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