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 Data
m/z

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

Spectra to Identify and Test Purity
LC with UV Absorbance Detection

Overlaid spectra from

Sample spectra overlaid with beginning, middle and end of
standard spectra eluting peak
Signal

Signal

190 230 270 310 360 390 190 230 270 310 360 390

Wavelength (nm) Wavelength (nm)

D. Wickam in A Practical Guide to HPLC Detection, D.
Parriott, ed., Academic Press, San Diego, CA, 1993, p67
 Fully Integrated Positive-Pressure
Driven Microfabricated Liquid Analyzer

The basic idea is to incorporate on-chip near real-time

sampling, capillary separation, and multivariate
detection 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
 Micro-fabricated Liquid Analyzer
Sample
Inlet
Channels in PDMS Mobile Sample
Phase By-pass
Inlet
10µm
Separation
100µm Channel

Detection
Region

Outlet
 Sub-Nanoliter Sample Injection by Flow Programming
Positive pressure driven with a pump.
An alternative to electrophoretic and electro-osmotic flow
Sample
Inlet

Block
Mobile By-pass
Phase Sample
Inlet By-pass

To
Separation Inject
Channel Sample
 Micro-fabricating a Channel

1. Spin Coat 2. UV Exposure 3. Develop

photoresist Channel Master

4. Cast PDMS 5. Remove PDMS 6. Affix PDMS

(stationary phase) to glass substrate
using thin PDMS coat
 Automation Provides Reproducible Injection

3 injections, 2 nL each 8 mM Bromocresol Green

Absorbance, mAU

80
5 mM phosphate mobile
phase, pH 7, 40 nL/min

PDMS channel
40 100 µm x 10 µm x 23 cm

Valcor Solenoid

0 Absorbance Detection
Ocean Optics SD2000

4 6 8 10
Time, min
 Micro-fabricated LC with Absorbance Detection

FD&C
Blue #1 Water m.p., 6 nL/min
Injected Volume 1 nL
FD&C
Separation Channel in PDMS
Red #3 100 µm x 10 µm x 6.6 cm
Absorbance Detection using
Equitech Spectrophotometer

800
750
700
Tim 700
e, 600
m
se
c
650 500 gth, n
l e n
600 400 ve
Wa
 Micro-fabricated LC Separation of Two Dyes
Temporal and Spectral Selectivity are Both Provided

800
Water m.p., 6 nL/min
FD&C
Red #3 Injected Volume 1 nL
750 Separation Channel in PDMS
100 µm x 10 µm x 6.6 cm
Time, sec

700 Absorbance detection using

Equitech spectrophotometer

FD&C
650 Blue #1

600
400 500 600 700
Wavelength, nm
 LC vs. Micro-fabricated LC
Lab 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.”

 Raman Spectroscopy:
Application to Process Analysis and HPLC Detection

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. Burgess

Analytical Chemistry, 1999, 71, 4808 - 4814.
 Light Scattering
• Two types of Electromagnetic Radiation scatter occur

•Elastic (Rayleigh scatter)

•Inelastic (Raman scatter)

Incident Beam
 Raman versus IR Spectroscopy

• 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
 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

Raman 785 nm
Detector Laser

10x
Sampling
Window

HPLC Teflon AF
Fitting Waveguide Sample
Flow Cell Outlet
Sample
Inlet
 Total Internal Reflection: Basics for LCW construction
• 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 - n22)1/2
• Critical angle for transmission of light, sin θc = n2/n1 ( n1 > n2 required)

acceptance cone core

cladding (tube) critical angle

n2
θa θc n1

eventually lost
 Index of Refraction for LCW Materials
Solvents and Cell Materials Index of Refraction (RI)

Teflon AF 2400 (capillary tube) 1.29 (good choice)

Teflon AF 1600 1.31
Methanol 1.326
Water (solvent) 1.333

FEP 1.338
Acetonitrile 1.342
Acetone 1.357

PTFE 1.35-1.38
1-Butanol 1.397
Chloroform 1.444

Quartz 1.458
Benzene 1.498
Bromoform 1.587

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