High performance liquid chromatography (HPLC) is a column chromatographic technique that uses a column packed with a sorbent (stationary phase), a liquid (mobile phase) passing through the packed column, sample solution being injected into the flow path of the mobile phase, sample components separating into individual components as they pass through the column, components are detected and presented in a chromatogram as peaks prior to peaks quantification [1,2].
Silica gel-based reverse phase chromatography involves the use of column packed with non-polar stationary phase (bonded silica gel to alkyl chain or phenyl groups) and polar mobile phase (water, Methanol, Acetonitrile etc.) in the analysis. Generally, retention time is proportional to both the chain length of the bonded phase and to the number and type of nonpolar groups on the drug molecule. This type of column has been used in the analysis of a myriad number of therapeutic agents (acidic, basic and neutral types). However, its hydrophilic selectivity, due to residual -OH groups can cause deviation from the true reverse phase behavior leading to basic drug molecules (polar in nature) to be retained longer than expected . Furthermore, basic drug molecules have been observed to show poor peak shapes, broad and tailing peaks, split peaks with silica-gel based columns with exposed silanols-Si-O-H. This is observed mainly with octyldecysilane (C18) chain that prevents reaction of all the silanol groups.
Basic drugs (pKa 6-12) are widely diverse in pharmacological actions but may have similar modes of absorption, distribution and elimination. Typical examples are amitriptyline (antidepressant), atropine (anticholinergic), chlorpheniramine (antihistaminic), chloroquine (antimalaria), chlorpromazine (antipsychotic), cimetidine (anti-ulcer), diazepam (anxiolytic), imipramine (antidepressant), lignocaine (local anaesthetic), morphine (opioid analgesic), nifedipine (antihypertensive), noradrenaline (adrenergic), olanzapine (atypical antipsychotic), oxprenolol (β- adrenoceptor blocker), physostigmine (cholinergic), piperazine (anthelmintic), timegadine (non-steroidal anti-inflammatory), verapamil (antihypertensive) etc. [4-6].
The variations in electronic and stereochemical characteristics of these drugs make them exhibit different ionization that is a pH dependent process. Thus, any change of the mobile phase pH will significantly affect the separation of any of these basic drug mixtures. A basic drug at a low pH will not be poorly retained on an exposed silanols-Si-O-H stationary phase. However, adjusting the pH of the mobile phase to be more basic, the equilibrium will be driven towards suppressing the ionization resulting in poor retention on an exposed silanols-Si-O-H stationary phase. But, it has also been noted that for some strongly basic drugs, the adjustment of the pH cannot be used for the ion suppression since they are fully ionized in the appropriate pH range, thus formation of ion pairs with appropriate counterions in the mobile phase is required to make it electrically neutral . Typical examples of such ion pairs are pentane, hexane, heptanes, octane sulfonate sodium salts.
The pKa or pKb of a drug molecule is used to determine the pH of the mobile phase. Analysts have observed that subtracting 1.5 from the lowest pKb of a basic drug gives the maximum pH of the mobile phase. Running at a pH close to the pKa of the basic drug can cause its peak shape to be broad, or in some cases split into to two peaks probably due to its existence in two different ionization states.
The retention dependence of basic molecule on the pH of mobile phase could be described as [8-10]:
(a) In its most hydrophilic form (fully protonated), it tends to be more solvated with water or methanol molecules and therefore exhibit more interaction with exposed silanols-Si-O-H resulting in a high retention.
(b) In its intermediate hydrophilic form (partial protonation-protonated and deprotonated) bad peak shape and unstable retention are generated. However, since the protinated form tends to have much stronger retention than deprotonated form and show greater interaction with exposed silanols-Si-O-H of the stationary phase, the ionization equilibrium in the mobile phase is shifted towards a formation of protonated molecules and further increase of overall retention.
(c) In its least hydrophilic form (the most hydrophobic) shortest retention is observed.
To reduce or eliminate the problems encountered while analyzing basic drugs on silica gel-based reverse phase column by high performance liquid chromatography a number of steps have been taken by analysts and they include [11-13]:
(i) Use of column that is packed with maximum coverage. Maximum coverage end-capped packings tend to be more chemically stable and show better peak symmetry than less fully covered packings (exposed silanols-Si-O-H). However, even after endcapping process is completed there are still free (unbonded) silanols (Si-O-H) present on the silica surface. These silanols are weakly acidic, and acidic pH (pH less than 3) is typically sufficient to protonate and neutralize most of them. Typical example of end-capping reagent is trimethylchlorosilane.
(ii) Buffers at low or intermediate pH generally provide better peak shapes. The nature of buffer cations and ionic strength, have also been found to affect retention and peak symmetry. Increasing buffer concentration has often led to improving peak symmetry and decreasing retention because of competition of buffer cations with the basic drug(s) for ion-exchange sites.
(iii) To employ acidic pH as the first choice for strong bases while pH 7 is recommended for weak bases.
(iv) Use of ion pairs or presence steric hinderance around basic center has helped to reduce worst asymmetry peaks produced by high pKa bases.
(v) Use of potassium salts rather than sodium salts to obtain the overall buffer concentration is preferred. It has been observed that higher buffer concentration can improve peak shape but can also lead to precipitation.
(vi) Use of low sample load for low pH values and higher sample loads for intermediate pH values are encouraged.
Poor peak shapes, broad and tailing peaks, split peaks, longer retention times have been observed when basic therapeutic agents (basic drugs) are analyzed on silica-gel based-reverse phase columns due to exposed silanol groups. The aforementioned analytical practices that have reduced or eliminated problems encountered with exposed silanol groups have contributed to making silica gel-based reverse phase columns the mainstay columns for high performance liquid chromatographic analysis of basic drugs. Finally, the interaction of basic therapeutic agents with silanol groups could be used to understand and explain the mechanism of binding of most basic drugs with plasma proteins in biological systems.
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