Purification of Lycopene by Reverse Phase Chromatography

Abstract

This work investigates the potential of various hydrophobic matrices for the separation and purification of lycopene from microbial biomass obtained from fermentation using Blakeslea trispora. The lycopene purification method was developed in two steps. In the first step, carotenoids were extracted from the biomass of B. trispora with petroleum ether/acetone (1:1). In the second step, this partially purified carotenoid extract was further purified by reverse phase chromatography technique. Various binding conditions were studied to achieve maximum amount of lycopene bound onto the chromatographic matrices. Column studies on the elution conditions of lycopene using linear and step gradient method were investigated. Purification of lycopene using packed bed column by reverse phase chromatography matrix HP20 was carried out using step gradient of 55% isopropyl alcohol in acetone followed by 65% isopropyl alcohol in acetone. The purity and recovery of lycopene was checked using Knauer high-performance liquid chromatography (HPLC) system. A 89% recovery of lycopene of HPLC purity 95% was achieved with substantial success. Proton magnetic resonance of the purified sample showed close resemblance with the chemical shifts (δ values) of the standard lycopene.

Appendix

Calculations for HETP and NTU for HP 20

• 1. Area under the curve $\left( {{\text{AUC}}} \right) = 60 \times 2 \times 5 = 600\;\mu {\text{g}}$ of lycopene

• 2. Total lycopene loaded during breakthrough (W)

  $$W = C_{\text{i}} \left( {V_{\text{E}} - V_{\text{B}} } \right) = 46.98\left( {42 - 18} \right) = 1127.52\;\mu {\text{g}}$$

• 3. Lycopene adsorbed during breakthrough (W _a)

  $$\begin{array}{*{20}c} {W_{\text{a}} = \left( {W - {\text{AUC}}} \right)} \\ { = \left( {1127.52 - 600} \right)} \\ { = 527.52\;\mu {\text{g}}} \\ \end{array} $$

• 4. Fraction of lycopene adsorbed

  $$f = \left( {{{W_{\text{a}} } \mathord{\left/ {\vphantom {{W_{\text{a}} } W}} \right. \kern-\nulldelimiterspace} W}} \right) = \left( {{{527.52} \mathord{\left/ {\vphantom {{527.52} {1127.52}}} \right. \kern-\nulldelimiterspace} {1127.52}}} \right) = 0.467$$

• 5. Length of adsorption zone (L _a)

  $$\begin{array}{*{20}c} {L_a = }{\frac{{L\left( {V_E - V_B } \right)}}{{V_E - \left( {1 - f} \right)\left( {V_E - V_B } \right)}}} \\ {L_a = }{\frac{{12\left( {42 - 18} \right)}}{{42 - \left( {1 - 0.467} \right)\left( {42 - 18} \right)}} = 9.86\;cm} \\ \end{array} $$

• 6. Number of transfer unit (N)

  $$N = 16\left( {\frac{{V_R }}{{V_E - V_B }}} \right)^2 = 16\left\{ {{{34} \mathord{\left/ {\vphantom {{34} {\left( {42 - 18} \right)}}} \right. \kern-\nulldelimiterspace} {\left( {42 - 18} \right)}}} \right\}^2 = 32.09 \approx 32$$

• 7. Height equivalent to theoretical plate (HETP)

  $${\text{HETP}} = {{L_{\text{a}} } \mathord{\left/ {\vphantom {{L_{\text{a}} } N}} \right. \kern-\nulldelimiterspace} N} = {{9.86} \mathord{\left/ {\vphantom {{9.86} {32.09}}} \right. \kern-\nulldelimiterspace} {32.09}} = 0.30\;{\text{cm}}$$

• 8. Dynamic binding capacity (DBC)

  $$\begin{array}{*{20}c} {{\text{DBC}} = \left( {V_{\text{B}} \times C_{\text{i}} } \right) + {{\left( {{\text{number}}\;{\text{of}}\;{\text{boxes}}\;{\text{above}}\;{\text{the}}\;{\text{curve}} \times {\text{scale}}} \right)} \mathord{\left/ {\vphantom {{\left( {{\text{number}}\;{\text{of}}\;{\text{boxes}}\;{\text{above}}\;{\text{the}}\;{\text{curve}} \times {\text{scale}}} \right)} {V_{\text{m}} }}} \right. \kern-\nulldelimiterspace} {V_{\text{m}} }}} \\ {92.67\;{{\mu {\text{g}}} \mathord{\left/ {\vphantom {{\mu {\text{g}}} {{\text{ml}}}}} \right. \kern-\nulldelimiterspace} {{\text{ml}}}}} \\ \end{array} $$

Where

C _i :

lycopene concentration of loaded sample (46.98 μg/ml)

V _E :

Exhaust volume (18 cm³)

V _B :

Breakthrough volume (42 cm³)

L :

Packing length (12 cm)

ɛ :

Void fraction (for spherical dumped packing ≈0.4)

V _R :

Retention volume (34 cm³)

V _m :

Volume of the matrix (15.92 cm³)