Modern Optical Spectroscopy

Shu-Ping Lin, Ph.D.

Institute of Biomedical Engineering E-mail: splin@dragon.nchu.edu.tw

Website: http://web.nchu.edu.tw/pweb/users/splin/

Introduction of AES, NMR, and Laser

Source: R. Thomas, “Choosing the Right Trace Element

Technique,” Today’s Chemist at Work, Oct. 1999, 42.

Atomic Absorption/Emission Spectroscopy Atomic absorption/emission spectroscopes involve e-’s changing energy

states

Most useful in quantitative analysis of elements, especially metals

These spectroscopes are usually carried out in optical means, involving

conversion of compounds/elements to gaseous atoms by atomization. Atomization is the most critical step in flame spectroscopy. Often limits the precision of these methods.

excitation of electrons of atoms through heating or X-ray bombardment

UV/vis absorption, emission or fluorescence of atomic species in vapor is measured

Instrument easy to tune and operate

Sample preparation is simple (often involving only dissolution in an acid)

Process of Atomization

固體 / 氣溶膠

樣品溶液

噴 入

氣態分子

原 子

原子離子

受激發分子

受激發原子

受激發離子

分子光譜

原子光譜

原子離子光譜

霧化

去溶劑化

揮發氣化

解離 ( 可逆 )

離子化 ( 可逆 )

Introduction Atomization – Atomic absorption is the absorption of light by

free atoms. An atomic absorption spectrophotometer is an instrument that uses this principle to analyze the concentration of metals in solution. The substances in a solution are suctioned into an excited phase where they undergo vaporization, and are broken down into small fragmented atoms by discharge, flame or plasma. Determination of 68 metals Ability to make ppb determinations on major components of a sample Precision of measurements by flame are better than 1% rsd. There are

few other instrumental methods that offer this precision so easily. AA analysis is subject to little interference. Most interference that occurs have been well studied and documented. Sample preparation is simple (often involving only dissolution in an acid) Instrument easy to tune and operate

Atomic Emission Spectroscopy: By exposing these atoms to such temperatures they are able to “jump” to high energy levels and in return, emit light. The versatility of atomic absorption an analytical technique (Instrumental technique) has led to the development of commercial instruments. In all, a total of 68 metals can be analyzed.

Atomic Emission Spectroscopy

原子化法 原子化溫度 ℃ 物理現象 方法簡稱

火焰 1700~3150 吸 收 原子吸收光譜法 (AAS)

發 射 原子發射光譜法 (AES)

螢 光 原子螢光光譜法 (AFS)

電熱 1200~3000 吸 收 電熱式原子吸收光譜法

(ETAAS)

螢 光 電熱式原子螢光 (ETAFS)

感應偶合氬電漿

4000~6000 發 射 感應偶合電漿光譜法 (ICP)

螢 光 感應偶合螢光光譜法 (ICF)

直流氬電漿 6000~10,000 發 射 DC氬電漿光譜法 (DCP)

電弧 4000~5000 發 射 弧光發射光譜法

電火花 40,000 發 射 電火花發射光譜法

Atomization – Flame Emission and Atomic Absorption

Spectroscopy (3 main types)

Atomic Emission (with thermal excitation), AES

Atomic Absorption, (with optical photon unit) AAS

Atomic Florescence, AFS

AES experiment set-up

Atomic Emission Spectrometer (AES)

Source

Sample

P Wavelength Selector

Detector Signal Processor

Readout

Type Method of Atomization Radiation

Source

arc sample heated in an electric arc sample

spark sample excited in a high voltage spark sample

argon plasma sample heated in an argon plasma sample

flame sample solution aspirated into

a flame sample

x-ray emission none required; sample

bombarded w/ e- sample

Atomic Absorption Spectrometer (AA)

Source

Sample

P P0

Chopper

Wavelength Selector

Detector Signal Processor

Readout

Type Method of Atomization Radiation

Source

atomic (flame) sample solution aspirated Hollow cathode into a flame lamp (HCL)

atomic (nonflame) sample solution HCL

evaporated & ignited

x-ray absorption none required x-ray tube

Atomic Fluorescence Spectrometer (AFS)

Source

Sample

P P0

90o

Wavelength Selector

Detector Signal Processor

Readout

Type Method of Atomization

Radiation

Source

atomic (flame) sample solution aspirated

into a flame sample

atomic (nonflame) sample solution sample

evaporated & ignited

x-ray fluorescence none required sample

Atomization – Three types of high-temperature plasmas

The inductively coupled plasma (ICP).

The direct current plasma (DCP).

The microwave induced plasma (MIP).

The most important of these plasmas is the inductively coupled plasma (ICP).

Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)

Use an inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element.

Advantages of ICP-AES: excellent limit of detection and linear dynamic range, multi-element capability, low chemical interference and a stable and reproducible signal.

Disadvantages: spectral interferences (many emission lines), cost and operating expense and the fact that samples typically must be in solution.

Solid and liquid samples must be prepared so that they can be easily evaporated and ionized by the instrument

ICP-AES is a destructive technique, but only a small bit of sample is necessary

Picture of an inductively-coupled plasma atomic emission spectrometer

Optics and Detectors

A typical inductively

coupled plasma source called a

torch

Characteristics of the ICP

High Temp.

Long residence times.

High electron number densities(few ionization interferences)

Free atoms formed in nearly chemically inert environment.

Molecular species absent or present at low levels.

Optical thin.

No electrodes.

The direct current plasma is created by the electronic release of the two electrodes. The samples are placed on an electrode. In the technique solid samples are placed near the discharge to encourage the emission of the sample by the converted gas atoms.

Qualitative analysis is done using AES in the same manner in which it is done using FES. The spectrum of the analyte is obtained and compared with the atomic and ionic spectra of possible elements in the analyte. Generally an element is considered to be in the analyte if at least three intense lines can be matched with those from the spectrum of a known element.

Quantitative analysis with a plasma can be done using either an atomic or an ionic line. Ionic lines are chosen for most analyses because they are usually more intense at the temperatures of plasmas than are the atomic lines.

The Direct Current Plasma Technique (DCP)

Quantitative Analysis

定量分析時，將樣品製成溶液形成氣溶膠，導入火焰中，選擇適當波長，測量吸收強度。可利用標準曲線法 ( 檢量線法 )；如圖所示；或用標準添加法來分析。以Cx代表樣品試液中待測定元素的濃度，Cs代表標準溶液的濃度， Cx+Cs為試液加入標準溶液後的濃度，Ax代表試液的吸光度，As代表標準溶液加試液的吸光度，依比耳定律

X X

S S X

XX S

S X

A kC

A k(C C )

AC C

A A

醫院中測定病患的血液或尿液中的鈉鉀含量，使用火焰分析法迅速而準確。在水污染方面也可使用火焰分析法，可測出很低的濃度。

其他如化學工業、石油工業、冶金工業等可利用火焰分析法來作元素的定量分析。

Atomization – AES WITH ELECTRICAL DISCHARGES

An electrical discharge between two electrodes can be used to atomize or ionize a sample and to excite the resulting atoms or ions. The sample can be contained in or coated on one or both of the electrodes or the electrode(s) can be made from the analyte. The second electrode which does not contain the analyte is the counter electrode.

Electrical discharges can be used to assay nearly all metals and metalloids. Approximately 72 elements can be determined using electrical discharges. For analyses of solutions and gases the use of plasmas is generally preferred although electrical discharge can be used. Solid samples are usually assayed with the aid of electrical discharges.

Typically it is possible to assay about 30 elements in a single sample in less than half an hour using electrical discharges. To record the spectrum of a sample normally requires less than a minute.

Atomization – ELECTRODES FOR AES

The electrodes that are used for the various forms of AES are usually constructed from graphite. Graphite is a good choice for an electrode material because it is conductive and does not spectrally interfere with the assay of most metals and metalloids. In special cases metallic electrodes (often copper) or electrodes that are fabricated from the analyte are used.

Regardless of the type of electrodes that are used, a portion of each of the electrodes is consumed during the electrical discharge. The electrode material should be chosen so as not to spectrally interference during the analysis.

Atomization – WAVELENGTH SELECTION AND DETECTION FOR AES

Arc and spark instruments normally contain non scanning monochromators. Either a series of slits is cut in the focal plane of the monochromator and a photomultiplier tube is placed behind each slit that corresponds to the wavelength of a line that is to be measured, or one or more photographic plates or pieces of film are placed on the focal of the monochromator.

Qualitative analysis is performed by comparing the wavelengths of the intense lines from the sample with those for known elements. It is generally agreed that at least three intense lines of a sample must be matched within a known element in order to conclude that the sample contains the element.

Quantitative analysis: Regardless of the type of detection used for the assay, the precision of the results can be improved by matrix-matching the standards with the sample. Use of the internal-standard method also improves precision. Usually a working curve is prepared by plotting the ratio or logarithm of the ratio of intensity of the standard's line to the internal standard's line as a function of the logarithm of the concentration of the standard. The corresponding ratio for the analyte is obtained and the concentration determined from the working curve.

How to Atomize??

Three steps are involved in turning a liquid sample into an atomic gas:

Desolvation – the liquid solvent is evaporated, and the dry sample remains

Vaporization – the solid sample vaporises to a gas

Volatilization – the compounds making up the sample are broken into free atoms.

AS deals with e transfer transition of valence electron between electronic states

AAS

I0 I

C A

) / log( T log A

εbC o

I I

吸收值與濃度呈線性關係

A：absorbance T：transmittance C：conc. ε：absorpivity b：path length

hν hν

ΦL = k′Φ0C ΦL C Φ0

C A

) / log( T log A

εbC o

I I

螢光源與入射光頂角成正比,且與濃度成正比

Light source 於P0°角看放出之螢光 (P0°乃因有散射)

激發態原子不穩定會降到ground state,而以光的形式放出,放出之光的強度與處於激發態的原子數目有關 (波茲曼係數)

Ej

Ei jE

jjijiE

n

VnhA

Nj/Ni = Pj*e-ΔEi/kT/Pi

AFS

AES

Temperature effect on the atomic spectra Boltzmann equation

AA吸收希望atoms在ground state,

AES溫度要高,在excited state’s atoms or ions ↑.

Spectral line intensity

Iem

λ

原子在excited愈多,強度愈高 (僅電流多點即可) 當conc.很低時,conc. ↑或原子在excited增加,則intensity會增強,最後不再增強而變寬

變寬效應

∴Iem C (但不會無限制增加)

Temperature Effect

j j j

o o

N P Eexp

N P kT

Poor Detection Limits on Certain Trace Elements

Examples of interferences include:

40Ar16O on the determination of 56Fe

38ArH on the determination of 39K

40Ar on the determination of 40Ca

40Ar40Ar on the determination of 80Se

Solution: the cold/cool plasma

Limits of Detection

Decrease in limits of detection over the course of time using examples of Perkinelmer ICP emission

Spectrometers ICP/5000 (1980), Optima 3000 (1993), Optima 3000 XL (1997)

All detection limits were determined by the blank method using the statistical factor K = 3 [concentrations in ppb]

1980

radial

1993

radial

1997

axial

As 193 150 50 5

Cd 214 3 2 0.3

Cr 267 5 2 0.2

Ni 231 10 5 0.7

Pb 220 50 10 0.8

Zn 231 2 1 0.1

Nuclear Magnetic Resonance (NMR) Spectroscopy

Molecular Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy: A spectroscopic technique that gives us information about the number and types of atoms in a molecule, for example, about the number and types of

hydrogen atoms using 1H-NMR spectroscopy.

carbon atoms using 13C-NMR spectroscopy.

phosphorus atoms using 31P-NMR spectroscopy.

Nuclear Spin States

An electron has a spin quantum number of 1/2 with allowed values of +1/2 and -1/2. This spinning charge has an associated magnetic field.

In effect, an electron behaves as if it is a tiny bar magnet and has what is called a magnetic moment.

The same effect holds for certain atomic nuclei. Any atomic nucleus that has an odd mass number,

an odd atomic number, or both, also has a spin and a resulting nuclear magnetic moment.

The allowed nuclear spin states are determined by the spin quantum number, I, of the nucleus.

Nuclear Spin States

A nucleus with spin quantum number I has 2I + 1 spin states; if I = 1/2, there are two allowed spin states.

Spin quantum numbers and allowed nuclear spin states for atoms common to organic compounds.

1H

2H

12C

13C

14N

16O

31P

32S

15N

19FElement

Nuclear spinquantum number ( I )

Number ofspin states

1/2 1 0 0 01/2 1

2 3 1 2 3 1

1/2

2 1

1/2

2

1/2

2

Nuclear Spins in H0

Within a collection of 1H and 13C atoms, nuclear spins are completely random in orientation.

When placed in a strong external magnetic field of strength H0, however, interaction between nuclear spins and the applied magnetic field is quantized. The result is that only certain orientations of nuclear magnetic moments are allowed.

Nuclear Spins in H0

for 1H and 13C, only two orientations are allowed.

Nuclear Spins in H0

In an applied field strength of 7.05T the difference in energy between nuclear spin states for

1H is approximately 0.120 J (0.0286 cal)/mol, which corresponds to a frequency of 300 MHz (300,000,000 Hz).

13C is approximately 0.030 J (0.00715 cal)/mol, which corresponds to a frequency of 75MHz (75,000,000 Hz).

Nuclear Spin in H0

The energy difference between allowed spin states increases linearly with applied field strength.

Values shown here are for 1H nuclei.

在較強磁場中，二自旋狀態之能量差△E，比在較弱磁場中之能量差更大。其能量差△E與磁場強度Ho成正比，即

o

hE H

2

(12-1)

其中 △E=α及β自旋狀態之能量差

h=蒲郎克常數

Ho=外磁場強度，高斯 (gauss)

γ=迴轉磁係數 (gyromagnetic ratio)，每一質子為

26,753-1高斯-1

當一質子在合適磁場接受一光子時，可由α-自旋狀態跳至β-自旋狀態，稱此原子核是共振 (resonance)。一光子的能量以E=hν表示，此式與式 (12-1) 合併可得

o

hE h H

2

(12-2)

解出為ν

o

1H

2

(12-3)

一質子之26,753-1高斯-1

1 1o o

1(26,753)H 4257.8 s H ( )

2

高斯 高斯 (12-4)

質子共振頻率發生於光譜的無線電頻率區。對於共振所需無線電頻率可由磁場Ho求出。

最常用的NMR操作頻率為60~300MHz(60~300百萬赫 ) 相當於14,092 高斯的磁場。對於較高解析度的NMR，則使用300~600MHz之頻率操作。

Nuclear Magnetic Resonance

When nuclei with a spin quantum number of 1/2 are placed in an applied field, a small majority of nuclear spins are aligned with the applied field in the lower energy state.

The nucleus begins to precess and traces out a cone-shaped surface, in much the same way a spinning top or gyroscope traces out a cone-shaped surface as it precesses in the earth’s gravitational field.

Nuclear Magnetic Resonance

If the precessing nucleus is irradiated with electromagnetic radiation of the same frequency as the rate of precession, the two frequencies couple

energy is absorbed

the nuclear spin is flipped from spin state +1/2 (with the applied field) to -1/2 (against the applied field).

Nuclear Magnetic Resonance

(a) Precession and (b) after absorption of electromagnetic radiation.

Nuclear Magnetic Resonance

Resonance: In NMR spectroscopy, resonance is the absorption of energy by a precessing nucleus and the resulting “flip” of its nuclear spin from a lower energy state to a higher energy state.

The precessing spins induce an oscillating magnetic field that is recorded as a signal by the instrument.

Signal: A recording in an NMR spectrum of a nuclear magnetic resonance.

Nuclear Magnetic Resonance

If we were dealing with 1H nuclei isolated from all other atoms and electrons, any combination of applied field and radiation that produces a signal for one 1H would produce a signal for all 1H. The same is true of 13C nuclei.

Hydrogens in organic molecules, however, are not isolated from all other atoms. They are surrounded by electrons, which are caused to circulate by the presence of the applied field.

The circulation of electrons around a nucleus in an applied field is called diamagnetic current and the nuclear shielding resulting from it is called diamagnetic shielding.

Nuclear Magnetic Resonance

The difference in resonance frequencies among the various hydrogen nuclei within a molecule due to shielding/deshielding is generally very small.

The difference in resonance frequencies for hydrogens in CH3Cl compared to CH3F under an applied field of 7.05T is only 360 Hz, which is 1.2 parts per million (ppm) compared with the irradiating frequency.

360 Hz

300 x 106 Hz

1.2= 1.2 ppm

106=

Nuclear Magnetic Resonance

Signals are measured relative to the signal of the reference compound tetramethylsilane (TMS).

For a 1H-NMR spectrum, signals are reported by their shift from the 12 H signal in TMS.

For a 13C-NMR spectrum, signals are reported by their shift from the 4 C signal in TMS.

Chemical shift (): The shift in ppm of an NMR signal from the signal of TMS.

Tetramethylsilane (TMS)

CH3

Si CH3

CH3

CH3

NMR Spectrometer

Schematic diagram of a nuclear magnetic resonance spectrometer.

NMR Spectrometer

Essentials of an NMR spectrometer are a powerful magnet, a radio-frequency generator, and a radio-frequency detector.

The sample is dissolved in a solvent, most commonly CDCl3 or D2O, and placed in a sample tube which is then suspended in the magnetic field and set spinning.

Using a Fourier transform NMR (FT-NMR) spectrometer, a spectrum can be recorded in about 2 seconds.

NMR Spectrum

1H-NMR spectrum of methyl acetate.

High frequency: The shift of an NMR signal to the left on the chart paper.

Low frequency: The shift of an NMR signal to the right on the chart paper.