add one more measurement to the tool box namely the resolution.
• Measuring quality of chromatogram- Tools 2.0
  

  In current post the discussion is on other features viz. peak asymmetry, peak shape, column plate number. and related aspects.
• Measuring quality of chromatogram – Tools 1.0
  

  Getting the controls that quantitatively measure the separation's quality is a crucial step in the method development. In the entire LC method development process, starting from the initial injection to the final stages of method validation and application, the quantitative measurements of separation quality is crucial.
• Selecting the tools
  

  Starting out with a sound strategy is one of the most important aspects of minimizing separation issues. Selecting the right tools is very critical to achieve the best results. By looking at the cause-and-effect diagram given below one can understand the factors that influence chromatographic separation.
• HPLC Method Development – prelude
  

  Many chromatographers develop HPLC separations on a daily basis. Today, chromatographic separation and how it varies depending on the sample and experimental conditions have a good practical understanding.

• #chiral_chromatography
• Chiral chromatography
  

  Chiral chromatography is now widely understood and applied in daily life. The correct phrase, however, is “enantioselective chromatography”.
• Chiral HPLC separation: strategy and approaches
  

  The basic aspect is to provide the right chiral environment where by the enantiomeric pair become distinguishable.
• Direct chiral HPLC separation on CSPs
  

  The most convenient and most popular analytical methodology to assess chiral purity is the direct separation of enantiomers on so-called chiral stationary phases (CSPs).
• Chiral selectors
  

  A wide range of chiral compounds have been examined as chiral selectors with regard to their ability to separate enantiomers using chromatography as part of the development of CSPs.
• Polysaccharide-based CSPs
  

  Surprisingly, chiral chromatography could not be performed in 1980 because there was no single chiral stationary phase on the market. But as the 1980s came to a close, chiral chromatography began to get more and more attention, thanks in large part to the efforts of Okamoto's institute in Japan, Pirkle and Armstrong's teams in the US, Schurig and König in Germany, Lindner in Austria, and Francotte in Switzerland.
• Cyclodextrin-based CSPs
  

  Cyclodextrin-based CSP belongs to the cavity type. Armstrong is considered the father of cyclodextrin-based CSPs.
• Donor-Acceptor (Pirkle)-type CSPs
  

  William H Pirkle developed these CSPs and are based on the ionic or covalent attachment of one enantiomer of an amino acid derivative to aminopropyl silica.
• Protein-based CSPs
  Proteins are complex, high-MW biopolymers composed of L-amino acids and possess ordered 3D-structure. They are known to bind /interact stereoselectively with small molecules reversibly, making them extremely versatile CSPs for chiral separation of drug molecules. Number of CSPs has been developed by immobilizing proteins.
• Separating mirrored molecules
  

  In nature, most chiral molecules only appear in one specific enantiomeric form. Moreover, the handedness of the enantiomer can have a powerful effect on how that molecule behaves.

• #drug_withdrawls
• Thalidomide
  

  Thalidomide was a racemic therapeutic and prescribed to pregnant women to control nausea and vomiting. The drug was withdrawn from world market when it became evident that the use in pregnancy causes phocomelia (clinical conditions where babies are born with deformed hand and limbs).
• Dilevalol
  

  Dilevalol is the (R,R)-diastereoisomer of labetalol. Labetalol has been in clinical use since 1977 for the treatment of hypertension. Soon after its introduction in the market in 1989, reports of hepatotoxicity began to appear. Although labetalol is known to manifest hepatotoxicity, the rate of incidence appeared to be lower than that associated with dilevalol. Hence dilevalol was withdrawn from market in 1990
• Terodiline
  

  (±)-Terodiline, the antianginal agent, perhaps represents the best authenticated example of a drug that had to be withdrawn from the world market as consequence of proven stereospecific toxicity. Terodiline has a close similarity to prenylamine from a structural and pharmacological view point.
• Prenylamine
  

  (±)-Prenylamine, an antianginal agent, was introduced in the market since early 1960. Reports associating Prenylamine with prolongation of the QT interval, ventricular tachycardia, and ventricular fibrillation started to appear. Some of these events had a fatal outcome and the drug was withdrawn from the market world-wide in 1988.

• #cMOTW
• Morphine
  

  The opioid agent morphine is a millennia-old-unichiral drug. Morphine has a highly demanding chemical structure. It is a pentacyclic 3°amine (alkaloid) with 5 stereogenic centers and exists in 32 stereoisomeric forms (2n = 25= 32 stereoisomers; n being number of stereogenic centers). But the desired analgesic activity resides exclusively in the natural product, the (-)-enantiomer with the configuration ((5R,6S,9R,13S,14R).
• Quinine
  

  Most fascinating old chiral drug, from historical point of view, is Quinine. It is known that by early 1600s quinine was being used by South American natives, in Peru, Ecuador, as a crude preparation from the bark of the Cinchona tree for the treatment of malaria
• Ascorbic acid
  

  L-(+)-Ascorbic Acid is another old chiral drug from natural source. It is a water-soluble vitamin (Vitamin C).
• Levofloxacin
  

  Levofloxacin, is the S-enantiomer of the previously marketed racemic antibacterial (±)-Ofloxacin. This is referred to as a chiral switch.
• Ethambutol
  

  (S,S)-(+)-Ethambutol is powerful and selective antitubercular drug. It is a classical example of an old drug that was introduced for clinical use in its chirally pure form.
• Esomeprazole
  

  Esomeprazole is the (S)-(−)-enantiomer of (±)-omeprazole. It is a chiral switch. Its chirality stems from the presence of stereogenic center at the sulfur atom of methylsulfinyl bridge between 1H-benzimidazole and the pyridine moieties.
• Salbutamol
  

  Salbutamol is formulated as a racemic mixture of the R- and S-isomers. Bronchodilator activity resides in R-(-)-Salbutamol (eutomer); S-Salbutamol (distomer) is inactive. The active (R)-enantiomer undergoes significantly faster metabolism than the inactive (S)-(+)-enantiomer after oral administration.
• Dexibuprofen
  

  The switch of ibuprofen is based on chiral inversion; (±)- and (S)-ibuprofen can be viewed as being essentially bioequivalent’. Ibuprofen carries a stereogenic center in the propanoic acid side chain and exists as a pair of enantiomers. Dexibuprofen is (S)-(+)-Ibuprofen.
• Propoxyphene
  

  Propoxyphene is a classical example of a synthetic chiral drug where the enantiomers of a chiral drug exhibit desirable but different therapeutic activity. Example of such biological behavior is exemplified by some of the natural products as well (e.g., quinine and quinidine) with anti-malarial and anti-arrhythmic activity.
• Levobupivacaine
  

  Bupivacaine is a racemic drug. This an example where the distomer shows toxic effect and the need for a switch. The chiral switch of (±)-Bupivacaine, (S)-(–)-enantiomer levobupivacaine, has been developed successfully as a safer local anaesthetic that has the same therapeutic indications as the racemate.
• Escitalopram
  

  The selective serotonin-reuptake inhibitor activity resides primarily in the (S)-enantiomer (eutomer) while the R-enantiomer is 30-fold less potent (distomer). By and large, (S)-citalopram appears to have advantages over (±)-citalopram and is a good example of the potential benefits of enantiopure drugs.
• Levocetirizine
  

  Levocetirizine, is chiral switch cetirizine, employed for allergies. It is a third generation, non-sedating, selective histamine H1 receptor antagonist, with antihistamine, anti-inflammatory activities.
• Indacrinone
  

  Indacrinone has one stereogenic center and exists as enantiomeric pair. (R)-(+)-enantiomer, the eutomer, is diuretic whereas the mirror-image version (S)-enantiomer antagonizes side effect of the eutomer. Indacrinone is an interesting example of an enantiomeric pair exhibiting therapeutic advantage. In this case both enantiomers contribute, in different ways, to the overall desired effect.
• L-Dopa
  

  L-dopa, the (S)-enantiomer, is a chiral drug with one stereogenic center. It exists as a pair of enantiomers. The initial use of racemic dopa for the treatment of Parkinson’s disease resulted in a number of adverse effects viz. nausea, vomiting, anorexia, involuntary movements and granulocytopenia. The use of L-dopa resulted in reducing the required dose, and adverse effects. The pharmacological activity resides in the (S)-enantiomer where as the (R)-version harbors adverse effects.
• Beta-Blockers
  

  Beta blockers, also spelled β-blockers, used as antihypertensive agents (lowering blood pressure), represent a class of chiral drugs that are marketed as racemates since the distomer exhibits no undesirable adverse effects. Three most important β-blockers viz. propranolol, atenolol, and metoprolol are marketed as racemic mixture.
• Levamisole
  

  Levamisole is used as anthelmintic agent to get rid of the intestinal worms. It is (S)-(-)-enantiomer of tetramisole. Tetramisole carries one chiral stereogenic center and exists as an enantiomeric pair. The anthelmintic activity resides in the (S)-(-)-enantiomer, levamisole. The (R)- isomer harbors undesirable side effects including vomiting. Hence the (S)-enantiomer was marketed as levamisole to replace the racemic tetramisole to treat intestinal worm infestations.

• #naming_system
• Naming enantiomers: the left-(or right-) handed?
  

  Enantiomers are pair of molecules that exist in two forms that are mirror images of one another but cannot be superimposed one upon the other. They are also referred to by chemists as chiral twins or handed molecules. Each twin is called an enantiomer. Naming of enantiomers is important to understand ‘which structure refers to which enantiomer?’.
• Fischer Projection: hassle free way to depict a stereoformula in 2D projection
  

  One of the major problems in organic chemistry is the representation of three-dimensional structures in a two-dimensional media (viz. sheet of paper, blackboard, etc.). Chemists sometimes represent structures for chiral molecules with two-dimensional formulas called Fischer projection formulas. These two-dimensional formulas are a quick way to show three dimensions without the hassle of having to draw 3-D.
• D-/L- system naming: the (left-) or (right-) hand side?
  

  D-/L-system of naming is still employed to designate the configuration of amino acids and sugars. So it is important to understand D-/L- system of naming enantiomers.
• Erythro- and Threo- prefixes: the (same-) or (opposite-) side?
  

  Other molecules with two different stereogenic centers, not necessarily on adjacent carbon atoms, are frequently designated by the prefix threo- and erythro-. These are interpreted as meaning that two identical (or similar groups are on the same side (erythro–) or on opposite side (threo–) of the carbon chain when expressed as a Fischer projection.
• Cis-trans and E-Z notation: choose your side
  

  When two atoms are connected by a single bond the barrier to free rotation about this bond is quite small. On the contrary, when two atoms are linked by multiple bonds then the barrier to rotation is much higher and where such compounds exist in two forms the passage from one to the other requires so much energy that each isomer has a stable and independent existence.
• Atropisomers: things are tight, single bond won't rotate
  

  Atropisomers are stereoisomers resulting from hindered rotation about single bonds where the steric strain barrier to rotation is high enough to allow for the separation of the conformers.
• The meso compounds: finding plane of symmetry
  

  Take note that the molecule (III’) has a horizontal plane of symmetry ( internal mirror plane indicated by dotted line) dividing the molecule into two identical parts such that one is reflection of the other.. The achiral, optically inactive stereoisomer (III) is called the meso-form, and it holds a diastereomeric relationship with the optically active stereoisomers (I and II).