MULTIDIMENSINALE SCHEIDINGEN IN DE GAS- EN VLEISTF CHRMATGRAFIE Hans-Gerd Janssen and many others Unilever Foods R&D Vlaardingen, Vlaardingen, the Netherlands University of Amsterdam, Amsterdam, the Netherlands
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The analytical tool kit
The analytical tool kit Many problems can be solved using one tool. Tighten a screw. More complex repairs often require a combination. Drill a hole to turn in a screw. Very complex operations require the use of two tools at the same time: Painting
Principle of Comprehensive 2D Chromatography Normal Chromatography Heart-cut 2D Chromatography Comprehensive 2D Chromatography Multidimensional techniques
Edible fats and oils (main constituents) Main compounds - Triglycerides (approx. 90%) - Diglycerides (approx. 5%) - Monoglycerides (approx. 0-5%) - Free Fatty Acids (approx. 0-1%) C H 2 C R C H C R ' C H 2 C R " R= CCCCCCCC.. R = CCCC=CCC.. R = CC=CCCC=CC.. C H 3 ( C H 2 ) 1 4 C H - Phospholipids (approx. 0-2%) - Sterols (approx. 0-2%) - Wax esters (approx. 0-300 mg/kg) - Contaminants (ppm levels) H R 2 R 3 CH 3 (CH 2 ) 14 C (CH 2 ) 15 CH 3 H R 1 R 3 H H R 3 C H 2 C R C H C R ' C H 2 P R " H
Introduction glycidyl esters CH 2 H CH 2 C R CH CH 2 CH CH 2 glycidol (2,3-epoxy-1-propanol) glycidyl ester Formation ccurrence Toxicity chem. synthesis not a typical food contaminant probably carcinogenic to humans IARC:2A vegetable oil refining (deodorization) refined vegetable oils/fats (ppm levels)??? (hydrolysis to glycidol in GI tract demonstrated!) Present in edible oils at < 1 mg/kg
Sample preparation method: heart-cut NPLC isolation Normal phase TLC, SPE and LC are widely used for isolating specific compound-classes from edible oils and fats. Glycidyl ester are slightly less polar than triacylglycerides. Detector signal (UV) GE-C18:1 Saturated GEs GE-C18:2 GE-C18:3 TAG DAG MAG.. GE reference solution Spiked palm oil 0 5 10 15 20 25 30 35 30 35 40 8 min = 8 ml Time [min.] The NPLC step provides an efficient isolation of the glycidyl-esters. Fractions are analysed by GC-MS (SIM) after evaporation/redissolution.
Stable oil/water emulsions require emulsifiers Sugar-ester emulsifiers (after silylation) Mayonnaise Margarines Dressings Non-polar tail, differing in length Polar head
Complementary nature of GC and LC I MW 190 180 Mono FA ester Mono FA SPAN GC separation: 170 160 150 140 130 120 Di Di FA SPANester Elution largely on MW (size) with some contribution of polarity. 110 100 90 80 70 60 Sugars Free sugars Fatty Acids Free FA Polarity Tri FA SPAN FA ester 50 40 30 20 Tetra FA ester Tetra FA SPAN 10 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Complementary nature of GC and LC II Polarity 35 NPLC separation: Weight [mg] 30 25 20 15 MW Elution largely on polarity (size) with some contribution of size / shape 10 5 0 0 5 10 15 20 25 30 35 40 45 50 55 Time [min]
Combination of LC and GC comprehensive ( ) LC fr. 50 LC fr. 1 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 GC time
Principle of GC GC
Comprehensive 2D Chromatography: Normal GC Vanilla flavour Approx. 15 compounds compose the flavour 13 25 38 50 63 75 Retention time (minutes)
And now in the GC GC mode second dimension relative retention times (seconds) 7.50 6.25 5.00 3.75 2.50 1.25 0.00 13 25 38 50 63 75 first dimension retention time (minutes)
3D-Representation of a GC GC separation (detail) reconstructed 1st dimension chromatogram iso propyl-benzene 1,2,4-trimethyl-benzene para + meta cymene 1,2,3-trimethyl-benzene 0.2 n-nonane iso butyl benzene tert butyl benzene indan 0.1 0 61 saturates olefins 62 63 1st dimension retention time (minutes) 64 65 mono-aromatics 66 0 1 2 3 2nd dimension retention times (s) 4 5
Nice, but also not without problems verloading of the 2 D will be difficult to avoid for samples containing trace- and high level components HS SPME of a green tea infusion. StabilWax-DA / BPX-35 30x250x0.25 1.1x100x0.1
Separation of a peptide sample using comprehensive LC LC (GPC RPLC) verlap 2 nd dimension retention time 5.0 5.0 4.5 4.5 4.0 4.0 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 verlap 0 20 40 60 80 100 120 140 160 180 200 220 (min) peak height 5.0 2.5 0 0 1 st dimension GPC fraction number D LC Time (min) 2 1 D SEC Time (min)
High peak capacity peptide separations: SEC RPLC 1. Initial studies used manual fraction collection / transfer. 2. Automated version based on stop-flow. Stop-flow communication Pump 1 Isocratic SEC column pump Injection loop Waste 6 1 Pump 2 Stop flow valve 5 3 2 RP column Pump A Mixer Restrictor Pump B UV/MS F. Bedani, W.Th. Kok and H.-G. Janssen, J. Chromatogr A, 1133 (2006) 126-134.
Comprehensive 2D Chromatography with intermediate conversion Comprehensive 2D Chromatography A, B, C, D, E, F, G, H, I, J, K The CHEMICAL modulator: stores, to separate different, but converts, related, compounds (refocusses), according to 2 dimensions. reinjects. P Q,R,S,T,U,V,W,X,Y,Z
Instrumentation for separation - modification - separation Auto injector LC column Fraction collector
Healthy frying oils Polymerisation during deep-frying High temperature GC with on-column injection allows us to study triglycerides. But a mass of 900 Da is really the limit. Response [mv] Liquid frying oils are healthy, but have you ever cleaned the pan? 500 450 400 350 300 250 rganic GPC (THF) with RI di-triglycerides tri-triglycerides polymerised triglycerides mono-triglycerides 200 10.0 12.0 14.0 16.0 18.0 20.0 Time [min]
Fatty acids in the polymerised triglycerides 18:2 / 18:3 region tri-acylglycerides = TAG = triglyceride 16:0 18:1 tr 18:2 trtr Chain length: 4-14-20-24 No. double bonds: 0-4- 8 Positions: 9,11-All rientation: sn-position: 1,3 or 2 cis and trans
What are Comprehensive Multi-dimensional Chromatography systems? Normal Chromatography Heart-cut Chromatography Hyphenation - Comprehensive 2D Chromatography Comprehensive XC Comprehensive 3D Chromatography XC XC
Instrumentation for Comprehensive (off-line) LC GC GC Injection valve Mobile phase delivery Column 1 st dimension Sequential GC GC analysis 2 nd dimension FID 3 rd dimension (ToF)-MS 4 th dimension
GC GC ToF MS vs LC-GC GC ToF MS LC GC GC ToF MS GC GC ToF MS Petrochemical product
Conclusions - Many samples around us are complex. - Single chromatographic techniques cannot resolve complex samples. - Multidimensional methods offer: - more peaks, - selective zooming. - Hyphenated couplings are preferred if you know what to look for. - Comprehensive methods provide you with the best possible separation. - Comprehensive methods are often simper than heart-cut methods. - Mass Spectrometry adds another dimension.