Micro-interferometric backscatter detection using a diode laser

Analytica Chimica Acta 400 (1999) 265–280

Micro-interferometric backscatter detection using a diode laser

Kelly Swinney, Dmitry Markov, Joseph Hankins, Darryl J. Bornhop∗Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA

Received 15 April 1999; accepted 21 July 1999

Abstract

Micro-interferometric backscatter detection (MIBD) is performed with a simple, folded optical train based on the interactionof a diode laser beam and a fused silica capillary tube allowing for refractive index (RI) determinations and detection ofoptically active molecules in small volumes. Side illumination of the capillary by a laser produces a 360◦ fan of scatteredlight that contains two sets of high contrast interference fringes. These light and dark spots are viewed on a flat plane in thedirect backscatter configuration. Signal interrogation for polarimetry is based on quantifying the relative intensities (depth ofmodulation (DOM)) of adjacent high frequency (HF) interference fringes for polarimetry and relative fringe position for RIdetection. Positional changes of the interference pattern extrema (fringes) allow for the determination of1nat the 10−7 level or5.3 pmol or 0.48 ng of solute. The MIBD-RI detection volume is just 5.0 nl. DOM changes allow for optical activity detectionlimits of 5.7× 10−5◦ (mandelic acid, [a]23 =−153◦, and D-glucose, [a]25 = +52.5◦), and a 2σ detection limit of 7.5× 10−4 M(D-glucose) and 1.14× 10−3 M (R-mandelic acid). The probe volume of MIBD-polarimetry was 38 nl, and within the probedvolume at the limit of detection, about 28.7 pmol of mandelic acid or about 43.7 pmol of D-glucose is present. Furthermore,DOM (polarimetry signal) is unchanged when a non-optically active solute is interrogated by the MIBD-polarimeter. Finally,an optical model was derived and used to evaluate the advantages and pitfalls of using diode laser for MIBD. ©1999 ElsevierScience B.V. All rights reserved.

Keywords:Polarimetry; Refractive index; Micro-interferometry; Optical activity; Micro-interferometric backscatter detection; Depth ofmodulation; Diode laser; Capillary tube; Small volume detection

1. Introduction

The advantages of miniaturized analysis schemesare numerous and include increased analysis speedand reduced cost. Further, micro-schemes have al-lowed applications such as DNA sequencing [1–4]and the study of cellular function [5–8]. A generaltrend towards instrumentation miniaturization and theproliferation of capillary-based separation schemeshas motivated researchers to seek out new, highly

∗ Corresponding author.E-mail address:[email protected] (D.J. Bornhop)

sensitive, small volume, optical detection techniques[9–26]. Probing nanoliter sample volumes for soluteconcentration is often difficult to achieve withoutsacrificing sensitivity because the background signalsand the small pathlengths (10–100mm) associatedwith capillary-based separations result in poor signalto noise ratios. Due to these difficulties, performinguniversal or polarimetric measurements in nanoliteror smaller probe volumes is still problematic. Thus, aneed exists for a polarimetric and a universal detectorwith minimal pathlength sensitivity.

Refractive index (RI) detectors are, in general bulkproperty, non-destructive sensors that are mass sensi-

0003-2670/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved.PII: S0003-2670(99)00627-3

266 K. Swinney et al. / Analytica Chimica Acta 400 (1999) 265–280

tive. As such, they are potential candidates for use asmicro-scheme universal detection, but pose a specialchallenge when small volume detection is desired. Forexample, the change in RI with temperature (dn/dT) islarge (8× 10−4 RIU/◦C for water) [26] for most flu-ids; thus, small changes in temperature result in appre-ciable RI signals. Furthermore, most conventional RItechniques are pathlength-sensitive, making detectionin capillaries intractable. Therefore, due to the limi-tations inherent in miniaturizing bulk property detec-tors to nanoliter volumes, RI measurement schemes,to date, have not been widely used for detection incapillary-scale separations.

Despite these difficulties, there have been manyattempts to develop RI detectors with volumes inthe nanoliter regime [19,21,29]. With interferometryallowing for the determination of very small phasechanges of coherent light, the most successful at-tempts have involved some form of interferometry[19–25]. The resulting RI sensitivity is high eventhough RI detectors based on interferometry normallyexhibit some level of pathlength dependency [19–22].The most promising attempts to make RI detectionsuitable for capillary scale separation schemes haveincluded the forward scatter technique developedby Bornhop and Dovichi [19] and further refinedby Krattinger et al. [21], a fiber optic-based deviceput forth by Buttry [27], and a technique based onSchlieren optics introduced by Pawliszyn et al. [28].

To circumvent previous limitations, a simplemicro-interferometric backscatter detector (MIBD)was developed by Bornhop and co-workers [23–26].This device uses an HeNe laser to probe a fused sil-ica capillary directly and can detect changes in RI at1.5 parts in 107 within a detection volume of 350 pl[23–25,29]. Among the important and unique featuresof MIBD are its relatively pathlength insensitivity forcapillaries ranging in tube size from 10 to 775mm[23–25], its optical simplicity, and its insensitivityto alignment. MIBD has been shown to be capableof providing picogram concentration detection lim-its in nanoliter probe volumes; it can be used as anon-column capillary electrophoresis detector [30–32]and can be used to perform non-invasive thermometry[25,33]. Using the unique optical properties of laserlight and the backscatter configuration for interferom-etry, it has also been shown that MIBD can facilitatepolarimetry in capillary dimensions [26].

MIBD also addresses a previously intractable prob-lem, the direct detection of chiral (optically active)species in capillaries. Just as RI detectors are oftenlimited in application to capillary-scale separationtechniques due to their large probe volumes, so arecurrent polarimeters. To date, there has been verylittle success in developing nanoliter sample vol-ume polarimeters for capillary-scale separations. Lowsignal-to-noise ratios, large-volume flow cells, andcomplex optical arrangements currently limit the useof polarimetry in capillary-based separations [34–38].While circular dichroism has been performed in pi-coliter volumes [39], to our knowledge, no detectionscheme for polarimetry has been possible ‘on-column’with capillary-based analysis techniques, except forthe capillary polarimeter based on MIDB developedby Hankins and Bornhop [26].

One drawback of MIBD as either a RI detector oras a polarimeter has been the need to use an HeNelaser. While HeNe lasers have excellent optical prop-erties, they are limited in applications that demandminiaturization by their bulky size. As a result, diodelasers are replacing HeNe lasers in many industrial,medical, and analytical applications. Diode lasers, ingeneral, are solid state, low-cost, and compact lightsources that possess many of the properties of gaslasers (HeNe’s). Among them are good beam quality(TEM00), low divergence, and some polarization pu-rity [39]. Furthermore, they have characteristic longlifetimes (in excess of 50,000 h), and provide reason-able coherent lengths (as great as 1 m) [39]. Diodelasers, however, differ from HeNe lasers in several im-portant ways, particularly when using them as inter-ferometry sources. Firstly, they emit elliptical insteadof circular Gaussian beams. Secondly, wavelength sta-bility of most diode lasers is generally poor due thedevice’s structure (small cavity size), resulting in a de-pendency on and sensitivity to current and temperaturechanges. Thirdly, while emitting light that is inher-ently linearly polarized, the polarity purity of a diodelaser’s beam is relatively low (100 : 1) [39]. Neverthe-less, if proper care is taken, diode lasers are low cost,coherent light sources that are adequate for interfero-metric detection schemes such as MIBD for both RIand polarimetric detection.

Here, we show that using a diode laser as the sourcefor MIBD, sensitive RI (DL = 0.48 ng glycerol) andpolarimetric (DL = 4.4 ng R-mandelic acid) measure-

K. Swinney et al. / Analytica Chimica Acta 400 (1999) 265–280 267

ments can be achieved in nanoliter volumes (5 and40 nl, respectively). The diode laser based MIBD sen-sitivity and characteristics will be compared to MIBDusing an HeNe laser. The inherent differences betweensolid state and gas lasers will be illustrated by thediode laser’s wavelength sensitivity to temperature andcurrent. Results generated from fundamental experi-ments conducted to quantify wavelength changes asa function of current will be presented and comparedwith results obtained under similar conditions from atheoretical optical ray trace model of MIBD. Finally,it will be demonstrated how the unique properties ofdiode lasers can be used advantageously in MIBD, fa-cilitating nanoliter volume polarimetry and RI mea-surements.

2. Experimental

2.1. Instrumentation

Both the polarimeter and the RI detector use asimilar optical train except for a difference in cap-illary inner diameter (RI: 100mm and polarimeter:250mm). The block diagram for the optical config-uration is shown in Fig. 1A and B. All componentswere mounted on massive aluminum risers which arebolted to a 4 ft× 4 ft vibrationally dampened opticalbread board (Newport Corp., CA). Illumination wasprovided by a 5 mW diode laser operating at 670 nm(Coherent, Inc. CA) and driven by an ILX powersupply (Model LDC-3722B, ILX, Bozeman, MT).An elliptical beam was produced by the integratedconditioning optics on the laser diode module. Diodelaser outputs are inherently linearly polarized, and tokeep the optical train as simple as possible, no addi-tional polarization purifying optics were employed.The specified exclusion level for the laser employedhave been estimated to range from 100 : 1 to 300 : 1[39]. Upon passing a neutral density filter, the po-larized beam side-illuminates a fused silica capillarytube (PolyMicro Technologies, Phoenix, AZ) withan outer diameter of 356mm, an inner diameter of249mm for the polarimetry detector or 100mm forthe RI detector. The polyimide outer coating is left inplace and it measures approximately 19mm. A neutraldensity filter was needed to reduce the intensity of the

Fig. 1. (A) Top view for the block diagram for the MIBD opticaltrain. CCD is charge coupled device (camera) and LBA is a laserbeam analyzer. (B) Side view for the block diagram for the MIBDoptical train. Components are the same as in Fig. 1A.

exciting laser beam, so the fringe pattern impingingon the CCD sensor does not saturate the detector. Therelative distances between optical components were4.0 cm from the laser’s aperture to a neutral densityfilter (optical density 0.5–1.5) and 36.0 cm from thefilter to the capillary tube. The total distance fromthe capillary tube to the laser head is 40 cm. A com-mercial camera (Cohu, San Diego, CA) based on aCCD detector, in communication with a laser beamanalyzer (LBA 100, Spiricon Inc.), was mounted ona micrometer driven translation stage (Newport Corp.CA). The CCD was positioned directly below theillumination plane of the laser beam at a distanceof 23.0 cm from the capillary tube. The plane po-larized electromagnetic radiation vector is orientatednormal to the long axis of the beam. For the MIBDexperiments, the plane of polarization of the beam isorientated parallel to the central axis of the capillaryand the long axis of the laser beam is orientated per-pendicular to the central axis of the capillary. Thisorientation is referred to as the perpendicular config-uration. As shown in the side view of the optical train(Fig. 1B), by slightly tilting the capillary, the foldedoptical configuration allows the backscatter radiationemanating from the tube to be directed onto the CCD.


Fig. 2. Intensity profile vs. pixel for a cross-sectional view of twoLF backscatter fringes (f0) which contain HF fringes (f1). Imax andImin refer to the maximum and the minimum intensity, respectively,of an HF fringe selected for polarimetric %DOM measurement.

A laser beam analyzer was employed to quan-tify and display the intensity distribution for a givencross-section of the backscatter interference patternand to quantitatively image the fringes. Functions ofthe LBA include 3-D intensity mapping, centroid po-sition determination and contour profiling. The CCDsignals were displayed in real-time on a CRT monitorand stored on a Pentium PC computer (Gateway, Inc.Austin, TX). The COHU CCD camera employed inthe detection scheme comprises a 678× 494 array of8.4× 9.8mm pixels.

The flow cell consists of a capillary tube mountedon a massive aluminum block (passive thermal sta-bilization) painted black, with a small circular holedrilled where the laser beam strikes the tube. The flowcell assembly was mounted on two stacked translationstages, for ease in positioning, and was tilted at an an-gle of about 7◦ relative to normal as shown in Fig. 1B.

2.2. Fringe interrogation for polarimetric and RImeasurements

Both the polarimetry and RI data were obtained us-ing the same optical configuration with the plane ofpolarization for the laser fixed so that it was perpen-dicular to the tube. In both cases, two frequencies ofinterference fringes were observed in the backscatterbeam profile. These are depicted graphically (Fig. 2)and shown as two low frequency (LF) fringes (f0) thatcontain five well defined high frequency (HF) fringes(f1) each (Fig. 3A). In contrast, in our observationwith an HeNe, [26] when rotating the diode laser to

Fig. 3. (A) Beam profile for the second fringe from the center asobserved in the backscatter configuration with the laser long axisorientated perpendicular to the central axis of the capillary. TheHF fringes are readily observable by comparison with the falsecolor contour scale. Red represents the highest intensity whileblack corresponds to little or no photon flux. (B) Beam profile forthe second fringe from the center as observed in the backscatterconfiguration with the laser long axis orientated parallel to thecentral axis of the capillary.

the parallel configuration compared to the perpendic-ular orientation, a backscatter pattern that is quite dif-ficult to interpret is produced (Fig. 3B). A cartoondepicting seven fringes (only part of one side of theinterference pattern) is shown in Fig. 3C. Selectionof the fringe is important as will be discussed later.The third fringe right of the central fringe was usedto evaluate the system’s performance in all but oneexperiment. The sixth fringe right (Fig. 3C) of thecentroid was selected for evaluation in order to de-termine if all fringes located in the direct backscattershift in response to changes in RI at the same level ofsensitivity.


Upon selecting a fringe, calibration involved aseveral-step process. First, a blank solution (distilledwater) was introduced into the capillary detectionzone with a manual injection valve connected directlyto the capillary (Valco, Inc. Houston, TX). The sam-ples were analyzed staticly after the capillary wasfilled and the solution had stabilized (>15 s) and thena fringe was imaged. Next, a solute solution was in-troduced into the capillary. Then, using the laser beamanalyzer, the fringe pattern was sampled. The solutionwas changed iteratively to allow the introduction of ahigher concentration of the analyte, followed in eachcase by beam profiling. Then, upon interrogating allcalibration standards, H2O was re-injected for fringepattern sampling of the blank solution.

Images of the fringes were interrogated by digitizingthe laser beam analyzer output and transferring it toa computer for storage. The beam profiles (intensityversus position) captured by the laser beam analyzerare quantitative in both position and intensity, allowingboth polarimetry and RI signals to be extracted.

2.2.1. RI signal quantitationAs described in earlier works for an MIBD using

a HeNe [23–25,29–33], the backscatter fringes shiftspatially with increasing solute concentration due tochanges in RI. These positional shifts of the fringesin response to changes in RI can be detected usingCCD camera in communication with a laser beam an-alyzer. By employing the centroid determination func-tion of the LBA, these positional shifts can be quanti-fied. The centroid determination function locates theX–Y coordinate pair that corresponds to the center ofthe backscatter fringe of interest. Knowing the col-lection angle of the camera and the resulting resolu-tion (9mm), positional changes can be converted fromthe coordinate system into distance or positional shifts(microns). All positional shift measurements are rela-tive to the initial position of the fringe for the blanksolution.

2.2.2. Polarimetric signal quantitationWhile RI changes result in fringe movement, solute

optical activity produces changes in the depth of mod-ulation (DOM) or relative intensity of the HF fringes.Fig. 4 qualitatively shows an example of this polari-metric response and how it is quantified. For optically

active solutes, the DOM of the HF fringes decreasesor nulls out (e.g. the difference between maximum andminimum intensities for a set of adjacent bright spotsor fringes decrease as the optical activity is increased)(Fig. 4). Experimentally, an individual LF fringe isimaged as successively increasing concentrated solu-tions of solute are introduced into the capillary. Theoptical activity of a solute is measured by integratingthe photon flux at particular positions on the fringe. Apercentage value, the DOM, results and is defined byEq. (1):

DOM = Imax − Imin

Imax× 100% (1)

whereImax is the maximum intensity for the selectedfringe andImin is the minimum intensity of an adja-cent null (Fig. 4). Subsequently, the DOM and the in-jected concentration are used to construct a calibrationcurve to define the response of the MIBD polarimetricsystem.

2.3. Chemicals

All chemicals were reagent grade and solutionswere prepared in distilled water that had not beendegassed or otherwise modified. Distilled water isalso used as a blank. Glycerol, a non-optically activesolute with an RI of 1.4746, was used to qualitativelydetermine the RI response of the system and also toshow that solute concentration changes in the absenceof optically active solutes do not influence the con-trast of the high-frequency fringes. Calibration curvesof RI and polarimetric response versus concentra-tion were generated using glycerol solutions rangingin concentration from 1 to 180 mM. Mandelic acid,the R enantiomer at 99.9% purity, [a]23 of −153◦,(Baxter Chemical, St. Louis, MO) and D-Glucose, at99.9% purity, [a]25 of +52◦ (Aldrich Milwaukee, WI)were used as chiral probe molecules to evoke changesin the high-frequency fringe pattern contrast to makepolarimetry calibration curves. The concentrations ofthe solutions in distilled water for Mandelic acid were1.28× 10−1, 8.52× 10−2, 4.28× 10−2, 1.28× 10−2,8.45× 10−3, 4.35× 10−3, 1.28× 10−3, and 0.0 M(distilled water) and for D-Glucose were 81.9, 41.0,20.0, 8.19, 5.88, 2.94, 1.50 and 0.0 mM (distilledwater). The polarimetry calibration curves for glyc-


erol were generated with the same solutions used togenerate the RI calibration curves.

3. Results and discussion

3.1. Optical signal production

In earlier works [23–26,29–33], it was demonstratedthat RI and polarimetric detection is possible withMIBD by directly probing fused silica capillary tubeswith a low power HeNe laser beam. One logical exten-sion of the detector, which would aid in instrumentalminiaturization, reduction in device cost, and systemruggidization, is to employ a diode laser as the excita-tion source. The block diagram for such an instrumentis shown in Fig. 1A and B. In the simple optical train,a diode laser produces an elliptical beam that is un-conditioned and impinges directly onto the surface ofa slightly tilted, unmodified fused silica capillary suchthat the long axis of the beam is orientated perpendic-ular to the central axis of the capillary. A fan of scat-tered light can be seen in 360◦ with a distinct set ofinterference fringes observable in the backscatter con-figuration (directly in-line with the laser either aboveor below the plane of the excitation source) (Fig. 1B).The fringe pattern has two distinct frequency compo-nents (Fig. 3A) which are easily seen when the inten-sity distribution of a cross-section of the backscatterfringe is plotted versus position (Fig. 2). A similarobservation was made when an HeNe was used eventhough the intensity distribution of the diode laser waselliptical pseudo-Gaussian instead of circular Gaus-sian like that of the HeNe. Rotation of the laser by 90◦does not produce the same reduction of the HF mod-ulations as observed previously [30] (Fig. 2B). Therelative position of the LF fringes provides a measureof the relative change in RI for the fluid containedon traversing the tube [23–25], while the intensity ofthe HF fringes provides a quantitative measurementof optical activity. It is also important to note that thepositional changes of the LF fringes do not interferewith the polarimetric determination.

3.2. RI results for MIBD using a diode laser

A calibration curve of fringe position versus glyc-erol concentration was generated to determine the RI

sensitivity of MIBD using a diode laser and the re-sults are shown in Fig. 5. A linear relationship existsbetween solute concentration and relative fringe shiftof the selected backscattered fringe. Based on posi-tional response of the fringe to RI changes, the masslimit of detection for MIBD using a diode laser is5.35 pmol or 0.48 ng based on a 5 nl probe volume.The low resolution of the CCD (9mm) significantlylimits the positional shifts that are measurable, result-ing in poorer detection limits than those reported pre-viously [24,25,29–32]. In fact, when using a detectionscheme based on an air slit/photodetector assembly inMIBD-RI, femtomole (picogram) mass detection lim-its are possible [30,32].

To directly compare the performance of the diodelaser to the performance HeNe’s, the CCD-LBA de-tection scheme was employed under identical con-ditions for both lasers. As shown in Fig. 6, usingCCD-LBA detection, the diode laser’s (670 nm) RIresponse is identical (within experimental error) tothat determined when using an HeNe laser (632.8 nm).Note that the difference in the slopes for the two wave-lengths is negligible, an important observation indi-cating that the cheaper smaller diode laser producesa sensitivity similar to that of the HeNe. Therefore,replacing the HeNe laser with the rugged diode laserwill not result in poorer performance. Furthermore,if an air/slit phototdetector assembly is employed fordetection in place of the CCD-LBA system, femto-mole (picogram) mass detection limits are expectedfor MIBD-RI using a diode laser.

To further evaluate the interference pattern forMIBD-RI, the motion sensitivity of the third and thesixth fringes (Fig. 3C) to changes in RI was compared.Fig. 7 shows the reproducible calibration curve resultsof fringe movement versus glycerol concentration us-ing the sixth fringe for signal interrogation. The slopeof the calibration curve (24.8mm/mM) is identical(within experimental error) to that of the responseproduced upon using the third fringe (24.9mm/mM)for signal evaluation. These data suggest that whenusing position sensing that is independent of intensitychanges, interference fringes located as far out as thesixth fringe respond to changes in RI with the samesensitivity. Therefore, it is not necessary that onebackscatter fringe be chosen over another when theLBA or other position sensitive methodology is usedfor detecting fringe movement.


Fig. 4. Fringe profiles produced when an increasingly concentrated D-glucose solution is contained in the capillary.

3.3. Polarimetry results of MIBD using a diode laser

Polarimetry calibration curves of DOM versusconcentration were generated for D-glucose and

R-mandelic acid (Fig. 8A and B). The calibrationcurve is derived from repetitive determinations, witheach point representing the average of two data setscollected at two different times. As expected, DOM


Fig. 5. Reproducible RI calibration curve of fringe position vs. glycerol concentration obtained with the diode laser. The third fringe rightof the central fringe was evaluated for positional information. Error bars are included in the graph but are smaller than the size of thedata points.

Fig. 6. Comparison of RI sensitivity as a function of probe beam wavelength. Reproducible calibration curves of fringe position vs. glycerolconcentration are shown for the diode laser (670 nm) and the HeNe laser (632.8 nm).

response is not linear over large concentration ranges.This effect is commonly seen in polarimetry mea-surements performed at higher concentrations andis due to solute–solute interactions and is discussedin detail later. In the region at low concentrations,the change in relative intensity of the HF fringesor the DOM scales linearly with solute optical ac-

tivity ( r = 0.995 for mandelic acid andr = 0.997 forD-glucose). This effect and further non-linearity isdue to both non-quantifiable photon fluxes at fringemaxima that exceeded the operation range of theCCD and solute–solute interactions. Using the ac-cepted relationships, the calculated 2σ detection lim-its for mandelic acid and D-glucose are, respectively,


Fig. 7. Reproducible RI calibration curve of fringe position for the sixth fringe right of the centroid vs. glycerol concentration obtainedwith the diode laser. Error bars are included in the graph but are smaller than the size of the data points.

7.5× 10−4 and 1.14× 10−3 M. Using an unmodified250mm inner diameter capillary and an unapertureddiode laser, the probe volume can be estimated to be38 nl. Within the probed volume, at the detection lim-its, there would be 28.7 pmol or 4.4 ng of mandelicacid and 43.7 pmol or 7.9 ng of D-glucose. At the 2σ

detection limit, 5.7× 10−5◦ in optical rotation can bequantified. The diode laser’s mass detection limits forD-glucose and mandelic acid are indistinguishablewhen compared to those obtained using an HeNe laser(D-glucose = 7.38 ng and R-mandelic acid = 6.7 ng[26]) as the probe source, and therefore, sensitivityis not sacrificed when using the diode laser. Further-more, upon comparison of MIBD-polarimetry withavailable commercial polarimeters, the sensitivity ofMIBD-polarimetry is comparable and is achieved indetection volumes that are three decades (1000 times)smaller (ml versus nl).

To ensure that DOM or relative intensity of the HFfringes respond only to chiral (optically active) so-lutes, a calibration curve of DOM versus concentra-tion was generated for a series of glycerol solutionsusing the exact same procedure that was employedfor D-glucose and R-mandelic acid (Fig. 9). As seenfrom the flat signal response in Fig. 9, DOM does notchange in response to changes in RI (glycerol concen-

tration). The lack of response by the HF fringes con-firms that a change in RI does neither attenuate norchange the HF modulations implying that only opti-cally active compounds produce these changes.

As mentioned, it is believed that the non-linear re-sponse over a somewhat limited dynamic range inconcentration for each solute can be attributed to twofactors. Firstly, it is well known that optically activemolecules do not act independently, particularly athigh concentrations (>10–15 mM) [40–42] in aqueoussolutions. Thus, chemical association or solute aggre-gation lead to a reduction in the observed or appar-ent signal. This limitation is somewhat analogous tonon-linear effects found in absorption measurements;all solutes do not interact with the light independently.Secondly, at low solute concentrations, because ofa relatively limited dynamic operation range for theCCD-based laser beam analyzer, saturation ensues forHF fringe maxima (the bright portion of the fringe)(Fig. 8A and B). Thus, saturation resulting in a lossof apparent change in DOM for solute concentrationor optical activity changes. At high solute concentra-tions (low DOM values), we approach the limit ofdetectability for the sensor and the optical technique,such that fringe maxima and minima are indistinguish-able by the CCD system.


Fig. 8. (A) Calibration curve for R-mandelic acid. (B) Calibration curve for D-glucose.

3.4. Fundamental diode laser investigations

Laser diodes continue to gain popularity as sourcesfor coherent illumination due to their relative low cost,small size, and long lifetimes. Among the advantagesof using laser diodes in interferometry is that theiroptical output can be modulated easily through thesupply current [43–45], opening a path to potentialalternative detection schemes in MIBD. But this mayalso excite some additional sources of noise, whichare not seen with HeNe laser (e.g. change in output

power). For most semiconductor lasers, the depen-dency of optical power and emitting wavelength fromdrive current is unique for each specific device andis rarely specified by the manufacturer. Also, for agiven laser diode, these properties may change withtemperature or age. So, in order to accurately modelpositional changes of the interference fringes as afunction of wavelength in MIBD with a diode laser,correlation between the wavelength generated anddriving current had to be estimated experimentally. Todo so, a simple method for calculating the wavelength


Fig. 9. Calibration curve of the polarimetric response of the non-optically active solute, glycerol, with varying concentration.

of the coherent light employing a diffraction gratingwas used.

There is a direct correlation between the angle atwhich the particular diffraction order is deflected andthe illuminating wavelength and the spatial frequencyof the grating, which can be expressed as [44]

Sin(2) = mfgλ (2)

where2 is the deflection angle,m= 0, ±1, ±2,. . . isthe diffraction order,fg is the frequency of the grat-ing, andλ is the wavelength of the light. By placinga diffraction grating in front of the laser diode andmeasuring the relative positiond of the first deflectedspot (first-order) to the undeflected spot (zero-order)at some distancez away from the grating, we can de-termine the wavelength of the incident light as

λ = d

fg√

d2 + z2(3)

whereλ is the emitted wavelength of the laser diode,dis the distance between the undiffracted and diffractedspots,z is the distance from the grating to the detectorplane, andm= +1 for the first-order of diffraction.

The experimental setup is shown in Fig. 10. At somedistancez away from the grating, a ruler was placed

Fig. 10. Experimental setup used for determining wavelength sen-sitivity to small changes in drive current.Z is the distance fromthe grating to the ruler;d is the relative distance between the zero-and first-orders of diffraction;2 is the deflection angle for the +1order.

in such a way so as to allow both diffracted and un-diffracted spots to be imaged on the scale, thus provid-ing an accurate and efficient way of determining therelative distanced between these spots. Simultaneousmeasurements of the spot position and current flowingthrough the laser diode produce a linear plot (r2 = 0.99)with the following relationship between current in mAand wavelength in nm:

λ(i) = 0.1165i + 672.5822 (4)

One should keep in mind that the above formula isspecific to the laser diode employed in these experi-ments and it may not apply to other laser diodes.


Fig. 11. Cross-sectional view of the optical ray trace model for MIBD using a diode laser with a 250mm inner diameter, 350mm outerdiameter capillary and a 20mm thick polyimide coating. Only three selected rays are shown with two splits.

3.5. Modeling

In order to better understand how MIBD functionsand to determine theoretical limits of the system, acomputer generated model was created using a sophis-ticated optical modeling program ASAP 6.0 (BRO Re-search, Inc). As this model allows us to directly simu-late MIBD using a laser diode at its operational wave-length and mimic the tube exactly, we can further val-idate our experimental data and predict future changesin the experimental output. The model was constructedusing a capillary with inner and outer diameters of250 and 360mm, respectively, and a 20mm-thick poly-imide coating. Shown in Fig. 11 is a cross-sectionalview of the capillary with a selected group of raystraced inside the tube. The long axis of symmetry ofthe tube coincides with theX-axis of our coordinatesystem. The light source located 3 cm away from thecapillary on theZ-axis has no beam divergence, andtherefore, the actual location of the emitter on that axisis not that important. This light source does not haveany physical or optical properties attached to it; thus,it can be viewed as a localized plane where the set ofspatially and temporally coherent rays originates. As a

result, the backscattered light traveling from the cap-illary to the detector passes through the emitter planeunperturbed, thus eliminating the need to tilt the capil-lary to gain access to the first orders of the interferencepattern. The backscattered intensity was observed onthe detector plane, which was placed at some distanceaway from the capillary in the +Z direction behind theemitter and perpendicular to theZ-axis.

In order to evaluate the behavior of MIBD usingthe model, changes in the fringe position as a functionof changes in the RI of the fluid inside the capillarywere simulated. A diode laser withλ = 670 nm wasused as a light source. A grid of seven coherent rayswas used to illuminate the capillary. Each ray was al-lowed to have no more than seven splits to account forreflection, refraction, and transmission at each opticalinterface. The detector was placed atz= 30 cm. As itwas expected, there was a significant change in posi-tions of maxima and minima associated with changein RI n. Shown in Fig. 12 are the superimposed pro-files of the intensity patterns calculated for the flu-ids inside the capillary with glycerol concentrationsof 10 mM (red curve) and 20 mM (blue curve). Theamount of shift for the third fringe corresponding was


Fig. 12. Fringe intensity profiles generated with the model depicting positional sensitivity of the fringes to small changes in RI. Solid redcurve = 10 mM glycerol solution; Dotted blue curve = 20 mM glycerol solution.

equal to 0.425 mm. Since these results appeared to bein good correlation with experimental data (the mea-sured shift = 0.431 mm), the relationship between thefringe position and the illuminating wavelength in theMIBD with laser diode as a source was investigatednext.

Eq. (4) illustrates one of the major differences be-tween diode and gas lasers; the magnitude of changein the wavelength produced by laser diode with respectto change in the drive current can be large and can bechanged easily. In order to further characterize MIBDwith diode lasers, wavelength changes with drivecurrent were evaluated with the computer-simulatedmodel. Noting from Eq. (4) that a current changeof 1i = 0.5 mA corresponds to a 0.058 nm change,wavelengths corresponding to 0.25 mA steps rangingfrom 43 to 50 mA were estimated and incorporatedinto the model. The calculated intensity distributionsas a function of current (wavelength) are depicted inFig. 13. As one can see, there is a good correlationbetween predicted and experimentally obtained fringemovements. Since effects of polarization were notincluded in this simulation, the HF fringes are notobserved as they are in the experimental results. Also,the model does not take into account changes in thediode laser’s optical power output with respect to thedrive current. This explains the absence of significantintensity changes in the theoretical results for differ-

ent values of the drive current when compared to theexperimentally obtained data. Clearly, this model canbe used to predict how MIBD will be influenced byvarious factors such as tube size, beam intensity andwavelength changes. Future investigations include po-larization effects and other experimental parameters.

3.6. Instrumental limitations and performanceimprovements

Various aspects and differences of diode laser radi-ation influence the resultant interference pattern pro-duced in MIBD relative to that produced by an HeNe.For example, an HeNe laser emits a constant wave-length with a relative stable optical power output.However, a diode laser’s wavelength and optical powerdepend on the current by which the device is driven.It was found experimentally that the operational rangefor the diode laser employed lies between the currentlevels of 38 and 50 mA. In this range, the device actsas a laser producing light with relatively long coher-ence lengths whose wavelength varies as a functionof current. When the current drops below the thresh-old current (∼37 mA), the device acts more as superluminescent diode (LED) with virtually no coherenceand the interference fringes extinguish. This observa-tion further verifies that coherent illumination is a re-


Fig. 13. (a) Positional shifts in the fringe pattern as a function of wavelength/current obtained theoretically. Numbers represent the orderof the fringe from the centroid. (b) Positional shifts in the fringe pattern as a function of wavelength/current obtained experimentally.Numbers represent the order of the fringe from the centroid.

quirement for the capillary polarimeter to function asa micro-interferometer.

Changing the current, at which the diode laser isdriven, results not only in a change in the output wave-length but also in a change in the optical power. Asa result, this places additional restrictions on the de-tection scheme used to interrogate the fringe pattern.Therefore, in order to avoid erroneous measurements,intensity detection schemes should avoided, thus leav-ing methods based on detecting changes in fringe po-sition, or intensity measurements that must be normal-ized for all values of drive current.

The detection scheme for monitoring fringe move-ment used in this work was limited by the resolu-tion of the CCD camera, restricting the lowest de-tectable amount of fringe movement to 9mm. Otherdetection schemes have proven to have better sensi-tivity to small positional shifts (<10mm) such as theair slit/photo-detector assembly used for RI detectionwith CE and micro-HPLC [30–32]. Ultimately, arraydetection should be implemented to accommodate themeasurement of small positional shifts of the fringe,

and if fringe counting techniques are employed to ex-pand the dynamic operational range.

When MIBD is used as a polarimeter, the opera-tional range of the CCD camera and the polarizationpurity of the incident beam are additional instrumen-tation parameters that currently limit the performanceof the detector. Firstly, absolute fringe contrast is af-fected critically (reduced) by polarization purity andshould be improved by increasing the extension co-efficient for the interrogating beam. Additional op-tics, such as a Glan-Thompson polarizer, can be usedin this regard. Secondly, the image acquisition sys-tem (CCD + LBA) introduces errors in two ways. One,the low levels of CCD saturation result in the lossof contrast between adjacent HF fringes with highintensities which drastically affects the DOM calcu-lations. Two, the limited intensity resolution of theLBA introduces additional error into the DOM cal-culation as well as in the location of fringe maxi-mum. Finally, using single point measurements to de-tect DOM results in the significant decrease in thesignal-to-noise ratio. Some of these instrumental lim-


itations can be compensated for by using photo-arraydetection with a wider operating range and is cur-rently under investigation. Array detection in conjunc-tion with a high-resolution data acquisition system(>8 bit) will also allow for full fringe analysis (DOMand position) with higher intensity and spatial resolu-tion, and thus, increase the signal-to-noise ratio of thedetector.

4. Conclusions

It has been demonstrated that RI and polarimetricdetection can be performed directly on a fused silicacapillary using a diode laser and a simple optical trainwithout reducing the sensitivity. With detection limitsin the range of 57 microdegrees for polarimetry and0.4 ng for RI using low nanoliter probe volumes, andconsidering the success of MIBD-RI detection withCE [30–32], applicability of diode laser-based MIBDto separation techniques such as capillary HPLC andcapillary electrophoresis seems quite tractable. Evenso, we believe that further improvements in the po-larimeter’s and the RI detector’s performance are pos-sible, including the use of higher polarization purityto increase the contrast in the HF fringes and a widedynamic range, multi-element detection system forreal time fringe interrogation. Further applications ofMIBD are underway in our labs including polarimet-ric detection to study solute–solute and solvent–soluteinteractions in micro-volumes [41,42] and RI de-tection to perform low temperature protein foldinginvestigations.

Acknowledgements

This research was supported by the Welch Founda-tion, the Dow Chemical Company and the ResearchEnhancement Foundation of Texas Tech University.The kind donation, from ILX Lightwave of BozemanMT, of the diode laser driver, power supply and thethermoelectric cooler controller is recognized. We alsothank Spiricon of Logan UT for donating the laserbeam analyzer and for assisting in manipulation of thebeam profile images. Breault Research Inc. of A2 isacknowledged for the donation of the ASAP Optical

Modeling Program. The help from the National Sci-ence Foudation is also acknowledged.

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Micro-interferometric backscatter detection using a diode laser

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