Egg White chromatogram analyses

An analysis similar to th a t c arried out for th e te s t chromatograms was carried out fo r th e egg white chrom atograms. Only those chromatograms which were su ccessfu lly deconvoluted and had th e ir c o n stitu en t peaks co rrectly identified were analysed - namely th e f ir s t (fig u re 2.17), second (fig u re 2.18) and fifth (not shown) egg white chromatograms.

Construction of eg g white fractionation diagrams

Fractionation diagram s were c o n stru cted as in section 4.2.1 for th e te s t chrom atogram s u sing th e programme in Appendix A1.5, th e peak functions param eters found by deconvolution, and th e extinction coefficients determ ined in section 2.sV The optimum fractio n s, both for high yield and high p u rity , were selected using th e programme d escribed in Appendix A1.6.

Results

The fractio n atio n diagram s obtained from th e eg g -w h ite chromatograms (fig u re 4.10) a re similar in n a tu re , ie. th ey all exhibit similar maximum purification fa c to rs and yields and total chromatogram areas. The c u rv e produced u sin g d ata from th e second egg white chromatogram d iffers th e most. The main d ifferen ces a re a shallower cu rv e, a h ig h er yield and a high d eg ree of c u rv a tu re a t th e beginning and end of th e fractionation cu rv e. This is due to th e high d eg ree of overlap of th e c e n tra l peak with th e th ird peak (see fig u re 2.18). These fac to rs will affect th e perform ance of th e sep aratio n in term s of th e selection of a high yield

The peak h eig h ts in Appendix A2.4 were co n v erted to absorbance u n its by dividing by 50 AU.mV ( th e d etecto r ra n g e was se t to 0.0 - 2.0 AU which re su lte d in a 0 to 100 mV o u tp u t ) and th en to a mass by dividing by th e len g th of th e flow-cell ( 2mm ) and th e extinction coefficient of th e component.

fractio n s which includes th ese sections of th e fractionation c u rv e (and chrom atogram). However th e selection of high p u rity fractio n s should be relativ ely unaffected since th is ty p e of fractio n only u se s cen tral portions of th e fractionation curve. The second and fifth egg white chromatogram contain e x tra visible peaks in addition to th o se peaks modelled. The optimum frac tio n s for th e th re e egg white chrom atograms a re shown in tab le 4.4.

Table 4.4 Optimum Fractms fw egg white chromatograms ( s e d m 2.3 )

Fraction start (ml) Fraction end (ml) PF ( - ) Observed Yield (mg) Yield (% )

Purity High Purity / High Yield 1st egg white chromatogram

82.0 94.4 3.5 3.1 90 52 High Yield

84.0 92.9 6.5 2.3 68 96 High Purity

2nd egg white chromatogram

80.3 89.1 2.5 3.3 75 53 High Yield

82.8 87.9 4.8 2.2 51 100 High Purity

. 5th egg white chromatogram

69.9 79.1 3.5 3.9 89 73 High Yield

72.3 78.2 4.8 3.2 71 99 High Purity

To a sse ss w hether th e c o rre c t optimum fractio n s had been determ ined a comparison of th e fractio n s ta r ts and en d s was made with th e data obtained by scanning SDS-PAGE gels. The data obtained by gel scanning cannot be used to a sse ss th e v alidity of th e p u rity and yield values since th e mass valu es a re unreliable. In stead th e re s u lts from SDS PAGE scanning a re used to indicate th e s ta r ts , maxima and ends of peak functions. This may be considered acceptable since th e combination of a low o b jectiv e function value (meaning a small difference betw een model and experim ental chrom atograms) to g e th e r with agreem ent on th e position of th e peak function s ta r ts and en d s should mean th a t th e elution profiles of th e peak fu n ctio n s a re co rrect.

Also su ch SDS d ata may be used off-line to e stab lish c o n stra in ts for th e deconvolution algorithm and th e selection of th e optimum fraction.

The high yield fractio n (described in tab le 4.4) for th e f ir s t egg white chrom atogram (fig u re 2.17) can be seen on fig u re 2.22 to include v irtu ally

all of th e fractio n s shown by th e SDS-PAGE analysis to contain Ovoglobulins (the p ro d u ct material) b u t none which do not contain any Ovoglobulins.

• f ir s t egg white chromatogram

The position of th e high yield fractio n has been successfully determ ined (ie. all of th e Ovoglobulins a re within th e selected fraction). The position of th e high p u rity fractio n for th is chromatogram is also found c o rre ctly since it only includes fractio n s containing Ovoglobulin which do not contain sig n ifican t amounts of o th er components, ie. only portions of th e c e n tra l p ro d u ct peak which do not overlap significantly with neighbouring peaks a re selected for th e high p u rity fraction.

• second egg white chromatogram

In comparison th e position of th e high yield optimum fraction (fig u re 2.18) is not determ ined precisely (see fig u re 2.23 for SDS-PAGE data). The high yield fraction is selected in accordance with th e peak function found by deconvolution, ie. th e s t a r t of th e fractio n o ccurs within th e f ir s t peak a t too low an elution volume and th e fractio n end o ccurs before th e end of th e peak function. This indicates th a t a poor high yield fractio n has been selected which contains some su b -fra c tio n s with no p ro d u ct whilst excluding some containing th e p ro d u ct material. In comparison th e high p u rity fractio n is found more satisfacto rily since th e positions of th e f ir s t peak end and th e th ird peak s ta r ts have been co rrectly determ ined by deconvolution. The reaso n fo r th e inability to c o rre c tly select th e optimum fractio n is th e ap p earan ce of e x tra peak which is highly resolved, in a valley.

• fifth egg white chromatogram

The position of th e optimum frac tio n s fo r th e fifth egg white chrom atogram were found c o rre ctly as fo r th e f ir s t egg white chrom atogram, d esp ite having an ex tra peak p resen t. This e x tra peak was not well resolved from th e c en tral peak and so made little impact on th e model peak function fo r th is peak.