The coccolith sedimentary and micropalaeontological archive has fostered great interest for palaeoclimate applications. Indeed, the geochemistry of coccolith calcite has the potential to reconstruct both palaeo-CO₂ concentrations and palaeo-temperature of seawater. Studying coccolith geochemistry aims at better understanding the changes in the vital effect of coccoliths with changes in environmental parameters, especially the carbonate chemistry of seawater. To this aim, we need to deconvolve the biological imprint from the environmental signals recorded in the composition of coccolith biominerals. We have undertaken large-scale culture experiments of four strains of coccolithophores of various sizes and growth rates, grown under eight CO₂/pH conditions typifying the long-term CO₂ evolution of the Cenozoic Era. We propose an assessment of the expression of the vital effects for <em>Emiliania huxleyi</em>, <em>Gephyrocapsa oceanica</em>, <em>Helicosphaera carteri</em> and <em>Coccolithus braarudii</em> with simultaneous changes in Dissolved Inorganic Carbon (DIC) and pH in the medium resulting in variations in CO_{2 aq} availability to the cells. We have identified a distinct isotopic response of <em>C. braarudii</em> to <em>p</em>CO₂ levels on either side of the 600 ppmv (pH 7.89) condition. We propose that this discrepancy is the result of a modification of the proton efflux across the plasma membrane (voltage-dependent proton channels). We further show that as the CO₂ level rises and pH decreases (from 200 to 500 ppmv and from 8.29 to 7.96 pH units, respectively), a significant increase in &delta;¹³C_coccolith of <em>C. braarudii</em> is expressed, along with a coeval decrease in &delta;¹³C_org. The constant physiological parameters of <em>C. braarudii </em>(growth rate, PIC, POC) across the 200 to 500 ppmv interval support the idea that the change in &delta;¹³C_coccolith is only a consequence of a lower fractionation between dissolved CO₂ and organic matter. Meanwhile, the small (less carbon-limited) cells of <em>E. huxleyi</em> and <em>G. oceanica</em> do not exhibit any change in their carbon vital effects with changes in carbonate chemistry of the environment across the whole CO₂ spectrum. Using this new biogeochemical framework, we have established a calibration between CO_{2 aq} concentration and the differential vital effect (∆&delta;¹³C) between isotopically-invariant small <em>G. oceanica</em> and large coccoliths <em>C. braarudii</em>, whose vital effect is CO₂-dependent at low CO₂. The CO₂-&Delta;&delta;¹³C transfer equation allows palaeo-<em>p</em>CO₂ reconstructions based on isotope changes explained by physiological processes, especially at low and medium CO₂ levels.