#𝐩𝐞𝐚𝐤: do you want to know how to handle me?
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭 : yes, i need you sharp not broad, without any tailing or fronting.
#𝐩𝐞𝐚𝐤: In chromatography, the principle, like dissolves like, applies, meaning nonpolar analytes interact well with nonpolar stationary phases, leading to higher distribution and improved separations. Neutral or (in non ionic form) analytes, being more nonpolar than ionized ones, exhibit enhanced retention on nonpolar reversed phase.
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: How can i know that analyte after injection will be in neutral form or in ionized form?
#𝐩𝐞𝐚𝐤: The pH of the mobile phase significantly affects the ionization state of ionizable analytes. mobile phase with pH equal to pKa of the analyte, makes analyte exist in both ionized and neutral states, resulting in poor chromatography with broad, tailing peaks as some of the analyte will transfer fast (ionized form) and others will transfer slowly (Neutral form), Therefore, all analytes of the same type will not be traveling through the column in the same manner resulting in very poor peak shapes .
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭 : Because of that, i can't use mobile phase with pH equal to or close to pKa of the analyte, then at what pH mobile phase need to be adjusted ??
#𝐩𝐞𝐚𝐤: Acidic analytes in acidic environments (pH at least two units below their pKa). retain their protons, leading to ion suppression and improved retention on reversed phase column, but in more basic environments, acidic analytes dissociate into their conjugate base, becoming ionized and resulting in reduced retention on reversed phase column.
Basic analytes, in basic environments, (pH at least 2 units above their pKa), remain in their ion-suppressed, neutral form, improving retention on reversed phase column. In more acidic environments, basic analytes become protonated and ionized, leading to reduced retention and earlier elution on reversed phase column.
A good rule of thumb is to fix the pH for an acidic compound at least 2 pH units below the pKa, and for a basic compound it should be set at 2 pH units above the pKa (to inhibit the ionization of the analyte and make it in neutral form).
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: Why 2 pH units , why not 1 pH unit ?
#𝐩𝐞𝐚𝐤: This ensures that the pH is far enough away from the pKa that any small changes in mobile phase pH will not have a dramatic effect on analyte retention and peak shapes.
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: but we have to take care, It is important to point out that it may not be as simple to follow the 2 pH units rule when working with bases that have pKa values above 8. If the rule is followed, the pH of the mobile phase would need to be at pH more than 10, which is outside of the working pH range of most silica columns. Similarly, if an acidic analyte has a pKa of 3, a mobile phase of pH may first well be too low for the column.
#𝐩𝐞𝐚𝐤 : you are right.
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: then what i have to do in this situation?
#𝐩𝐞𝐚𝐤: In the case of weak bases, the first option relies on exploiting any additional hydrophobic interactions between the analyte and the reversed phase column to maintain sufficient retention. If the basic moiety is part of a large nonpolar molecule, it may be possible to analyze it at a low pH in the ionized state. The hydrophobic interactions should overcome enough of the polarity introduced by the protonation of the base to ensure adequate retention.
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: also The added benefit of analyzing bases at low pH is the ion-silanol groups, SiOH, on the silica support will be ion-suppressed. At a pH above their pKa, about pH 4, ion-silanol groups act like an acid and will dissociate to give ionized SiO that interacts strongly with basic moieties and results in bad peak tailing.
#𝐩𝐞𝐚𝐤: yes you are right, At low pH, the SiOH remains neutral.
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: If the additional hydrophobic interactions of basic moiety from ionized weak bases do not provide sufficient retention for the analyte, what we have to do?
#𝐩𝐞𝐚𝐤: at this case, one alternative would be to use a polar embedded or polar end-capped column. These columns have polar groups within or end capped onto the silica backbone of the column and so allow very highly aqueous 100% mobile phases to be used without the risk of phase collapse self-association. The highly polar ionized bases are more likely to be retained by the increased polarity provided by these more polar reversed phase column.
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: what will we do if samples contain mixes of weak acids and bases where at least one functional group will be ionized at whatever pH is chosen. Or analysis of strong acids and bases, or analytes that have multiple functional groups??
#𝐩𝐞𝐚𝐤: In cases such as these, the pH of the mobile phase is used to fix the relevant analytes in the ionized state, so that they will interact ion-pair with a modifier added to the mobile phase that carries the opposite charge. This interaction leads to an overall neutral molecule that will then be successfully retained by reversed phase column.
-If you use #trifluoroacetic acid, TFA, in your mobile phases, it lowers the pH to ion-suppress any acids, while also ion-pairing with any ionized bases. Other common ion-pair reagents are #quaternary_ammonium_compounds, for example tetrabutylammonium phosphate, to ion-pair with acids and suppress weak bases. And alkyl sulfonic acids to ion-pair with bases and suppress weak acids.
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: why we use #Triethylamine commonly in mobile phase?
#𝐩𝐞𝐚𝐤: Triethylamine, TEA, is another commonly used modifier, often referred to as a sacrificial base that will ion pair with ionized silanol groups on the stationary phase, reducing peak tailing effects.
#𝐀𝐧𝐚𝐥𝐲𝐬𝐭: what is the disadvantages of using ion pair reagents?
#𝐩𝐞𝐚𝐤: 1-you are chemically modifying your stationary phase and so should always have ion pair dedicated columns.
2-Some ion-pair reagent also have significant UV activity, and so may absorb light at the analytical wavelengths being used with UV detection, for example the UV cutoff for TFA is 210 nanometer.
3-any neutral analytes present in the sample having limited access to stationary phase, resulting in decreased retention.