explorations in capillary reverse phase a …
TRANSCRIPT
EXPLORATIONS IN CAPILLARY REVERSE PHASE
LIQUID CHROMATOGRAPHY
by
THOMAS SCOTT KEPHART, B.S.
A DISSERTATION
IN
CHEMISTRY
Submitted to the Graduate Faculty of Texas Tech University in
Partial Fulfillment of the Requirements for
die Degree of
DOCTOR OF PHILOSOPHY
Approved
May, 2001
ACKNOWLEDGMENTS
I would like to thank my advisor, Sandy Dasgupta for his patience and
guidance these last five years. I would also like to thank my wife, Nicole Kephart
and my son. Hunter Kephart. I could not have done it with out their support. I
would also like to thank my father, Bill Kephart. The acknowledgements page
would not be complete with out thanking Bill Shumway for all the conversations
at lunch that kept me going the last few years.
This research was partially supported by the U. S. Environmental Protection
Agency through STAR Grant R82-5344-01-0. This manuscript has not, however,
been reviewed by the agency and no endorsements should be inferred. General
Assistance from Dionex Corporation is also acknowledged. We also gratefully
acknowledge the gift of the ZirChrom-PBD by ZirChrom Separations Inc. We
would also like to thank Dr. Matthias Pursch of the National Institute of Standards
and Technology for the Ti02 based packings.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF FIGURES v
LIST OF ABBREVIATIONS viii
CHAPTER
I. INTRODUCTION 1
Micro Column Liquid Chromatography 3
Instrument Requirements 4
Research Presented in this Dissertation 8
Literature Cited 13
II. AN AFFORDABLE HIGH PERFROMANCE PUMPING SYSTEM FOR GRADIENT CAPILLARY LIQUID CHROMATOGRAPHY 16
I ntroduction 16
Experimental 18
Results and Discussions 22
Literature Cited 28
III. HOT ELUENT CAPILLARY LIQUID CHROAMTOGRAPHY 37
Introduction 37
Experimental 38
Results and Discussions 42
Literature Cited 50
IV. SUPERHOT WATER ELUENT CAPILLARY LIQUID CHROMATOGRAPHY 61
Introduction 61
Experimental 64
Results and Discussions 67
Literature Cited 78
V. FUTURE APPLICATIONS AND POTENTIAL IMPROVEMENTS 89
iii
Portable RPLC System 89
Super-Hot Water LC-MS System 90
Supercritical Water Chromatography 91
Liquid Ionization Detector 92
Experimental Verification of Boiling Point in the Column.... 93
Literature Cited 94
VI. CONCLUSION 95
APPENDIX
A. RETENTION MECHANISM THEORY 98
B. HPLC COLUMN PERFORMANCE 100
C. COMPUTER CONTROL OF PUMPING SYSTEM 104
IV
LIST OF FIGURES
2.1. Layoutof the capillary HPLC system 30
2.2. Stainless steel syringe end seal design 31
2.3. Pressure stability during a typical isocratic run. 75% acetonitrile in water flowing at 5 ^iL/rnin, through a 5 |j,m C-18 silica column... 32
2.4. Illustrative isocratic system reproducibility. RSD in retention time is < 1%. All analytes are 500 |JM, 20 nL injection. 5-|am PRP-1 column, 2 i^L/min. Peak identities from left to right: cytosine, uracil, adenine, uridine, thymidine, adenosine, xanthosine, and inosine.... 33
2.5. Gradient chromatogram for the same sample and same column as in Figure 2.4. Flow 2 |aL/min 34
2.6. Illustrative gradient reproducibility, 5 |a,m C-18 silica column, flow 5 I^L/min. Average RSD in peak retention time is 0.545%. Amounts injected are as follows: peaks 1-3: -100 pg, 4: 120 pg, 5, 7-10: 85 pg, 6: 150 pg. Peak identities from left to right: (1) phenol, (2) benzaldehyde, (3) benzonitrile, (4) nitrobenzene, (5) benzene, (6) bromobenzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, and (10) t-butylbenzene 35
2.7. Chromatogram on a 12 cm long ZirChrom-PBD column, flow rate 6.3 pL/min, 6500 psi. All samples were in 75% acetonitrile, 25% water. Before injection, the eluent is 37% ACN, at injection it is switched to 55% ACN. The time for the step change in gradient to reach the head of the column is ~ 30 seconds. -100 pg of each analyte is injected 36
3.1. Experimental set up of chromatographic system 52
3.2. Viscosity of 50:50 v/v ACN:H20 as a function of temperature temperature. The points represent experimental data; the best fit line is shown. The error bars on the experimental points are smaller than the dimensions of the symbols plotted 53
3.3. Retention time of benzene and homologs as a function of temperature. The error bars on the experimental points are smaller than the dimensions of the symbols plotted 54
3.4. Computed diffusion coefficient of the 50:50 ACN:H20 eluent as a function of temperature. The best-fit data from Figure 2 was used to calculate Dm 55
3.5. Separations of benzene, toluene, ethylbenzene, and propylbenzene at four different temperatures. A flow rate of 2 ^iL/min was used throughout 56
3.6. Knox plot of ethylbenzene at four different temperatures 57
3.7 Knox A, B, and C terms for ethylbenzene as a function of temperature 58
3.8. a) Isocratic separation of (from left to right) benzaldehyde, benzene, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, n-amylbenzene, 1-phenylhexane and 1-phenylheptane. A flow rate of 3.5 (^L/min and a column temperature of 200 °C were used. b) Gradient separation of the same mixture, same flow rate and temperature 59
3.9. High-speed gradient separation on C-18 modified Ti02 column of benzene, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, n-amylbenzene, 1-phenylhexane and 1-phenylheptane. Flow rate 25 fj,L/min; column temperature 160 °C 60
4.1. Hot water chromatography system, schematically shown. CV: Check valve; PG: pressure sensor and gauge; SSC: silica saturation column; CH: column heater; BPC: back pressure column; 81
4.2. 60 times magnification of capillary column after structural failure.... 82
4.3. Dielectric constant and viscosity of water at 7200 psi along with the viscosity and dielectric constant of both pure ACN and a 50% ACN 83
4.4. Dielectric constant of water as a function of pressure 84
4.5. Separations on (left) ZirChrom-Carb and (right) ZirChrom-PBD columns; detection at 195 nm, flow rate of 8.6 |iL/min. s: solvent (acetonitrile), 1: phenol, 2: benzene, 3: toluene, 4: nitrobenzene, 5: ethylbenzene, and 6: n-propylbenzene. All subsequent figures has the same numerical identification for analytes 85
VI
4.6. High speed separations (a) ZirChrom-Carb and (b) ZirChrom-PBD columns at 300 °C and 240 °C, respectively 86
4.7. Van't Hoff plots for the retention of benzene derivatives 87
4.8. Thermal gradient performed in GC oven using a FID detector. 180 i m i.d. 13 cm silica capillary with ZirChrom-Carb packing, flow rate 8.6 nL/min. Temperature gradient started at 100 °C and was ramped to 250 °C @ 50 °C/min 88
Vll
LIST OF ABBREVIATIONS
ACN - Acetonitrile
CE - Capillary electrophoresis
FID - Flame ionization detector
GC - Gas Chromatography
HPLC - High Performance liquid chromatography or high pressure liquid chromatography
HTLC - High temperature liquid chromatography
LC - Liquid Chromatography
LC-MS - Liquid chromatographic instrument coupled to a mass spectrometric detector
MS - Mass spectrometry
NMR - Nuclear magnetic resonance
ODS - Octadecyl silicate, C-18 packing
PAH - Polycyclic aromatic hydrocarbon
PBD - Polybutadiene
PEEK - Poly(ether-etherketone)
PC - Personal computer
PCB - Polychlorinated biphenyls
PRP - Polymeric reverse phase
PSDVB - Polystyrene divinylbenzene
PTFE - Polytetrafluoroethylene (Teflon®)
Vll l
Rl - Refractive index
RPLC - Reverse phase liquid chromatography
RSD - Relative standard deviation SFC - Supercritical fluid chromatography
UV - Ultraviolet
Vis - Visible light
IX
CHAPTER I
INTRODUCTION
The origin of chromatography can be traced back to 1906 when Tswett
separated chlorophyll pigments using a glass column packed with calcium
carbonate particles. ' Tswett coined the term "chromatography" by combining
two Greek words, chroma, color and graphein, write and defined it as a method
in which different components of a mixture can be separate on an absorbent
column in a flowing system.
From Tswett's original work until the 1930's, little research was done in the
field of chromatography. Starting in the 1930's, however, many new advances
came: thin-layer, ion-exchange and electrophorefic chromatography were all
developed during this decade. Paper, partition, and gas chromatography were
developed in the 1940's. The 1950's brought the development of gas-liquid
chromatography and gel based chromatography. Supercritical fluid
chromatography made its appearance in the 1960's.^
The evolufion of high performance liquid chromatography (HPLC)
spanned many decades. The promise of faster and more efficient separations
propelled the discipline ever fonward. Research in HPLC started in the early
1960's and continued to evolve until it became a well-accepted method by the
1980's. The advances in HPLC involved the development of high pressure
pumping systems, small diameter particles with controlled porosity, new
stationary phases, injectors and small volume detectors.'* Modern HPLC may be
divided into five different classes according to the retention mechanism utilized:
adsorption, partition, size exclusion, affinity, and ion exchange. Adsorption and
partition separations are based on the competition of neutral analytes between a
liquid mobile phase and a neutral, solid (adsorption) or liquid-like (partifion)
stationary phase. Size exclusion chromatography is based on molecular sieving,
affinity chromatography is based on specific detention like a 'lock and key'
mechanism, and ion exchange is based on the competifion of charged analytes
for the oppositely charged sites on the stationary phase.
Adsorption and partition chromatography can be broken down further into
normal phase and reverse phase. Normal phase chromatography was the first to
be performed and is thus, for historical reasons, called "normal phase." This type
of separation uses a polar stationary phase and a less polar mobile phase. The
origin of reverse phase type separations (the opposite of normal phase using a
non-polar stafionary phase and a more polar eluent) can be traced back to the
late 1940's.^'^ The term "reverse phase chromatography" can be traced back to
Howard and Martin who coined the phrase in 1950. ^ Due to its speed, efficiency
and diversity, reverse phase chromatography has grown to be the most widely
used chromatographic technique.'* Only reverse phase type separations are
considered in this dissertafion.
Micro Column Liquid Chromatography
The first work in micro-column chromatography is credited to Horvath and
co-workers in 1967.°'^ These studies used 0.5-1.0 mm stainless steel capillaries
packed with pellicular particles to separate ribonucleotides. Starting in 1977, Ishii
and co-workers described, in a series of publications, the use of slurry packed
Teflon micro-columns (0.5 mm i.d.); these papers are generally regarded as the
pioneering studies in micro-column liquid chromatography.^°' ^^ ' ' '*' ^ Shortly
after Ishii a group led by Scott published papers on their work with 1.0 mm
columns.*^'^^*°'^^'^° The inifial work by Ishii and Scott's groups along with the
work of Novotny^*' ' ^ and Yang '* are all regarded as key publications in the
development of micro-scale liquid chromatography.
The research into the capillary scale has been driven by the fundamental
advantages that this small scale has over the conventional (4.6 mm) columns.
The capillary scale provides; (1) excellent solvent economy, (2) small sample
requirements, (3) high mass sensitivity, and (4) ease of coupling to sophisticated
detectors especially mass spectrometers.^^'^^ However, the development of
capillary based separation systems mirrors the original development of HPLC.
The technique is in an ongoing process of maturation and has once again been
dependent on the development of adequate pumping systems, injectors,
columns, and detectors.^^
Instrument Requirements
Pumping system
Capillary based HPLC requires a pumping system that is able to deliver
reproducible, pulse-free flow rates in the 1 |aL/min range. Split flow methods
have been used to reduce the flow rate of a conventional HPLC pump. This
method uses a flow splitter that splits off most of the flow through a restrictor
capillary and produces a flow rate in the i^L/min or nL/min range for the capillary
column.^^ The problems with this type of pumping system are: (1) solvent
consumption is equal to a conventional system and (2) the percent of the flow
that is sent to the capillary is based on the split ratio, which is dependent on the
viscosity of the eluent. Split flow based pumping systems are not an appropriate
pumping method to use when changes in the viscosity are expected. This
includes solvent gradients or temperature gradients.
Today, pumps are commercially available which allow flow rates in the low
iL /min range. The Cap-LC system by Waters^® uses a pumping system capable
of gradient flow rates in the 1-40 |j,L/min range; however, the system costs
$70,000. Other less expensive commercial systems exists;^° however, these
utilize split flow method. The pressure limits of most of these systems are
around 5000 psi.
Custom made pumping systems have been developed to handle extreme
pressures. Jorgenson's group has developed an ultra high pressure pumping
system capable of pressures up to 130,000 PSI.^* There are, however, no on
line injectors that can be used with the system.
Injector
To avoid column overloading, the volume of sample injected onto the
column has to be smaller than 1/100 the column volume.^^ In the capillary scale,
this corresponds to an injection volume from one nanoliter (nL) to one microliter
(|iL). Commercial injectors are available with injection volumes down to 20 nL.
Another concern with an injector is its ability to handle high pressures. Vaico has
introduced an injector (model C2XL) which is capable of operating at pressure up
to 12,000 psi while the convenfional VaIco injectors are rated at 5000 psi.
Capillary columns
The column is the heart of any chromatographic system. It is the column
that separates the analytes allowing analytical information to be obtained from
the detector. Micro-LC columns have commonly been classified into four
categories based on the internal diameter of the column. This classification
invokes: micro-bore columns (1-2 mm i.d.), packed capillary columns (0.1-0.5
mm), semi-packed capillaries (0.02-0.1 mm i.d.), and open tubular capillaries
(0.01-0.075 mm i.d.).^^ Only packed capillaries with an internal diameter of 0.18
mm i.d. are used in this work.
The column, in modern capillary liquid chromatography, is typically made
from fused silica capillaries; however, other column materials have been used
such as poly(ether-etherketone) (PEEK), Teflon tubes, and stainless steel
capillaries. At the tail of the column, a frit is used to keep the packing particles
from being pushed out of the column. Frits have been made from many different
materials including metal screen, porous glass either from glass wool or sintered
glass particles, or polymeric materials. Frits are also used at the head of the
column to prevent particles from escaping back towards the injector. In this
work, no frit was used at the head of the column. This allows the column to be
easily trimmed as the packing settles.
The packing process is crucial to the performance of the capillary column.
Many different techniques have been reported using methods ranging from slurry
packing, dry packing, and electrokinetic packing. '*' ^ The most popular
technique is wet slurry packing. Slurry packing consists of suspending the
packing particles in an appropriate solvent based on the chemical and physical
properties of the packing. The packing slurry is placed into a chamber to which
the head of the column is attached. A high pressure pump is used to deliver the
packing into the column. Unfortunately the process of packing capillary columns
is still an art and different techniques are required for each different type of
packing particle. Many papers have been published on the aspects of packing
capillary columns.^ ' ' ' ' ^
Detectors
Numerous detectors have been developed for use with capillary scale
HPLC. In the past 15 years, detection methods in the capillary scale have been
a major research area due to the concurrent grow/th of capillary electrophoresis
(CE) and capillary HPLC. The most widely used detecfion method is UV
absorbance.'*" Other detection methods have been developed using refractive
index (Rl) detectors,'*^ laser based Rl detectors,'*^ fluorescence detectors,''^
electrochemical detectors'*^'*^'*^ and gas chromatographic (GC) detectors.^''
There has also been research into hyphenated detection systems such as
nuclear magnetic resonance spectrometry (NMR)'*^ and mass spectrometers
(MS).'*^ Carbon containing eluents cannot be used with flame ionizafion detector
(FID). Recent research into superheated water based reverse phase liquid
chromatography has enabled the use of FID's in capillary LC.'*^
Extra column band broadening
The small peak volume in capillary chromatography magnifies any
sources of dispersion present in the system. Dispersion in liquid
chromatography, either conventional scale or micro scale, has three main
sources: the injector, connecting tubing, and the detector. Minimizing the
volumes as much as possible can minimize the dispersion due to the injector and
the detector. Extra-column broadening due to connecting tubing increases
linearly with the length of the tubing and with the fourth power of the inner
diameter.^^ Throughout this work, peak broadening has been reduced to a
minimum by using small injection volumes, direct connection when possible, and
on column detecfion.
Research Presented in this Dissertation
The primary goal of this research project was to explore methods to
decrease analysis time in reverse phase liquid chromatographic separations by
creating a capillary scale instrument that was capable of operafing at high
pressures as well as high temperatures. When compared to a conventional
system, the instrument needed to provide significant improvements in initial cost,
operating costs, and analysis times. These requirements were met by
developing an inexpensive gradient pumping system capable of generating
12,000 psi along with a column heating system capable of heating the column to
400 °C. Heating the column had two effects: (1) the high temperature lowers the
viscosity of the eluent, which allows higher linear velocifies, and (2) the increase
in the diffusion coefficient increases the efficiency of the separation. Normally
the use of higher linear velocities reduces the analysis time but leads to a major
loss of efficiency. An increase in diffusion coefficient mifigates this problem while
the decrease in viscosity allows high flow rates. The use of high temperatures
also allowed the use of superheated water as a reverse phase eluent. Typical
reverse phase eluents use organic modifiers that are harmful and expensive. A
system capable of using superheated water can eliminate the organic solvent
8
replacing it with inexpensive and harmless water. The elimination of the organic
solvent also allows the coupling of the system to new detection methods like
fiame ionization detection which, is a universal detector for carbon containing
analytes.
An Affordable High Performance Pumping System for Gradient Capillary Liquid Chromatography
As previously noted an important part of the capillary HPLC system is the
pumping system. Convenfional HPLC pumping systems are capable of
generating constant reproducible flow rates only in the ml/min range. In order to
uses such pumping systems in the capillary scale you need to use a spilt flow
method. The commercial capillary scale pumps that are now available are
expensive and are limited to pressures around 5000 psi which limits the choice of
column lengths, particle sizes, and flow rates.
Chapter II describes an inexpensive, dual syringe, gradient pumping
system developed in this work which is capable of producing constant,
reproducible flow rates from 500 nL/min up to 30 |LiL/min with a 250 i L syringe.
Different syringe sizes can easily be used to produce flow rates from 50 nL/min
(25 |j.L syringe, 1000 msec delay, and 1 step) up to 350 |j,L/min (1 mL syringe,
10 msec delay, and 10 steps). The upper pressure limit of the system is over
10,000 psi, which is over double that of the commercial conventional or capillary
scale pumps. System performance is demonstrated using 3 capillary columns
that were packed in-house. Reproducibility of the chromatographic system, in
both isocratic and gradient modes, is reported.
Hot Eluent Capillary Liguid Chromatography
Although maximum efficiency attainable with a given column (plate
counts) is commonly published very few columns are operated at maximum
efficiency due to the long elution time needed to minimize the increase in
theorefical plate height due to slow mass transfer. The required minimum
separafion time of liquid based separafion systems, unlike GC and SFC, is
usually dictated by the back pressure produced by the column. Two methods
are currenfiy used to reduce the analysis time in LC systems: (1) increase the
pressure limit of the system, or (2) decrease the back pressure produced by the
column. Jorgenson has explored the limit of high pressure by developing a
pumping system that is capable of 130,000 psi.^^ This approach, although
elegant, is limited by injector pressure limits and the physical size of the pumping
system. The second approach is to lower the back pressure produced by the
column usually accomplished by lowering the viscosity of the eluent. Several
groups have explored operating near the supercritical domain with reverse phase
eluents or modifying supercritical fluids with significant amounts of reversed
phase eluents to dramatically lower eluent viscosity. This is an interesting
technique with limited commercial acceptance due to the complexity of the
system required to keep the mixed eluent at or near the supercritical state.
10
Chapter III explores the use of high temperatures along with the high
pressure pumping system to accomplish efficient high-speed separations. The
use of high temperatures lowers the viscosity of the eluent so that at maximum
practical pressures, the system can operate at a very high linear velocity. Also,
due to an increase in the diffusion coefficient, the deterioration of the plate height
at high linear velocities is ameliorated. Separations are performed at
temperatures up to 200 °C, back pressures up to 10,000 psi, and flow rates up to
25 ^L/min. A separation of several alkylbenzenes is performed in less than 2
minutes.
High-Pressure. High-Speed. Superheated Water Eluent. Capillary Liguid Chromatography
A modern day laboratory has to deal with many issues that were not
present 10 years ago. The environmental impact of organic solvents and its
economic consequences have led many research groups to explore methods to
limit or to eliminate the consumption of these solvents which can be harmful to
health and expensive to dispose of. One of the advantages of the capillary scale
is the dramatic decrease in the consumpfion of these organic modifiers. In 1995
Hawthorne and co-workers demonstrated another alternative. They showed that
supercritical water at temperatures of 400 °C and pressures of 350 bar can be
used as a replacement for organic solvents for extraction.^°'^^ This discovery
lead to the use of very hot water as an eluent for reverse phase liquid
chromatography (RPLC).^^ Although the capillary scale has not previously been
11
used to perform separations using superheated water as an eluent, the small
scale should prove to be ideal due to the excellent thermal conductivity and low
thermal mass.
Chapter IV reports on the use of a high pressure, capillary scale, reverse
phase liquid chromatography system that uses superheated water as an eluent
to separate non-polar alkylbenzene derivatives. Separafions are shown on 180
)am columns packed with 3 jxm zirconia particles modified with elemental carbon
or polybutadiene at temperatures up to 400 °C. A high speed separation is
shown separating six benzene derivatives in less than 100 seconds.
12
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15
CHAPTER II
AN AFFORDABLE HIGH PERFORMANCE PUMPING SYSTEM FOR
GRADIENT CAPILLARY LIQUID CHROMATOGRAPHY
Introduction
Since the fundamental work of Howard and Martin^ nearly 50 years ago,
reverse phase liquid chromatography (RPLC) has grown to become one of the
most widely used liquid phase separation methods. RPLC has been used
successfully in diverse applicafions ranging from the separation and purification
of peptides and the determination of protein structures,^ to purity analysis in
quality controP'* and process monitoring.^ RPLC is a broadly applicable
technique that can use many different combinations of columns and eluents to
analyze a variety of sample types. Gradient elufion capability adds a great deal
to such systems in terms of fiexibility, separafion efficiency, and analysis time.
The advantages of moving from conventional HPLC into the capillary
domain have been known for over 20 years. As shown by Ishii,^ these benefits
include lower solvent and sample consumpfion, greatly increased mass
sensitivity, and higher peak efficiencies. A conventional scale LC instrument
consumes nearly 500 L of eluent a year; a capillary system can be run for a year
with what would be consumed in one day by a conventional system. This allows
the use of expensive (ultrapure, deuterated, etc.), exotic (e.g., chiral), or toxic
and even environmentally hazardous solvents in the capillary domain. Many
16
current biological invesfigations are carried out in a scale that provides an
amount of sample sufficient only for analysis by capillary based systems.
Hyphenated analysis methods, especially LC-MS, proven invaluable in the
characterizafion of proteins and peptides in complex mixtures, are particularly
well suited to the capillary scale. LC-MS is also indispensable in drug
development where peak confirmation can be legally mandated.'^ Operation at
the capillary scale not only improves the performance of an LC-MS interface, but
it makes the use of a buffered mobile phase without complex desalting
procedures possible and generally greatly reduces the ion source fouling
problems.®'
Finally, the small power and space requirements make capillary HPLC an
ideal candidate for portability. A portable instrument needs to be light and
compact, have manageable power requirements, and match the performance of
the convenfional counterpart. Capillary scale instrumentation should be
inherently compact due to the small ancillary components needed (although this
still remains a wishful thought for the optical detectors in use); in particular, the
power requirements for the pumping system are very low (less than 1 mW of
power is needed to pump 1 |j,L/min at 4000 psi).
Unfil fairly recently, hardware for capillary LC was not plentiful. At the
present time, nanoliter-scale valves and packed capillary microcolumns are
commercially available. In the wake of popular interest in capillary
electrophoresis (CE), capillary-scale opfical detectors have been much improved
17
in terms of their sensitivity and are also widely available. The cost remains
relatively high; affordable dedicated wavelength alternatives were previously
described by this laboratory.^" Relative to the detectors, there are far fewer
vendors that offer a gradient pumping system capable of nL-|aL/min flow rates;
the available products are expensive.
This paper describes a capillary-scale liquid delivery system based on two
independent syringe pumps. The pumps are controlled by custom software and
are capable of reproducible gradients at constant ^L/min flow-rates.
Experimental
The system layout is shown in Figure 2.1. The pumps are 48 000 step,
motor driven syringe-type dispensers (Model 50300, Kloehn Inc., Reno, NV) that
operate under internal jiProcessor control or can be controlled by serial port
connecfion from a personal computer (PC). Any PC capable of running
Windows™ (3.1 or higher, Microsoft, Redmond, WA) is suitable.
A stainless steel (type 316) block was machined in-house with
appropriately sized ports to accommodate a low leak dual ball and seat inlet
check valve (P/N 44541, Dionex Corp., Sunnyvale, CA), the threaded head of the
syringe, and a liquid outlet port. A small volume (25 mL) glass vial functioned as
the eluent reservoir. It was affixed next to the syringe dispenser and was
connected to the check valve with Teflon tubing. Porous polypropylene inlet
filters (nominal pore size 20 i m) were used in each solvent reservoir.
18
Glass syringes are commonly available and can be used up to -1000 psi ^
but the present applicafion requires higher pressures. Stainless steel syringes
were constructed in-house (Figure 2.2). The syringe barrel is a stainless steel
cylinder. We ufilized commonly available 316 grade stainless steel column blank
used for packing HPLC columns, (2.1 mm i. d., 6.35 mm o. d., cut to a 83 mm
length); it need not be polished internally. The pistons are 1.27 mm dia. 316
stainless steel rods press fit into a metal stub that attaches to the pump
movement. The syringe is of the end-seal type. A Teflon® high-pressure seal
was made by machining polytetrafluoroethylene (PTFE) stock to the appropriate
dimensions (5.56 mm o. d., 1.27 mm i. d., and 11.4 mm long) and press fitting it
into the bottom of the barrel and the end cap by tightening the end-cap. We have
used end caps that were machined in-house from aluminum, any suitable
material can be used since the end cap is not a wetted part. The Teflon® piece
molds itself into the correct shape under pressure to form a high-pressure seal.
Similar other applicafions, in which a piece of Teflon® cold-flows to form a seal
are in the literature; pieces of PTFE tubes can be used as ferrules, for example.^^
Because of the end-seal design, the syringe acts as a displacement pump where
the displacement volume is dependent on the piston rod diameter and not the
barrel internal diameter. This design also obviates the need for having a
polished barrel. With a 1.27 mm dia. piston rod, the total displacement is 76 ^L
for the maximum linear travel of 60 mm allowed by the pump.
19
A pressure sensor (0-10000 psi, model SP70-A10000, Senso-Metrics,
Simi Valley, CA) was connected to the liquid output port of the stainless steel
block on pump A using 0.25 mm i. d. stainless steel or fused silica tubing. The
column back-pressure was confinuously monitored to insure proper system
performance. For isocratic or gradient eluent production in the capillary HPLC
system, the output from pump A and pump B were coupled to a tee (microvolume
tee, MT.5XCS6, VaIco Instruments, Houston, TX). The tee is connected to the
injector via a 400 ixm id . 2 cm long stainless steel tube that functions as a 2 fxL
volume mixing chamber. To produce a constant flow rate in the gradient mode,
custom software was written in Microsoft Visual Basic® and provided the user
interface for instrument control. During actual pump operation, the pump does
not need the PC, and the latter is completely free to execute other tasks.
An electrically actuated injection valve equipped with a 20 nL internal
sample loop (VaIco Instruments, Houston, TX) was connected to the mixing
chamber and used for sample introduction. The injector is rated by the
manufacture to have a pressure limit of 7000 psi.
The analytical columns used were 360 im o.d. and 180 |Lim i.d. fused
silica capillaries (Polymicro Technologies, Phoenix, AZ) packed in-house. Two
columns were - 50 cm long and were packed with reverse phase 5 ^m PRP-1
(Hamilton Co., Reno, NV) and 5 i m Adsorbosphere HS C-18 (Alltech Associates,
Deerfield, IL) particles, respectively. A third column was 12 cm in length and
packed with ZirChrom-PBD, a Zr02-based 3 inm dia. packing (ZirChrom
20
Separafions, Anoka, MN). A frit was made by packing glass wool (-0.25 mm
thick bed) in a nominally 0.3 mm i.d., 1.6 mm o.d. PTFE tube which served as a
butt-joint connector between the column and a 75 |am i.d., 365 |um o.d. fused
silica exit capillary. The silica tubes must be force fitted into the PTFE sleeve,
the frictional resistance is sufficient to prevent uncoupling from the connector.
Detection was made through a window created on the exit capillary, 4 cm from
the frit.
All columns were packed by a slurry packing technique. For the 5 i m
PRP-1, a 10 w/v% slurry in methanol was used with the slurry reservoir in a
sonicator. The pressure was increased from 0 to 6000 psi in 10 min and
maintained at that pressure for 24 hours. For the Adsorbosphere HS CI 8, the
packing material was air dried, slurried in acetone (3.6% w/w), sonicated for 10
min, shaken for 2 hours on a mechanical shaker and transferred to the slurry
reservoir. The slurry was stirred throughout the packing process. Methanol was
used as the push solvent. The initial pressure was set to 100 psi, and after the
back pressure increased beyond 100 psi, it was set to 1000 psi. From there it
was increased in 150 psi increments to 5000 psi and held at that pressure for 15
hours. For the 3 |Lim ZirChrom-PBD, a 10 w/v% slurry was made in 10 mM
aqueous sodium dodecyl sulfate containing 10% glycerol. The slurry reservoir
was constantly agitated and the pressure was increased from 0 to 7000 psi in 2
minutes. The pressure was maintained at 7000 psi for 24 h.
21
A Linear model UVIS 200 absorbance detector (Spectra-Physics /
Thermoseparation systems), designed for on-column detection with capillaries,
was used for detection. A detection wavelength of 254 nm was used in all work.
Results and Discussion
There are two popular methods to achieve constant flow rates in the low
or sub-|j,L/min scale: (a) Conversion of conventional systems to operate in the
correct flow rate range and (b) the use of syringe pump based system.^^.
Modificafion of conventional systems usually requires the use of a flow-splitter,^'*
which directs the majority of the flow to waste and fonwards a |al/min flow rate to
the column. This approach fails to reduce solvent consumption and flow can
vary with the changing viscosity of the eluent during gradient operation.
The use of a syringe based pumping system for a dedicated capillary
scale pump is attractive because such pumps deliver pulseless flow in the flow
rate range required by a capillary LC system. The flrst commercial syringe
pumps for use with microbore or larger capillary columns were introduced by
Brownlee Labs. Dual syringes of 10 mL volume were used with the seal at the
usual place, the head of the plunger and an acfive motor driven valve to switch
between refill and dispense modes.^^ The minimum recommended flow rate for
gradient applicafions was 10 |j,L/min and the minimum attainable fiow rate was 1
)LiL/min. The original pressure limit of 5000 psi was upgraded to 5500 psi after
the pump was sold under the Applied Biosystems name.^^ The present Perkin-
22
Elmer model 140 D pump^^ is derived from the original Brownlee MPLC pump
design, it uses 2.5 mL volume syringes with useful flow rate ranges of 2-100
|j,L/min and a pressure limit of 5000 psi.
The fundamental problem with designs that put the pump seal at the head
of the plunger is that it is difficult to reduce the plunger diameter (and therefore
the total available displacement volume) past a certain point, because of the
difficulty of machining a seal. In addition, polishing the interior of a narrow
syringe barrel can also be demanding. In contrast, the presently developed
pumps with end-seals enable reproducible flow rates down to 500 nL/min with
the stated plunger diameter and will allow smaller flow rates with a smaller
diameter plunger and a smaller volume mixing chamber. The pressure capability
is better than any of the commercial pumps cited above. At under 8 lbs, it is a
fraction of the weight of the original 45 lbs. Brownlee Pump.*^
The performance of the syringe and seal design was examined by
connecting the output of pump A to one side of the pressure gauge and the
output of pump B to the other side of the pressure gauge. The closed system
was then pressurized, the pumps were shut off and the pressure was monitored.
It took 1 h for the pressure in the system to drop from 4030 to 3960 psi (1.75%
change). The pressure dropped to 3550 and 3200 psi in 8 and 24 h,
respectively. This corresponds to a leak rate of 700 pL/min, this can probably be
attributed entirely to the leakage through the two check valves. In actual use
configurafion, the system has been pressure tested to 8000 psi, the injector.
23
which is pressure rated by the manufacturer to 7000 psi, leaks past this point. By
shutting off the tee output to the injector by a plug, we have pressurized the
system to the maximum limit of the pressure gauge (10000 psi) and verified the
pumping system itself holds pressure at this point with very low leak rates as
menfioned above. If suitable injectors were available, it is likely that this simple
pump design can be adapted for use at even higher pressures. The pressure
stability of the system under more typical operating conditions is shown in Figure
2.3.
The performance of the mixing chamber and the dual pump system were
tested by comparing the retention time relative standard deviation (RSD) of the
dual pump system to that of a single syringe pump system (same system as here
with only one operafing pump containing a premixed eluent) under othenwise
identical conditions. The average RSD of retention fimes for an 8 component
mixture was 0.825% (n= 30) with the dual pump system operating to produce a
constant eluent composifion throughout the run. Using only a single syringe
pump pumping the same eluent, the average RSD of retention times was actually
0.921% (n= 30). The fact that the dual pump system has a lower average RSD
indicates that the main source of retention irreproducibility is not from the
pumping system.
The isocratic evaluafion of the system was performed on the PRP-1
column with samples of biochemical/biological interest. A solution of 100 mM
ammonium formate (pH 4.25) was filled in pump A and the same solufion
24
containing 10% acetonitrile was filled in pump B. Figure 2.4 illustrates system
reproducibility for isocrafic elufion of 8 sample components with a 50:50 A and B
mix. Figure 2.5 shows the same analytes separated under gradient condifions.
The concentration (|aM) (and mass, pg) detection limits computed based on the
S/N=3 criterion for the gradient analysis are as follows: Cytosine 5.4 ]xM (12 pg),
Uracil 6.8 i M (15 pg). Adenine 11.2 ^M (30 pg). Uridine 2 (xM (10 pg), Thymidine
6.7 ^M (32 pg). Adenosine 5.4 |aM (32 pg), Xanthosine 11.5 |uM (74 pg), and
Inosine 7.6 |LIM (41 pg).
The gradient capabilities of the system were examined by separafing a
series of benzene derivatives on the HSC-18 column. Pump A contained a
mixture of acetonitrile and water (50:50); pump B contained only acetonitrile.
Figure 2.6 shows four overlaid sample chromatograms. The overall performance
of a pumping system is ultimately judged by its ability to provide reproducible
gradient elution, in terms of solvent flow rate and temporal composition. The
average retention fimes (n = 6) and the % RSD values for the individual analytes
in a 10-component mixture separated on the HSC-18 column were as follows:
phenol (5.06 min, 1.28%), benzaldehyde (5.91 min, 0.80%), benzonitrile (6.08,
0.72%), nitrobenzene (6.40 min, 0.64%), benzene (6.83 min, 0.52%),
bromobenzene (7.28 min, 0.37%), toluene (7.40 min, 0.33%), ethylbenzene (7.65
min, 0.30%), propylbenzene (8.16 min, 0.24%) and f-butylbenzene (8.30 min,
0.24%). The average RSD of retenfion fimes under gradient condifions was
0.545%. This corresponds to a variation of 2.05 (+ .88) seconds for 10 peaks
25
elufing in - 8.5 minutes. It is interesfing that note that the absolute standard
deviation itself decreases with retention time in an exponential fashion, -
log(standard deviation) is linearly correlated with the retention time with a
correlation coefficient of 0.9928. Column re-equlibrafion between gradient runs
require -5 min.
CE is presently one of the most actively researched areas in separation
science. While very large efficiencies are often reported for CE separations,
there is no equivalent of gradient elufion in CE. Although peak efficiencies are
not usually computed for gradient mode separations in chromatography because
intrinsic column efficiencies of different columns cannot be compared in this
manner, we feel that the most valid way to compare separation efficiencies
between the two techniques is to nevertheless compare in terms of the effective
plate numbers obtained during gradient elufion, the most common mode of LC
practice. In the chromatogram of Figure 2.6, the maximum peak efficiency was
observed for toluene, which exhibited 168 500 theoretical plates in the gradient
mode. The average peak efficiency for the 10 components was 111 000
theoretical plates. This correlates to an average of 14 000 plates per minute.
The plate counts for each peak under the gradient run conditions are as follows:
phenol (16 500), benzaldehyde (34 000), benzonitrile (89 500), nitrobenzene (131
000), benzene (167 500), bromobenzene (142 500), toluene (168 500),
ethylbenzene (125 000), propylbenzene (133 000), and t-butylbenzene (101 500).
26
For small molecules with substanfial diffusion coefficients, this is comparable to
CE efficiencies.
Fast separations were investigated with the short ZirChrom-PBD column
with 3 fxm packing, at a relafively fast flow rate. Five benzene derivatives could
be separated in less than 2 min (Figure 2.7).
In summary, we have described here a gradient capable pumping system
for capillary LC. With a fabrication cost under $3000, this would be affordable by
most laboratories interested in exploring this excifing area.
27
Literature Cited
1. G. A. Howard and A. J. P. Martin, Biochem. J. 46, (1950), 532.
2. J. F. Hancock, W. Henzel, and C. S. Horvath, HPLC of Biological Macromolecules, Methods and Applications; K. M. Gooding and F. E. Regnier, Eds. Marcel Dekker: New York, 1990. p 145.
3. S. Borman, Anal. Chem. 59, (1987), 969A.
4. R. L. Garnick, N. J. Solli and P. A. Papa, Anal. Chem. 60, (1988), 2546.
5. P. A. De Phillips, S. Yamazaki, F. S. Leu, B. C. Buckland, K. Gbevonyu, and R. D. Sitrin, Proceedings of the 9'" International Meeting on HPLC of Proteins, Polypeptides, and Polynucleotides; Philadelphia, November 1989, Paper No. 501.
6. D. Ishii, K. Asai, K. Hibi, T. Yonokuchi, and M. Nagaya, J. Chromatogr 144, (1977), 157.
7. Y. Hieda, S. Kashimura, K. Hara and M. Kageura, J. Chromatogr. 667, (1995),
241
8. A. Cappiello and F. Bruner, Anal. Chem. 65, (1993), 1281.
9. A. Cappiello, and G. Famiglini, Anal. Chem. 69, (1997), 5136.
10. C. B. Boring and P. K. Dasgupta, Anal. Chim. Acta., 342, (1997), 123. 11. C. B. Boring, P. K. Dasgupta and A. Sjogren, J. Chromatogr 804, (1988),
45.
12. J. L. Meek, Anal. Chem., 56, (1984), 1752.
13. F. Andreolini and A. Trisciani, J. Chromatogr Sci., 28, (1990), 54.
14. S. Van Der Wal and F.J. Yang, J. High Resolut. Chromatogr Chromatogr Commun,. 6, (1983), 216.
15. MPLC Micropump Technical Note 923. October, 1983. Brownlee Labs Inc., Santa, Clara, CA.
28
16. MicroGradient™ System: High Performance Microbore LC Product Bulletin 804. September, 1987. Applied Biosystems, Santa Clara Analytical Division, CA.
17. http://www2.perkin-elmer.com/pa/347203/347203.html
29
I '
Pump A
6547 PSI
Mixing Chamber ^ .„ UV Detector
Capillary Pressure >s I /rrs. Column
I I
PumpB
o
Computer
Figure 2.1 Layout of the capillary HPLC system.
30
^ Syringe barrel
[I ^ Teflon high pn\ pressure seal
Aluminum end cap
^ Stub (connects to motor drive)
Figure 2.2. Stainless steel syringe end seal design.
31
3000
2500
Q. 2000
3 1500 (/)
^ 1000
500
0 0 1 2 3 4 5 6 7 8 9 10
Time, min
Figure 2.3. Pressure stability during a typical isocratic run. 75% acetonitrile in water
flowing at 5 |j,L/min, through a 5 inm C-18 silica column.
32
0.01 AU
0 2 4 6 8 10 12
Time (min)
Figure 2.4 Illustrative isocratic system reproducibility. RSD in retention fime is
<1%. All analytes are 500 pM, 20 nL injecfion. 5-(xm PRP-1 column, 2 (iL/min. Peak identities from left to right: cytosine, uracil, adenine,
uridine, thymidine, adenosine, xanthosine, and inosine.
33
0.005 AU
Figure 2.5 Gradient chromatogram for the same sample and same column as in Figure 2.4.
Flow 2 laL/min.
34
m 98% c
85%^ 75%^ 65% o
CO
60% o
CD
-| r-
0 1 2 3 4 5 6 7 8 9 Tinne (min)
Figure 2.6 Illustrative gradient reproducibility, 5 |4,m C-18 silica column, flow 5 \x\Jm'm. Average RSD in peak retention time is 0.545%. Amounts injected are as follows: peaks 1-3: -100 pg, 4: 120 pg, 5, 7-10: 85 pg, 6: 150 pg. Peak identities from left to right: (1) phenol, (2) benzaldehyde, (3) benzonitrile,
(4) nitrobenzene, (5) benzene, (6) bromobenzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, and (10) t-butylbenzene.
35
0.005 AU
1. Benzaldehyde 2. Nitrobenzene 3. Benzene 4. Toluene 5. Ethylbenzene
I I , — — r — T — y — — I — I [ I I —I r I r 1 r
2.5 3.0 0.0 0.5 1.0 1.5 2.0 Tinne, nnin
Figure 2.7. Chromatogram on a 12 cm long ZirChrom-PBD column, flow rate 6.3 pL/min, 6500 psi. All samples were in 75% acetonitrile, 25% water. Before injection, the eluent is 37% ACN, at injection it is switched to 55% ACN. The time for
the step change in gradient to reach the head of the column is - 30 seconds. -100 pg of each analyte is injected.
36
CHAPTER III
HOT ELUENT CAPILLARY LIQUID CHROMATOGRAPHY
Introduction
In liquid chromatographic separations that demand either high speed or
high efficiency, mobile phase viscosities ultimately govern what can be attained.
Cui and Olesik added CO2 to a methanol/water eluent to dramatically lower the
viscosity; however, a more complex system was required.^ The other obvious
means to lower eluent viscosity is to increase temperature. Due to the excellent
thermal conductivity of silica, the small dimensions and the resulting small
thermal mass, high temperature liquid chromatography (HTLC) will be most
suitable in the capillary scale.^ Takeuchi et al. first demonstrated HTLC but this
was in a open tubular format where the thermal stability of the stafionary phase
bonded to the silica column surface is a major problem.^ Dynamic coating of the
capillary can sometimes be used, but it is not a general solution.'* Packed
column capillary HTLC has been explored by several investigators. Sheng et al.
studied capillary HTLC at temperatures approaching 100 °C.^ Trones et al. have
studied the applicability of various detectors to reversed phase separations,
using octadecyl silica up to temperatures of 150 °C. "^ Present silica based
packings are more thermally stable than the older packings which degraded at
temperatures under 100 °C.^ However, at temperatures well above 100 °C,
degradation due to the increased solubility of silica in water can still be a
37
problem. Zirconia based packings have exceptional thermal stability, they are
reported to be stable at least up to 200 °C.^° Although conventional scale HTLC
studies using zirconia based packings have been reported, performance at the
upper temperature limits remains an unknown.^^'^^ At this time no studies, to our
knowledge, are available on titania-based packings; they too would be expected
to be thermally stable.
In the present study, we have investigated the utility of capillary HTLC
using zirconia and titania based stationary phases to separate alkyl benzene
derivatives using an acetonitrile-water eluent at pressures up to 10,000 psi and
operating temperatures up to 200 °C.
Experimental
Instrument
The system layout shown in Figure 3.1 is similar to that described in
Chapter II. The outputs from pump A and pump B were coupled via 0.25 mm i.d.
stainless steel tubing to a four way cross (microvolume cross, MX1XCS6, VaIco
Instruments, Houston, TX). The third port of the union is connected to the injector
via a 300 ^m i.d. 2 cm long stainless steel tube that functions as a 1.4 [iL volume
mixing chamber. The pressure sensor (0-10000 psi, model SP70-A10000,
Senso-Metrics, Simi Valley, CA) is connected to the fourth port of the union using
0.25 mm i. d. stainless steel tubing.
38
A newly designed electrically actuated injection valve equipped with a 20
nL internal sample loop (model C2XL, VaIco Instruments, Houston, TX) was
connected to the mixing chamber and used for sample introduction. This new
injector design has a pressure limit of 12,000 psi. This extraordinary pressure
limit is obtained by passing the pressurized eluent behind the internal loop rotor
before flowing through the sample loop. As the pressure increases more force is
applied to the rotor sealing surface. The preheated eluent passes through the
injector thus heating the injector. To prevent the sample from boiling before
injection, the injector loop was cooled externally by closed-loop pumping of cold
ethanol through 2.5 mm i.d. flexible tubing (Tygon, S-50-HL) wrapped around the
sample loop casing with a variable-speed pump drive (Model 75225, Cole
Parmer, Vernon Hills, IL). The cooling loop incorporated a 100 ml vial
submerged in an ice bath. Due to the limited residence time in the injector, the
preheated eluent cools very little while passing through the cooled injector.
The analytical column was housed in a custom heating enclosure
consisting of a 450 |xm i.d. stainless steel tube into which the column was
inserted. A heating tape surrounded the stainless steel tube. A layer of
aluminum foil, an 1-inch thick layer of glass wool, and another layer of aluminum
foil then complete the heating enclosure. The temperature was monitored by a
platinum RTD in contact with the steel tube and is controlled by a temperature
controller (Micromega CN770, Omega Engineering, Stamford, CT).
39
An absorbance detector (Linear UVIS 200, Spectra-
Physics/Thermoseparation systems), designed for on-column detection with
capillaries, was used for detection. A detection wavelength of 254 or 195 nm
was used in all work. In order to keep the superheated eluent from boiling while
passing through the detector, 10 jxm id . capillaries of variable lengths were used
to maintain a back pressure of 1000 - 2000 psi at the detector exit.
Analytical columns
The analytical columns used were 360 ^m o.d. and 180 ^m i.d. fused
silica capillaries (Polymicro Technologies, Phoenix, AZ) packed in-house.
Results are described herein for two column types: (a) a 25 cm long column
packed with polybutadiene coated ZrOa-based 3 |j,m dia. packing, ZirChrom-PBD
(ZirChrom Separafions, Anoka, MN), (b) a 15 cm long column packed with 3 inm
dia. C18-modifiedTi02.
Frits were made at the column outlet with a procedure based on a method
developed by Kennedy and Jorgenson.^^ The fused silica tubing was taped end
downward onto a pile of 5 )Lim silica particles until a 0.5 mm section was packed.
Since about a 10 cm section is required after the frit for a detection window and
exit plumbing, the silica particles were pushed about 10 cm into the column by
using a 150 (xm o.d. 10 )xm i.d. silica capillary as a plunger. This process was
repeated several times until the frit was - 2.5 mm long. Once the particles were
in place and the bed was of the desired length, the bed was sintered to form a frit
40
by passing the section of the column containing the frit through a small propane
flame 2-3 times. Frit robustness was tested prior to packing by producing a 5000
psi pressure drop across the frit. The sintering process also produces a 1 cm
detection window directly after the frit.
A slurry packing technique was used to pack both types of columns. A 10
w/y% slurry of either the zirconia or titania based packing in 10% aqueous Triton
X-100 was used. The slurry was agitated and a portion was transferred to the
slurry reservoir. The columns were then packed using a Shandon (Shandon
Southern Instruments Inc., Model 628 x51, Sewickley, PA) HPLC packing pump,
using water as the packing liquid. During the packing process, the slurry
reservoir was constantly agitated and the pressure was increased from 0 to 7000
psi in 1 minute. Due to the large pressure drop produced when packing 3 (xm
particles, the entire column was placed in a 0.6 mm stainless steel tube that was
heated with heating tape to 200 °C. The increased temperature greatly
decreased the viscosity of the packing solvent thus allowing much (3-4 times)
longer columns to be packed than what was possible when packing at room
temperature. After the desired column length was achieved, the packed column,
in the case of the zirconia based particles, was then placed in an ultrasonic bath
and sonicated for 3 hours with the column under 7000 psi. The titania based
packing was not placed into the ultrasonic bath due to potential fragmentation
risks. The pressure was then maintained at 7000 psi for 24 h.
41
We prefer not to use frits at the head of the column. This configuration
allows the front of the column to be trimmed if compression of packing material
over a period of time leads to a void at the head of the column.
Materials and reagents
Acetonitrile (HPLC grade) was obtained from EM Science (Gibbstown,
NJ). Water was distilled then purified in a Barnstead Nanopure system. All
fused silica capillaries were from Polymicro Technologies, inc. (Phoenix, AZ).
Benzaldehyde, toluene, ethylbenzene (all from J. T. Baker), benzene (EM
Science), n-propylbenzene (Ethyl Corporation), n-butylbenzene, n-amylbenzene,
1-phenylhexane and 1-phenylheptane (all from Sigma-Aldrich) were used as
received.
Results and Discussion
Dependence of viscosity on temperature
There is very limited data in the literature on the viscosity of mixed liquid
systems, especially above their normal boiling temperature. Since
acetonitrile:water is probably the most commonly used LC eluent, we measured
the viscosity of an eluent that was prepared at room temperature to be 50%
acetonitrile and 50% water (v/v). The temperature dependence of the viscosity of
the eluent was measured by monitoring the pressure drop through a narrow bore
capillary. The eluent was pumped at 0.5 |aL/min through a 32 cm long 9 jj.m i.d.
42
fused silica capillary placed in the column heater, followed by a second capillary
maintained at room temperature that prevented boiling. Low volume flow-
through pressure gauges were placed both at the head and tail of the
measurement capillary. The differential pressure between the two pressure
transducers was used to compute the viscosity according to the Hagen-Poiseuille
equation.^'*'^^ The results are shown in Figure 3.2. The viscosity r| decreases
exponentially with absolute temperature according to:^^
T1=A exp (E / RT) (3.1)
where A is a constant and E is the effective activation energy for molecular
displacement. For our data. In r\ is plotted against 1/T, a straight line is obtained
with a linear r value of 0.9999. The best fit values for A and E are, respectively,
0.2097 cP and 16.59 kJ/mol. This data, shown in Figure 3.2, shows an order of
magnitude decrease of viscosity between ambient temperature and 200 °C; this
would allow a proportionately increased flow rate or column length.
Chromatographic performance
The performance of the zirconia based packing as well as the durability of
the packing at high temperature was verified. Destructive studies were
performed to determine the onset of failure. While long-term testing showed that
both packing and capillary are stable at 200 °C and can operate for long periods
if undisturbed, the capillary itself becomes brittle after several days at 200 °C. It
is possible that this embrittlement is caused by the degradation of the polyimide
43
coating and aluminized capillaries, as used in high temperature gas
chromatography will provide greater handling durability. However, by about 300
°C, dissolution of silica from the interior wall of the column leads to catastrophic
failure of the capillary in approximately 15 hours, typically exhibiting picturesque
helical cracking patterns. At 400 °C, the Zirchrom-PBD packing lifetime is
shortened to < 1 h, likely due to the decomposition of the bonded PBD.
The effect of temperature on column performance was studied for the
separation of a series of alkybenzenes on the Zirconia-PBD phase with the
above 50% acetonitrile-50% water eluent. The temperature of the system was
varied from 25-200 °C in 25 °C increments. The performance of the column at
different temperatures was evaluated by comparing the retention time, theoretical
plate count, and peak asymmetries for each peak. By any of these measures,
the separation improved continuously with increasing temperature from 25-175
°C with an apparent small drop in performance past this temperature.
The reduction in the retention time is shown in Figure 3.3 and follows the
trend reported by others. In our judgment, the reduced retention is likely best
interpreted in terms of the solvophobic theory of Sinanoglu and Pullman as
adapted later to reverse phase liquid chromatography by Horvath et al. ' ' ^
An increase in temperature also increases the diffusion coefficients of the
mobile phase and the analytes. According to the Stokes-Einstein relafionship,
the diffusion coefficient is proportional directly to the absolute temperature and
the reciprocal of the viscosity:^^
44
Dm=D, ,25(T/298)(Tl25/Tl) (3.2)
Where Dm,25 and T125 are the molecular diffusivity and the viscosity of the eluent at
25 °C. The value of 6.1 x 10'^ cm^/s was used for Dm,25 of the 50% acetonitrile
50% water eluent.^° Figure 3.4 shows the estimated change of the diffusion
coefficient as a function of temperature based on the measured viscosity data.
Enhanced mass transfer due to increased diffusivity will be expected to
significantly improve the quality of the separafions. Figure 3.5 shows the
separation of 4 benzene derivatives as a function of temperature at the same
flow rate; the separafion improves dramatically as the temperature is increased
from 25-200 °C. Figure 3.6 shows Knox plots for ethylbenzene at four different
temperatures.^^ It is interesting to note that the best efficiency at the opfimum
velocity at each temperature in the 25-200 °C range actually deteriorates with
increasing temperature. Molander et al. made similar observations with a 5 ^m
Hypersil-ODS™ stationary phase at temperatures up to 175 °C, noting that the
efficiency at the optimal linear velocity actually decreases above 100 °C. ^ This
was hypothesized to be due to extra-column dispersion. In the present study,
detection occurs on-column immediately after the column frit; extra-column band
broadening is not likely to be a major factor in the observed column efficiency.
An alternative possibility is that the contribution of sites accessible through
very small pores becomes more significant as the eluent viscosity is decreased
and/or due to actual thermal expansion of the packing. An alternative way of
stating this is that in regard to the accessibility of its adsorption sites, the packing
45
becomes more heterogeneous at higher operating temperatures. Consider the
A, B, and C terms in the Knox equafion for ethylbenzene as a funcfion of
temperature (Figure 3.7). The B and C terms behave predictably, as may be
expected from the positive temperature dependence of diffusivity. The A-term,
however, shows a -25% increase between 100 and 150 °C; this is consistent
with increased heterogeneity of the accessible sites.
Figure 3.7 also highlights the fact that it is the decrease in the C term that
becomes the determinant of the plate height at higher linear velocities while at
the lower velocities, the plate height is mostly affected by the increase in the A
and B terms. Of particular importance to high speed separations, the loss of
efficiency with increasing velocity is less in the 100-200 °C range than at ambient
temperature.
Thermodynamic data
The standard enthalpy and entropy of the solute transfer were calculated
from the slope and intercept of the Van't Hoff plots respectively.^^ The calculated
AH° and (AS°) values for benzene, toluene, ethylbenzene, and propylbenzene
were (in J/mol and and J/ °K, respectively), -4046 (-12.93),-4081 (-11.79), -4183
(-10.84), and -4383 (-9.954). The absolute values of the entropy terms are
dependent on the precise value of the Vs/Vm ratio; however, the relative patterns
of the enthalpy and entropy terms are worthy of closer inspection. The
enthalpies of adsorption on the stationary phase increases exponentially with
46
increasing alkyl chain length. The observed data can be fitted to a relation of the
type:
Enthalpy (J/mol) = 17.5 exp (alkyl chain carbon number) + 4037 (3.3)
The predicted enthalpies then correlate with the experimental enthalpies with a
linear ^ value of 0.9998.
Regarding the entropy terms, it is not surprising that the system is more
organized when the solute is in the stationary phase because the association of
the solute with the solvent is likely much smaller. It is interesting, however, that
the entropy change decreases from benzene to the higher homologs.
High temperature gradient chromatography
Ultimately, the important advantage of a system like this is the ability to
perform rapid separafions. The particular advantage of the capillary scale is
operation at very high linear velocities without consuming large amounts of
eluent. Figure 3.8a shows an isocratic separation of various benzene
derivatives. Even at 200 °C and a relatively high linear velocity (a flow rate of 3.5
I L/min corresponds to 2.3 mUmin through a 4.6 mm i.d. column), the isocrafic
separation took 18 min and the efficiency was unsatisfactory. In contrast. Figure
3.8b shows the same mixture separated under same temperature and fiow,
except under gradient conditions. The present system allows step changes in
solvent composition with time steps of > 6 s, allowing for sharp gradients. The
47
entire separation took under 4 min, generating the equivalent of 7500 plates/min
for the last elufing peak 1-phenylheptane.
The average retention times (n = 4) and the % RSD values of the retention
times for the individual analytes in the 9-component mixture on the Zirchrom-
PBD column were as follows: benzaldehyde (1.69 min, 0.83%), benzene (1.92
min, 0.73%), toluene (2.17 min, 0.64%), ethylbenzene (2.50 min, 0.69%), n-
propylbenzene (2.95 min, 0.82%), n-butylbenzene (3.28 min, 0.88%), n-
amylbenzene (3.53 min, 0.88%), 1-phenylhexane (3.75 min, 0.80%) and 1-
phenylheptane (3.96 min, 0.92%). The average RSD in peak retenfion times is
0.79%, corresponding to a variation of 1.80 s for an elution window of -4 min.
High speed gradient chromatography
The column packed with 3 im C-18 modified Ti02 exhibited high
permeability and was particularly suitable for studying the effect of very high flow
rates. Figure 3.9 shows a gradient separafion of various alkylbenzenes at 160
°C with a flow rate of 25 ^L/min generating a back pressure - 9500 psi. The
separation took under 2 min. Note that this linear velocity is equivalent to 16
ml/min for a conventional 4.6 mm packed column, this would be impossible to
attain at ambient temperature or in conventional scale.
We have shown that attractive high-speed separations are possible in
zirconia and titania based stationary phases at temperatures up to 200 °C; this
temperature is also likely the maximum practical temperature for performing such
48
separations. Capillary scale gradient HTLC for thermally stable analytes with
such packing is a viable and very attractive proposition when speed is of
paramount importance.
49
Literature Cited
1. Y. Cui, S. V. Olesik, J. Chromatogr. 691 (1995) 151
2. H. Poppe, J.C. Kraak, H.H.M. Vandenberg, Chromatographia 14 (1981) 515.
3. T. Takeuchi, M. Kumaki, D. Ishii, J. Chromatogr 235 (1982) 309.
4. D. Pyo, P. K. Dasgupta, L. S. Yengoyan. Anal. Sci. 13 (SuppI) (1997) 185.
5. G. Sheng, Y. Shen, M. L. Lee, Journal of Microcolumn Separations 2 (1997) 63.
6. R. Trones, A. Iveland, T. Greibrokk, J. Microcol. Sep. 7 (1995) 505.
7. R. Trones, T. Anderson, I. Hunnes, T. Greibrokk; J. Chromatogr. A., 814 (1999)55.
8. R. Trones, A. Tangen, W. Lund, T. Greibrokk; J. Chromatogr. A., 835 (1999) 105.
9. K. D. Nugent, W. G. Burton, T. K. Slattery, B. F. Johnson, L. R. Snyder, J. Chromatogr. 443 (1988) 381.
10. J. Li, P. W. Carr, Anal. Chem. 69 (1997) 837.
11.. N. M. Djordjevic, P. W. J. Fowler, F. Houdiere, J. Microcol. Sep. 11(6) (1999)
403.
12. P. Molander, E. Ommundsen, T. Greibrokk, J. Microcol. Sep. 11(8) (1999) 612.
13. R. T. Kennedy, J. W. Jorgenson, Anal. Chem. 10 (1989) 1128.
14. F. M. White, Viscous Fluid Flow, McGraw-Hill, New York, (1978).
15. A. E. Scheidegger, The Physics of Flow Through Porous Media, University
of Toronto Press, Toronto, (1960).
16. H. Colin, J.C. Diez-Masa, G. Guiochon, J. Chromatogr. 167 (1978) 65.
17. O. Sinanoglu, B. Pullman, Molecular Associations in Biology, Academic
Press, New York, 1968, pp 427-445.
50
18. Cs. Horvath, W. Melander, I. Molnar, J. Chromatogr. 125 (1976) 129.
19. F. Daniels, R. A. Alberty, Physical Chemistry, Wiley, New York, 1955. pp503-
506.
20. L. R. Snyder, J. Chromatogr. Sci. 15 (1977) 441.
21. G. H. Kennedy, J. H. Knox, J Chromatogr. 10 (1972) 549.
22. P. Molander, R. Trones, K. Haugland, T. Greibrokk, Analyst, 124 (1999) 1137.
23. S. O. Akapo, C. F. Simpson, Chromatographia, 44 (1997) 135.
51
Pressure Gauge
Pump A PumpB
Column Heater UV Detector
1546 PS 5 Back Pressure Capillary
1 Pressure Gauge
Computer
Figure 3.1 Experimental set up of chromatographic system.
52
300 350 400 450 500
Temperature, K
Figure 3.2 Viscosity of 50:50 v/v ACN:H20 as a function of temperature temperature.
The points represent experimental data; the best fit line is shown. The error bars on the experimental points are smaller than the dimensions of the
symbols plotted.
53
16
.E 14 E - 12
i - 10
q
CD
CD
8
6
4
2 0
•— Benzene V - Toluene «— Ethylbenzene o— Propylbenzene
50 100 150 200
Temperature, C
Figure 3.3 Retention time of benzene and homologs as a function of temperature. The error bars on the experimental points are smaller than the dimensions of the
symbols plotted.
54
CO CM
E o o
0 50 100 150
Temperature, C 200
Figure 3.4 Computed diffusion coefficient of the 50:50 ACN:H20 eluent as a function of
temperature. The best-fit data from Figure 2 was used to calculate D^.
55
200 C
-A"^
150 C
3 < o d
100 C
25 C
JLJ\ ^ — 1 J—
0 2 4 6 8 10 12 14
Time, nnin
Figure 3.5 Separations of benzene, toluene, ethylbenzene, and propylbenzene at
four different temperatures. A flow rate of 2 ^L/min was used throughout.
56
0 1 2 3 4 5 6
Reduced Linear Velocity
Figure 3.6 Knox plot of ethylbenzene at four different temperatures.
57
0 50 100 150 200
Temperature, C
Figure 3.7 Knox A, B, and C terms for ethylbenzene as a funcfion of temperature.
58
- « ^
2 0
10 0 z 8 0 O 6 0 'i 4 0 ^
Figure 3.8 Isocratic and gradient chromatograms of same mixture
a) Isocratic separation of (from left to right) benzaldehyde, benzene, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, n-amylbenzene, 1-
phenylhexane and 1-phenylheptane. A flow rate of 3.5 ^iL/min and a column temperature of 200 °C were used, b) Gradient separafion of the same
mixture, same fiow rate and temperature.
59
0.0 0.5 1.0 1.5 2.0
Time, min
2.5 3.0
Figure 3.9 High-speed gradient separation on C-18 modified TiOj column of
benzene, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, n-amylbenzene, 1-phenylhexane and 1-phenylheptane. Flow rate 25
laL/min; column temperature 160 °C.
60
CHAPTER IV
SUPER-HOT WATER ELUENT CAPILLARY
LIQUID CHROMATOGRAPHY
Introduction
The environmental impact of organic solvents and the consequent
economics of their disposal has provided much impetus to limit their consumpfion
or to eliminate their use altogether. In 1995, Hawthorne et al. demonstrated that
supercritical water at 400 °C and 350 bar can be used to extract non-polar
analytes such as polycyclic aromafic hydrocarbons (PAHs) and polychlorinated
biphenyls (PCBs) from contaminated soil samples.^'^ Under these extreme
conditions, the dielectric constant of water is greatly reduced.^ Supercritical
water is a very aggressive solvent that readily oxidizes or decomposes many
substances; supercritical water treatment has been studied as a method to
decompose toxic waste.'* Throughout the temperature range above its normal
boiling point up to the supercrifical temperature, liquid water exhibits a much
lower dielectric constant than room temperature water and is likely less
aggressive than supercritical water. The dielectric constant, viscosity and
hydrogen bonding of water changes continuously from room temperature up to
supercrifical conditions; these are the properties that are likely to influence the
behavior of water as a liquid chromatographic eluent.
61
Foster and Synovec were the first to exploit the use of pure water as an
LC eluent by using highly polar stationary phases and by decreasing the
stationary/mobile phase ratio. Separations could be accomplished at room
temperature but efficiencies for nonpolar analytes were modest.^ Miller and
Hawthorne first used a traditional reversed phase (in a 2 mm column format) and
utilized the temperature effect on the solvent properties of water, with thermal
gradients to 175 °C to separate alcohols, polyhydroxybenzenes, and amino
acids.^ About the same fime. Smith and Burgess separated phenols,
barbiturates, and parabens on tradifional PS-DVB and ODS-bonded silica
columns with water as hot as 200 °C.^ Yang et al. were the first to report the
separation of non-polar aromatic hydrocarbons on traditional reversed phases
using water up to 200 °C.^ It has been shown that buffering components can
also be put in the hot water eluent.^
The absence of carbon containing compounds in a pure water eluent
allows the use of detection methods that are difficult or impossible to use with
convenfional eluents. The use of a flame ionization detector (FID) can be
particularly attractive.^'^° In addition to the traditional UV/VIS detector. Smith et
aL showed the facile applicability of NMR and MS detectors to a superhot D2O
eluent LC system with temperatures to 190 °C. ^ They also authored a thorough
review documenting the manifold utility of a water eluent as hot as 240 °C.^^ One
particularly noteworthy aspect is that of many analytes investigated, very few
actually decomposed under the condifions of the separation.
62
A few quesfions remain: For thermally stable analytes, what is the
practical upper limit of the separation temperature given the most thermally
stable stationary phase currently available? How fast can such separations be
carried out if one takes advantage of the decreased viscosity? We attempt to
answer these questions using an inexpensive capillary scale system. The low
flow rates and the small thermal mass in the capillary scale allow rapid
temperature ramps and greatly facilitate radial heat transfer, thus minimizing
radial temperature gradients.^^ The complexity of interfacing to detectors that are
intrinsically compatible with a low flow rate (e.g., mass spectrometers) is also
reduced. Capillary scale water eluent liquid chromatography has not been
previously reported. We report here the capillary scale separation of hydrophilic
and hydrophobic benzene derivatives at temperatures up to 400 °C and
pressures up to 11,000 psi with water as an eluent. When no backpressure is
applied at the column exit with an FID as a detector, the effluent is obviously in
the gas phase. At what point does the liquid turn into gas? Early studies of gas
chromatography with FID detectors that use steam as the mobile phase are well
known. ' Do the present separafions with FID detecfion represent gas, or liquid
phase separations, or both?
63
Experimental
The pumping system shown in Figure 4.1 is similar to the home-built
gradient pumping system described in chapter 1 except that only a single pump
is required in the present work. The output from the pumping system is
connected with 0.25 mm i.d. stainless steel tubing to a high pressure inline check
valve CV (cartridge CV«3000; Upchurch scientific. Oak Harbor, WA). The check
valve housing was machined in-house out of poly(etheretherketone) (PEEK) to
handle pressures over 10,000 psi. The high-pressure side of the check valve is
then connected to a pressure sensor and gauge PG (0-10000 psi, model SP70-
A10000, Senso-Metrics, Simi Valley, CA) to confinuously monitor system
pressure.
To minimize the dissolufion of silica from the capillary wall by the superhot
water, a silica saturator (4.6 x 40 mm stainless steel guard column packed with
200 mesh silica gel) was placed ahead of the injector. A separate siliconized
band heater was used to heat the presaturator. This column was heated to a
temperature approximately 50 °C lower than the column temperature:
overheating the presaturator can result in dissolved silica subsequently
depositing in the injector which is cooler. An electrically actuated injecfion valve
equipped with a 20 nL internal sample loop (model C2XL, VaIco Instruments,
Houston, TX, rated at 15000 psi) was connected to the guard column and used
for sample introducfion. To prevent the sample from boiling before injecfion, the
injector loop was cooled externally by closed-loop pumping of cold ethanol from a
64
100 mL reservoir kept in an ice bath through 2.5 mm i.d. Tygon tubing, wrapped
around the sample loop casing with a variable-speed pump drive (Model 75225,
Cole Parmer).
The analytical column was inserted in a 450 \xm i.d. stainless steel tube
around which heating tape was wrapped. A layer of aluminum foil, a 1-inch thick
layer of glass wool, and another layer of aluminum foil, each tightly wrapped,
completed the column heafing enclosure CH. The temperature was monitored
by a platinum RTD in contact with the steel tube and was controlled by a PID
temperature controller (Micromega CN770, Omega Engineering, Stamford, CT).
An absorbance detector (Linear UVIS 200, Spectra-Physics /
Thermoseparation systems), designed for on-column detecfion with capillaries,
was used for detection. A detection wavelength of 195 nm was used in all work.
In order to keep the superhot liquid eluent from boiling while passing through the
detector, 10 i m i.d. capillaries of variable lengths were attached to the exit of the
column to maintain a back pressure of 1000 ~ 3000 psi.
For thermal gradient separations using an FID detector, the column was
placed in a gas chromatography oven (Model 4300SX, Varian Inc., controlled by
Varian Star 4.5 software) with the column outlet being directly connected to the
FID inlet (held at 275 °C). Others have previously looked into opfimizing the
hydrogen and air flows to the FID for maximizing the signal to noise rafio. We
made only crude adjustments to obtain reasonable signals, no detailed sensifivity
optimization was carried out since improving detecfion sensitivity was not an
65
objective of this work. No backpressure regulator was used after the column and
no modifications were made to the FID.
Water used in the experiments was disfilled and then further purified in a
Barnstead Nanopure system. Fused silica capillaries (Polymicro Technologies,
Phoenix, AZ), hydrogen (ultrapure grade, Airgas, Lubbock, TX), benzene
derivatives (reagent grade, Aldrich) were obtained as indicated.
Fused silica capillary columns (360 nm o.d. and 180 |am i.d., Polymicro
Technologies, Phoenix, AZ) were packed in-house with frits made after Kennedy
and Jorgenson as previously described.^^ For use with the UV detector, the frit
was placed -10 cm from the exit end of the capillary. With the FID, the frit was
placed at the very end. The bed length was 13 cm for all reported results. Two
different 3 |Lim diameter zirconia based packing materials were used: (a) Zr02
modified with polybutadiene, and (b) Zr02 modified with elemental carbon
(ZirChrom-PBD and ZirChrom-Carb, ZirChrom Separafions, Anoka, MN). Only
for prolonged high temperature studies with the FID, stainless steel capillaries
(0.014 inch o.d. 0.007 in. i.d. tubing, x 20 cm. Small Parts Inc., Miami Lakes, FL)
were packed with 3 i m dia. ZirChrom-Carb. A frit was made by packing glass
wool (~0.25mm thick bed) in a 0.016 inch i.d., 0.020 inch o.d. stainless steel tube
which served as a butt-joint connector between the column and an exit tube with
the same dimensions as the column.
To pack columns, a 40% w/v slurry of the zirconia based particles in a
10% aqueous Triton X-100 solution was agitated and a portion was transferred to
66
the slurry reservoir. A Shandon HPLC packing pump (Shandon Southern
Instruments Inc., Model 628 x51, Sewickley, PA) was used with water as the
packing liquid. During the packing process, the slurry reservoir was constantly
agitated and the pressure was increased from 0 to 7000 psi in 30 s, with
complete column packing taking less than 3 min. After the desired column length
was achieved, the packed column was then placed in an ultrasonic bath and
sonicated for 3 h with the column under 7000 psi. The pressure was then
maintained at 7000 psi for 24 h. We did not use frits at the head of the column.
Results and Discussion
Silica Presaturator
The solubility of silica is greatly increased in superhot water. Water was
continuously pumped through the column at a fiow rate of 8.5 i^L/min and the
temperature was alternately maintained at 250 °C and 25 °C for 12 h periods.
Without the silica presaturator, the column burst due to dissolufion of the capillary
wall In -3 days. With the silica presaturator, the column maintained its structural
integrity and no voids were seen in 30 days of continuous operation. At
temperatures closer to supercritical conditions, e.g., at 370 °C, a fused silica
column fails catastrophically in 1 h, generally developing picturesque helical
cracking patterns that separate in sawtooth patterned hemicylindrical pieces
(Figure 4.2). The presence of the silica presaturator increased the operafional
67
period under these condifions to 10 h; of course, this is still not long enough to be
practical and the use of some other column material is warranted.
Thermally stable stationary phases
The selection of a stationary phase compatible with superhot water is
obviously an issue. If dissolufion of silica from the very limited surface area of a
fused silica column is of concern at temperatures above 250°C, the dissolution of
silica from the core of any silica based stationary phase will obviously be a much
greater problem, especially since it is impossible to provide quantitative surface
coverage of whatever type of funcfionality is put on. Polymeric phases,
especially poly(styrenediyinylbenzene) (PSDVB), do not have solubility problems
but these phases generally exhibit too great a retenfion for nonpolar aromatic
analytes for use with a water eluent. Further, at temperatures above 250°C, the
confinually increasing absorbance of the column effluent suggests that the
packing is beginning to depolymerize. Any chemical modification that is made to
the surface functional group must also be compatible with both the temperature
and an aqueous environment. As a core material, zirconia is excellent due to its
exceptional thermal stability. Its aqueous solubility, even at elevated
temperatures, is negligible. Both elemental carbon and polybutadiene modified
zirconia based stationary phases are commercially available and both functional
groups have good thermal stability and are compafible with aqueous eluents.
The elemental carbon phase is fully stable at the maximum temperature used in
68
this work; it is likely that other commercially available carbon based phases will
also be stable.
Properties of Superhot Water
An oft-cited paper by Hawthorne et al. ^ contains an informative figure on
the variation of dielectric constant of water as a function of temperature and how
these values compare with common eluent compositions used in reversed phase
LC. Similar informafion has been subsequently published by others. ^ In Figure
4.3, we show both the viscosity and the dielectric constant of water over a more
extended rage of temperature. Both viscosity and the dielectric constant
decrease with increasing temperature, approximately in an exponential
fashion.^''•^^ For liquid water, the viscosity is only weakly pressure dependent;
we have chosen the viscosity data at a pressure of 7200 psi, typical of our
column operations in this study. Over the entire range of temperature of interest,
the relationships below satisfactorily express the dielectric constant £-and the
viscosity rj of water:
f = 85.31-0.2901 f+2.572x10"^/^, r2=0.9965 (4.1)
; ,ater(@ 7200 psi) = (f°^''^^'Y 18906, r = 0.9947 (4.2)
where t is the temperature in degrees Celsius. The majority of the change in the
viscosity occurs between room temperature and 200 °C. The viscosity of water
changes by 400% within this range,^^ permitting a four fimes longer column or a
4-fold greater flow rate at the higher temperature. The Stokes-Einstein
69
relationship^" invokes that the diffusion coefficient varies in proportion to the ratio
of the absolute temperature to the viscosity. Further gains in chromatographic
performance may therefore be attainable at high flow rates at temperatures
beyond 200 °C, albeit no further significant gains are likely to be realized in terms
of decreased column pressure beyond this temperature.
The dielectric constant of water is pressure-dependent; this is more
pronounced at higher temperatures. Figure 4.4 shows the pressure dependence
of the dielectric constant at 200, 300, 400 and 550 °C. ®' ^ Even when an exit
backpressure is applied to keep the effluent in the liquid state with UV absorption
detectors, the pressure at the column exit is far smaller than the column head
pressure. As such, there is a confinuous and significant pressure gradient
across the column. If the column is at the same temperature along its length,
then the dielectric constant/polarity also varies confinuously along the length of
the column. A solvent polarity gradient along the column has the same effect as
decreasing the stationary phase capacity from the start to the end. The solvent
is more polar at the head of the column than towards the end; the difference can
be especially pronounced at low pressures, just above the critical pressure.
However, for high-speed separations driven by an increased flow rate, most of
the column will not experience such a low pressure.
Most previous studies using superhot liquid water have been limited to
separating polar organic analytes. For superhot liquid water to compete with
organic modifiers in RPLC, the system has to be able to elute polar as well as
70
non-polar analytes. A series of benzene derivatives were separated using both
the elemental carbon and polybutadiene modified 3 ^m zirconia phases. The
injected sample contained a mixture of 7 analytes, 0.1% (v/v) phenol, and 0.5%
(v/v) benzene, nitrobenzene, toluene, ethylbenzene, n-propylbenzene, and
n-butylbenzene. A complete mixture was injected in every run; however, all the
analytes did not elute from the column at lower temperatures. Figure 4.5 shows
the nature of these separations. At 370 °C, n-butylbenzene elutes in under 7 min
(not shown in the figure). The temperature stability of ZirChrom-PBD is lower
and 300 °C is the maximum practical operating temperature. At all temperatures,
the PBD-phase was observed to be more retentive for the alkylbenzene analytes
than the carbon phase. This behavior is opposite to that observed for ambient
temperature hydroorganic eluent separations on these phases.^ ' ^
The retention times over the studied range of temperatures vary over such
a large range that the chromatograms are plotted with a logarithmic time axis for
best viewing. Because the sample solvent (acetonitrile) elutes essentially in the
void volume, it does not fully attain the column temperature by the time it exits
the column. The response from the solvent is primarily due to refractive index
effects. As such, the solvent peak increases in magnitude as the operating
temperature increases. It is interesfing to note that nitrobenzene and toluene
changes retenfion orders with temperature on the carbon phase but not on the
PBD-phase. In a tradifional hydroorganic eluent system, similar behavior has
been observed by others as the polarity of the eluent is increased.^^ The
71
opfimum temperature for the carbon phase for this particular separafion appears
to be ~170 °C while the separation confinues to improve with increasing
temperature on the PBD phase.
Figure 4.6 shows high speed separations on (a) the ZirChrom-Carb phase
at 300 °C and a flow rate of 20 ^L/min (equivalent to 13 mL/min on a 4.6 mm
column) and (b) the ZirChrom-PBD column at 240 °C at a flow rate of 27 ^L/min
(equivalent to 17.5 mL/min on a 4.6 mm column). To our knowledge, these
separafions are faster than any other isothermal isocrafic liquid chromatographic
separation performed on comparable compounds, not just with water as eluent.
Faster separafions are indeed possible with hydroorganic eluent gradients; this
also helps maintain chromatographic efficiency for late eluting peaks. The
equivalent to this in the present system is a thermal gradient, and is discussed in
a later section.
Does Separafion Mechanism Change with Temperature? Thermodynamics of Solute Transfer.
A sensitive probe for any changes in the separation mechanism as a
function of temperature is a Van't Hoff plot (log k' vs. 1/T). A recfilinear plot
indicates that the same separation mechanism prevails across the entire
temperature range of interest. Recently, Yang et al. reported that Van't Hoff plots
for the separation of substituted benzene derivatives on a Nucleosil C-18 phase
using a superhot water eluent were non-linear, thus suggesfing a change in
72
retenfion mechanism.^^ Figure 4.7 illustrates that recfilinear Van't Hoff plots are
obtained for all solutes on both the ZirChrom phases over an even greater range
of temperature than studied by Yang et al. The difference in the obsen/ations is
therefore phase related. The behavior observed by Yang et al. could indeed be
due to a shift from an adsorpfion to a partition-like process if the C-18 chains
unfold as the dielectric constant of the water decreases with increasing
temperature. Unlike the C-18 packing used by Yang et al., the polybutadiene
modifier always lies on the surface due to extensive cross linking and thus can
not collapse in an aqueous environment or open up in a non-polar environment.
The nature of the carbon phase also likely remains unaltered throughout the
temperature range studied.
The standard enthalpy and entropy of the solute transfer can be calculated
from the slope and the intercept of the plots in Figure 4.7, respectively.^^ The
highly parallel nature of the alkyl benzene behavior in the plots is readily
observed. Detailed data are presented in Table 4.1. The entropy terms should
be considered merely relafive because we can only estimate the gross (as
opposed to the active) volume of the stationary phase. Nevertheless, the data
readily show that while the retention of the alkylbenzenes, especially with
increasing carbon number, is promoted due to entropic reasons, the retenfion of
the more polar phenol and especially nitrobenzene is largely governed by
enthalpy.
73
Thermal Gradients
One clear advantage of the capillary scale in high temperature liquid
chromatography is its ability to facilitate rapid and uniform heating throughout the
column. Figure 4.8 shows a thermal gradient separafion with flame ionizafion
detection. The average retention times (n = 4) and the % RSD values for the
individual analytes in a 6-component mixture were as follows: phenol (1.20 min,
0.80%), benzene (1.35 min, 0.96%), nitrobenzene (1.65 min, 0.86%), toluene
(2.02 min, 1.10%), ethylbenzene (2.44 min, 1.55%), and propylbenzene (3.16
min, 1.56%). The average RSD of retenfion fimes under gradient conditions was
thus 1.20%. It should be noted that this performance is obtained with a
homemade pumping system that costs under $1500 to fabricate.
When Does Liquid Water Turn into Gas?
With a detection technique like flame ionizafion or mass spectrometry, that
actually handles the sample in the gas phase, the issue of using a backpressure
capillary after the detector is moot. A small diameter exit restrictor between the
column and the detector can be used to keep the mobile phase in the liquid state
in the column but coupling such a restrictor between the column and the detector
generates unavoidable and undesirable post column band broadening. If the
column terminus is directly coupled to the detector, some of the end portion of
the column is effectively the restrictor and the mobile phase will go from liquid to
the gas phase at some point in the column. In terms of experimental
74
performance and simplicity, we found that the best results are indeed obtained
with the column directly coupled to the detector.
The interesfing quesfion that then arises is at what point does the eluent
boil in the column and how does it affect the resulfing separation? Is it merely all
a continuum between liquid chromatography with superheated water and gas
chromatography with hot steam?^'* In chemical engineering pracfice, the
backpressure created by fluid flow through a packed bed is traditionally
computed by the Ergun equafion:^"*
AP/AL = (1-(D)p(/[(150Ti(1-<D))/(pwdp) + 1.75]/{0%) (4.3)
Where AP is the pressure drop across a portion of a packed column of
length AL, p, u, and r| are respectively the density, nominal flow velocity and the
viscosity of the fluid, dp is the particle diameter and O is the void fraction in the
column. The fluid velocity u is most conveniently calculated from the known
mass flow rate G pumped by the pump, the density of the fluid at the location of
the column under considerafion (p) and the inner radius of the column r.
u = G/{nf^p) (4.4)
A program written to calculate the Ergun equation values was obtained on
the web. ^ We used the Ergun equation in an iterative fashion as follows.
Consider that a column is being operated at fC. The vapor pressure of water at
this temperature is known to be Pb atm. At the point in the column the pressure
drops below this value, liquid water is converted into the vapor phase. A
constant given temperature is assumed in any calculation. Starting then at fC
75
and Pb atm we use an iterative BASIC routine that utilizes a software
addressable version of the steam tables^^ to calculate p and r|. The Ergun
equation is then used to calculate the pressure drop AP over a small length AL (1
[am was used in our calculations). The calculafion is then repeated for a new
pressure P-AP This procedure is repeated until the new pressure becomes
equal to the ambient pressure. If the total number of iterations to reach this point
is n, the total length of the column from the end in which the liquid water has
turned into vapor is then nAL. At a flow rate of 8.6 mg/min of water (same
condifions as in Figure 4.8), we calculate for our columns (void fracfion in these
columns is typically 0.56) that the transifion to vapor phase begins respectively 0,
0.044, 0.35, 2.38, 8.15, 28.3 and 48.0 cm at temperatures of 100, 150, 200, 250,
300, 350, and 370 °C. The relationship of the length with temperature is
approximately exponential (r = 0.9858). If the packing contains small pores, the
resulting capillary effect will reduce vapor pressure. The length of the column
filled with gaseous eluent as computed above will be a small overestimation.
Also, these computations are meaningful only if a given column is sufficiently
long and the head pressure on the column is greater than P/,. Othenwise, if a 20
cm column at 370 C is used with a H2O mass flow rate of 8.6 mg/min, the enfire
column will contain water in the gaseous state. In actual experiments with a 20
cm stainless steel capillary column and connected directly to the FID, separafion
of injected analytes was observed up to temperatures of 300 °C; however, as the
temperature was raised to 350 °C, only a single undifferentiated response was
76
observed. These results, therefore, suggest that in this particular packed
capillary mode, without a backpressure on the column, operafions should be
limited to temperatures at which the majority of the column contains the eluent in
the liquid phase. It is interesfing to note that these calculafions also suggest that
as long as mixtures of gases and liquids are not formed within the detector cell
causing consequent problems, even with an optical detector it is not essential to
have a backpressure capillary, absorbance can be measured just as well in the
gas phase. However, due to a greater refractive index mismatch; light
throughput through the cell will be poorer.
In summary, we have described an affordable, environmentally friendly
capillary scale reverse phase liquid chromatography system using superheated
water eluent that can operate in isothermal and temperature gradient modes and
is capable of separafing both polar and non-polar compounds. There is, of
course, no barrier to using a solvent gradient at the same fime if a binary
pumping system is used.
77
Literature Cited
1. Hawthorne, S.B.; Yang, Y.; Miller, D. J. Anal Chem., 1994, 66, 2912.
2. Yang, Y.; Bowadt, S.; Hawthorne, S. B.; Miller, D.J., Anal Chem., 1995, 67,
4571.
3. Arkelof, G. C; Oshry, H. I.; J. Am. Chem. Soc, 1950, 72, 3844.
4. Onobu, M.; Suzuki, A., Journal of the Surface Finishing Society of Japan. 2000,51, 11.
5. Foster, M. D.; Synovec, R. E.; Anal Chem, 1996, 68, 2838.
6. Miller D.J.; Hawthorne, S.B., Anal Chem. 1997, 69, 623.
7. Smith, R. M.; Burgess, R. J., J. Chromatogr., A 1997, 785, 49.
8. Yang, Y.; Belghazi, M.; Lagadec, A.; Hawthorne, S.B.; Miller, D.J., J. Chromatogr, A 1998, 810, 149.
9. Smith, R. M.; Chienthavorn, O., Chromatographia 1999, 50, 485-489.
10. Ingelse, B. A.; Janssen, H. G.; Cramers, C. A. J. High Resolut. Chromatogr. 1998, 21, 613.
11. Smith, R.S.; Chienthovorn, O.; Wilson, I.D.; Wright, B.; Taylor, S.D., Anal Chem, 1999,71,4493.
12. Smith, R. M.; Burgess, R. J.; Chienthavorn, O; Bone, J. R. LC GC Mag. 1999, 17, 938.
13. Poppe, H.; Kraak, J. C; Vandenberg H. H. M., Chromatographia , 1981 14, 515.
14. Nonaka, A. Anal. Chem. 1972, 44, 271-276; Rudenko, B. A.; Baydarovtseva, A.; Kuzovkin, V. A.; Kucherov, V. F. J. Chromatogr. 1975, 104, 271-275.
15. Hawthorne, S. B.; Yang, Y.; Miller, D. J. Anal. Chem. 1994, 66, 2912.
16. Yang, Y; Jones, A. D.; Eaton, C. D., Anal Chem., 1999, 71, 3808.
78
17. Heger, K.; Uematsu, M.; Franck, E. U., BerBunseges. Phys. Chem. 1980, 84, 758.
18. Uematsu, M.; Franck, E. U., J. Phys. Chem. Ref Data. 1980, 9, 1291.
19. Haar, L.; Gallagher, J. S.; Kell, G. S., National Bureau of Standards/National Research Council Steam Tables; Hemisphere Publishing corp.; Bristol, P. 1984.
20. F. Daniels, R. A. Alberty, Physical Chemistry, Wiley, New York, 1955. pp503-506.
21. Weber, T. P.; Jackson, P. T.; Carr, P.W., Anal Chem, 1995, 67, 3042.
22. Li., J.; Carr, P. W. Anal. Chem. 1996, 68, 2857
23. Akapo, S. O.; Simpson, C. F. Chromatographia, 1997, 44, 135.
24. Ergun, S. Chemical Engineering Progress, 1952, 48, 89.
25. http://www.processassociates.com/process/fluid/rx 1 .htm
26. www.Taftan.com
79
Table 4.1. Thermodynamic Properties for Solute Transfer
ZirChrom-Carb ZirChrom-PBD
Analyte Enthalpy Entropy Enthalpy Entropy (kJ/mol) J/(mol°K) (kJ/mol) J/(mol °K)
Benzene -8.56 -12.29 -8.00396 -7.36
Toluene
Ethylbenzene
Propylbenzene
Phenol
Nitrobenzene
-9.49
-9.10
-9.02
-12.48
-16.89
-4.41
3.12
12.74
-25.69
-23.26
-8.19004
-7.93
NM
-8.72
-7.55
-5.02
13.01
NM
-21.41
5.31
80
Pump Computer
Figure 4.1 Hot water chromatography system, schematically shown. CV: Check
valve; PG: pressure sensor and gauge; SSC: silica saturation column; CH: column heater; BPC: back pressure column.
81
Figure 4.2 60 times magnification of capillary column after structural failure.
82
100.00
80.00
•2 60.00 c o
O o
o -^ 40.00 Q
20.00
0.00
o 0
n
A
•
Dielectric constant, water
Dielectric constant, acetone
Dielectric constant, acetonitrile
Dielectric constant, 50:50 ACN:H20
Viscosity of water at 7200 psi
viscosity, acetonitrile
viscosity, 50:50 ACN:H20
1000
800
600
400
200
0.00 200.00 400.00 Temperature, degrees C
600.00
V)
a o o CM
V)
-a o o <n "m o CO a. o o
to o o CO
Figure 4.3 Dielectric constant and viscosity of water at 7200 psi along with the viscosity
and dielectric constant of both pure ACN and a 50% ACN.
83
Pressure, psi
Pressure, MPa
Figure 4.4 Dielectric constant of water as a function of pressure.
84
i ZirChrom-Carb s,i \ 2 A , 370C ZirChrom-PBD
Time, min ^o""
I M I r M r
100 Time, min 1000
Figure 4. 4.5 Separafions on (left) ZirChrom-Carb and (right) ZirChrom-PBD columns;
detecfion at 195 nm, flow rate of 8.6 ^iL/min. s: solvent (acetonitrile), 1: phenol, 2: benzene, 3: toluene, 4: nitrobenzene, 5: ethylbenzene, and 6:
n-propylbenzene. All subsequent flgures has the same numerical identification for analytes. 5
85
E c in
8 c CO
o IB
0.00
E c
UO C35
8 c CD
JQ
o (0
<
'1 ZirChrom-Carb, 300 °C, 20 juL/min 10150 psi
120.00 40.00 80.00 Time, s
ZirChrom-PBD, 240 °C, 27 ^L/min 10750 psi
40 80 Time, s
120
Figure 4.6 High speed separations (a) ZirChrom-Carb and (b) ZirChrom-PBD
columns at 300 °C and 240 °C, respectively.
86
4.00 —
0.00 —
# Phenol • Benzene X Toluene
VNitrobenzene O Ethylbenzene
O Propylbenzene
4.00 —I
2.00 —
0.00 —
-2.00
0.0016
ZirChrom-Carb
0.0012 0.0016 0.0020 0.0024 0.0028 0.0032 Reciprocal Absolute Temperature
ZirChrom-PBD
0.0020 0.0024 Reciprocal Absolute Temperature
0.0028
Figure 4.7 Van't Hoff plots for the retenfion of benzene derivatives.
87
5,00
4,00
p 3 , 0 0 -
S . 2.00 </> a>
1,00
0,00 - -
0.00
s(ACN) A
1,00 2.00 Time, min
Figure 4.8 Thermal gradient performed in GC oven using a FID detector. 180 |xm
i.d. 13 cm silica capillary with ZirChrom-Carb packing, flow rate 8.6 j^L/min. Temperature gradient started at 100 °C and was ramped to
250 °C @ 50 °C/min.
88
CHAPTER V
FUTURE APPLICATIONS AND POTENTIAL IMPROVEMENTS
Details of a capillary scale liquid chromatography system have been
described in the preceding chapters. The system was shown to provide superior
performance over conventional reverse phase systems by utilizing the capillary
scale, an inexpensive, easy to construct pumping system with much higher
pressure rafing than available commercially, and high operafing temperatures.
These factors allowed the new system to perform separations in a fraction of the
time required by convenfional scale instruments while consuming a very small
quantities of sample and eluent. The lower solvent and sample consumpfion
along with reduced analysis time alone can pay for such a system in a short
period.
Portable RPLC System
Capillary scale HPLC systems are inherently capable of being portable.
From a practical standpoint, a portable instrument needs to be light weight, small,
have low power requirements, and have performance close to or comparable to
their bench top counterparts. The advantage of a portable instrument is the
ability to analyze a sample immediately. This gives one, in environmental
studies, the flexibility and the feedback on where to pick the sample site while in
the field running the analysis. Capillary scale HPLC instruments require limited
89
quantities of eluent, have low power requirements, the ancillary components are
or can be made small and compact, and the overall performance can come close
to that of the larger conventional systems. The one component that is not readily
portable is the UVA/IS absorbance detector. Recent research in this laboratory
into inexpensive high performance optical absorbance detectors for capillary
systems suggest that compact capillary scale optical detectors, at least in a
dedicated wavelength format can be built easily.^ The use of compact
electronics, low-power, high-output light emitting diodes (LEDs), and photo
detectors will allow optical detectors to be built which are small enough in terms
of physical size and weight as well as power consumption to be portable. The
high pressure RPLC system described in Chapter II has the capability of being
made portable. The components used in the system are similar to a portable IC
system, which was developed in this laboratory.^ The development of such a
portable capillary scale reverse phase LC system would reduce the required
analysis fime and increase the flexibility while running the analysis.
Super-Hot Water LC-MS System
Recently, there has been a lot of research into the hyphenated systems
using LC, specifically LC-MS systems.^'^ Capillary LC systems have also been
used due to the small flow rate produced by these systems.^'^ Systems using
super-heated water as an eluent have also been used LC-MS systems which
take advantage of the fact that there are no organic modiflers in the eluent to
90
interfere with the organic analytes.^ However, there has been no published
research that combines the low flow rate of the capillary scale with the
advantages of using super-heated water as an eluent in a LC-MS system. The
system described in Chapter IV can easily be adapted to a commercial MS. The
capillary scale will enable direct coupling and the high operafing temperature and
low backpressure at the tail of the column will allow the effluent to go into the gas
phase before entering the MS system. This system would combine the
advantages of a capillary LC system along with the low interference of a pure
water eluent to produce a very sensitive and powerful LC-MS system for drug
development analysis as well as many other applicafions.
Supercritical Water Chromatography
The system described in Chapter IV proved that chromatography can be
done at extremely high temperatures. However, this author feels that the upper
temperature limit has not been achieved. A system which uses a column that is
made from a material, which is insoluble and unreacfive to supercrifical water
would allow studies to be made at higher temperatures with greater stability.
However, the study would also have to incorporate a feasibility study looking at
what effect these super high temperatures have on real world analytes.
91
Liquid Ionization Detector
An interesting and novel detection method developed in this laboratory® is
an oxidative ionization detection system. The system, designed to be coupled to
a hot water LC system, uses high temperature, oxygen saturated water, and UV
light to fully oxidize the organic analytes. Ozone is produced as an intermediate
with low wavelength UV and most organic analytes are at least partially oxidized
to produce ionizable products. Many are fully oxidized to CO2.
Organic Analyte + O^^^^^ """•"" ) H,0 + CO^
CO^^^^^+H^O >2H^ +C0-'
Ionized products can be detected by a simple conductivity detector. The initial
research done has proved the concept showing a conductivity signal even from
an analyte such as benzene; however, it has not been adapted to the LC system
and much work is needed to get a fully operating system with acceptable
detecfion limits. However, once operational such a system has the potential to
be a sensitive universal detector for organic analytes with a hot water eluent.
Another potential configuration for this setup is to allow the CO2 saturated water
to go into the gas phase then to use a simple IR absorbance measurement of
CO2 in the gas stream.
92
Experimental Verification the Boiling Point in the Column
In Chapter IV, a theoretical evaluation of the point at which the water in
the super-heated water system will boil if there is no backpressure on the column
was presented. This was, however, not explored experimentally. This is an
interesting and important point about hot water chromatography that needs to be
explored. Experimentally this boiling point can be verified by using an open tube
capillary of sufficiently small dimensions. A setup using a 10 )Lim open capillary is
proposed. It would be put into a heating enclosure and operated at temperatures
that will boil the water at some point in the capillary. The optical properties of
liquid water and high-pressure steam should be different enough to allow a
simple absorbance or refractive index measurement to determine which is
present. Moving the measurement device to different points on the column, or
changing the temperature and pressure until a change is detected at a single
point should experimentally verify the theoretical calculation made in Chapter IV.
93
Literature Cited
1. C.B. Boring, P.K. Dasgupta, Anal. Chima. Acta, 342 (1997) 123
2. C.B. Boring, P.K. Dasgupta, A.J. Sjogren, J. Chromatogr. A., 804 (1998) 45
3. G.S. Rule, A.V. Mordehai, J. Henion, Anal Chem 66 (1994) 230
4. K. Fujii, Y. Ikai, H. Oka, M. Suzuki, K. Harada, Anal. Chem 69 (1997) 5146
5. T. Trones, A. Tangen, W. Lund, T. Greibrokk, J. Chromatogr. A. 835 (1999) 105
6. A. Cappiello, F. Bruner, Anal. Chem. 65 (1993) 1281
7. R.M. Smith, O. Chienthavron, I.D. Wilson, B. Wright, S.D. Taylor, Anal. Chem 71 (1999)4493
8. Sakai, H.; Dasgupta, P.K.; Personal Communication, 1998
94
CHAPTER VI
CONCLUSION
The research work presented in this dissertation developed a capillary
based reverse phase system capable of separating non-polar based analytes in
substantially less time than what is required from current commercial based
instruments. This time savings provides a substantial cost savings per run in
addition to the cost saved in the reduction of expensive organic modifiers.
Throughout this work benzene based homologs were used as example analytes
because of their availability, cost, and non-polar characteristics. Benzene and its
homologs can be separated more efficiently by gas chromatography, and thus
GC is the preferred method of separation. The purpose of this research was not
to develop a new method for separating benzene derivatives but to use the
benzene derivatives as an example of what could be achieved.
The high pressure gradient pumping system, discussed in Chapter II
surpassed the capillary scale commercial pumping systems available at the fime
for a fracfion of the cost. The merits of the pumping system were comparable to
the commercial pumps with the exception of the pressure limit which was
improved by a factor of 2. This allowed separations to be performed in half the
time or allowed longer columns or columns packed with smaller particles to be
used.
The high temperature reverse phase system described in Chapter III used
the high operating pressures along with high temperatures to perform high speed
95
efficient separations. The elevated temperatures, up to 200 °C, lowered the
viscosity of the eluent along with increasing the diffusion rate. The study
discussed utilized the properties to reduce greatly the required analysis time,
thus increasing the number of plates achieved per minute. The efficient
separation of 8 benzene derivatives was achieved in less than 2 minutes. This
research focussed primarily on minimizing the separation time or maximizing the
number of theoretical plates per minute; however, the same system can be
configured to maximize total efficiency or the number of theoretical plates per
meter. By using longer columns packed with smaller particles, more efficient
separations could have been achieved at the expense of analysis time.
The setup described in Chapter IV is the first report of a capillary based
reverse phase chromatography system that uses super-hot water as an eluent.
The system was able to perform high speed separations that surpassed the
performance of the currently reported conventional scale systems using super-
hot water. By using the excellent thermal conductivity of the capillary scale, fast
thermal gradients were possible, allowing complete, efficient, and fast
separations to be performed. The system also demonstrated the ease of
coupling the capillary scale to non-traditional LC detection methods such as the
flame ionization detector used in gas chromatography.
The instrument described herein can provide substantial cost savings to
an analytical laboratories. The cost of the instrument on the commercial market
could easily be made competitive with the current commercial capillary scale and
96
convenfional scale instruments. The savings in eluent consumption compared to
the conventional scale instruments and reduction in analysis fime compared to
the capillary systems that operate at ambient conditions would provide significant
cost savings.
97
APPENDIX A
RETENTION MECHANISM THEORY
Even though reverse phase liquid chromatography (RPLC) is the most
utilized separation method, it has, perhaps, the least understood retention
mechanism.^ The two widely accepted theories which explain the separation
mechanism present in RPLC are the solvophobic theory developed by Horvath
and co-workers, •^•"* and the partifioning model.^' °' ''• ® In the solvophobic theory
the stationary phase is thought to behave like a solid. The solute interacts with
the surface of the stationary phase and the retention mechanism is only
dependent on the interactions between the solute and the mobile phase. When
the solute is adsorbed onto the surface of the stationary phase, the surface area
of the hydrophobic analyte exposed to the hydrophilic mobile phase is reduced.^
In the partition model, the stationary phase plays a more active role in the
retention process. The solute is thought of as being able to fully penetrate the
stationary phase, rather being limited to surface adsorption. Therefore the
stationary phase is considered more "liquid-like" than in the solvophobic theory.
Although the exact retention is still a matter of debate, there is a general
agreement that as the chain length of the bonded material becomes longer, the
retention mechanism is better describe as partition like. As the chain becomes
shorter, the retention mechanism becomes more similar to the adsorption
mechanism.
98
Literature Cited
1. P.W. Carr, J. Li, A.J. Dallas, D.I. Eikens, and LC. Tan, J. Chromatogr, 656,
(1993), 113.
2. Cs. Horvath, and W. Melander, J. Chromatogr Sci., 15, (1977), 393.
3. Cs. Horvath, W. Melander and I. Molnar, J. Chromatogr., 125, (1976), 129. 4. W. Melander and Cs. Horvath, in Cs. Horvath (editor). High Performance
Liquid Chromatography - Advances and Perspectives Vol 2, Academic Press, New York, 1980, pp201.
5. D.E. Martire, D.E. and R.E. Boehm, J. Phys. Chem., 87, (1983), 1045.
6. K.A. Dill, J. Phys. Chem., 91, (1987), 1980.
7. K.A. Dill, J. Naghizadeh, and J.A. Marqusee, Annu. Rev. Phys. Chem., 39,
(1988), 425.
8. J.G. Dorsey, and K.A. Dill, Chem. Rev., 89, (1989), 331.
9. A. Weston, and P.R. Brown, HPLC and CE: Principles and Practice, Academic Press, San Diego, 1997.
10. H. Colin and G. Guiochon, J. Chromatogr, 141, (1977), 289.
99
APPENDIX B
HPLC COLUMN PERFORMANCE
The performance of a chromatographic column is based on two
parameters. The chromatographic selectivity reflected by the distance between
two peak maxima, and the peak band-broadening\ The concept of theorefical
plates was developed to describe band-broadening by analyzing the shape of the
chromatographic peaks. Column efficiency can be expressed as the theoretical
plate number (N) or the height equivalent to a theorefical plate (HETP). The
number of theoretical plates can be calculated by the following equation^:
N=5.54(tr/wi/2)^ (B.I)
where tr is the retention time of the peak and W1/2 is the full peak width at half
height (FWHH). The HETP is calculated b / ° :
H (HETP)=L/N (B.2)
where L is the length of the column and N is the number of theorefical plates.
Band dispersion in a well-packed column arises from several independent
kinetic processes. Van Deemter et. al. developed the original rate theory
describing the process of band-broadening^ which, was later modified by Knox"*' ^•
^•^. The Knox equation is as follows:
1/ R h = Av'^+ — + Cv. (B.3)
V
The terms containing the constants A, B, and C will be discussed later while h is
equal to the reduced plate height and v is the reduced velocity.
100
Reduced Velocity v = ud„ ( L\( d
p
D^ - (B.4)
Reduced Plate Height h n
d. 5.54 J , yh J (B.5)
Where Dm is the diffusion coefficient, L is equal to the column length, to is the
unretained peak fime, dp is the packing particle diameter, tr is the retenfion fime
of peak and Wi/2 is equal to the full peak width at half height. The advantage of
using the reduced Knox equafion, versus the typical van Deemter equafion, is
that it enables column to column comparisons to be made more accurately by
taking into account the ratio of the column diameter to that of the packing
diameter, referred to as the Knox-Parcher rafio.
The A Term
The A term of the Knox equation accounts for the anisotropic flow that is
encountered in the column. Flow anisotropy arises from the different paths and
flow rates that the eluent experiences as it flows around the packing material.
Analytes traveling in the different flow paths will reach the end of the column at a
different fime, thus producing a broader peak. The A term affects the maximum
optimal efficiency that can be attained on the column.
The B Term
The B term is used to describe the axial molecular diffusion. The axial
molecular diffusion is dependent only on the effective diffusion coefficient of the
101
solute while it is in the mobile phase. The B term affects the column efficiency at
flow rates lower than the optimal flow rate.
The C Term
The C term in the Knox equation accounts for the slow kinetics of the
mass transfer between the stationary phase and the mobile phase. The kinetics
of the slow mass transfer is dependent on the kinetics of the adsorpfion or
partition mechanism and on the diffusion rate through the stagnant pools of
eluent that surround the stationary phase. The C term affects the efficiency of
the column at flow rates greater than the opfimal flow rate.
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Literature Cited
1. G. Szepsi, "How To use Reverse Phase HPLC", VCH Publishers, Inc., New York, 1992.
2. G. Szepesi, "How To Use Reverse-Phase HPLC", VCH Publishers, Inc., New York, NY, 1992.
3. J.J. van Deemter, F.J. Zuiderweg, and A. Klinkenburg, Chem, Eng. Sci., 5,
(1956), 271.
4. J.H. Knox, H.P Scott, IJ. Chromatogr., 282, (1983), 297.
5. J.H. Knox, G.J. Kennedy, J. Chromatogr Sci., 10, (1972), 549.
6. E. Grushka, L.R. Snyder, J.H. Knox, J. Chromatogr Sci., 13, (1975), 25.
7. Knox, J. H. J. Chromatogr. Sci. 15, (1977), 352.
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APPENDIX C
COMPUTER CONTROL OF PUMPING SYSTEM
The program, written in Visual Basic™ 4.0, used to control the custom
gradient pumping system utilizes a six-step gradient where each step has an
individual module. Pump flow rate is controlled by varing the fime delay between
pump steps. Delays can be as short as 1 microsecond and as long as 500
microseconds producing a wide variety of flow rates. The module calculates the
time delay between steps by dividing the total flow rate desired between the two
pumps based on eluent composition desired. The length of time required for the
step is converted into a specific number of steps. The module then creates a
command string for that module with the appropriate number of steps and delays
and attaches it to the end of the previous module's command string. When the
pumps are told to start the entire command string is downloaded into the resident
memory of each pump hardware via an RS-232 serial communication port and
told to run. Once the gradient has started the pumps will work independently of
the computer and of each other.
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