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Como escolher a colunaideal para LCMS
Celso Blatt, Ph.D.Agilent Technologies Brasil
COLACRO XIV - Florianopolis
Colunas para LCMS
Page 2
Colunas ZORBAX RP-HPLC para a máximaestabilidade em faixas de pH específicas
StableBond, pH 1-61. Uses bulky silanes
2. Non-endcapped
Eclipse XDB, pH 3-81) eXtra Densely Bonded
dimethylalkylsilanes
2) proprietary double-endcapping
*
Si
O
NH O
Zorbax Rx-SIL Silica Support Surface
Bonus-RP, pH 3-81) polar alkyl phase
2) triple endcapped
3) uses bulky silanes
Extend-C18, pH 2-11.51) unique bidentate structure
2) double endcapped
Si
O
Si
O
C18C18
At low pH, silica breaks down by hydrolyzing the siloxane* bond. This
breakdown can be almost entirely eliminated by using bulky silanes, found in
StableBond columns (left).
At mid-range and high pH, breakdown occurs from silica dissolution, and is
minimized by using endcapping procedures such as those in Eclipse XDB, Bonus-
RP and Extend.
*
3
Diferentes seletividades com a ZORBAX RRHT C18
1 2
3
4
1 2
3
4
1 2,3 4
1 234
Eclipse Plus C18
Eclipse XDB C18
Extend C18
StableBond C18
Mobile phase: (69:31) ACN: water
Flow 1.5 mL/min.
Temp: 30 °C
Detector: Single Quad ESI positive
mode scan
Columns: 4.6 x 50 mm 1.8 um
Sample:
1. anandamide (AEA)
2. Palmitoylethanolamide (PEA)
3. 2-arachinoylglycerol (2-AG)
4. Oleoylethanolamide (OEA)
1 2 3 4 5
250000
500000
750000
1000000
1250000
MSD1 TIC, MS File (C:\CHEM32\1\DATA\120506\ETAMIDE000075.D) ES-API, Pos, Scan, Frag: 70, "scan"
1 2 3 4 5
250000
500000
750000
1000000
1250000
1 2 3 4 5
250000
500000
750000
1000000
1250000
min1 2 3 4 5
250000
500000
750000
1000000
1250000
1
2
3
4
Page 4
Reduzindo o diâmetro da coluna para aumentar a sensibilidade
min0 2.5 5 7.5 10 12.5 15 17.5
mAU
0
200
400
600
800
1000
1200
1400
200ng Bifenil
Coluna: Zorbax SB C18
0.3mm0.5mm1.0 mm4.6 mm
Page 5
Efeito do tamanho da partícula
Curva de Van-Deemter para diferentes tamanho de partículas
16
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14
ZORBAX Eclipse XDB-C18
4.6 x 50mm
85:15 ACN:Water
1.0µL Octanophenone
0.05 – 5.0 mL/min
20°C
5.0 µµµµm
3.5 µµµµm
1.8 µµµµm
HETP
[µ
m/
pla
te]
ue [mm/s]
Particle Hmin
5.0 µm 9.3 µm
3.5 µm 6.0 µm
1.8 µm 3.8 µm
H = A + B/u + Cu
Comprimento da coluna e tamanho de partícula na eficiência da coluna
Coluna
Comp.
(mm)
Coluna
Eficiência
N(5 µm)
Coluna
Eficiência
N(3.5 µm)
Coluna
Eficiência
N(1.8 µm)
150 12,500 21,000 35,000
100 8,500 14,000 23,250
75 6000 10,500 17,500
50 4,200 7,000 12,000
30 N.A. 4,200 6,500
15 N.A. 2,100 2,500
Eficiência(N)
Pressão
TempoAnálise
VolumePico
Tempo de
análise
-
-33%
-50%
-67%
-80%
-90%
Uso de Solvente
Page 7
Aumento da Resolução
4.6 x 150, 1.8µm490 barN = 28669 R_S = 1.80 (+57%)S/N = 44
7 Impurezas
todas 7 separadas!
4.6 x 150, 3.5µm165 barN = 14862R_S = 1.37S/N = 50
7 Impurezas
6 nãoseparadas!
4.6 x 150, 5µm93 barN = 7259R_S = 1.15S/N = 42
4 Impurezas
2 nãoseparadas!
Page 8
Colunas para LC/MS
Coluna Coluna I.D. Comp. colunaTamanhopartícula
Fluxo ideal
Nano 0.075, 0.10 mm 50, 150 mm 3.5 um100 – 600
nL/min
Capillary 0.3, 0.5 mm35, 150, 250
mm3.5, 5 um 1 – 10 uL/min
MicroBore 1.0 mm 30, 50, 150 mm 1.8, 3.5, 5um 30 – 60 ul/min
Narrow Bore 2.1 mm 15 – 250 mm 1.8, 3.5, 5 um0.1 – 0.3 mL/min
Solvent Saver 3.0 mm 50 – 250 mm 1.8, 3.5, 5um 0.3 – 1 mL/min
Analytical 4.6 mm 15 – 250 mm 1.8, 3.5, 5um 1 – 1.5 mL/min
Page 9
Escolha colunas longas para ter a máxima resolução cromatográfica 2.1 x 150 mm Column
0 1 2 3 4 5
Bromazepam
m/z = 316
Oxazepam
m/z = 287
Lorazepam
m/z = 321
Diazepam
m/z = 285
Clobazamm/z = 301
Total Ion Chromatogram
4.58
4.07
3.26
3.01 4.07
2.26
Time (min)
Diazepam
3.01
2.26 3.28
4.07
4.57
160 180 200 220 240 260 280 300 320 340
260
261 281
301
303
285
287
321
323
287
289
316 318
m/z
Bromazepam
Oxazepam
Lorazepam
Diazepam
ClobazamColumn: ZORBAX SB-CN
2.1 x 150 mm, 5 umMobile Phase: 60% water + 0.2% formic acid
40% ACNFlow Rate: 0.25 mL/minDetection: Electrospray Positive IonSample: Benzodiazepines
1. Bromazepam2. Oxazepam3. Lorazepam4. Diazepam5. Clobazam
Page 10
Escolha colunas curtas para análises rápidas com identificação por MS 2.1 x 50 mm Column
m/z = 316
m/z = 287
m/z = 321
Diazepamm/z = 285
Clobazam
m/z = 301
2.19
1.94
1.63
1.47(1.94)
1.16
Time (min)
(Diazepam)
1.15 1.94
0 1 2 3 4 5
1.49
12,3
4,5
160 180 200 220 240 260 280 300 320 340
260318
301
303
285
287
321
323
287
289
316 318
319
m/z
Diazepam
Clobazam
Total Ion Chromatogram
Bromazepam
Oxazepam
Lorazepam
Bromazepam
Oxazepam
Lorazepam
Single Ion Chromatograms
Column: ZORBAX SB-CN2.1 x 50 mm, 5 um
Mobile Phase: 60% water + 0.2% formic acid
40% ACNFlow Rate: 0.20 mL/minDetection: Electrospray Positive IonSample: Benzodiazepines
1. Bromazepam2. Oxazepam3. Lorazepam4. Diazepam5. Clobazam
Page 11
Longitudinal diffusion
Partículas menores aumentam a eficiência da colunaP
late
Heig
ht
H
Linear Velocity u
Eddy Diffusion
Sum Curve: van-Deemter
Resistance to Mass Transfer
H = A + B/u + C u
Large Particle
Small
Particle
u opt
H min
Page 12
Sub 2um Totally Porous Particle
Superficially Porous Particle
Melhor transferência de massaMelhor transferência de massa
SOLID CORE
Page 13
Colunas Poroshell 120 para HPLC e UHPLC
• 80-90% efficiency of sub 2um
• At ~40-50% lower pressure
• 2X efficiency of 3.5um (totally porous)
• A 2.7um particle size
• A 2um frit to reduce clogging
• A 600 bar pressure limit
• The particle has a solid core (1.7um) and
porous outer layer with a 0.5um diffusion
path
Poroshell 120 columns have:
1.7um
0.5um
0.5um
14
Análise de 15 analgésicosMesmo método para ambas colunas
ZORBAX RRHD Eclipse Plus C18, 3 x 100 mm, 1.8 µm
ZORBAX Poroshell 120 EC-C18, 3 x 100 mm, 2.7 µm
Ibuprofen:PW1/2=0.012S/N=353nc=54
Ibuprofen:PW1/2=0.012S/N=256nc=56
2 min
Page 15
Poroshell 120 Comparada com Sub 2umDesempenho similar
min0 2 4 6 8 10
min0 2 4 6 8 10
RRHT Eclipse Plus C18
1.8 um
P = 510 Bar
Poroshell 120
EC-C18
2.7 um
P = 332 Bar
Columns: 4.6 x 100 mmA: Water 0.1% Formic Acid
B: Acetonitrile 0.1% formic Acid
Gradient 2 ml/min
Initial 8 % B
10 min 30% B
275 nm 2mm flow cell
Injection: 10 uL
Agilent 1200 SL
40 °C
1. Hydroquinone
2. Resorcinol
3. Catechol
4. 4-Nitrophenol
5. p-cresol
6. o-cresol
7. 2-Nitrophenol
8. 2,3 Dimethyl phenol
9. 2,5 Dimethyl phenol
10. 1-Naphthol
10
4 56 7
8
9
10
12
3
12
34 5
67
8
9
Page 16
Tamanho de partícula constante, gradiente, mais resolução
Page 17
min0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
min0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
min0 2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
RRHD SB-C18 2.1 x 50 mm, 1.8umPmax=366 barnc = 424
RRHD SB-C18 2.1 x 100 mm , 1.8umPmax=595 bar nc = 485
RRHD SB-C18 2.1 x 150 mm, 1.8umPmax=768 bar nc = 589
Rs: 2.40
Rs: 1.37
Rs: 0
Page 17
0 2 4 6 8 10 12 14
mAU
0
50
100
150
200
250
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\070809SBC180003.D)
1.4
74
3.3
23
5.0
64
5.6
51
6.1
14
6.9
64
8.3
37
9.6
90
10.
982
Coluna convencional - 4.6 x 150mm, 5µm, SB-C18
Flow Rate 1.0 ml/minInjection Volume 15uL
Temperature 30°CWavelength 246nmSample rate 2.5 Hz
Time (min) % Acetonitrile
0 50
10 90
13.5 90
13.6 50
15 50
Initial Pressure: 69 barFinal Pressure: 38 bar
Page 18
Coluna mais curta, partícula menor e gradiente mais rápido1/3 Comprimento- 1/3 Gradiente
Coluna – 4.6 x 50mm, 1.8µm, SB-C18
min0 1 2 3 4 5 6
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-09 17-25-25\070809SBC180009.D)
1.1
76
2.0
04
2.2
54
2.4
64
2.8
11
3.3
57
3.8
71
4.3
43
Flow Rate 1.0 ml/min
Injection Volume 5uL
Temperature 30°CWavelength 246nm
Sample rate 13.74 Hz
Time (min) % Acetonitrile
0 50
3.33 90
4.5 90
4.53 50
5 50
Initial Pressure: 132 barFinal Pressure: 74 bar
Page 19
Ancrease Column Flow-Reduce Gradient TimeDouble Flow (2mL/min) – ½ Gradient Time
RRHT 4.6 x 50mm, 1.8µm, SB-C18
0 0.5 1 1.5 2 2.5 3
mAU
0
20
40
60
80
100
120
140
VWD1 A, Wavelength=246 nm (D:\SAMPLE TEST\RRHT-1100\HDS 2007-08-10 08-22-16\070810SBC180004.D) 0
.58
7
1.0
20
1.1
46
1.2
60
1.4
37
1.7
15
1.9
70
2.2
01
Flow Rate 2.0 ml/min
Injection Volume 5uL
Temperature 30°CWavelength 246nm
Sample rate 13.74 Hz
Time (min) % Acetonitrile
0 50
1.67 90
2.25 90
2.27 50
3.34 50
Initial Pressure: 266 barFinal Pressure: 146 bar
Page 20
Poroshell 120 150mm Columns – HPLC or UHPLC
2 mL/min
min0 2 4 6 8 10 12 14 16 18
mAU
0
20
40
60
80
100
120
min0 5 10 15 20 25 30 35
mAU
0
20
40
60
80
100
120
1 mL/minP = 285 bar
P = 538 bar
Conditions: Column: Poroshell 120 EC-C18, 4.6 x 150mm, 2.7um Mobile Phase: Solvent A: Water with 0.1% Formic Acid
Solvent B: Acetonitrile 1200 SL controlled temperature at 25 °C 2 ul flow cell
1. Hydroquinone2. Resourcinol3. Catechol4. Phenol5. 4-Nitrophenol6. p-cresol7. o-cresol8. 2-Nitrophenol9. 3,4 di methyl phenol10. 2,3 di methyl phenol11. 2,5 di methyl phenol12. 1-napthol
Gradient: 1mL/min
Time%B
6.0 5%
51 60%
Gradient: 2mL/min
Time%B
3.0 5%
25.5 60%
Page 21
Comparison of 4.6 x 250 mm 5 um to Poroshell 120 EC-C18 4.6 x 100 mm, 2.7um
min5 10 15 20 25 30
mAU
0
20
40
60
80
100
9.7
12
11
.11
6
11
.59
6
12
.67
4
15
.24
8
16
.15
11
6.4
35
20
.68
7
23
.07
6
29
.29
0
min5 10 15 20 25 30
mAU
0
50
100
150
200
250
1.7
19
2.1
89
2.3
11
2.6
06
3.8
67
4.4
37 4
.55
8
5.4
50
5.9
20
7.0
37
325 bar
110 bar
Sulfadiazine,Sulfathiazole SulfapyridineSulfamerazine,Sulfamethazine, Sulfamethazole, Sulfamethoxypyridazine,SulfachloropyridazineSulfamethoxazole, Sulfadimethoxine
Time %B0 833 3334 33Column: Eclipse Plus C184.6 x 250mm, 5umFlow Rate: 1 mL/min
Mobile Phase:A: 0.1% formic acid in WaterB: 0.1% formic acid in ACN
Time %B0 812 3313.2 33Column: 4.6 x 100mmPoroshell 120 EC-C18, 2.7umFlow Rate: 1 mL/min
Page 22
Expand High Speed Chromatograms for True Comparison to Slower Separation
min5 10 15 20 25 30
mAU
0
20
40
60
80
100
9.7
12
11
.11
6
11
.59
6
12
.67
4
15
.24
8
16
.15
11
6.4
35
20
.68
7
23
.07
6
29
.29
0
110 bar
5
mAU
0
50
100
150
200
250
1.7
19
2.1
89
2.3
11
2.6
06
3.8
67
4.4
37 4
.55
8
5.4
50
5.9
20
7.0
37
325 bar
min
Page 23
Max Performance RRHD - UHPLC/TOF (1290/6230)Identify More Compounds in Very Short Run Time
1 .5
min
Time Composition
0.0 10% ACN
1.5 100% ACN
224 pesticides at 50 pg each217 ionized & detected in positive mode(97%, Find by Formula) 2.1 x 50 mm x 1.8 micron
Eclipse Plus C-18900 bar1.5 mL/min1290 Infinity
Page 24
Colunas para diferentes modos de operação emLCMS
• SCAN – (SQ, QQQ, TRAP) – coluna muito eficiente
• SCAN – (TOF, QTOF) – picos extreitos
•SIM – (SQ, QQQ) – coluna eficiente
•MRM – (QQQ) – picos extreitos
Page 25
Escolhendo a coluna e condições para LC/MS
Configuração da coluna
• Comprimento da coluna – resolução cromatográfica
• Tamanho de partícula da coluna – resolução cromatográfica
• Diâmetro interno da coluna – fluxo, quantidade de amostra
Escolha da fase móvel
• Melhora a ionização do soluto – baseado na técnica usada – ESI, APCI, APPI
• Melhora a sensibilidade
• Uso de aditivos voláteis
Fase ligada
• Escolha do pH da fase móvel para aumentar a vida útil e diminuir o sangramento
Page 26
Tampões indicados para ESI
Positive ion detection of basic
analytes
Buffer choices (10 mM or less)
• Acetate pKa 4.8
• Propionic acidpKa 4.8
• Formate pKa 3.8
• TFA highly acidic
Typical analytes – amines, amides,
antibiotics
Negative ion detection of acidic analytes
Buffer choices (10 mM or less)
• Ammonia pKa 9.2
• Diethylamine pKa 10.5
• Triethylamine pKa 10.7
• Piperidine pKa 11.1
Typical analytes – acids, hydroxyls,
phosphates, sulfates
Mantenha 1 – 2 pH acima ou abaixo do pKa do analito.
Page 27
Adaptando um método LC existente para um método LC/API-MS
Substituia os tampões não voláteis por tampões voláteis na concentração
de <10 mM para ESI ou <100 mM para APCI
• Substitua os fosfatos e boratos com acetato de amonia, formiato de
amonia ou TFA
• Se um tampão não volátil tiver que ser usado, escolha o tampão com
somente uma parte anionica ou cationica não volátil (ex. fosfato de
amônia e mantenha a concentração bem baixa) e mantenha o diâmetro
e o fluxo baixo (2.1 ou 1.0 mm id)
Mantenha o mesmo pH do método original incluindo os aditivos voláteis –
ácido fórmico, ácido acético, TFA e hidróxido de amônia
Page 28
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