Liquid Chromatography

Mass Spectrometer

液相層析質譜儀

speaker: 應用化學系黃立心

date: 2015/4/9

LC-MS of 貴重/共同儀器中心

設備

LC-QqQ MS

液相層析-三段四極柱式質譜

LC-Q-TOF MS

液相層析-四極柱-飛行時間式質譜

游離方式

ESI電灑游離、APPI大氣壓力光游離

田家炳光電大樓 617室

2

chromatography

separation (+ enrichment)

mass spectrometry

separation + detection + identification

isotope analysis

qualitative analysis ; quantitative analysis

3

LC - MS 強調儀器硬體上的連接LC / MS 強調方法、技術上的結合

Why are they combined together ?

Part I

Mass Spectrometer

basic concepts

ion sources

mass analyzers

tandem MS

4

used in LC-MS

Mass spectrometer overview

5

sample

inlet

ion

source

mass

analyzer

ion

detector

data

system

gas phase

ions

vacuum

mass spectrum

ion sorting

by m/z

Ion & spectrum

mass to charge ratio , m/z

single charged, multiple charged

mass of atom/molecule

accurate mass & monoisotopic mass

average mass & nominal mass

mass resolution

parent ion

daughter (product, fragment) ion

6

7

8

rela

tive

in

ten

sity

m/z

A

B 單一同位素質譜圖

原始質譜圖

986.42 100 987.56 100

987.42 50.1 988.57 50.7

988.42 21.1 959.57 17.7

989.42 6.9 990.57 4.9

990.42 1.9 991.57 1.1

991.42 0.4 992.57 0.1

mass massrelative

abundance

relative

abundance

A = C41H66N10O14S2 B = C41H77N15O11S

0

50

100

985 986 987 988 989 990 991 992 993 994

0

50

100

985 986 987 988 989 990 991 992 993 994

保留訊號峰A = 986.42

B = 987.56

Ion sources used in LC-MS

ionization

high-energy exciting or attach other ion to the sample

hard or soft ionization

properties of sample : mass , stability…

other concerns

continuous sample introducing

large amount of solvent large amount of vapor

operate under atmospheric pressure

9

what kind of data

you want ??

Electrospray ionization 電灑游離 , ESI

10

1 atm

vacuum

1.5 - 2 kV

11

ESI

needle tip

sampling

cone (to

analyzer)

solvent of high polarity :

water, ACN (acetonitrile, CH3CN), methanol…

with volatile acid (H+ source) :

formic acid, acetic acid…

min. flow rate :

~20 nL/min

ESI :

M + nH+ [M + nH]n+

multiple charged ions

advantage?

disadvantage?

very soft

van der Waals complex

ion is possible !!

sample

small to macro molecules

high polarity

12

sample : 17 kDa protein

Atmospheric pressure photoionization

大氣壓光游離 , APPI

13

霧化器

analyzer

14

solvent dopant

dopant ion react with sample

ionization of sample

energy of photon

enough to ionize

sample or dopant

not enough to ionize

O2 , N2

atmospheric pressure chemical

ionization , 大氣壓化學游離 , APCI

corona電暈放電

inductively coupled plasma ,

感應耦合電漿 , ICP

8,000 - 10,000 K argon plasma

inorganic elemental analysis

15

16

ESI

APCIAPPI

can be applied to

small & medium molecules

that insoluble in water

small molecules to macro molecules

(peptide, protein, DNA… )

that soluble in water

Mass analyzer used in LC-MS

principles of analyzing ions with different m/z

different trajectories in electric/magnetic field

the same kinetic energy , different velocity

motion in electric/magnetic field induced current

concerns

m/z range

resolution

scanning or not , scanning speed

17

Quadrupole 四極柱

18

mass “filter”

ions of appropriate m/z

can pass through

19

+(U+V∙cosw t) –(U+V∙cosw t)

high-pass filter low-pass filter

if RF (AC) only no filter function

Time of flight 飛行時間式 ,TOF

20

getting the

same kinetic

energy

m/z = 2eV(t / L)2techniques for reducing

space & energy spread

5-30 kV

flight tube : 1 - 2 m

reflectron , electrostatic reflector

correcting the energy dispersion

longer flight distance

21

Quadrupole vs TOF

22

Quadrupole Time of Flight

mass range < 4,000 ; usually < 2,000 > 100,000

resolution usually set for unit resolution linear: ~3000

reflector: > 30,000 ; usually ~15,000

mass accuracy 50-100 ppm 2-50 ppm

scan speed < 4,000 Th/sec 106 Th/sec

kinetic energy of ion low high

accelerating voltage ~ 5-20 V ~ 5-30 kV

operating pressure < 10-3 Torr < 10-6 Torr

other features continuous pulse

compact size large size

$ $($$)

cylindrical quadrupole ion trap 圓柱形四極離子阱

orbitrap 軌道阱

exciting trapped ions induced current

23

Tandem MS 串聯質譜

why tandem MS ??

single or multiple analytes

different operating modes

precursor ion product ion

occur during hard ionization

collision-induce dissociation, CID

precursor ion inert gas (N2, Ar,…)

photo dissociation & other techniques

24

?

MS/MS operating modes

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操作模式 目的 操作方法

子離子（產物離子）product ion

獲得母離子的結構資訊

MS1 選擇單一母離子MS2 掃描記錄所有子離子

母離子（前驅物離子）precursor ion

尋找會產生相同子離子的母離子

MS1掃描所有母離子MS2選擇單一子離子

中性丟失neutral loss

尋找會產生相同中性碎片的母離子

MS1和MS2維持相同質荷比差，同時掃描

選擇反應監測selected reaction

monitoring

監測特定反應MS1選擇單一母離子MS2選擇單一子離子

product ion mode

26

other modes

27

precursor ion

28

neutral loss

selected reaction monitoring

Triple-quadrupole, QqQ

29

MS1 collision chamber MS2

(AC only q)

collision gas

Quadrupole-time-of-flight, Qq-TOF (Q-TOF)

30

collision gas

MS1

(Q)

ion guide

precursor ion product ions

collision chamber

(AC only q)

ion guide

MS2

(TOF)

detector

reflectron

QqQ & Q-TOF

Why there is a quadrupole in the collision chamber ?

comparison

operating modes : QqQ – all 4 modes

TOF can not be a filter Q-TOF – product ion mode

product ion sensitivity , resolution , mass accuracy : Q-TOF

simple & easy : QqQ

other MS/MS ? ion traps , TOF-TOF , TOF-Q

31

not for LC/MS

pressure , ion guide , energy

32

quadrupole or TOF ??

33

Part II

Liquid Chromatography

principle of chromatography

GC or HPLC

HPLC , LC/MS

34

MS (even tandem MS) is not enough

sample complexity

multiple analytes have to be analyzed

number , variety , concentration of analytes & matrixes

most samples, especially “real world” samples, vary widely in

complexity

sample – MS compatibility

matrixes/impurities , concentration

more information about a complex analyte

macro biomolecules : protein, DNA, polysaccharide

35

Principle of chromatography

36

mobile

phase

stationary

phase

flow of mobile phase

A

B

detecting

time

A B

chromatograph

層析圖

B

B

B

A

A

Gas vs liquid chromatography

GC

volatile molecules : low molecular weight , low-polarity

+ thermal stable

easy to couple with various ion sources of MS

LC (HPLC , HP = high performance or high pressure)

difficult to couple with MS

nonpolar to ionic , small to Marco molecules

various mechanism of separation alternatives

37

most versatile

technique !!

HPLC

38

PUMP column detector

sample

injector -

manual or

automatic

eluent 沖提液

(mobile phase)

separation according to :

polarity – normal or reverse phase

ion interaction – ion exchange

size of molecule

39

mobile

phase

auto sampler

pump HPLC

column

electrospray

ion source

QqQ MS

40

LC/MS/MS

time

TIC (total ion counts)

chromatograph

m/z

precursor ion

spectra

m/z

product ion

spectrainformation

of molecule

quantitation

analyzer

Considerations

41

sample

column

& eluent

ion

source

flow rate

solvent of eluent

additive in eluent

separation efficiency

amount of sample

recovery

molecular weight

ionization efficiency

soft or hard ionization

number of charge

Part III

Application Examples

melamine 三聚氰胺 additive in food

protein identification

peptide mass fingerprinting 胜肽質量指紋

peptide sequence tag 胜肽序列標籤

42

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Separating melamine from foods

foods : complex , variety can

to be analysis by MS directly

melamine

polar , slightly dissolved in water

using reverse phase HPLC + ESI

44

45

food sample (eg milk powder)

+ (water + CAN + 2.5% formic acid)

removing insolubles , proteins

HPLC separation

MS/MS analysis

hydrophilic

interaction

operate manually

on line analysis

46

– H2NCN – NH3

precursor ion

m/z = 127

product ion

m/z = 85

product ion

m/z = 68

selected reaction monitoring : m/z 127 85

quantification

Amino acid peptide protein

amino acid

20 essential a-amino acids with different side groups (R)

average molecular weight ~110 Da

peptide

50 or fewer amino acids linked together by peptide bonds

47

protein

> 50 amino acids

function

may have a few modifications on side group of amino acid

structure

primary structure – sequence of amino acid

▲ sequencing, identification, quantification…

secondary , tertiary , quaternary structures

48

MS

Protein identification

MS analysis

Why MS data from

peptides

is usually useful than

that

from intact protein ?

49

protein

peptides

Peptide mass fingerprinting , PMF

50

protease

(蛋白酶)

database search ,

identification

purified protein

AB

D

C

EF

mixture of peptides

A

B D

CE

F

MS

mass fingerprint

…….……………….….……

m/z

separation is not required!!

database search setting

51

result of search (extracted)52

Peptide analyzed by MS/MS

53

x3 y3 z3 x2 y2 z2 x2 y2 z2

a1 b1 c1 a2 b2 c2 a3 b3 c3

peptide : SIMAETLK

precursor ion ([M+H]+)

monoisotopic mass

= 892.5

product ionscollision condition !!

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protein mixture

A BC

DE

a

b g d

23

45

1

6

peptide mixture

A

B

C

D

E

a

b

g

d

2

3

4

5

1

6

MS/MS

LC separation

time

chromatograph

m/z

spectra of peptide

precursor ions

m/z

spectra of

product ions

database search

identify

multiple

proteins

protease

(蛋白酶)

vs PMF

proteins do NOT need to be separated

spectrum of product ions

sequence of peptide

find an unique peptide the protein can be identified

▲ multiple peptide is needed in PMF

type & position of modification

55

database

by computation

genomic data protein peptide product ion of peptide

56

2 matched peptide

data of product ions

(computed & found)