Applications. Overview of applications of elevated temperature and temperature programmed liquid chromatography
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1 Applications Overview of applications of elevated temperature and temperature programmed liquid chromatography The applications are arranged by field of interest Fundamental page 2 Pharmaceutical page 4 Biochemical page 11 Environmental page 12 Chemical page 15 Food page 22 Polymers page 24 1
2 FUNDAMENTAL 1 Increasing efficiency and resolution by coupling columns at elevated temperature A test mixture is used to demonstrate the effect of coupling columns on efficiency and resolution. (F. Lestremau, A. Cooper, R. Szucs, F. David, P. Sandra, J. Chromatogr. A 1109 (2006) ) Increasing efficiency/resolution Column Zorbax StableBond C18, 250x4.6 mm, 5 µm Mobile phase Water/acetonitrile Isocratic 60/40 v/v UV 210 nm C 25 cm (1 column) 6 Rs 6/7 = 19 7 Sample: 1 Uracil 2 Caffeine 3 Pyridine 4 Phenol 5 Aniline 6 Benzene 7 Toluene C 25 cm (1 column) 6 Selectivity Rs 6/7 = 15 N7 = 20, C 100 cm (4 columns) Rs 6/7 = 30 N7 = 78, C 200 cm (8 columns) Efficiency/Resolution Rs 6/7 = 45 N7 = 162,
3 Maintaining analysis time C 25 cm (1 column) 1 ml/min Rs = C 100 cm (4 columns) 2 ml/min Rs =
4 PHARMACEUTICAL 1 Fast analysis of benzalkonium chloride N + R Column Selerity Blaze200 C18, 100x2.1 mm, 3 µm Mobile phase A= 0.5% ammoniumformate/0.1% formic acid B= acetonitrile Gradient 20 to 90% B in 4.5 min 1.1 ml/min Temperature 140 C C12 UV 262 nm CH 3 CH 3 - Cl R ~ C 8H 17 C 22H 45 C14 C16 C18 C The method enables fast analysis of the various benzalkonium chloride homologues. Elevated temperature is combined with high flow rate. A volatile mobile phase is used enabling detection methods like mass spectroscopy, corona aerosol discharge, and evaporative light scattering. 2 Determination of benzalkonium chloride in pharmaceutical formulation Temperature was used for the analysis of various benzalkonium chloride homologues in a pharmaceutical formulation. Selectivity is significantly affected by analysis temperature. Additionally, the use of high temperature speeds up the analysis. 4
5 Column Zorbax StableBond C18, 150x3 mm, 3.5 µm Mobile phase Buffer/acetonitrile Isocratic 50/50 v/v 2 ml/min UV 214 nm BAC C12 Polymer Polaratherm: 60 C BAC C14 Polaratherm: 80 C Polymer Polaratherm: 100 C Polymer (Min) 3 Analysis of pharmaceutical compounds Increasing resolution I A mixture of a pharmaceutical compound and impurities is analyzed in a conventional and high resolution set-up. Significantly higher resolution is obtained with the same analysis time. Column Zorbax StableBond C18, 150x3 mm, 3.5 µm Mobile phase A= formic acid in water B= formic acid in acetonitrile Gradient UV 239 nm 90 C 300 (2x150) mm column 0.45 ml/min Sample: Mixture of main compound and impurities 90 C 600 (4x150) mm column 0.9 ml/min
6 4 Analysis of pharmaceutical compounds Increasing resolution II Temperature and selectivity Column Zorbax SB300-C18, 250x4.6 mm, 5 µm Mobile phase Formic acid in water/acetonitrile Isocratic 50/50 v/v 1 ml/min UV 254 nm Sample: Mixture of active and impurities 1 3 Impurities elute after compound Room temperature 25 cm (1 column) Impurities elute in front of compound C 25 cm (1 column) Normal and high resolution 60 C 25 cm (1 column) C 125 cm (5 columns) A complete separation of all compound is obtained with the high resolution set-up (5 columns coupled in series). 6
7 5 Influence of temperature on the analysis of sulfonamides Selectivity and speed Column Zorbax StableBond C18, 150x3 mm, 3.5 µm Mobile phase A = 0.1% acetic acid in water B = 0.1% acetic acid in acetonitrile Gradient 20 to 50% B in 2 min UV 270 nm 40 C 0.6 ml/min Sample: 1 Sulfamethizole 2 Sulfamethazine 3 Sulfachlorpyridazine 4 Sulfamethoxine 60 C 0.6 ml/min 2 Selectivity 80 C 0.6 ml/min 2 Speed 60 C 1.2 ml/min Temperature programming - GREEN CONDITIONS 7
8 Column Zorbax StableBond C18, 150x3 mm, 3.5 µm Mobile phase A = 0.1% acetic acid in water B = 0.1% acetic acid in ethanol Gradient 17 to 50% B in 3 min 0.6 ml/min UV 270 nm Isotherm 80 C T-program C 20 C/min
9 6 Comparison of solvent and temperature programming for the analysis of sulfonamides Column Hypercarb, 100x3 mm, 5 µm Mobile phase A = 0.1% acetic acid in water B = 0.1% acetic acid in ethanol 0.5 ml/min UV 273 nm MS ESI+, scan m/z Isocratic 50%B Isotherm 50 C Sample: 1 Sulfamethizole 2 Sulfamethazine 3 Sulfachlorpyridazine 4 Sulfamethoxine 4 Gradient 0 to 5 min 50 to 100%B Isotherm 50 C Isocratic 50%B T-program 0 to 2 min 40 C hold 2 to 9 min 40 to 180 C (20 C/min) Peak area (MS) 3.E+07 2.E+07 MS sensitivity Temperature program Mobile phase program 1.E+07 0.E Peak number A sulfonamide standard mixture is analyzed with LC-MS using a solvent gradient or a temperature gradient. A temperature gradient provides better peak shape and detectability. The ionization efficiency is not affected by changes in mobile phase composition when a temperature program is used instead of a solvent gradient. (G. Vanhoenacker, P. Sandra, J. Sep. Sci. 29 (2006) ) 9
10 7 Analysis of sulfonamides on a temperatureresponsive stationary phase Column Mobile phase Home-made PNIPAA (poly(n-isopropylacrylamide))- modified aminopropyl, 150x4.6 mm, 5 µm 100% water 1 ml/min UV 254 nm % water 25 C Sample: 1 Sulfamethizole 2 Sulfamerazine 3 Sulfamethoxazole 4 Sulfadimethoxine 5 Sulfaquinoxalin % water 55 C The surface properties and functions of a temperature-responsive stationary phase are controlled by temperature. Retention and selectivity can thus be altered by changing the analysis temperature. (G. Vanhoenacker, P. Sandra, J. Sep. Sci. 29 (2006) ) 10
11 BIOCHEMICAL High efficiency separation of tryptic digest High efficiency separations of tryptic digest samples were obtained on conventional LC equipment by coupling eight 25 cm columns in series at 60 C. A peak capacity of ca. 900 was obtained using this set-up. (P. Sandra, G. Vanhoenacker, J. Sep. Sci. 30 (2007) ) Column Zorbax SB300-C18, 2000(=8x250)x2.1 mm, 5 µm Mobile phase A=0.1% TFA in water/acetonitrile 98/2 v/v B=0.1% TFA in water/acetonitrile 30/70 v/v Gradient 0 to 70%B in 520 min 0.2 ml/min Temperature 60 C UV 214 nm BSA Digest Serum Digest Peak Capacity ~ Serum Digest Detail min
12 ENVIRONMENTAL 1 Fast analysis of phenylurea pesticides Significant temperature dependent selectivity changes can be observed for the mixture of a phenylurea pesticides. At elevated temperature, the flow rate can be increased to shorten the analysis time. Column Zorbax StableBond C18, 50x2.1 mm, 1.8 µm Mobile phase Water/acetonitrile Isocratic 70/30 v/v UV 245 nm C ml/min 7 (tr 11.4 min) (tr 4.9 min) 60 C 0.35 ml/min 80 C 0.35 ml/min Sample: 1 Fenuron 2 Metoxuron 3 Chlortoluron 4 Diuron 5 Isoproturon 6 Linuron 7 Chloroxuron (tr 2.1 min) 80 C 0.85 ml/min
13 2 Influence of temperature on selectivity for the analysis of phenylurea and triazine pesticides (G. Vanhoenacker, P. Sandra, J. Sep. Sci. 29 (2006) ) Column Zorbax StableBond C18, 150x4.6 mm, 1.8 µm Mobile phase A=water B=acetonitrile Gradient 20 to 55%B in 30 min 1 ml/min UV 230 nm 50 C T1 T2 P1 P2 T3 T4 P3 T5 P6 P5 P4 P7 P8 T6 T7 T8 +P9 P10 T9 T10 90 C T1 T2 T4 P1 P2 T3 P5 T5 P8 P3 P4 P6 P7 T6 +P9 T7 T8 T9 T10 P10 T-program T1 T2 P1 P2 T3 T4 P3 P4 T5 P5 P6 P7 P8 T6 T7 P9 T8 P10 T9 T Triazine pesticides Peak Phenylurea pesticides Peak Desisopropylatrazine T1 Fenuron P1 Desethylatrazine T2 Metoxuron P2 Simazine T3 Methabenzthiazuron P3 Cyanazine T4 Chlortoluron P4 Atrazine T5 Monolinuron P5 Sebuthylazine T6 Diuron P6 Propazine T7 Isoproturon P7 Terbuthylazine T8 Metobromuron P8 Prometryn T9 Linuron P9 Terbutryn T10 Chloroxuron P10 13
14 3 High efficiency separation of PCB mixture A high efficiency separation of a polychlorinated biphenyl (PCB) mixture is obtained on conventional LC equipment by coupling eight 25 cm columns in series at 80 C. A peak capacity of over 300 was obtained using this set-up. (F. Lestremau, A. Cooper, R. Szucs, F. David, P. Sandra, J. Chromatogr. A 1109 (2006) ) Column Zorbax StableBond C18, 250x4.6 mm, 5 µm Mobile phase A=water, B=acetonitrile Gradient 50 to 100%B Temperature 80 C UV 214 nm Sample: Mixture of Arochlor 1242, 1254, and 1260 (1/1/1 ratio) 25 cm (1 column) 1 ml/min Peak Capacity ~ cm (4 columns) 2 ml/min Peak Capacity ~ cm (8 columns) 1 ml/min Peak Capacity ~
15 CHEMICAL 1 Analysis of naphthylamine isomers Mobile phase selection Column Zorbax SB-CN, 150x3 mm, 3.5 µm Mobile phase A= water B= methanol, acetonitrile, or ethanol Gradient 0.6 ml/min Temperature 40 C UV 220 nm B=acetonitrile 20 to 60% in 20 min 6 7 B=methanol 30 to 80% in 20 min B=ethanol 25 to 75% in 20 min Green chromatography Sample: 1 Aniline 2 α-naphthylamine 3 β-naphthylamine 4 α-naphthol 5 β-naphthol 6 N-phenylα-naphthylamine 7 N-phenylβ-naphthylamine 15
16 Column Zorbax SB-CN, 150x3 mm, 3.5 µm Mobile phase Temperature 80 C UV 220 nm A= water B= ethanol Gradient 0.6 ml/min: 10 to 60% ethanol in 20 min 1.2 ml/min: 10 to 60% ethanol in 10 min 80 C water/ethanol 0.6 ml/min 1.2 ml/min
17 2 Polar compounds I Resolution and speed Column Blaze C8, 150x4.6 mm, 3 µm Mobile phase Phosphate buffer in water/acetonitrile Gradient UV 290 nm 70 C 1 ml/min Sample: Mixture of wide variety of polar compounds 3 4 Resolution 100 C 1 ml/min Speed 100 C 1.8 ml/min
18 3 Polar compounds II Temperature programming The use of a temperature program leads to a reduced analysis time and an improved signal-to-noise for the late eluting compounds. Column Zorbax StableBond C18, 150x3 mm, 3.5 µm Mobile phase Acetic acid in water/isopropanol 94/6 v/v Isocratic 0.42 ml/min UV 254 nm Sample: Mixture of polar compounds Isotherm 40 C T-program 0-4 min 40 C, hold 4-6 min 40 to 80 C (20 C/min) 6 min 80 C, hold
19 4 Polysulfides An isothermic solvent gradient method for the analysis of a polysulfide sample is compared to an isocratic temperature-programmed method. Selectivity is significantly influenced by temperature and mobile phase composition (e.g. relative position in chromatogram of sulphur peak). Column Zorbax StableBond C18, 150x3 mm, 3.5 µm 0.42 ml/min DAD, 254 nm Sample Polysulfide mixture containing free sulphur Internal standard Free sulphur Polysulfides 40 C Ternary solvent gradient water/acetonitrile/ isopropanol Free sulphur T-program min 40 C min C (10 C/min) Isocratic 100% acetonitrile Free sulphur T-program 0-4 min 50 C 4-8 min C (10 C/min) Isocratic Ethanol/water
20 5 Increased resolution for the analysis of a complex reaction mixture The resolution for a separation of various compounds in a complex reaction mixture is significantly improved by coupling columns in series. Column Zorbax Eclipse XDB C18, 750(=3x250)x4.6 mm, 5 µm Mobile phase A=ammonium acetate in water B=acetonitril Gradient 50 to 100% B 1 ml/min Temperature 50 C UV 234 nm 250 mm (1 column) 50 to 100% B in 40 min mm (3 columns) 50 to 100% B in 120 min Sample: Reaction mixture
21 6 High efficiency analysis of phenones The theoretical chromatographic efficiency(n) of an LC setup can be calculated as follows: N = L/2dp (L=column length, dp = particle size) Eight 25 cm long, 5 µm dp columns coupled in series should ideally yield ca. 200,000 plates. Column Zorbax SB300-C18, 250x2.1 mm, 5 µm Mobile phase A=water, B=acetonitrile 0.2 ml/min Temperature 60 C Sample: phenone mixture UV 245 nm Ac Acetanilide C2 Acetophenone C3 Propiophenone C4 Buterophenone C5 Valerophenone Efficiency (N): C6 Hexanophenone C7 Heptanophenone Theory Bz Benzophenone 200,000 Isocratic 70% acetonitrile Ac C2 C4 C3 Bz C5 C6 C7 Chromatogram 201, ,000 C4 C5 C6 C7 Bz Ac C2 C3 Gradient 50 to 90% acetonitrile in 90 min
22 FOOD 1 High resolution analysis of citrus extracts Conventional columns were coupled in series to increase the efficiency and resolution for the analysis of a mixture of lemon and orange oil. The calculated peak capacity was approximately 260 for a 60 min gradient. (G. Vanhoenacker, P. Sandra, J. Sep. Sci. 29 (2006) ) Column Zorbax StableBond C18, 1000(=4x250)x4.6 mm, 5 µm Mobile phase A= water, B= acetonitrile Gradient 40% B 0 to 10 min 40 to 100% B 10 to 70 min 1.5 ml/min Temperature 80 C UV 315 nm Lemon/orange oil Detail
23 2 Sub-ambient temperature programming for the analysis of triglycerides Sunflower oil is analyzed on a reversed-phase column under isocratic conditions using a sub-ambient temperature program to increase resolution. Column Hypersil ODS, 200(=2x100)x 4.6, 3 µm Mobile phase Acetonitrile/Isopropanol/Hexane Isocratic 55/40/5 v/v Flow-rate: 1 ml/min : UV 214 nm Diglycerides Sample: Sunflower oil Triglyceride composition: Isotherm 25 C L = linoleic acid O = oleic acid P = palmitic acid S = stearic acid T-program 0 to 25 C 1.3 C/min T-program -20 to 5 C 1.3 C/min PLL OLO SLL PLO LLL OLL PLL OLO SLL PLO LLL OLL PLL OLO SLL PLO LLL OLL
24 POLYMERS Influence of temperature on the analysis of octylphenol ethoxylates Selectivity for the analysis of octylphenol ethoxylates was significantly affected by temperature in reversed phase LC. The elution order of the oligomers was reversed comparing separations at ambient and elevated temperature. (G. Vanhoenacker, P. Sandra, J. Chromatogr. A 1082 (2005) ) Column Zorbax StableBond C18, 150x3 mm, 3.5 µm Mobile phase Water/acetonitrile Isocratic 50/50 v/v 0.6 ml/min UV 225 nm Polaratherm: 20 C High MW Low MW Sample: Standard solution of Triton X-100 (O ) n OH Polaratherm: 50 C n ~1-20 Polaratherm: 90 C Low MW High MW
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