Mikrochimica Aeta [Wien] 1972, 288--297 �9 by Springer-Verlag 1972

Contribution from the Research Center, International Flavors & Fragrances, Union Beach, ~T. J., and Spex Industries, Inc., Metuchen, •. J., U. S. A.

Mierosampling Techniques in Laser Raman Spectroscopy By

Stanley K. Freeman*, P. R. Reed, Jr.**, and Donald 0. Landon**

With 9 Figures

(Received July 13, 1971)

Introduction

More than forty years ago, Raman observed a spectrum of an organic compound using the sun as a source, a telescope as a receiver, and his eye as ~ detector. During the 1930's, Raman spectroscopy proved to be a powerful tool for molecular structure investigations. However, many problems confronted the experimenter. For example, the low energy light sources which produced l~aman radiation necessitated using relatively large samples (ca. 105-10 ~ nl) ~nd often required photographic exposure times of several days to produce a line spectrum. Furthermore, the fluorescence of many substances swamped the extremely weak g a m a n emissions, and sample decomposition was not uncommon. In spite of these severe limitations, Raman spectroscopy was a simpler method than infrared spectroscopy at that time. Development of commercial automatic recording infrared instrumentation about 1940 eclipsed Raman spectral studies, but the recent advent of laser technology has brought about a Raman renaissance. The laser supplied chemists and spectroscopists with a nearly ideal light source and essentially emancipated this spectral discipline from the disadvantages accompanying mercury are excitation. Of particular interest is the fact tha t a laser beam can be focused on extremely small samples. Powers and related data of various lasers appear in Table I. The signal intensity values listed are based on a band appearing in the Raman spectrum of linalool,

* Research Center, International Flavors 85 Fragrances, Union Beach, N . J . 07735.

** Spex Industries, Inc., !V[etuchen, ~ . d., U. S. A.

Freeman et al. : Microsampling Techniques in Laser Raman Spectroscopy 289

an organic chemical displaying "average" in tens i ty R a m a n bands. This b a n d s t rength a t 1685 cm -1 varies for different laser exci ta t ion frequencies

of the same power. Not only do short wavelengths (blue - - 4 8 8 nm) have greater molecular scat ter ing efficiencies t h a n long ones (red

- - 632.8 rim), propor t ional to the 4th power of the exci tat ion frequency, bu t photomul t ip l ier detectors incorporated in R a m a n spectrometers are more sensit ive to blue light. I n actual practice, one usual ly a t t empts to record a spect rum first with blue or green rad ia t ion and then switches to red exci ta t ion if sample fluorescence or photodecomposi t ion is observed to occur. I t has been our experience t ha t circa 500 m W in the blue (measured at the sample) general ly is the m a x i m u m useable power for

Table I. C o m p a r i s o n s of Some C o m m e r c i a l l y A v a i l a b l e L a s e r s

Type Excitat ion Average of Wavelength Power at Laser (nm) Sample

(roW)

Peak Signal* Peak to Peak Time (rain) to (counts per l~loise in Record Ra- sec) Background man Spec-

(% of full t rum 0 to scale) 4000 cm -1 at

4 cm -1 resolu- tion

Cd-He Cd 441.6 20 3. 104 ~ 0.5 15

Ion Ar or A r - K r Ar 488.0 400** 6. 10 ~ ~ 0.1 4***

Ion Ar or A r - K r Ar 514.5 400** 3- 105 --~ 0.2 4***

He-l~e Ne 623.8 50 1.5. 10 a ,~ i 30

Ion tKr or A r - K r Kr 647.1 200 5. 104 ,~ 0.3 16

* With reference to the C=C stretching vibration at 1685 cm -1 of linalool.

** Maximum useable power for microsamples in capillary cells using a focused laser beam.

*** Limited by response of standard chart recorder. A 1 minute scan is possible with fast recorders.

l iquid and solid materials. Higher energies often br ing about sample decomposit ion. Laser powers in the neighborhood of one wa t t can be to lera ted by solutions. Li t t le success has been achieved in rou t ine ly genera t ing spectra of organic compounds in the gaseous state. Since the dens i ty of a l iquid is several orders of magn i tude greater t h a n its

Mikrochim. Aeta 197213 19

290 S.K. Freeman, P. R. I~eed, Jr., and D. O. Landon: [Mikrochim. Ac~a

vapor, one requires lasers with outputs in the neighborhood of 10 to 50 watts to produce good spectra of organic gases whose molecular weights exceed circa 100. The maximum power of commercial lasers is approximately 5 watts and most of the activity with respect to recording laser Raman spectra of gases is still at the research stage. Nevertheless, in view of rapid developments in Raman spectroscopy it is probable that instrumentation will become available within, a few years, enabling the chemist and spectroscopist to conveniently obtain vapor spectra.

DouD/e ,~r

z~sr ]

De/ec/o," H De/gOc'OP Lc/CCl~POnl'CS H /~'ecoPdel; I

Fig. 1. Diagramatie sketch of a modern laser Raman spectrometer

Experimental In s t rumen ta t ion

A modern laser Ram~n spectrometer includes the basic features depicted in the diagramatie sketch shown in Fig. 1.

Fluorescence

The presence of small amounts of impurities sometimes causes sample fluorescence. The incidence of this phenomenon, which can frustrate generating an acceptable Raman spectrum, is significantly reduced by compound purification via gas or thin-layer chromatography. Also, appreciable fluorescence decay may be attained by exposing the sample to 200-500 mW blue radiation in a matter of a few minutes to several hours. The extent of fluorescence at times differs with the excitation frequency, and a minimum spectral background can be selected if a multi-line laser is available. Another approach makes use of the fact that fluorescence eurves sometimes peak at different spectral frequencies for different excitations. By selecting "windows" for each source, a spectrum can be pieced together.

1972/3] Microsampling Techniques in Laser l~aman Spectroscopy 291

Techniques /or Small Sample Handling

The beam diameter of laser wavelengths em- ployed in Raman spectroscopy is approximately 2 mm and it is reduced to approximately 20#m after focussing. The focussed beam volume is circa 10 -2 nl, which represents the minimum sample volume expected to yield a useful spectrum. The first publication concerned with laser Raman examination of small samples appeared in 1966. Lau and Hertz 1 obtained acceptable spectra on 80/zl of material with a modified Ulbrieht scat- tering globe cell (Fig. 2). In the following year, Baily, KAnt, and Seherer 2 reduced the sample volume to 40 nl (CC14) by adopting an axial ex- citation/transverse viewing geometry suggested by Daman, Leite, and Porto 8. The focused laser beam was directed along the axis of a cylinder and the scattered radiation viewed horizontally at capillaries were

Fig. 2. Modified Ulbricht scattering cylinder in which minimum sample size is

around 80 td

90 ~ from the main cylinder axis (Fig. 3). Individual prepared for each sample by carefully heat-forming a

I,

,,, , i , ,

L~,SER

Fig. 3. Axial excitation/transverse viewing cell of dBailey, Kint , and Soberer in which minimum sample

size is 40 nl

i i r

"" Jl

J

t

<3:5:> LASER

Fig. 4. Transverse excitation/transverse viewing cell of Pez in which minimum

sample size is 8 nl

hemispherical lens at one end of the tube, the spherical surface serving as a lens to collimate the light. Pez 4 chose transverse excitation/transverse viewing for liquids in 1 mmi . d. borosilieate glass capillary tubes (Fig. 4).

19"

IO

6 4- 0

35

00

3

00

0

25

00

2

00

0

RA

MA

N

SH

IFT

(C

M")

18

00

1600

14

00

I

--

L

I~0

0

IO0

0

80

0

GO

0

40

0

20

0

--I

I .

.~

--

]

[ ]

Fig

, 5.

S

pec

tru

m

of

2 n

l li

n~

loo

l (0

.1 m

m

cap

illa

ry)

--

18 r

ain

. st

un

, 4

cin

-1

reso

luti

on

, 1

00

raW

, 5

14

.5 m

n

exci

tati

on

o

b~

b9

*r

�9 r ~ 9:

1972/3] Microsampling Techniques in Laser :Raman Spectroscopy 293

This method was extended by Freeman and Landon 5 to smaller bore capillaries containing solids as well as liquids. They obtained a spectrum of 8 nl linalool in 0.1 m m i. d. tubing.

Alignment of a capillary cell in the pa th of a focussed laser beam is a relatively simple operation. The x, y, and z translational motions of a goniometer may be used to accomplish this operation. A capillary is inserted into the goniometer and the device adjusted to maximize the sample's scattered radiation passing through the entrance slit. Many laser t~aman spectrometers now are equipped with a sampling compart-

30tO0 2 8 0 0 I0 I ~

RAMAN SHIF'T (CM 4) 1800f 16001 1400f I2P O0 FOOOr 8 0 0 5010 4010 2001

_ 4

2

Fig . 6. Spec t rum of 2 nl Indene (0.1 m m capi l lary) -- 3 rain. scan, 8 cm ] resolution, 250 roW, 514.5 nm exc i t a t ion

merit which Mlows more rapid positioning of the sample by means of a movable stage. In contrast to the goniometer technique, once a small diameter capillary is properly set at the focussed region of the laser, another sample in a cell of the same size can readily replace it s.

Liquids. The smallest quant i ty of liquid which can be examined conveniently employing approximately 200 m W blue or green laser energy is approximately 2 nl - - a circa 0.3-mm continuous liquid column in a 0 . l - ram bore capillary tube. A lower limit of about 5 nl (0.2-ram bore cell) accompanies the use of a 50 mW He-Ne source. Ordinarily, a respectable spectrum (4 cm -1 resolution) of a clear liquid can be produced within twenty minutes (Fig. 5). For a rapid survey, the resolution is halved, while the source energy is increased, permitt ing a reduction in total recording t ime to three minutes (Fig. 6). McLachl in ~ obtained a 2.5 cm -1 resolution spectrum on 0.3 nl of benzyl alcohol in a 0.48-mm i. d. cell (Fig. 7). This pa th length closely approaches the minimum required to yield an interpretable spectrum. Highly polar compounds such as

)- P

z W P z

294 S.K. Freeman, P. IL Reed, Jr., and D. O. Landort: [Mikrochim. Aeta

water and methanol are poor Raman scatterers and therefore are excellent solvents. A spectrum of 2 nl of a 20% aqueous solution of cysteine hydrochloride is shown in Fig. 8.

IO

8

R A M A N S H I F T ( C M - t )

6

4-

2

O

1 6 0 0 1 4 0 0 I I

I P O 0 1 0 0 0

:Fig. 7. Par t ia l spectrum of beitzyl alcohol (0.048 mm capillary) -- 100 cm-1/mii~, 2.5 c n y 1 resolution, 250 roW, 488.0 nm excitat ion

Increasing the cell path length above 1 mm does not result in a more intense signal because the usable slit height of most laser Raman instru- ments is approximately 10 ram. However, for capillary diameters less than 1 mm, signal strength is proportional to path length. This means that a spectrum gained on a sample contained in an 0.l-ram capillary has one-tenth the intensity observed in a 1.0-ram cell, and at least a ten-fold signal amplification is required to restore this energy loss. This, of course, adversely affects the signal to noise ratio necessitating spectral recording at slower speeds or with wider slits.

Solids. Solids weighing a few mg can be ground to a powder in a small mortar and transferred to a capillary by tamping. Smaller samples,

1972/3] 295

1 9 0 0

Microsampling Techniques in Laser Raman Spectroscopy

RAMAN SHIFT (CM -z)

1700 f 5 0 0 1300 1100 900 700 500 f 1 I I i =

300

b

Z M

o

Fig. 8. Par t ia l spectrum of a 20% aqueous solution (2 nl) of cysteine ItCI (0.1 m m capillary) -- 100 cm-1/min, 5 cm -~ resolution, 300 mW, 514.5 nm excitat ion

1600 I0

8

6

.4

o

R A M A N S H I F T (CM ' : )

1 4 0 0 1 2 0 0 I 0 0 0 8 0 0 6 0 0 4 0 0 2 0 0 I I I I I I I

Fig. 9. Par t ia l spectrum of a single crystal of chlordiazepoxide--HC1 (ca. 3ttg) -- 100 cm-1/min., 2 cm 1 resolution, 200 roW, 568.2 nm excitat ion

296 S.K. Freeman, P. ]%. lqeed, Jr., and D. O. Landon: [Mikrochim. Acta

as little as one ng, are best handled by dissolving in a low boiling solvent, picking up the solution in a fine capillary, and allowing the solvent to evaporate while the cell is suspended in a vertical positionh

Wyss s obtained a spectrum on a few #g of a single crystal (Fig. 9) and McLachlin 7 has recorded l%aman spectra of single crystals weighing circa 0.5#g.

Gas Chromatographic Fractions. We have found tha t capillaries down to 0.2-ram i. d. are compatible with most gas chromatographs for t rapping both liquids and solids. BulIcin, Dill, and Dannenberg ~ recently reported isolating G.C. fractions in 0.2-ram tubes. A flow reduction of only 15% occurs when a 0.2-ram bore glass capillary is inserted into a gas chromatograph equipped with a 1/s" i. d. column operated with a helium flow of 50 ml/min. A negligible decrease is observed when this bore capillary is used for trapping fractions from a support coated open tubular (SCOT) column. For low boiling materials, capillaries can be cooled with a cold stream of air or nitrogen.

Routinely, sample isolation for R a m a n examination is achieved in our laboratory (SKF) by trapping into inexpensive, s turdy 1 mm i. d. borosilicate glass melting point capillaries. One end is then heat sealed and the material centrifuged to the closed end. Depending on sample size, a 0.1- to 0.3-ram bore capillary is threaded into the 1-mm tube and capillary action effects the transfer. Alternately, the opposite end section of the sealed 1-mm container is drawn to approximately 0.2 m m i .d . sealed, and then the sample is either centrifuged to the drawn end, or gently heated and condensed into the constricted section by cooling with a thin metallic strip or with metal forceps. Isolated solids are concentrated to a narrow band by heat "chasing" the material while the capillary is maintained in a vertical position.

The difficulty of containing a neat sample in a volume less than 0.3 nl militates against recording Raman spectra of quantities as little as 0.0I nl, which is the lower limit dictated by laser beana diameters. The solution to this problem appears to lie in spectrally examining vapors with high power laser sourees.

Summary Microsampling Techniques in Laser Raman Spectroscopy

Laser sources for Raman spectroscopy make it possible for the chemist and spectroscopist to routinely and rapidly record the spectra of a few pg of liquids and solids. Taking more care, as little as 0.5 ~g sample suffices to yield a good Raman spectra. The techniques for handling liquids, solids, and gas chromatographic fractions are described.

1972/3] ]Ylierosampling Techniques in Laser Raman Spectroscopy 297

Zusammen[assung

Mit Hilfe yon Laser-Strahlen lassen sich Raman-Spoktren rontinemiil]ig und rasoh unter Verwendung yon wenigen Mikrogramm fliissiger oder fester Substanz ermitteln. Mit etwas mehr Sorgfalt geniigen sogar 0,5/~g, urn ein gutes Raman-Spek t rum zu erhalten. Die Arbeitsweise fiir die Bearbei tung von Fliissigkeiten, festen Stoffen und gaschromatographischen Frakt ionen wnrde beschrieben.

References

1 A . L a u and J . H. Hertz, Spectrochim. Acta 2~, 1935 (1966). 2 G . F . Bailey, S . K i n g and J . R. Scherer, Analyt . Chemistry 39, 1040

(1967). 3 T. C. Daman, R. C. C. Leite, and S. P . S. Porto, Physic. Rev. Letters

14, 9 (1965). 4 G. Pez, McMaster University, pr ivate communication (1968). 5 S . K . Freeman and D.O. Landon, Analyt . Chemistry 41, 398 (1969). 6 S .K ._Freeman and P. R. Reed, Jr. , Unpublished work (1971).

R. McLaehl in , Dow Chemical Co., pr ivate communication (1971). s H. Wyss , I tofmann La Roche, pr ivate communication (1971). 9 B. J . Bullcin, K . Dill , and J . J . Dannenberg, Analyt . Chemistry 4~, 974

(1971).