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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 m
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
(mi n)
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
30C
25 cm (1 column)
80C
25 cm (1 column)
80C
200 cm (8 columns)
80C
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
30C
25 cm (1 column)
1 ml/min
80C
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 m
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 140C
Detection UV 262 nm
Time
(min)
0 1 2 3 4 5
C10
C12
C14
C16
C18
N
+
R
CH
3
CH
3
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 m
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: 60C
Polaratherm: 80C
Polaratherm: 100C

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 m
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
90C
300 (2x150) mm column
0.45 ml/min
90C
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 mm
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 temperature
25 cm (1 column)
60C
25 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
60C
25 cm (1 column)

60C
125 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 m
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
40C
0.6 ml/min
60C
0.6 ml/min
80C
0.6 ml/min
60C
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 m
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
80C
T-program
70-90C 20C/min


9

6 Comparison of solvent and temperature
programming for the analysis of sulfonamides
Column Hypercarb, 100x3 mm, 5 m
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 50C
Gradient 0 to 5 min 50 to 100%B
Isotherm 50C
Isocratic 50%B
T-program 0 to 2 min 40C hold
2 to 9 min 40 to 180C (20C/min)
MS sensitivity
0.E+00
1.E+07
2.E+07
3.E+07
1 2 3 4
Peak number
P
e
a
k

a
r
e
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 m
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
25C
100% water
55C
1
2
4
3
5
1
2
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 60C. 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 m
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 60C
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 mm
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
40C
0.35 ml/min
80C
0.35 ml/min
60C
0.35 ml/min
80C
0.85 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 mm
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
5
0

C

9
0

C

T
-
p
r
o
g
r
a
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 80C. 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 m
Mobile phase A=water, B=acetonitrile
Gradient 50 to 100%B
Temperature 80C
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 m
Mobile phase A= water
B= methanol, acetonitrile, or ethanol
Gradient
Flow rate 0.6 ml/min
Temperature 40C
Detection UV 220 nm

Sample:
1 Aniline
2 a-naphthylamine
3 b-naphthylamine
4 a-naphthol
5 b-naphthol
6 N-phenyl-
a-naphthylamine
7 N-phenyl-
b-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
80C
water/ethanol
0.6 ml/min
1.2 ml/min
Column Zorbax SB-CN, 150x3 mm, 3.5 m
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 80C
Detection UV 220 nm


17

2 Polar compounds I Resolution and speed
Column Blaze C8, 150x4.6 mm, 3 m
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
70C
1 ml/min
100C
1 ml/min
100C
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 m
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
40C
T-program
0-4 min 40C, hold
4-6 min 40 to 80C
(20C/min)
6 min 80C, 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 m
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
F
r
e
e

s
u
l
p
h
u
r

F
r
e
e

s
u
l
p
h
u
r

F
r
e
e

s
u
l
p
h
u
r

Polysulfides
40C
Ternary solvent gradient
water/acetonitrile/
isopropanol
T-program
0-3.5 min 40C
3.5-8.5 min 40-90C (10C/min)
Isocratic
100% acetonitrile
T-program
0-4 min 50C
4-8 min 50-90C (10C/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 m
Mobile phase A=ammonium acetate in water
B=acetonitril
Gradient 50 to 100% B
Flow rate 1 ml/min
Temperature 50C
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 m dp columns coupled in series should ideally yield ca. 200,000
plates.


Column Zorbax SB300-C18, 250x2.1 mm, 5 mm
Mobile phase A=water, B=acetonitrile
Flow rate 0.2 ml/min
Temperature 60C
Detection UV 245 nm
Time
(min)
0 10 20 30 40 50 60 70
A
c

C
2

B
z

C
4

C
3

C
5

C
6

C
7

A
c

C
2

B
z

C
4

C
3

C
5

C
6

C
7

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 m
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 80C
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 m
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
25C
T-program
0 to 25C 1.3C/min
T-program
-20 to 5C 1.3C/min
D
i
g
l
y
c
e
r
i
d
e
s

L
L
L

O
L
L

P
L
L

O
L
O

S
L
L

P
L
O

L
L
L

O
L
L
P
L
L
O
L
O

S
L
L

P
L
O

L
L
L

O
L
L

P
L
L
O
L
O

S
L
L

P
L
O





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 m
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: 20C
Polaratherm: 90C
Polaratherm: 50C
High MW Low MW
Low MW High MW
(O )
OH
n
n ~1-20

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