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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
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) 191-196) Increasing efficiency/resolution
Column Zorbax StableBond C18, 250x4.6 mm, 5 µm Mobile phase Water/acetonitrile
Isocratic 60/40 v/v Detection UV 210 nm
Sample: 1 Uracil 2 Caffeine 3 Pyridine 4 Phenol 5 Aniline 6 Benzene 7 Toluene
0 20 40 60 80 100 Time(min)
0 5 10 15 20 25
1
2
4+5
3
6 7
0 10 20 30 40 50 60
0 2 4 6 8 10 12 14
1
2 3
6 7
4 5
30°C 25 cm (1 column)
80°C 25 cm (1 column)
80°C 200 cm (8 columns)
80°C 100 cm (4 columns)
Selectivity
Efficiency/Resolution
Rs 6/7 = 19
Rs 6/7 = 45 N7 = 162,000
Rs 6/7 = 30 N7 = 78,000
Rs 6/7 = 15 N7 = 20,000
3
Maintaining analysis time
0 5 10 15 20 25
1
2
4+5
3
6 7
0 5 10 15 20 25 Time(min)
1
2 3
6 7
4 5
30°C 25 cm (1 column) 1 ml/min
80°C 100 cm (4 columns) 2 ml/min
Rs = 19
Rs = 34
4
PHARMACEUTICAL
1 Fast analysis of benzalkonium chloride
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 Flow rate 1.1 ml/min Temperature 140°C Detection UV 262 nm
Time(min)
0 1 2 3 4 5
C10
C12
C14
C16 C18
N+ R
CH3CH3
Cl-
R ~ C8H17 – C22H45
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.
5
Column Zorbax StableBond C18, 150x3 mm, 3.5 µm Mobile phase Buffer/acetonitrile
Isocratic 50/50 v/v Flow rate 2 ml/min Detection UV 214 nm
Time (Min)
0 1 2 3 4 5 6
BAC C12
BAC C14
Polymer
Polymer
Polymer
Polaratherm: 60°C
Polaratherm: 80°C
Polaratherm: 100°C
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 Detection UV 239 nm
Sample: Mixture of main compound and impurities
Time(min)
0 2 4 6 8 10
90°C 300 (2x150) mm column 0.45 ml/min
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 Flow rate 1 ml/min Detection UV 254 nm Sample: Mixture of active and impurities
Time(min)
0 2 4 6 8 10
Room temperature25 cm (1 column)
60°C25 cm (1 column)
1
2
3
7
5 6
4
5
6
Impurities elute after compound 3
Impurities elute in front of compound 3
Normal and high resolution
0 2 4 6 8
Time(min)
0 10 20 30 40
60°C25 cm (1 column)
60°C125 cm (5 columns)
A complete separation of all compound is obtained with the high resolution set-up (5 columns coupled in series).
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
Detection UV 270 nm
Sample: 1 Sulfamethizole 2 Sulfamethazine 3 Sulfachlorpyridazine 4 Sulfamethoxine
Time(min)
0 1 2 3 4 5 6 7
40°C 0.6 ml/min
60°C 0.6 ml/min
80°C 0.6 ml/min
60°C 1.2 ml/min
1 2 3
4
2
2
Selectivity
Speed
Temperature programming - GREEN CONDITIONS
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
Flow rate 0.6 ml/min Detection UV 270 nm
Time(min)
0 1 2 3
Isotherm 80°C
T-program 70-90°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 Flow rate 0.5 ml/min Detection UV 273 nm MS ESI+, scan 180-400 m/z
Sample: 1 Sulfamethizole 2 Sulfamethazine 3 Sulfachlorpyridazine 4 Sulfamethoxine 1 2
3 4
0 Time(min)
10 20 30 5 15 25
Isocratic 50%B Isotherm 50°C
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)
MS sensitivity
0.E+00
1.E+07
2.E+07
3.E+07
1 2 3 4Peak number
Peak
are
a (M
S)
Temperature program
Mobile phase program
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) 1822-1835)
10
7 Analysis of sulfonamides on a temperature-responsive stationary phase
Column Home-made PNIPAA (poly(N-isopropylacrylamide))- modified aminopropyl, 150x4.6 mm, 5 µm
Mobile phase 100% water Flow rate 1 ml/min Detection UV 254 nm
Sample: 1 Sulfamethizole 2 Sulfamerazine 3 Sulfamethoxazole 4 Sulfadimethoxine 5 Sulfaquinoxalin
0 10 20 30 Time(min)
100% water 25°C
100% water 55°C
1 2
4
3
5
12
4
3
5
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) 1822-1835)
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) 241-244)
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
Flow rate 0.2 ml/min Temperature 60°C Detection UV 214 nm
BSA Digest
Serum Digest
Peak Capacity ~ 900
Serum Digest
Detail 230-330 min
240 260 280 300 320
50 100 150 200 250 300 350 400 450
Time(min)
Time(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 Detection UV 245 nm
Sample: 1 Fenuron 2 Metoxuron 3 Chlortoluron 4 Diuron 5 Isoproturon 6 Linuron 7 Chloroxuron
Time(min)
0 2 4 6 8 10 12
0 1 2 3 Time(min)
4 5
40°C0.35 ml/min
80°C0.35 ml/min
60°C0.35 ml/min
80°C0.85 ml/min
1 2
3 4 5
6 7 (tR 11.4 min)
4 5 7 (tR 4.9 min)
4 5 7 (tR 2.1 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) 1822-1835)
Column Zorbax StableBond C18, 150x4.6 mm, 1.8 µm Mobile phase A=water
B=acetonitrile Gradient 20 to 55%B in 30 min
Flow rate 1 ml/min Detection UV 230 nm
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
Time(min)
5 10 15 20 25
T3 T7
T1 T2 T4 T5 T6 T8 T9 T10
P1
P2
P3
P4
P5 P6
P7 P8 P9
P10
T3 T1 T2 T4 T5 T6
+P9 T7 T8 T9 T10
P1
P2
P3
P4
P5
P6 P7
P8
P10
P8
T1 T2
T3
T4 T5 T6 T7 T8
+P9 T9 T10
P1
P2
P3
P4
P5 P6
P7 P10
50°C
90
°C
T-pr
ogra
m
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) 191-196)
Column Zorbax StableBond C18, 250x4.6 mm, 5 µm Mobile phase A=water, B=acetonitrile
Gradient 50 to 100%B Temperature 80°C Detection UV 214 nm
Sample:Mixture of Arochlor 1242, 1254,
and 1260 (1/1/1 ratio)
0 10 20 30 40
Time(min)
0 100 200 300
0 20 40 60 80
25 cm (1 column) 1 ml/min
100 cm (4 columns) 2 ml/min
200 cm (8 columns) 1 ml/min
Peak Capacity~100
Peak Capacity~200
Peak Capacity~320
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 Flow rate 0.6 ml/min Temperature 40°C Detection UV 220 nm
Sample: 1 Aniline 2 α-naphthylamine 3 β-naphthylamine 4 α-naphthol 5 β-naphthol 6 N-phenyl-
α-naphthylamine 7 N-phenyl-
β-naphthylamine
Time(min)
0 4 8 12 16
B=acetonitrile 20 to 60% in 20 min
B=ethanol 25 to 75% in 20 min
B=methanol 30 to 80% in 20 min
1 2 3
4
5
6 7
Green chromatography
16
Time(min)
0 4 8 12 16
80°C water/ethanol
0.6 ml/min
1.2 ml/min
Column Zorbax SB-CN, 150x3 mm, 3.5 µm Mobile phase 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 Temperature 80°C Detection UV 220 nm
17
2 Polar compounds I – Resolution and speed
Column Blaze C8, 150x4.6 mm, 3 µm Mobile phase Phosphate buffer in water/acetonitrile Gradient Detection UV 290 nm
Sample:Mixture of wide variety
of polar compounds
Time(min)
0 5 10 15 20
1
2
3 4
5
6
7 8
9
10 11
3
4
Resolution
0 2 4 6 8 10 Time(min)
Speed
70°C 1 ml/min
100°C 1 ml/min
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 Flow rate 0.42 ml/min Detection UV 254 nm
Sample:Mixture of polar
compounds
Time(min)
0 10 20 30
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 Flow rate 0.42 ml/min Detection DAD, 254 nm Sample Polysulfide mixture containing free sulphur
Time(min)
0 5 10 15 20
0 2 4 6 8 10 Time(min)
Internal standard
Free
sul
phur
Fr
ee s
ulph
ur
Free
sul
phur
Polysulfides
40°CTernary solvent gradient
water/acetonitrile/isopropanol
T-program0-3.5 min 40°C 3.5-8.5 min 40-90°C (10°C/min)
Isocratic100% acetonitrile
T-program0-4 min 50°C 4-8 min 50-90°C (10°C/min)
IsocraticEthanol/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 Flow rate 1 ml/min Temperature 50°C Detection UV 234 nm
Sample:Reaction mixture
0 5 10 15 20 25 30 35
Time(min)
0 20 40 60 80 100
250 mm (1 column) 50 to 100% B in 40 min
750 mm (3 columns) 50 to 100% B in 120 min
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 Flow rate 0.2 ml/min Temperature 60°C Detection UV 245 nm
Time(min)
0 10 20 30 40 50 60 70
Ac
C2
Bz
C4
C3
C5
C6
C7
Ac C
2
Bz
C4
C3
C5 C6
C7
Isocratic 70% acetonitrile
Gradient 50 to 90% acetonitrile in 90 min
Efficiency (N): Theory 200,000
Chromatogram 201,000 - 210,000
Sample: phenone mixture Ac Acetanilide C2 Acetophenone C3 Propiophenone C4 Buterophenone C5 Valerophenone C6 Hexanophenone C7 Heptanophenone Bz Benzophenone
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) 1822-1835)
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
Flow rate 1.5 ml/min Temperature 80°C Detection UV 315 nm
10 20 30 40 50 60 70
10 20 30 40 50 60 Time
(min)
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 Detection: UV 214 nm
Sample: Sunflower oil Triglyceride composition: L = linoleic acid
O = oleic acid P = palmitic acid S = stearic acid
Time(min)
0 10 20 30 40
Isotherm 25°C
T-program 0 to 25°C 1.3°C/min
T-program -20 to 5°C 1.3°C/min
Dig
lyce
rides
LLL
OLL
PL
L O
LO SLL
PLO
LLL
OLL
PLL
OLO
SL
L PL
O
LLL
OLL
PLL
OLO
SLL
PLO
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) 193-202)
Column Zorbax StableBond C18, 150x3 mm, 3.5 µm Mobile phase Water/acetonitrile
Isocratic 50/50 v/v Flow rate 0.6 ml/min Detection UV 225 nm
Sample: Standard solution of Triton X-100
0 Time (min)
5 10 15 20
Polaratherm: 20°C
Polaratherm: 90°C
Polaratherm: 50°C
High MW Low MW
Low MW High MW
(O )OHn
n ~1-20