Chem. 230 – 11/18 Lecture. Announcements I Exam 3 Results –Lower Average (72%) –Distribution New Homework Posted Online (long problems due next week)

Chem. 230 – 11/18 Lecture

Announcements I• Exam 3 Results

– Lower Average (72%)– Distribution

• New Homework Posted Online (long problems due next week)

Announcements II• Special Topics Presentations

– Need to prepare reading material (link to journal or photocopies in folder) one week before presentations (due today for group going 11/25)

– Besides presentation, will need Homework Problems (I request 4 per group) on day of presentation

11/25 Group Stephanie & Diana

Topic MEKC

12/2 Brenden & Leo

Theo & Chris

Nancy & Maria

SMB chromatogr.

SPME-HPLC

Ion-pairing HPLC

12/9 Sam & Luis Morgan & Nicole

Dai & Olga Emily & Adriana

Dustin & Rich

Thao & Addison

Fluid Flow Fractionation

SF extraction

Chiral Separations

Zirconia in HPLC

2D LC Zwitterionic HPLC

Announcements III• Today’s Lecture

– Quantification• Methods of Calibration

– Mass Spectrometry• Applications• Instrumentation• Use as Chromatographic Detector• Interpretation• Other Topics

Quantitation in Chromatography Calibration Methods

• External Standard– most common method– standards run separately and

calibration curve prepared– samples run, from peak areas,

concentrations are determined– best results if unknown concentration

comes out in calibration standard range

• Internal Standard– Common for GC with manual injection

(imprecisely known sample volume)– Useful if slow drift in detector response– Standard added to sample; calibration

and sample determination based on peak area ratio

– F = constant where A = area and C = conc. (X = analyte, S = internal standard)

SX

SX

CC

AAF

/

/

Area

Concentration

AX/AS

Conc. X (constant conc. S)

Quantitation in Chromatography Calibration Methods

• Standard Addition– Used when sample matrix

affects response to analytes– Commonly needed for LC-MS

with complicated samples– Standard is added to sample

(usually in multiple increments)– Needed if slope is affected by

matrix– Concentration is determined by

extrapolation (= |X-intercept|)• Surrogate Standards

– Used when actual standard is not available

– Should use structurally similar compounds as standards

– Will work with some detector types (FID, RI, ABDs)

Area

Concentration Added

Analyte Concentration

0 bmXA

mbX /

standards in water

QuantitationAdditional (Recovery Standards +

Questions)• Recovery Standards

– Principle of use is similar to standard addition– Standard (same as analyte or related

compound) added to sample, then measured (in addition to direct measurement of sample)

– Useful for determining losses during extractions, derivatization, and with matrix effects

expected

unknowntotal

expected

recovered

amount

100amount - amount

amount

100amount recovered%

QuantitationSome Questions/Problems

1. Does increasing the flow rate improve the sensitivity of a method?

2. Does the use of standard addition make more sense when using a selective detector or a universal detector?

3. Is a matrix effect more likely with a simple sample or a complex sample?

4. Why is the internal standard calibration more common when using manual injection than injection with an autosampler?

QuantitationSome Questions/Problems

5. A scientist is using GC-FID to quantitate hydrocarbons. The FID is expected to generate equal peak areas for equal numbers of carbons (if substances are similar). Determine the concentrations of compounds X and Y based on the calibration standard (1-octanol). X = hydroxycyclohexane and Y = hydroxypentane.

Compound

1-octanol cC6-OH cC5-OH

Area 3520 299 1839

Conc. (ug mL-1)

10.0 ? ?

QuantitationSome More Questions/Problems

6. A chemist is using HPLC with fluorescence detection. He wants to see if a compound co-eluting with a peak is quenching (decreasing) the fluorescence signal. A set of calibration standards gives a slope of 79 mL μg-1 and an intercept of 3. The unknown gives a signal of 193 when diluted 4 mL to 5 mL (using 1 mL of water). When 1.0 mL of a 5.0 μg mL-1 standard is added to 4.0 mL of the unknown, it gives a signal of 265. What is the concentration of the unknown compound and is a significant quenching (more than 10% drop in signal) occurring?

QuantitationSome More Questions/Problems

7. A chemist is testing an extraction process for removing DDT from fish fat. 8.0 g of fat is first dissolved in 50 mL of 25% methylene chloride in hexane. The 50 mL is divided into two 25 mL portions, one of which is spiked by adding 2.0 mL of 25.0 ng mL-1 DDT. Each portion is run through a phenyl type SPE cartridge and the trapped DDT is eluted with 5.0 mL 100% methylene chloride. The methylene chloride is evaporated off, and the sample is redissolved in 0.5 mL of hexane and injected onto a GC. The un-spiked sample gives a DDT conc. (in 0.5 mL of hexane) of 63 ng mL-1, while the spiked sample gives a DDT conc. of 148 ng mL-1. What is the % recovery? What was the original conc. of DDT in the fat in ppb?

Mass SpectrometryOverview

• Applications of Mass Spectrometry• Mass Spectrometer Components• GC-MS• LC-MS• Other Applications

Mass SpectrometryApplications

• Direct Analysis of Samples– Most common with liquid or solid samples– Reduces sample preparation– Main problem: interfering analytes

• Off-line Analysis of Samples– Samples can be separated through low or high

efficiency separations– More laborious

• Chromatographic Detectors– generally most desired type since this allows

resolution of overlapping peaks


• Purposes of Mass Spectrometry– Quantitative Analysis (essentially used as any

other chromatographic detector)• Advantages:

– selective detector (only compounds giving same ion fragments will overlap)

– overlapping peaks with same ion fragment can be resolved (through deconvolution methods)

– semi-universal detector (almost all gases and many solutes in liquid will ionize)

– very good sensitivity• Disadvantages

– cost– requires standards for quantification


• Purposes of Mass Spectrometry - continued– Qualitative Analysis/Confirmation of Identity

• With ionization method giving fragmentation, few compounds will produce the same fragmentation pattern

• Even for ionization methods that don’t cause fragmentation, the parent ion mass to charge data gives information about the compound identity.

• Some degree of elemental determination can be made based on isotopic abundances (e.g. determination of # of Cl atoms in small molecules)

• Additional information can be obtained from MS-MS (further fragmentation of ions) and from high resolution mass spectrometry (molecular formula) if those options are available.

– Isotopic Analysis• Mass spectrometry allows analysis of the % of specific isotopes

present in compounds (although this is normally done by dedicated instruments to get good enough precision for use as source tracers)

• An example of this use is in drug testing to determine if testosterone is naturally produced or synthetic

Mass SpectrometryInstrumentation

• Main Components:– Ion source– Analyzer– Detector– Data Processor


• Ion Sources– For Gases

• Electron Impact (EI):– electrons from heated

element strike molecules– M + e- => M+* + 2e-

– M+ is the parent ion– Because M+* often has

excess energy, it can fragment further, usually producing a smaller ion and a radical

– Fragmentation occurs at bonds, but electronegative elements tend to keep electrons

e-

+

e-

gas stream M

CH3-Br+*

CH3+ + Br∙

Main fragment

CH3∙ + Br+

Minor or unobserved fragment

Mass SpectrometeryInstrumentation

• Ion Sources– For Gases

• Chemical Ionization (CI):– Can produce positive or negative ions– First, a reagent gas reacts with a corona

discharge to produce a reagent ion: CH4 => => CH5

+ (more likely CH4∙H+)

– Then the reagent ion transfers its charge to a molecule: M + CH5

+ => MH+ (one of largest peak has mass to charge ratio of MW + 1)

– Less fragmentation occurs, so more useful for identifying the parent ion


• Ion Sources– For Liquids

• Earlier Methods (particle beam and thermospray) suffered from poorer efficiency and ability to form ions from large molecules

• Electrospray Ionization (ESI):– Liquid is nebulized with sheath gas– Nebulizer tip is at high voltage (+ or –), producing charged droplets– As droplets evaporate, charge is concentrated until ions are expelled– Efficient charging of polar/ionic compounds, including very large

compounds– Almost no fragmentation, but multiple charges possible– For positive ionization, major peak is often M+1 peak; or for multiply

charged compounds, peak is [M+n]n+ where n = charge on ion

Liquid in

Nebulizing gas High voltage

++

+++

M+


• Ion Sources– For Liquids (continued)

• Atmospheric Pressure Chemical Ionization– Liquid is sprayed as in ESI, but charging is from a

corona needle nearby- More restricted to smaller sized molecules

• Atmospheric Pressure Photoionization– UV light causes photoionization of molecules


• Ion Sources– For Solids (common

off-line method)• Matrix Assisted Laser

Desorption Ionization– Sample plus strong

absorber placed on substrate

– solvent removed– laser focused on sample– heat causes desorption

and ionization of analytesM+

Mass Spectrometry Instrumentation

• Analyzers– Separates ions based on mass to charge ratio– All operate at very low pressures (vacuums) to avoid

many ion – ion or ion – molecule collisions– Analyzers for chromatographic systems must be

fast. (If a peak is 5 s wide, there should be 4 scans/s)

– Most common types (as chromatographic detectors):• Quadrupole (most common)• Ion Trap (smaller, MS-MS capability)• Time of Flight (higher speed for fast separations and can be

used for high resolution applications)


• Mass Spectrometer Resolution– R = M/ΔM where M = mass to charge ratio and is ΔM

difference between neighboring peaks (so that valley is 10% of peak height).

– Standard resolution needed:• To be able to tell apart ions of different integral weights (e.g.

(CH3CH2)2NH – MW = 73 vs. CH3CH2CO2H – MW = 74)– High Resolution MS:

• To be able to determine molecular formulas from “exact” mass • example: CH3CH2CO2H vs. CHOCO2H; both nominal masses are 74

amu but CHOCO2H weighs slightly less (74.037 vs. 74.000 amu) because 16O is lighter than 12C + 41H (Note: need to use main isotope masses to calculate these numbers – not average atomic weights). Needed resolution = 74/0.037 = 2000

• To separate similar ions requires very high resolution > 104 to 105 • However, to obtain “accurate” mass (error in mass under 5 ppm)

is not quite as hard in terms of resolution but requires internal standards and clean peaks

Mass Spectrometry Instrumentation

• Analyzers – how separation works– Analyzers can act as filters (only passing a

specific m/z at a time) – e.g. in quadrupoles and ion traps, can give full spectrum in a short time (time of flights), or can give full information over an acquisition (Fourier Transform ion cyclotron resonance)

– Control of ion throughput makes sense in ion traps or in quadrupoles but in time of flight full spectrum comes (whether desired or not)


• Detectors:– Faraday Cup (simple, but not sensitive)– Electron Multiplier (most common)– Array Detector (Multichannel Analyzer)

Anode

Cathode

Dynodes

M+

e-e-

I

Detection Process:

Ion strikes anode

Electrons are ejected

Ejected electrons hit dynodes causing a cascade of electron releases

Current of electrons hitting cathode is measured

M+

I

Mass SpectrometeryUse with GC

• MS matches well to capillary GC flow rates• With EI gives good qualitative information• CI used if compound fragments too much• Total Ion and Selective Ion Modes:

– Total Ion Current (TIC) gives full mass spectra at every point (better for qualitative analysis)

– Selective Ion Monitoring (SIM) only determines signal at several ions (the fragments of interest) (better for quantitative analysis because of better sensitivity)

Mass SpectrometeryUse with GC - Example

• Example of examination of co-eluting peaks

• Synthetic diesel sample shows large number of peaks – mostly alkanes and alkenes

min0 5 10 15 20 25

pA

20

40

60

80

100

120

140

160

180

FID1 B, (YVONNE\08081301.D)

1.6

34

1.7

07

1.7

56

1.8

24

1.9

61

2.0

40

2.0

87

2.1

43

2.1

94

2.2

67

2.7

37

2.7

68

2.8

09

2.8

84

2.9

74

3.1

20

3.1

84

3.2

44

3.3

17

3.3

69

3.5

04

4.3

78

4.4

07

4.5

19

4.5

94

4.6

43

4.7

84

4.8

58

5.0

13

5.1

42

5.2

89

5.6

58

6.0

08 6

.16

2 6

.29

2 6

.42

3 6

.53

7 6

.62

6 6

.70

6 6

.76

4 6

.86

5 6

.99

4 7

.16

4

7.6

32

7.7

23

7.7

60

7.8

59

7.9

40

8.0

69

8.1

51

8.2

69

8.3

49

8.4

68

8.6

62

8.9

69

9.0

39

9.0

78

9.1

25

9.2

09

9.2

62

9.4

72

9.5

73

9.6

38

9.7

51

9.8

59

9.9

08

10

.23

7 1

0.2

84

10

.33

2 1

0.4

12

10

.58

5 1

0.6

53

10

.74

2 1

0.8

00

10

.90

8 1

1.2

63

11

.33

4 1

1.3

86

11

.43

5 1

1.5

09

11

.66

9 1

1.7

33

11

.81

3 1

1.8

64

11

.97

5 1

2.3

24

12

.35

6 1

2.4

04

12

.45

4 1

2.5

27

12

.80

9 1

2.8

56

12

.96

5 1

3.2

77

13

.31

5 1

3.3

69

13

.41

7 1

3.4

90

13

.76

4 1

3.8

10

14

.20

6 1

4.2

59

14

.31

4 1

4.3

64

14

.43

9 1

4.7

14

15

.13

7 1

5.2

01

15

.25

8 1

5.3

08

15

.38

6 1

5.6

55

16

.07

0 1

6.1

46

16

.20

6 1

6.3

36

16

.60

4

17

.00

9 1

7.1

58

17

.29

1 1

7.5

56

18

.50

2

19

.43

8

20

.35

4

21

.24

9

22

.12

0

22

.96

4

23

.83

4

24

.78

4

25

.85

0

peak cluster = (mostly) same number carbons

min10.2 10.4 10.6 10.8 11

pA

10

20

30

40

50

60

FID1 B, (YVONNE\08081301.D)

10

.23

7

10

.28

4

10

.33

2

10

.41

2

10

.58

5

10

.65

3

10

.74

2

10

.80

0

10

.90

8

Alkane

2-Alkenes1-Alkene

mostly branched alkanes

C12s

Mass SpectrometeryUse with GC – Example – Cont.

• Analysis didn’t match manufacturer’s assessment of 4% alcohols

• However, alcohols are hard to determine by MS due to loss of H2O in fragmentation– CH3(CH2)6OH → CH3(CH2)5CH·+ (MW = 98 – same as expected

for alkene M peak)

• Linear Alcohols found to elute at time of branched C10 alkanes

mass spectrum shows alkyl chains


• Careful examination of fragmentation shows differences between right and left sides of peak with right side close to that of C7 alcohol standard

right shoulder

1-heptanol


• Ion Extraction allows separation of chromatographic peaks based on 70 vs 71 fragments

• Could improve by: using CI, using slight difference in column polarity• Identification stronger due to water washing fuel

70 (alcohol) fragment

71 (branched alkane) fragment

Mass SpectrometeryUse with HPLC

• One disadvantage is the volume of gas developed as solvent evaporates

• For this reason, HPLC flows must be low (e.g. semi-microbore), or splitters are needed

• With most common ionization (ESI), little fragmentation occurs, making identification of unknown compounds harder

• Because of little fragmentation, MS-MS is more common

• In MS-MS, ions leaving mass analyzer are then fragmented (by collisions with molecules) before entering a second mass analyzer or re-entering the mass analyzer

• Also, some compounds are hard to ionize efficiently

Mass SpectrometeryInterpretation

• Fragmentation Analysis– Focus on possible structure of fragments (low end of

spectrum) or of fragments lost (high end of spectrum)

• Isotopic Analysis– For elements with more than 1 isotope in abundance– Average MW not useful, MW of specific isotopes

determines charge– Formation of M+1, M+2, M+3 ... peaks to predict

elements present

• Determination of Charge– Important for interpreting MALDI and ESI peaks where

multiple charges are possible

Mass SpectrometryIsotope Effects

• It also may be possible to distinguish compounds based on isotopic composition

• Average MW is not useful (except for very large MW compounds), but abundance of each isotope gives each element a “fingerprint”

• Compounds in high resolution example will have different expected M+1/M and M+2/M ratios (which will NOT require high resolution to see)

• Go over calculations on board for CH3SSCH3

• Main difficulty is accurately determining ratios (plus effects of contaminants, variation in ratio, etc.)

Mass SpectrometryOther Topics – Multiple Charges in ESI

• In ESI analysis of large molecules, multiple charges are common due to extra (+) or missing (-) Hs (or e.g. Na+)

• The number of charges can be determined by looking at distribution of big peaks

• For + ions m/z = (M+n)/n (most common)

• For – ions m/z = (M–n)/n

Ion

curr

ent

m/z

m/z

m/z = (M+n)/n – (M+n+1)/(n+1) = (M+n)(n+1)/[n(n+1)] – (Mn+n2+n)/[n(n+1)] = M/[n(n+1)] = 141.9, (94.5, 67.7)

(M+n)/n

(M+n+1)/(n+1)

Example: m/z peaks =711.2, 569.3, 474.8, 407.1

Do rest on board

Mass Spectrometry Other Topics – Multiple Charges in ESI

• Another way to find charge on ions is to examine the gap in m/z between isotope peaks (0 13C vs. 1 13C)

• The +1 mass difference will be ½ if charge is +2 or 1/3 if charge is +3

Glycodendrimer core Glycodendrimer core

gap = 405.73 – 405.23 = 0.50

Mass SpectrometryOther Topics - MS-MS

• In LC-ESI-MS, little fragmentation occurs making determination of unknowns difficult

• In LC-ESI-MS on complicated samples, peak overlap is common, with interferants with the same mass possible (e.g. PBDPs)

• In both of above samples, using MS-MS is useful

• This involves multiple passes through mass analyzers (either separate MSs or reinjection in ion-trap MS) and is termed MS-MS

• Between travels through MS, ions are collided with reagent gas to cause fragmentation

Mass SpectrometeryQuestions I

1. Which ionization method can be achieved on solid samples (without changing phase)

2. If one is using GC and concerned about detecting the “parent” ion of a compound that can fragment easily, which ionization method should be used?

3. For a large, polar non-volatile molecule being separated by HPLC, which ionization method should be used?

Mass SpectrometeryInterpretation Questions

1. Determine the identity of the compound giving the following distribution:

m/z Abundance(% of

biggest)

25 14

26 34

27 100

35 9

62 77

64 24


2. Determine the identity of the compound giving the following distribution:

m/z Abundance(% of

biggest)

29 9.2

50 30.5

51 84.7

77 100

93 16

123 39


3. From the following M, M+n ions, determine the number of Cs, Brs and Cls:

m/z Abundance(% of

biggest)

117 100

118 1.4

119 98

121 31.1

123 3

Documents

Chem. 230 – 11/18 Lecture. Announcements I Exam 3 Results –Lower Average (72%) –Distribution New Homework Posted Online (long problems due next week)