Hyphenated Techniques

Hyphenated Techniques combine chromatographic and spectral methods to exploit the advantages of both.

Chromatography - Produces pure or nearly pure fractions of chemical components in a mixture.

Spectroscopy – Produces selective information for identification using standards or library spectra.

LC-Absorbance Data

James K. Hardy and The University of Akron http://ull.chemistry.uakron.edu/chemsep/hyphen/

LC-MS Datam

/z

JEOL HIGHTECH LTD http://www.datum.jeol.co.jp/hightech/ai-msdata-3.html

Spectra to Identify and Test Purity

Sample spectra overlaid with standard spectra

Sign

al

Wavelength (nm)190 230 270 310 360 390

Sign

al

Wavelength (nm)190 230 270 310 360 390

Overlaid spectra from beginning, middle and end of

eluting peak

LC with UV Absorbance Detection

D. Wickam in A Practical Guide to HPLC Detection, D. Parriott, ed., Academic Press, San Diego, CA, 1993, p67

Fully Integrated Positive-PressureDriven Microfabricated Liquid Analyzer

The basic idea is to incorporate on-chip near real-timesampling, capillary separation, and multivariatedetection in a single integrated microfabricated device. Soft lithography is used for rapid prototyping for proof of principle design and testing.

References:

(1) Initial analyzer development - P. G. Vahey, S.H. Park, B. J. Marquardt, Y. Xia, L. W. Burgess and R.E. Synovec, Talanta, 2000, 51, 1205 - 1212.

(2) Incorporation of GLRS detection - P.G.Vahey, S. A. Smith, C. D. Costin,Y. Xia, A. Brodsky, L.W. Burgess and R. E. Synovec, Analytical Chemistry,2002, 74, 177 - 184.

Photo of Microfabricated Liquid Analyzer

Separation Channel

SampleBy-pass

SampleInlet

Mobile Phase

Inlet

Outlet

Detection Region

100µm10µm

Channels in PDMS

Micro-fabricated Liquid Analyzer

BlockBy-passMobile

Phase Inlet

SampleBy-pass

Sub-Nanoliter Sample Injection by Flow ProgrammingPositive pressure driven with a pump.

An alternative to electrophoretic and electro-osmotic flow

Inject Sample

SampleInlet

To Separation

Channel

Micro-fabricating a Channel

1. Spin Coatphotoresist

2. UV Exposure 3. Develop Channel Master

4. Cast PDMS 5. Remove PDMS(stationary phase)

6. Affix PDMSto glass substrateusing thin PDMS coat

3 injections, 2 nL each

4 6 8 10

0

40

80

Time, min

Abso

rban

ce, m

AUAutomation Provides Reproducible Injection

8 mM Bromocresol Green

5 mM phosphate mobile phase, pH 7, 40 nL/min

PDMS channel 100 µm x 10 µm x 23 cm

Valcor Solenoid

Absorbance Detection Ocean Optics SD2000

Micro-fabricated LC with Absorbance Detection

Water m.p., 6 nL/min

Injected Volume 1 nL

Separation Channel in PDMS 100 µm x 10 µm x 6.6 cm

Absorbance Detection using Equitech Spectrophotometer

400500

600700

600650

700750

800

Time, secWavelength, nm

FD&C Red #3

FD&C Blue #1

Micro-fabricated LC Separation of Two DyesTemporal and Spectral Selectivity are Both Provided

Water m.p., 6 nL/min

Injected Volume 1 nL

Separation Channel in PDMS 100 µm x 10 µm x 6.6 cm

Absorbance detection using Equitech spectrophotometer

FD&C Red #3

FD&C Blue #1

400 500 600 700600

650

700

750

800

Wavelength, nm

Tim

e, s

ec

LC vs. Micro-fabricated LCLab Scale Microfabricated

Column Diameter 4.6 mm 10 µm x 100 µm

Flow Rate 1 mL/min 10 nL/min

Waste Generated 500 L/year 5 mL/year

Sample Used 10 µL 1 nL or less

Pump Pressure 1000 psi 5 psi

Cost per column $400 and up $10

“A disposable, plug and play LC device.”

The basic idea is to utilize optical waveguide principles to produce a low volume (~ 2 µL) detection cell that simultaneously provides over a 1000-fold enhancement in the limit of detection relative to a typical Raman measurement. A Teflon AF 2400 tube provides a lower refractive index than just about any liquid of interest, so the tube functions as a long optical path Raman detection cell. The flow cell is suitable for HPLC, FIA or direct process monitoring.

Reference:

B. J. Marquardt, P. G. Vahey, R. E. Synovec and L. W. BurgessAnalytical Chemistry, 1999, 71, 4808 - 4814.

Raman Spectroscopy:

Application to Process Analysis and HPLC Detection

Light Scattering• Two types of Electromagnetic Radiation scatter occur

•Elastic (Rayleigh scatter)

•Inelastic (Raman scatter)

Incident Beam

• Raman spectroscopy provides information about vibrational energies of molecules

• The Raman mechanism is emission from a momentarily induced dipole versus a change in dipole vibrational mode for molecular IR

• Raman is complementary to IR, producing relatively stronger peaks for symmetrical stretching versus anti-symmetrical stretching modes

Raman versus IR Spectroscopy

Benefits of Raman Spectroscopy

• Multicomponent Analysis

• Useful for measuring aqueous phase chemistries

• Identification of analytes by molecular signature

• Spectra obtained in seconds

• Applicable to in situ analysis

• Ease of sampling ! no sample pretreatment needed! only require optical access to sample

Raman-Waveguide Detector: Using a Liquid Core Waveguide (LCW) Flow Cell

10x

785 nmLaser

RamanDetector

Sampling Window

Teflon AF Waveguide Flow Cell

Sample Outlet

HPLC Fitting

Sample Inlet

• Basics of light transmission in an optical fiber/waveguide

• Fibers/waveguides consist of a core, RI = n1, and a cladding, RI = n2

• Numerical aperture (NA) describes the acceptance cone

• NA = sin θa = (n12 - n2

2)1/2

• Critical angle for transmission of light, sin θc = n2/n1 ( n1 > n2 required)

θθθθa

cladding (tube) critical anglecore

eventually lost

θθθθc

Total Internal Reflection: Basics for LCW construction

acceptance cone

n1

n2

Index of Refraction for LCW Materials

Values from: Applied Spectroscopy, 1990, 40, 163-5 and Applied Optics, 1997, 36, 8992-8

Solvents and Cell Materials Index of Refraction (RI)

Teflon AF 2400 (capillary tube) 1.29 (good choice)Teflon AF 1600 1.31Methanol 1.326Water (solvent) 1.333

FEP 1.338Acetonitrile 1.342Acetone 1.357

PTFE 1.35-1.381-Butanol 1.397Chloroform 1.444

Quartz 1.458Benzene 1.498Bromoform 1.587

Excitation and Raman Scatterin an Optical Waveguide

Benefits of the liquid core optical waveguide(1) Enables the excitation of more molecules since total internal reflection

in sampling tube (flow cell) increases pathlength within a small volume (2) Since Raman scatter is isotropic it allows very efficient collection of the

Raman scattered light

Not all resolved along time axis

All analytes resolved using both dimensions