The Need for Multidimensionality in Analytical Thinking. Chromatographic Developments as an Example
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1 The Need for Multidimensionality in Analytical Thinking. Chromatographic Developments as an Example Hernan J. Cortes, Ph.D. H.J. Cortes Consulting, LLC Midland, MI, USA
2 Outline Theoretical considerations GCxGC analysis of ethylene LCxLC MS for protein post translational modifications LCxLC characterization of epoxy resins Low thermal mass GC Low thermal mass LC Conclusions
3 R = (N ½ /4) (α 1/ α) (k 2 /1+k 2 ) where α = (k 2 /k 1 ) Adapted from P. Sandra, F. David in H. Cortes, ed. Multidimensional Chromatography Marcel Dekker, New York. 1990
4 Peak Capacity n = (1 + N ½ /r) ln (1 + k) N = theoretical plates r = standard deviations equaling peak width (4) k = capacity factor of the last peak in a series J. Davis, C. Giddings. Anal. Chem. 55, (1983) 418.
5 Statistical Model of Component Overlap (Poisson Statistics with Random Distribution) α = m/n m= Hypothetical max. number of Separable Components n=peak Capacity Probability component is a singlet. P 1 = (e α ) (e α ) = e 2α Probability component is a doublet. P 2 = (e 2α ) (1 e α ) In general Pn = (e 2α ) (1 e α ) n 1
6 P= Peak Number = Sum of mean number of singlets, doublets, etc P= n α e α Number of visible peaks P = 0.37 Number of single component peaks S = 0.19
7 J. C. Giddings in H. J. Cortes, ed. Multidimensional Chromatography Marcel Dekker, New York. 1990
8 J. C. Giddings in H. J. Cortes, ed. Multidimensional Chromatography Marcel Dekker, New York. 1990
9 A B A A 1D B B 2D
10 Comprehensive Two Dimension Gas Chromatography First Dimension Comprehensive nd Col(sec) Transfer Device st Col (min) vs traditional heartcutting
11 Impurities in Ethylene Process Samples Problem: Low polyethylene catalyst efficiency Frequent regeneration of clean up (adsorbent) beds (3 ~ 5 days) Routine analysis shows the problem was not from common poisons. P. Eckerle, M. Pursch, H. J. Cortes, K. Sun, B. Winniford, J. Luong. J. Sep. Sci. 31 (2008)
12 Major Polar Impurities
13 Learnings from GC x GC analysis of Ethylene Variety of oxygenated impurities not effectively captured by clean up (adsorbent) beds Indicates that the clean up bed media is not effective for these impurities test/try other media
14 Multidimensional Liquid Phase Separations and Mass Spectrometry for Detailed Characterization and Quantification Proteins Development of a low flow (microbore/capillary) LCxLC MS/MS system capable of resolving, identifying, quantifying, and structurally characterizing a targeted protein. Applications of LCxLC MS/MS to address: Stability Structure/Function Efficacy Post translational modifications Batch to Batch variations T. Kajdan, H. J. Cortes, S. Young, K. Kuppannan., J. Chromatogr. A, 1189 (2008)
15 One Dimensional Reversed Phase Separation. Co elution of Peptide Fragments (BSA). 100 T47 48 T57 58 Relative Intensity (%) m/z Time (min.) T. Kajdan, H. J. Cortes, S. Young, K. Kuppannan., J. Chromatogr. A, 1189 (2008)
16 Configuration of Comprehensive LCxLC System 1 st Dimension Pump 2 nd Dimension Pump Splitter BioBasic SCX 50 x 0.5 mm i.d. 5 µm, 300 Å Valve Position A Valve Position B Manual Injector Small Molecule CapTrap 2 x 0.5 mm Zorbax SB C x 0.5 mm i.d. 3.5 µm, 80 Å Detector (UV or MS) To Waste
17 SPIKE GRADIENT vs. STEP GRADIENT Spike Gradient Profile Mobile Phase % B Time Step Gradient Profile Mobile Phase % B Time
18 Two Dimensional Separation. Peptide Fragments Separated. 100 T57-58 T47-48 Relative Intensity (%) m/z 0 m M 10 m M 15 m M 20 m M 30 m M 40 mm 1000 m M T47-48 T Time (min.)
19 100 Relative Intensity (%) TVMENFVAFVDK TVMENFVAFDK M = unoxidized Met [M +H ] +1 = m/z 10 m M 15 m M Time (min.) 100 Relative Intensity (%) TVM ENFVAFVDK M = oxidized M et [M + O x] +1 = m/z T im e (m in.)
20 40 RP HPLC (retention time, min.) 20 T Ion Exchange (spike gradient ammonium formate mm) RP HPLC (retention time, min.) 20 T75ox Ion Exchange (spike gradient ammonium formate mm)
21 RNASE A AND RNASE B 2D Contour Plots RNase A RNase B RP HPLC (time = min) RP HPLC (time = min) 2 Ion Exchange (spike gradient ammonium formate mm) 1 Ion Exchange (spike gradient ammonium formate mm) 3 Pancreatic Ribonuclease K ETAAAK FER QHMDSSTSAA SSSNYCNQMM K SR NLTK DR C KPVNTFVHES LADVQAVCSQ 3,4 3 1 K NVACK NGQT NCYQSYSTMS ITDCR ETGSS K YPNCAYK TT QANK HIIVAC EGNPYVPVHF DASV 1,2 2,5,6 7 N Glycosylated; N Deamidated
22 Summary: LCxLC has advantages of unique selectivity and high efficiency of separation methods combined with mass specificity and sensitivity of MS. LCxLC has the ability to resolve individual post translational modifications better than 1 dimensional separations. LCxLC/UV allows for quantitative evaluation of area percent purity and impurities of recombinant proteins. Meaningful quantitative evaluation of product quality that is not possible with other current analytical approaches. One set of conditions essentially applicable to most proteins
23 LCxLC of Epoxy Resin Samples 1st dimension = Size exclusion chromatography Molecular weight 2nd dimension = LC at critical conditions (LCCC) End group functionality LCCC LCCC LCCC LCCC LCCC LCCC LCCC LCCC LCCC LCCC LCCC LCCC LCCC LCCC 1.5 min Regenerated SEC separation profile S. Julka, H. J. Cortes, R. Harfmann, A. Schweizer, M. Pursch, D. West. Anal Chem. 81 (2009)
24 Size Exclusion Chromatography Adsorption Chromatography Liquid Chromatography at Critical Conditions no interaction with stationary phase retention caused by entropy change. 0 < k < 1 Segments of the macromolecule interact with stationary phase. retention dominated by enthalpy changes 1 < k < oo End groups interact with stationary phase entropy and enthalpy are balanced Retention by enthalpy changes of defects
25 SEC Chromatogram Regenerated From Successive LCCC Analyses Stacked Side by side Into The Plane Of This Plot MW distribution is clearly evident (higher MW range) no longer buried under DGEBA SEC
26 2nd Dimension Liquid Chromatogr aphy At Critical Conditions (LCCC) (min.) Comprehensive 2DLC Separation (SEC x LCCC) of Solid Epoxy Resin (110) Two Dimensional HPLC Runs Flow Rate: 2.1ml/min 1st Dimension Size Exclusion Chromatography (min.) Flow Rate: 12µL/min.
27 The development of a new platform in fast gas Chromatography Low Thermal Mass GC J. Luong, R. Gras, R. Mustacich, H. J. Cortes. J. Chrom. Sci. 44 (2006) 253
28
29 RVM LTM GC Technology
30 Key Features of LTMGC Ideal attributes for fast GC Low power consumption Rapid cooling Fast heating
31 Temperature programming rate: 100, 500, 1000 C/min Column technology: 5 meter, 0.1 mm id, 0.12 micron PDMS, helium at 44 cm/sec 100C/min 500C/min 1000C/min Exxon Norpar 12 fluid in Hexane
32 Conventional and LTM GC Throughput Comparison of Throughput Between Conventional GC (CGC) and LTMGC LTMGC: 2 metre, 0.1 mm id, 0.12 micron DB-1 Column Technology LTMGCEXT: Extended run time to remove wax Time (minutes) CGC LTMGCF LTMGCEXT GC Technology Cool down time Run time autosampler
33 Determination of volatiles in Styrene/butadiene Latexes vinylcyclohexene 2 ethylbenzene 3 styrene 4 i-propylbenzene 5 n-propylbenzene 6 4-cyanocyclohexene ethylhexanol 8 undecane 9 2-ethylhexyl acetate 10 2-ethylhexyl acrylate 11 4-phenylcyclohexene
34 Trends in LC Sub 2 µm particle (d p ) H A B / u Cu Fused core (d p ) Monolith (C) Temperature (D) C f ( k) D d 2 p
35 LTMLC Capillary column 0.25 mm vs 4.6 mm i.d. (300 times lower in mass) Housing and end fittings Inlet frit Micro column LTM assembly Ending frit B. Gu, H. J. Cortes, J. Luong, M. Pursch, P. Eckerle, R. Mustacich. Anal Chem. 81 (2009)
36 Temperature Programming G 1800 o C/min 1600 G 200 o C/min 1400 F 100 o C/min F Response (mv) E D C B 50 o C/min 24 o C/min 18 o C/min 12 o C/min Response (mv) E D C B 400 A 6 o C/min 400 A Retention time (min) Retention time (min)
37 Selectivity Tuning o C D Response (mv) C B o C o C Column: 250 um x 25 cm; Zorbax SB C18, 5 um Mobile phase: 45/55% v/v ACN/40 mm phosphate, ph (2.30) UV: 220 nm 300 A o C Analytes: neutral, acidic and basic Retention time (min)
38 Oscillated Thermal Gradient Response (mv) o C C B 50 o C A 25 o C o C 100 o C to 25 oc at a rate of 25 oc/min was used, followed by 1 min hold at 25 oc and then ramped to 100 oc at 25 oc/min resolution between analytes 3 and 4 was better (1.81 vs. 1.62), 50 oc for 4.5 min, then ramped to 100 oc at a rate of 12 oc/min, and hold at 100 oc for 3.5 min 25 o C Retention time (min) B. Gu, H. J. Cortes, J. Luong, M. Pursch, P. Eckerle, R. Mustacich. Anal Chem. 81 (2009)
39 Ultrafast LTMLC 800 E 100 o C/min 700 D 75 o C/min Response (mv) 600 C 40 o C/min 500 B 20 o C/min 400 A 25 o C isothermal R etention tim e (m in)
40 Reproducibility (n = 5) 400 Response (mv) Retention time (min)
41 Conclusions Multidimensional Chromatography is very powerful technique that provides information and knowledge not previously attainable. Comprehensive 2DGC offers unique opportunities for problemsolving (ethylene polar impurities, extruder contamination). Comprehensive 2DLC MS established for protein characterization and emerging as a polymer characterization technology. LTMLC was realized by the combined use of a mini oven and a capillary column, both of which have low thermal mass. Very fast temperature gradients (both increasing and decreasing) cab be applied. Replaces gradient elution and very fast recovery times. Oscillated temperature gradient was demonstrated, for the 1 st time, for fine tuning separation. Ultra fast and reproducible LTMLC was also demonstrated. Second dimension for LCxLC
42 Acknowledgements S. Julka, R. Harfmann, B. Gu, M. Pursch, J. Luong, P. Eckerle Shimadzu Corporation M. Nishimura, R. Ludwig, M. Takahashi University of Messina, Italy. L. Mondello, P. Dugo, T. Kumm
43
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