Fundamental Principles of GC/MS & Introduction to Shimadzu GC/MS
Customer Support CentreShimadzu Asia Pacific Pte. Ltd.
Singapore
2
Outline
Objective: to understand how a GC/MS works
Topics:Gas chromatography basics Mass spectrometry basicsBasic GC/MS data
3
What is GC/MS
The technique
GC/MS stands for Gas Chromatography Mass Spectrometry, an analytical technique which allows separation and identification of organic compounds in a sample
GC/MS analysis starts by the separation of the compounds by using gas chromatographic technique, which makes use of a polymeric material contained in a column (called ‘stationary phase’) and an inert gas (called ‘carrier gas’) flowing through the column
The separated compounds are detected and further analyzed by mass spectrometric technique. In mass spectrometry, a compound is ionized and the resulting ions are separated according to their mass-to-charge ratio, and the ion abundances are measured.
4
What is GC/MS
The instrument
The instrument used for gas chromatographic mass spectrometric analysis is known as a gas chromatograph mass spectrometer (also called GC/MS)
Sample Analysis Report
SampleAnalysis
5
Why we need GC/MS
The information
By using GC/MS, we can:Identify what compounds are contained in a sampleDetermine whether certain substances/compounds are present in a sampleDetermine the amount (% weight, % composition) of specified compounds in a sample
6
Why we need GC/MS
Samples for GC/MS
Strictly speaking, samples for GC/MS are organic compounds withsufficient volatility (guideline: is in vapor form or can be turned to vapor at 400oC or less), sufficient thermal stability (does not decompose on heating)
In practice, some compounds which do not fulfill the criteria above can be made to fulfill the criteria by means of chemical reaction (‘derivatization’), thus making it possible for these compounds to be analyzed by GC/MS
7
Why we need GC/MS
Typical applications of GC/MS:
Pesticide residues and pollutants in water, agricultural products, foodstuffOrganic solvents in packaging materials, ink, etc.Drugs of abuse in urine, blood, tabletsFatty acid contents in edible oils, fat, etc.Essential oil characterizationAllergens in fragrancePolymer characterizationetc…
Food industry Agriculture industry
Environmental field Forensic field
Biotechnology field Fragrance industry
Chemical industry
8
Gas Chromatograph-Mass Spectrometer
Gas flow controller
Injection Port (Injector)
Column
Detector (MS)
Carrier Gas
Sample
Data System
• Separates mixture of compounds into individual components
• Interface between sample introduction device (e.g. syringe) and column
• Turns liquid sample into vapor by heating• Mixes sample vapor with carrier gas
• Controls the supply of gas(es) to the GC• Two types: manual flow controller and electronic flow controller
• Detects separated compounds and send signal to data system
• Controls GC system (more advanced, software-based system)• Converts signal from detector into human-readable format• Data analysis (process data)
9
Mass Spectrometer
Ion source
Mass analyzer
Ion detector
• Turns sample molecules into fragment ions• Transmits ions to mass analyzer
• Separates ions according to their mass-to-charge ratio
• Measures abundance of ions
Sample
Vacuum system • Evacuates the system
10
Gas Chromatograph-Mass Spectrometer
Injection port
Instrument controlData acquisitionData processingData storage
Control & DataSystem
GC OvenCapillary Column
Gas Chromatograph
Vacuum System
Ion SourceMass Analyzer
Mass Spectrometer
Detector
Gas
su
pply
(H
eliu
m)
Analyst
Interface
Detector of GC
Flow controller
11
Shimadzu GC/MS
GCMS-QP2010GCMS-QP2010S
QP2010 QP2010SGC GC-2010 GC-2010
Ionization modeEI, CI, NCI (dedicated ion sources). NCI has simulated EI and CI.
EI only
Ion source temp. Variable 100 - 260 degC Variable 100 - 260 deg C
Mass range 1.5 - 1024 a.m.u. 1.5 - 900 a.m.u.
High vacuum system Dual TMP (65 L/s + 260 L/s)
Single TMP (65 L/s)
Low vacuum system Rotary pump Rotary pumpMaximum column flow rate
15 mL/min 2 mL/min
Sensitivity at installation (OFN 1 pg, m/z 272, EI)
S/N > 60 S/N > 30
Gas ChromatographMass Spectrometer
12
GC Components of GCMS-QP2010GC Detector (optional)
Oven
Column
Injector
GC
Car
rier
G
as
Hyd
roge
n
Air
Mak
e up
Detector Gas(es) Flow Controller
Carrier Gas Flow Controller
Column
Oven
For GC/MS, carrier gas is normallyHelium
Electronic Flow Controllers
13
MS Components of GCMS-QP2010
Ion Detector
Vacuum chamberIon source
Quadrupole mass analyzer
Direct Interface to GC
Dual TMP system
14
Gas Chromatography Basics
Separation of compounds occurs inside the GC column
t2
Mixture
t1
Mobile (moving) phase
Stationary phase
t3
15
Separation of compounds
When analytes are introduced into the column, the molecules distribute between the stationary and mobile phases
The molecules in the mobile phase are carried down the column
Those in the stationary phase are temporarily immobile and do not move down the column
M
S
M = mobile phase (carrier gas)
S = stationary phase
16
Separation of compounds
The molecules in the mobile phase are carried down the column
Those in the stationary phase are temporarily immobile and do not move down the column
M
S
17
Separation of compounds
Molecules in the mobile phase re-enter the stationary phase when they collide with the stationary phase
At the same time span, molecules leave the stationary phase and enter the mobile phase
M
S
18
Separation of compounds
The molecules in the mobile phase are carried down the column
The process is repeated many many times inside the column
While the process is repeated, separation takes place
M
S
19
Separation of compounds
All molecules of the same compound travel through the column at nearly the same rate and appear as a band of molecules (called sample band)
M
S
20
Separation of compounds
Sample band of compound which is less ‘soluble’ in the stationary phase moves faster, because more of the molecules spend more time in the mobile phase (carrier gas)
M
S
21
From Injection Port
To Detector
Retention Time = time spent by a compound inside the column
Column
Separation in column
From Injection Port
To Detector
Column
From Injection Port
To Detector
Column
22
Chromatographic Data
Analysis time
Chromatogram
Intensity
Peak area (response)Depends on amount of sample
Retention time (measured from time of injection)Depends on compound and analytical conditions
23
Goal of Gas Chromatography
No overlap between adjacent sample bands as they exit the column
Make each sample band travel at a different rateMinimize the width of the sample band
Analysis time
Good chromatography
Analysis time
Poor chromatography
Coeluting peaks
24
Migration rates of compounds in column (1)
Different migration rates of compounds can be achieved if these compounds have different interaction strengths with the stationary phase
Weaker interactionStronger interaction
Stationary phase
25
Migration rates of compounds in column (2)
Migration rate of compounds in column depend on:
Compound chemical structure
Stationary phase chemical structure
Column temperature
26
Effect of compound chemical structure on migration rate
M
S
M
S
GC Column GC Column
27
Effect of stationary phase on migration rate
M
S
M
S
GC Column 1 GC Column 2
28
Effect of column temperature on migration rate
M
SLower T
M
SHigher T
GC Column GC Column
29
Sample Band Width
Sample band width depends on:
Operating conditions
Dimensions of the column
30
GC Parameters
Retention time (tR)Retention time of unretained compound (tM)Retention factor (k)Distribution constant (K)Phase ratio (β)Separation factor (α)Resolution (R)Number of theoretical plates (N)Height equivalent to a theoretical plate (HETP)Carrier gas linear velocity (v)
31
Retention Time (tR)
The time an analyte takes to travel through the column
A measure of the amount of time an analyte spends in the column
Sum of the time spent in the stationary phase and the mobile phase
32
Retention time of an unretained compound (tM)
The time an unretained compound takes to travel through the column
Unretained compound travels down the column at the same rate as the mobile phase (carrier gas)
Equivalent to the time a compound spends in the mobile phase
33
Retention factor (k)
Another measure of retentionRatio of the amount of time a compound spends in the stationary and mobile phasesA measure of retention by the stationary phasePreviously called capacity factor, or partition factor
tR - tMk = tM
34
Distribution constant (K)
Ratio of analyte concentration in the stationary phase and mobile phaseK is constant for a given compound, stationary phase, and column temperature
cSK =
cM
cS = concentration in stationary phasecM = concentration in mobile phase
35
Phase Ratio (β)
The change in the phase ratio can be used to calculate the change in a compound’s retention, provided that the same stationary phase and column temperature (program or isothermal) are maintained
An increase in phase ratio results in a decrease in retention (k), since K is constant; and vice versa
rK = kββ =
2df
r = column radius (µm)df = film thickness (µm)
36
Separation Factor (α)
A measure of the time or distance between the maxima of two peaks
α = 1 means the two peaks have the same retention and co-elute
α =k2
k1
37
Resolution (R)
A measure of overlap between two peaks; the higher the resolution, the less the overlapSeparation (α) is only the distance between two peak maxima; resolution takes both α and the width of the peaks into accountBaseline resolution usually occurs at R = 1.50
Analysis time
Analysis time
Analysis condition 1
Analysis condition 2
Same α, different R
R = 1.18tR2 – tR1
wh1 + wh2R = 2
tR2 – tR1
wb1 + wb2
wh = peak width at half peak heightwb = peak width at base
38
Number of theoretical plates (N) or Column Efficiency
Theoretical plates is a conceptTheoretical plates numbers are an indirect measure of peak width for a peak at a specific retention timeColumns with high N are considered to be more efficient than those with lower NA column with a high N will have a narrower peak at a given retention timeColumn efficiency is a function of:
Column dimensionsType of carrier gas and its average linear velocityCompound and its retention
tR
wb
tR
wh
N = 16N = 5.545
39
Height equivalent to a theoretical plate (HETP or H)
Another measure of column efficiency
Small plate heights indicate higher efficiencyL
H =N
L = column length (mm)N = theoretical plates number
40
Carrier Gas Linear Velocity (v)
Affects the chromatographic resolution (i.e. separation)
For each gas there is a linear velocity where optimum separation can be achieved (minimum HETP)
Van Deemter Curve
Linear Velocity (cm/s)
41