Subject: Sci.chem FAQ - Part 5 of 7
Date: Sat, 18 Nov 1995 21:56:34 GMT
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Subject: 19. Physical properties of chemicals
     
19.1  Rheological properties and terminology

Contributed by Jim Oliver
                                 
RHEOLOGY

What is RHEOLOGY ?
RHEOLOGY describes the deformation of a material under the influence of 
stresses. Materials in this context can be solids, liquids or gases. In this 
FAQ we will be concerned only with the rheological properties of liquids.[1]
Perry discusses the some aspects of the behaviour of gases, and Ullmann
discusses elastic solids.

When liquids are subjected to stress they will deform irreversibly and flow. 
The measurement of this flow is the measurement of VISCOSITY. IDEAL liquids 
are very few, whereas non-ideal examples abound. Ideal liquids are : water 
and pure paraffin oil. Non-ideal examples would be toothpaste or cornflour 
mixed with a little water. [2]

What is VISCOSITY ?
VISCOSITY is expressed in Pascal seconds (Pa.s) and to be correct the 
conditions used to measure the VISCOSITY must be given. This is due to the 
fact that non-ideal liquids have different values of VISCOSITY for different 
test conditions of SHEAR RATE, SHEAR STRESS and temperature. [3,4]

A graph describing a liquid subjected to a SHEAR STRESS (y axis) at a 
particular SHEAR RATE (x axis) is called a FLOW CURVE. The shape of this 
curve reveals the particular type of VISCOSITY for the liquid being studied. 
[3]

What is a NEWTONIAN LIQUID ?
NEWTONIAN LIQUIDS are those liquids which show a straight line drawn from the 
origin at 45 degrees, when graphed in this way. Examples of NEWTONIAN liquids 
are mineral oil, water and molasses.  (Issac NEWTON first described the laws 
of viscosity) [1] All the other types are NON NEWTONIAN.

What does NON NEWTONIAN mean ?
a. PSEUDOPLASTIC liquids are very common. These display a curve starting at 
   the origin again and curving up and along but falling under the straight 
   line of the NEWTONIAN liquid. In other words increasing SHEAR RATE results 
   in a gradual decreasing SHEAR STRESS, or a thinning of viscosity with 
   increasing shear. Examples are toothpaste and whipped cream.
b. DILATANT liquids give a curve which curves under then upward and higher 
   than the straight line NEWTONIAN curve. (Like a square law curve) Such 
   liquids display increasing viscosity with increasing shear. Examples are 
   wet sand, and mixtures of starch powder with small amounts of water. A car 
   may be driven at speed over wet sand, but don't park on it, as the car may 
   sink out of sight due to the lower shear forces (compared to driving over) 
   the wet sand.

There are other terms used which include :

THIXOTROPY - this describes special types of PSEUDOPLASTIC liquids. In this 
case the liquid shows a YIELD or PLASTIC POINT before starting to thin out. 
What this means is the curve runs straight up the y axis for a short way then 
curves over following ( but higher and parallel to ) the PSEUDOPLASTIC curve. 
This YIELD POINT is time dependant. Some water based paints left overnight 
develop a FALSE BODY which only breaks down to become usable after rapid 
stirring. Also: the curve describing a THIXOTROPIC liquid will be different 
on the way up (increasing shear rate) to the way down (decreasing shear rate). 
The area inside these two lines is a measure of it's degree of THIXOTROPY. 
This property is extremely important in industrial products, e.g to prevent 
settling of dispersed solids on storage. [3]

A RHEOPECTIC liquid is a special case of a DILATANT liquid showing increasing 
viscosity with a constant shear rate over time. Again, time dependant but in 
this case _increasing_ viscosity.

Why do some liquids become solid ?
A few special liquids (dispersions usually) display  extraordinary DILATANT 
properties. A stiff paste slurry of maize or cornflour in water can appear to 
be quite liquid when swirled around in a cup. However on pouring some out 
onto a hard surface and applying extreme shear forces (hitting with a hammer) 
can cause a sudden increase in  VISCOSITY due to it's DILATANCY. The 
VISCOSITY can become so high as to make it appear solid. The "liquid" then 
becomes very stiff for an instant and can shatter just like a solid material.

It should be noted that the study of viscosity and flow behaviour is 
extremely complex. Some liquids can display more than one of the above 
properties dependant on temperature, time and heat history.

19.2  Flammability properties and terminology 

There are several properties of flammable materials that are frequently
reported. It should be remembered that most discussions concerning
flammable liquids usually consider air as the oxidant, but oxygen and 
fluorine can also be used as oxidants for combustion, and they will result
in very different values. 

The Flammability Limits in air, are usually reported as the Upper and Lower 
limits in volume percent at a certain temperature ( usually 25C ), and 
represent the concentration region that the vapour ( liquid HCs can not burn ) 
must be within to support combustion. Hydrocarbons have a fairly narrow range, 
( n-hexane = 1.2 to 7.4 ) whereas hydrogen has a wide range ( 4.0 to 75  ).

The minimum ignition energy is the amount of energy ( usually electrical ) 
required to ignite the flammable mixture. Some mixtures only require a very 
small amount of energy (eg hydrogen = 0.017mJ, acetylene = 0.017mJ ), 
whereas others require more (eg n-hexane = 0.29mJ, diethyl ether = 0.20mJ, 
ammonia = >1000mJ ).  

The Flash Point is the most common measure of flammability today, especially
in transportation of chemicals, mainly because most regulations use the Flash
Point to define different classes of flammable liquids. The Flash Point of a
liquid is the temperature at which the liquid will emit sufficient vapours
to ignite when a flame is applied. The test consists of placing the liquid
in a cup and warming it at a prescribed rate, and every few degrees applying
a small flame to the air above the liquid until a "flash" is seen as the
vapours burn. Note that the flame is not applied continuously, but is
provided at prescribed intervals - thus allowing the vapour to accumulate.

There are a range of procedures outlined in the standard methods for 
measuring Flash Point ( ASTM, ISO, IP ) and they have differing cup 
dimensions, liquid quantity, headspace volume, rate of heating, stirring 
speed, etc., but the most significant distinction is whether the space above 
the liquid is enclosed or open. If the space is enclosed, the vapours will be 
contained, and so the Flash Point is several degrees lower than if it is 
open. Most regulations specify closed-cup methods, either Pensky-Martens 
Closed Cup or Abel Closed Cup. It is important to remember that these methods 
are only intended for pure chemicals, if there is water or any other volatile 
non-flammable compounds present, their vapours can extinguish or mask the 
flash. For used lubricants, this may be partially overcome by using the TAG 
open cup procedure - which is slightly more tolerant of non-flammable 
vapours. A material can be flammable, but may not have a flash point if other
non-flammable volatile compounds are present. For alkane hydrocarbons, Flash 
Point increases with molecular weight.

There an older measure, called the Fire Point, which is the temperature at
which the liquid emits sufficient vapours to sustain combustion. The Fire
Point is usually several degrees above the Flash Point for hydrocarbons. 

The minimum Autoignition Temperature is the temperature at which a material 
will autoignite when it contacts a surface at that temperature. The procedure
consists of heating a glass flask and squirting small quantities of sample
into it at various temperatures until the vapours autoignite. The only 
source of ignition is the heat of the surface. For the smaller hydrocarbons 
the autoignition temperature is inversely related to molecular weight, but
also increases with carbon chain branching. Autoignition temperature also
correlates with gasoline octane ratings ( refer to Gasoline FAQ available in
rec.autos.tech, which lists octane ratings and autoignition temperatures for
a range of hydrocarbons.) 
                     Flash Point    Autoignition   Flammable Limits 
                                    Temperature     Lower     Upper
                       ( C )           ( C )        ( vol % at 25C)
methane                -188             630          5.0      15.0
ethane                 -135             515          3.0      12.4
propane                -104             450          2.1       9.5
n-butane                -74             370          1.8       8.4
n-pentane               -49             260          1.4       7.8
n-hexane                -23             225          1.2       7.4
n-heptane                -3             225          1.1       6.7
n-octane                 14             220          0.95      6.5
n-nonane                 31             205          0.85       -
n-decane                 46             210          0.75      5.6
n-dodecane               74             204          0.60       -             
          
19.3  Supercritical properties and terminology? 

Supercritical fluids have some very unusual properties. When a compound is
subjected to conditions around the critical point ( which is defined as
the temperature at which the gas will not revert to a liquid regardless how
much pressure is applied ), the properties of the supercritical fluid become
very different to the liquid or the gas phases. In particular, the solubility
behaviour changes. The behaviour is neither that of the liquid or that of the 
gas. The transition between liquid and gas can be completely smooth.

The pressure-dependant densities and corresponding Hildebrand solubility 
parameters show no break on continuity as the supercritical boundary is 
crossed. Physical properties fall between those of a liquid and a gas. 
Diffusivities are approximately an order of magnitude higher than the 
corresponding liquid, while viscosities are an order of magnitude lower. 
These ( along with the lack of  surface tension ) allow SCFs to have 
liquid-like solvating power with the mass transport characteristics of a gas.

Potential Supercritical Fluids
Compound          Critical      Critical
                 Temperature    Pressure
                   ( C )        ( atm )
Ammonia            132.5         112.5
Carbon dioxide      31.3          72.9
Methanol           240.1          82.0
Nitrous oxide       36.5          72.5
Propane             96.8          43.1
Water              374.4         224.1
Xenon               16.6          58.4   

Note that using liquid CO2 at pressure ( as for the commercial extraction
of hops ) is still just liquid CO2 extraction, not supercritical CO2 
extraction. There are several good general introductions to supercritical
fluids [5,6]

------------------------------

Subject: 20. Optical properties of chemicals
     
20.1  Refractive Index properties and terminology 

When light passes between media of different density, the direction of the
beam is changed as it passes through the surface, and this is called
refraction. In the first medium, the angle between the light ray and the
perpendicular is called the angle of incidence (i), and the corresponding 
angle in the second medium is called the angle of refraction (r). The
ratio sine i / sine r is called the index of refraction, and usually the
assumption is that the light is travelling from the less dense (air) to more
dense, giving an index of refraction that is greater than 1. Although the
theoretical reference is a vacuum, air ( 0.03% different ) is usually used.
The refractive index of a compound decreases with increasing wavelength
( dispersion ), except where absorption occurs, thus the wavelength should 
be reported. The D lines of sodium are commonly used. 

The refractive index of a liquid varies with temperature and pressure, but 
the specific refraction ( Lorentz and Lorentz equation ) does not. The molar 
refraction is the specific refraction multiplied by the molecular weight,
and is approximately and additive property of the groups or elements 
comprising the compound. Table of atomic refractions are available in the
literature, as are descriptions of the common types of refractometers [1].
 
20.2  Polarimetry properties and terminology 

Supplied by: Vince Hamner <vinny@vt.edu>

     Polarimetry is a method of chemical analysis that is concerned
with the extent to which a beam of linearly polarized light is rotated
during its transmission through a medium containing an optically active
species.[2]  Helpful discussions regarding polarized light may be found
elsewhere.[3,4]  In general, a compound is optically active if it has
no plane of symmetry and is not superimposable on its mirror image.
Such compounds are referred to as being "chiral".  Sucrose, nicotine,
and the amino acids are only a few of these substances that exhibit
an optical rotary power.

     A simple polarimeter instrument would consist of:

     1).  a light source -- typically set to 589 nm (the sodium "D" line)
     2).  a primary fixed linear polarizing lens (customarily called the
          "polarizer")
     3).  a glass sample cell (in the form of a long tube)
     4).  a secondary linear polarizing lens (customarily called the
          "analyzer") and
     5).  a photodetector.[5]

     Biot is credited with the determination of the basic equation
of polarimetry.[6,7]  The specific rotation of a substance (at a given
wavelength and temperature) is equivalent to the observed rotation (in
degrees) divided by the pathlength of the sample cell (in decimeters)
multiplied by the concentration of the sample (for a pure liquid,
-density- replaces concentration).  Influences of temperature,
concentration, and wavelength must always be taken into consideration.
If necessary, it is possible to apply corrections for each of these
variables.[8]  A few early contributors to our understanding of optical
activity and polarimetry include:  Malus, Arago, Biot, Drude, Herschel,
Fresnel, and Pasteur.

------------------------------

Subject: 21. Molecular and Structural Modelling

Supplied by: Dave Young (young@slater.cem.msu.edu)

21.1  What hardware do I need to run modelling programs? 

.There are two types of programs that are referred to as molecular
modeling programs.  This first is a program which graphically displays
molecular structures as Lewis structures, ball & stick, etc.  The second
is a program which does a calculation to tell you something about the
molecule, such as it's energy, dipole moment, spectra, etc.

.For an introductory description of various types of computations,
see http://www.cem.msu.edu/~young/topics/contents.html

.There are many programs of both sorts available for a large range 
of machines.  The speed, memory, graphics and disk space on the machine 
will determine how big of molecules can be modeled, how accurately and
how good the images will look.  There are a few programs that will run 
on a 286 PC with windows.  There are some fairly nice things that can be 
done on a 386 with about 8 MB of RAM and windows.  The professional
computational chemists are generally using work stations and larger machines.

.Currently many computational chemists are using machines made by
Silicon Graphics (SGI) ranging from the $5,000 Indy to the $1,000,000
power challenge machines.  These are all running Irix, which is SGI's
adaptation of Unix.  SGI is popular for two reasons; first that the power
is very good for the price, second that SGI's run the largest range
of chemical software.  However, you will find some computational chemistry 
software that can run on almost any machine.

.As far as graphics quality, the SGI Onyx (about $250,000) is about 
the top of the line.  Even if you find a machine that claims to have better 
graphics than this, chances are you won't find and chemistry software that 
can utilize it.  

.For chemical calculations there is no limit to the computing
power necessary.  There are some calculations that can only be done
on the biggest Cray's or massivly parallel machines in the world.  There
are also many calculations which are too difficult for any existing
machine and will just have to wait a few years or a few centuries.

21.2  Where can I find a free modelling program?

.The single best place for public domain modelling software
is probably the anonymous ftp server at ccl.osc.edu in the directory
pub/chemistry/software.  "ccl" stands for "computational chemistry
list server" and is a list frequented mostly by professional 
computational chemistry researchers.  This machine contains their 
archives with quite a bit of information as well as software.

.For work stations and larger, the program GAMESS (General Atomic
and Molecular Electronic Structure System) can be obtained as source
code from Mike Schmidt at mike@si.fi.ameslab.gov   GAMESS is a quantum
mechanics, ab initio and semiempirical program.  It is powerful but
not trivial to learn how to use.

.The COLUMBUS program for work stations and larger can be obtained 
by anonymous ftp from ftp.itc.univie.ac.at   It is a HF, MCSCF and 
multi-reference CI program.  This is probably the most difficult program
to use that is in use today since it requires the user to input EVERY 
detail manually.  However, because you control everything there are some
calculations that can only be done with COLUMBUS.

.CACAO is an extended Huckel program available by anonymous
ftp at cacao.issecc.fi.cnr.it

21.3  Where can I find structural databanks? 

21.4  Where can I find ChemDraw or ChemWindows 

For ChemDraw (Macintosh, Windows, UNIX)
     CambridgeSoft Corporation
     875 Massachusetts Avenue
     Cambridge, MA 02139
     Phone: (800) 315-7300 or (617) 491-2200
     Fax: (617) 491-7203
     Internet: info@camsci.com
     http://www.camsci.com

For ChemIntosh or ChemWindows
     SoftShell
     1600 Ute Avenue
     Grand Junction, CO 81501
     Phone: (970) 242-7502
     Fax: (970) 242-6469
     Internet: info@softshell.com
     http://www.softshell.com

------------------------------

Subject: 22. Spectroscopic Techniques
     
All of these are covered in texts on instrumental Analysis [1-4], and I 
will eventually include a paragraph about each.

22.1  Ultra-Violet/Visible properties and terminology 
22.3  Nuclear Magnetic Resonance properties and terminology 
22.4  Mass Spectrometry properties and terminology 
22.5  X-Ray Fluorescence properties and terminology 
22.6  X-Ray Diffraction properties and terminology 
22.7  Fluorescence/Phosphorescence properties and terminology 
   
------------------------------

Subject: 23. Chromatographic Techniques   

23.1  What is Paper Chromatography? 

Paper chromatography was the first analytical chromatographic technique 
developed, allegedly using papyrus (Pliny). It was first published by Runge
in 1855, and consists of a solvent moving along filter or blotting paper.
The interaction between the components of the sample, the solvent and the 
paper results in separation of the components. Most modern paper
chromatography is partition chromatography, where the cellulose of the
paper is the inert support, the water adsorbed ( hydrogen bonded ) from air 
onto the hydroxyl groups of the cellulose is the stationary phase. If the
mobile phase is not saturated with water, then some of the stationary phase
water may be removed from the cellulose resulting in a separation that is
a mixture of partition and adsorption. Paper chromatography remains the 
method of choice for a wide range of coloured compounds, and is used 
extensively in flower colour research. The technique is suitable for any 
molecules that are significantly less volatile than the solvent, and many
examples and references are provided in Heftmann [1]. 

23.2  What is Thin Layer Chromatography?

Thin layer chromatography involves the use of a particulate sorbent on an
inert sheet of glass, plastic, or metal. The solvent is allowed to travel
up the plate with the sample spotted on the sorbent just above the solvent.
Depending on the sorbent, the separation can be either partition or 
adsorption chromatography ( cellulose, silica gel and alumina are commonly
used ). The technique came to prominence during the late 1930s, however it 
did not become popular until Merck and Desaga developed commercial plates 
that provided reproducible separations. The major advantage of TLC is the 
disposable nature of the plates. Samples do not have to undergo extensive 
cleanups as they would for HPLC. The other major advantage is the ability 
to detect a wide range of compounds cheaply using very reactive reagents 
( iodine vapours, sulfuric acid ) or indicators. Non-destructive detection 
( fluorescent indicators in the plates, examination under a UV lamp ) also
means that purified samples can be scraped off the plate and analyzed by 
other techniques. There are special plates for such preparative separations, 
and there are also high-performance plates that can approach HPLC resolution.
The technique is described in detail in Stahl [2] and Kirchner [3].  

23.3  What is Gas Chromatography? 

Gas chromatography is the use of a gas to carry the sample through a column 
consisting of an inert support and a stationary phase that interacts with 
sample components, thus it is usually partition chromatography, however
there are also a range of materials, especially for permanent gas and
light hydrocarbon analysis that utilise adsorption. The simplest partition
systems consisted of a steel tube filled with crushed brick that had been 
coated with a high boiling hydrocarbon. Today the technique uses very narrow 
fused silica tubes ( 0.1 to 0.3mm ID ) that have sophisticated stationary 
phase films ( 0.1 to 5um ) bonded to the surface and also cross-linked to 
increase thermal stability. The ability of the film to retard specific 
compounds is used to ascertain the "polarity" of the column. If benzene 
elutes between normal alkanes where it is expected by boiling point ( midway
between n-hexane and n-heptane ), then the column is "non-polar" eg
squalane and methyl silicones. If the benzene is retarded until it elutes
after n-dodecane, then the column is "polar" eg OV-275 ( dicyanoallyl 
silicone ) and 1,2,3-tris (2-cyanoethoxy) propane. In general polar columns
are less tolerant of oxygen and reactive sample components, but the ability
to select a select different polarity columns to obtain satisfactory peak
resolution is what made GC so popular. 

The column is placed in an oven which has exceptional temperature control, 
and the column can be slowly heated up to 350-450C ( sometimes starting at 
-50C to enhance resolution of volatile compounds ) to provide separation of 
wide-boiling range compounds. The carrier gas is usually hydrogen or helium, 
and the eluting compounds can be detected several ways, including in flames 
( flame ionisation detector ), by changes in properties of the carrier 
( thermal conductivity detector ), or by mass spectrometry. The availability 
of "universal" detectors such as the FID and MS, makes GC a popular tool in 
laboratories handling organic compounds. There are also columns that have a 
layer of 5-10 um porous particulate material (such as molecular sieve or 
alumina ) bonded to the inner walls ( PLOT = Porous layer open tubular ), 
and these are used for the separation of permanent gases and light 
hydrocarbons.  GC is restricted to molecules ( or derivatives ) that 
are sufficiently stable and volatile to pass through the GC intact at the
temperatures required for the separation. Specialist books on the production 
of derivatives for GC are available [4,5]. 

There are several manufacturers of GC instruments whose catalogues and 
brochures provide good introduction to the technique. (eg Hewlett Packard, 
Perkin Elmer, Carlo Erba ). The catalogues of suppliers of chromatography 
consumables also contain explanations of the criteria for selection of the 
correct columns and conditions for analyses, and they provide an excellent 
indication of the range of applications available. Well-known suppliers 
include Alltech Associates, Supelco, Chrompack, J&W, and Restek. They also 
sell most of the standard GC texts, as do the instrument manufacturers.    
Popular GC texts include "Basic Gas Chromatography" [6], "High-Resolution
Gas Chromatography" [7], and "Open Tubular Column Gas Chromatography" [8].
There are Standard Retention Index Libraries available [9], however they
really only complement unambiguous identification by mass spec. or 
dual-column analysis.

23.4  What is Column Chromatography? 

Column chromatography consists of a column of particulate material such as 
silica or alumina that has a solvent passed through it at atmospheric or low 
pressure. The separation can be liquid/solid (adsorption) or liquid/liquid
(partition). The columns are usually glass or plastic with sinter frits to
hod the packing. Most systems rely on gravity to push the solvent through.
The sample is dissolved in solvent and applied to the front of the 
column. The solvent elutes the sample though the column, allowing the 
components to separate based on adsorption ( alumina, hydroxylapatite) or
partition ( cellulose, diatomaceous earth ). The mechanism for silica
depends on the hydration. Traditionally, the solvent was non-polar and the 
surface polar, although today there are a wide range of packings including 
bonded phase systems. Bonded phase systems usually utilise partition 
mechanisms rather than adsorption. The solvent is usually changed stepwise, 
and fractions are collected according to the separation required, with the 
eluate usually monitored by TLC. 

The technique is not efficient, with relatively large volumes of solvent 
being used, and particle size is constrained by the need to have a flow of 
several mls/min. The major advantage is that no pumps or expensive equipment 
are required, and the technique can be scaled up to handle sample sizes
approaching a gram in the laboratory. The technique is discussed in detail
in Heftmann [1].

23.5  What is High Pressure Liquid Chromatography? 

HPLC is a development of column chromatography. it was long realised that
using particles with a small particle size ( 3,5,10um ) with a very narrow 
size distribution would greatly improve resolution, especially if the flow 
rate  and column dimensions could be adjusted to minimise band-broadening. 
Pumps were developed that could handle both the chemicals and pressures 
required. Traditional column chromatography ( nonpolar solvent and
polar surface ) is described as "normal" and, as well as silica, there are
columns with amino, diol, and cyano groups. If the system uses a polar
solvent ( water, methanol, acetonitrile etc. ) and a non-polar surface it
is described as "reversed phase". Common surface treatments of silica include
octadecylsilane ( aka ODS or C18), and it has been the development of 
reverse-phase HPLC that has experienced explosive growth. Reverse-phase HPLC
is the method of choice for larger non-volatile biomolecules, however it is 
only recently that a replacement "universal" detector ( evaporative 
light-scattering ) has emerged. The most popular detector (UV), places 
constraints on the solvents that can be used, and the refractive index 
detector can not easily be used with solvent gradients. There are several 
excellent books introducing HPLC, including the classic "Introduction to 
Modern Liquid Chromatography" [10]. HPLCs can be a pain to operate, and 
novices should borrow "Troubleshooting LC Systems" by Dolan and Snyder [11].
There is also a handy basic primer on developing HPLC methods by Snyder [12],
however, unlike GC, you need to search the journals ( Journal of 
Chromatography, Journal of Liquid Chromatography  ) to find relevant examples 
to assist method development. 
 
23.6  What is Ion Chromatography?

Ion chromatography has become the method of choice for measuring anions 
( eg Cl-, SO4=, NO3- ) in aqueous solutions. It is effectively a development
from ion-exchange systems ( which were extensively developed to deionise
water and aqueous process streams ), and brings them down to HPLC size. 
IC uses pellicular polymeric resins that are compatible with a wide pH range. 
The sample is eluted through an ion-exchange column using a dilute sodium 
hydroxide solution. The eluent is passed through self-regenerating 
suppressors that neutralise eluant conductance, ensuring electrochemical 
detectors ( conductivity or pulsed amperometric ) can detect the ions down 
to sub-ppm concentrations. The major manufacturer of such systems is Dionex, 
who hold several patents on column, suppression, and detection technology. 
There are several books covering various aspects of the technique [13,14].
  
23.7  What is Gel Permeation Chromatography?

Gel Permeation chromatography ( aka Size Exclusion chromatography ) is based 
on the ability of molecules to move through a column of gel that has pores of 
clearly-defined sizes. The larger molecules can not enter the pores, thus 
they pass quickly through the column and elute first. Slightly smaller 
molecules can enter some pores, and so take longer to elute, and small 
molecules can be delayed further. The great advantage of the technique is
simplicity, it is isocratic ( single solvent - no gradient programming ),
and large molecules rapidly elute. The technique can be used to determine 
the molecular weight of large biomolecules and polymers, as well as 
separating them from salts and small molecules. The columns are very 
expensive and sensitive to contamination, consequently they are mainly used 
in applications where alternative separation techniques are not available, 
and sample are fairly clean. The best known columns are the Shodex 
cross-linked polystyrene-divinylbenzene columns for use with organic solvents, 
and polyhydroxymethacrylate gel filtration columns for use with aqueous 
solvents. "Modern Size Exclusion Chromatography" [15], and Heftmann [1],
provide good overviews, and there are some good introductory booklets from
Pharmacia.

23.8  What is Capillary Electrophoresis? 

Capillary electrophoresis uses a small fused silica capillary that has been
coated with a hydrophilic or hydrophobic phase to separate biomolecules, 
pharmaceuticals and small inorganic ions. A voltage is applied and the 
materials migrate and separates according to charge under the specific
pH conditions,as happen for electrophoresis.The capillary can also be used 
for isoelectric focusing of proteins. The use of salt or vacuum mobilization 
is no longer required.  

23.9  How do I degas chromatographic solvents?

One major problem with pressurising chromatography systems using liquid 
solvents is that pressure reductions can cause dissolved gases to come out
of solution. The two locations where this occurs are the suction side of the
pump ( which is not self-priming, consequently a gas bubble can sit in the
pump and flow is reduced ), and at the column outlet ( where the bubbles
then pass through the detector causing spurious signals).Note that the 
problem is usually restricted to solvents that have relatively high gas 
solubilities - usually involving an aqueous component, especially if a 
gradient is involved where the water/organic solvent ratio is changing.
As water usually has a higher dissolved gas content, then a gradient 
programme may cause the gases to come out of solution as the mobile phase
components mix. 

There are three traditional strategies used to remove problem dissolved 
gases from chromatographic eluants. Often they are used in combination to 
lower the dissolved gases.
a. Subject the solvent to vacuum for 5-10 mins. to remove the gases.
b. Subject the solvent to ultrasonics for 10-15 mins. to remove the gases. 
c. Sparge the solvent with a gas that has a very low solubility compared
   to the oxygen and nitrogen from the atmosphere. Helium is the preferred
   choice - 5 minutes of gentle bubbling from a 7um sinter is usually 
   sufficient, although maintaining a positive He pressure is even better.
Note that most aqueous-based solvents usually have to be degassed every
24 hours. Also remember that solubility of gases increases as temperature
decreases, so ensure eluants are at instrument temperature prior to 
degassing. 

Some HPLCs are sold with a "solvent degassing module" that removes 
undissolved gases automatically.  
 
23.10  What is chromatographic solvent "polarity"?

There are four major intermolecular interactions between sample and solvent 
molecules in liquid chromatography, dispersion, dipole, hydrogen-bonding,
and dielectric. Dispersion interactions is the attraction between each pair
of adjacent molecules, and are stronger for sample and solvent molecules 
with large refractive indices. Strong dipole interactions occur when both
sample and solvent have permanent dipole moments that are aligned. Strong
hydrogen-bonding interactions occur between proton donors and proton
acceptors. Dielectric interactions favour the dissolution of ionic 
molecules in polar solvents. The total interaction of the solvent and
sample is the sum of the four interactions. The total interaction for a 
sample or solvent molecule in all four ways is known as the "polarity" of 
the molecule. Polar solvents dissolve polar molecules, and for normal
phase partition chromatography solvent strength increases with solvent
polarity, whereas solvent strength decreases with increasing polarity.
The subject is discussed in detail in Snyder and Kirkland [10].

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Subject: 24. Extraction Techniques   
     
24.1  What is Solvent Extraction? 

Solvent extraction is usually used to recover a component from either a solid
or liquid. The sample is contacted with a solvent that will dissolve the
solutes of interest. Solvent extraction is of major commercial importance
to the chemical and biochemical industries, as it is often the most efficient
method of separation of valuable products from complex feedstocks or
reaction products. Some extraction techniques in involve partition between two 
immiscible liquids, others involve either continuous extractions or batch
extractions. Because of environmental concerns, many common liquid/liquid
processes have been modified to either utilise benign solvents, or move to
more frugal processes such as solid phase extraction. The solvent can be a 
vapour, supercritical fluid, or liquid, and the sample can be a gas, liquid 
or solid. There are a wide range of techniques used, and details can be found 
in Organic Vogel, Perry as well as any textbook on unit operations. 

24.2  What is Solid Phase Extraction? 

Solid Phase Extraction (SPE) is an alternative to liquid/liquid extraction,
which has been the method of choice for the separation and purification of
a wide range of samples in the laboratory. The sample is usually dissolved in 
an appropriate solvent and passed through a small bed of appropriate 
particulate adsorbent. The compounds are eluted off with small amounts of 
different solvents. The major advantage is that solvent volumes are greatly
reduced. There is a newer, modified technique that is used in analytical
laboratories, called Solid Phase MicroExtraction. This immerses a fused
silica fibre coated with a stationary phase into the sample solution for 
several minutes, The analytes adsorb onto the stationary phase, which is
subsequently pushed into a hot GC injector to rapidly desorb the sample.  

24.3  What is Supercritical Fluid Extraction? 

Supercritical fluids have been investigated since last century, with the 
strongest commercial interest initially focusing on the use of supercritical 
toluene in petroleum and shale oil refining during the 1970s. Supercritical 
water is also being investigated as a means of destroying toxic wastes, and
as an unusual synthesis medium [1]. The biggest interest for the last decade
has been the applications of supercritical carbon dioxide, because it has
a near-ambient critical temperature (31C), thus biological materials can
be processed at temperatures around 35C. The density of the supercritical
CO2 at around 200bar pressure is close to that of hexane, and the solvation 
characteristics are also similar to hexane, thus it acts as a non-polar 
solvent. Around the supercritical region CO2 can dissolve triglycerides at 
concentrations up to 1% mass. The major advantage is that a small reduction 
in temperature, or a slightly larger reduction in pressure, will result in 
almost all of the solute precipitating out as the supercritical conditions
are changed or taken to subcritical. Supercritical fluids can produce a 
product with no solvent residues. Examples of pilot and production scale 
products include decaffeinated coffee, cholesterol-free butter, low-fat meat, 
evening primrose oil, squalene from shark liver oil. The solvation 
characteristics of supercritical CO2 can be modified by the addition of an 
entrainer, such as ethanol, however that then remains as a solvent residue 
in the product, negating some of the advantages of "residue-free" extraction.
 
There are other near-ambient temperature supercritical fluids, including 
nitrous oxide and propane, however there are safety issues with both of them. 
There are several introductory texts on supercritical fluid extraction, 
including some the ACS Symposium series [2-4]. There are also a large 
number of articles on applications of the technique, including processing [5],
extraction of natural products [6], and chemical synthesis [7]. The major 
concentration of information occurs in the various proceedings of the 
International Symposium on Supercritical Fluids [8].  There is also a Journal 
of Supercritical Fluids.

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Subject: 25. Radiochemical Techniques
     
25.1  What is radiochemistry?

------------------------------  

Subject: 26. Electrochemical Techniques
     
26.1  What is pH?

The pH scale determines the degree of acidity or alkalinity of a solution,
but as it involves a single ion activity it can not be measured directly.

pH = - log10 ( gammaH x mH)

     gammaH = hydrogen ion single ion activity coefficient
         mH = molality of the hydrogen ion.

As pH can not be directly measured, it is defined operationally according to
the method used to determine it. IUPAC recommend several standardised methods
for the determination of pH in solution in aqueous solutions. There are 
seven primary reference standards that can be used, including 0.05 mol/kg
potassium hydrogen phthalate as the Reference Value Standard. There is an
ongoing debate concerning the relative merits of having a multiple primary
standard scale ( that defines pH using several primary standards, and their
values are determined using a cell without a liquid junction ) or a single
primary standard ( that requires a cell with a liquid junction ). Interested
readers can obtain further information on the debate in [1]. Bates [2], is a 
popular text covering both theory and practise of pH measurement. 
   
26.2  How do pH electrodes work?     

Contributed by Paul Willems <Paul.Willems@rug.ac.be>

The most common type of pH electrodes are the so called glass electrodes.
A special glass membrane is sensitive to variations in pH and a pH
variation creates a variation in the potential over the glass. In order
to be able to measure this potential, a second electrode, the so called
reference electrode is required. Quite often both electrodes are combined
to one "combined" pH electrode.

The glass electrode consists of a glass shaft on which a bulb of a special
glass is mounted. The inner is filled with KCl, most often at a
concentration of 3 Mol/liter and sealed. Electrical contact is provided
by the way of a silver wire immersed in the KCl.

Normally this glass electrode is surrounded by a concentric reference
electrode. This reference electrode can consists of a silver wire in
contact with the almost insoluble AgCl. The electrical contact with
the meter is through the silver wire. The contact with the solution
to be measured is by way of a KCl filling solution which is physically
in contact with the solution to be measured. In order to minimise
mixing of the solution to be measured and the filling solution, a porous
sealing, the diaphragm, is used. Alternatively other devices which
allow a slow mixing contact can also be used. Besides the "normal"
KCl solutions, often solutions with an increased viscosity, and hence
lower mixing rate are used. In stead of a liquid KCl filling, also
gel filling is used. This eliminates the necessity of low mixing devices.

The glass electrode in contact with some solution gives in respect to
the reference electrode a voltage of about 0 mV at pH 7, increasing with
59 mV per pH above 7 or decreasing with 59 mV per pH unit below 7.
Both the slope and the intercept of the curve between pH and generated
potential are temperature dependent. In fact, the potential of the
electrode is roughly given by the Nernst equation :

E = E0 - RT log [H+] = E0 + RT pH

In which E is de generated potential, E0 is a constant, R is universal
gas constant and T is the temperature in degrees Kelvin.

All pH dependent glasses are also susceptible to other ions, such as
Na or K. This gives an correction on the above equation. By this reason
the relation between pH and generated voltage becomes nonlinear at
high pH values.

Also the slope tends to diminish as the electrode wears out. At high pH the 
slope tends also to diminish. As the electrode has a very high impedance, 
typically 250 Mega Ohms to 1 giga Ohm, it is absolutely necessary to use a 
very high impedance measuring apparatus.

The reference electrode has a potential that does normally not vary
too much. However the potential is also temperature dependent and can
also vary if the activity of the silver ions in the reference electrode
would vary. This can be the case if a pollutant enters the reference
electrode.

Calibration

From the preceding, it is obvious that a frequent calibration and
adjustment of pH meters are necessary. To check the pH meter, one
should verify if the pH shown does not differ from the "real" pH
of so called buffer solutions. At least two such solutions are required,
e.g. pH 7 and pH 4. If the difference is not acceptable, one should
adjust the reading.

To adjust, one should take care not to work too fast, so as to be sure
that the system is in equilibrium. Also the pH meter should be already
powered on for some time so as to ensure that all components are in a
thermal steady state. On should first use the buffer at pH 7 and adjust
the zero (or the intercept). Thereafter, one should use the buffer at
a different pH to adjust the slope. This cycle in repeated at least once
or until no further adjustments are necessary. Note many modern pH meters
have an automatic calibration feature. In this case one only needs to
use each buffer only once.

Errors

Although many people take a pH measurement for granted, many errors are
possible. Those can have different causes. There can be errors of the
pH dependent glass, errors on behalf of the reference, errors in the
electrical part as well as externally generated errors.

Errors of the pH dependent glass

The pH dependent glass can break or crack. Sometimes such a break is
obvious, but sometimes such a break is hard to find. If there is a
connection between the internal liquid of the pH measuring part and
the external environment, one will find a pH value close to 7, which
does not change when the electrode is put is a solution of a known
different pH. Also if one measures the electrical resistivity over
the glass membrane, one find a value which is typical below 1 mega ohm.
In that case one can only replace the electrode with a new one.

A similar case can develop if the glass wall between the inner and the
outer part of a combined electrode break. This is possible eg. in case the
outer part is made of a plastic material, which is bent. The inner part
can crack without any marks on the outside. The electrical resistivity
is over the glass electrode itself intact, but actual measuring between
both electrodes reveals as in previous case a low resistivity. The remedy
is the same as in previous case : replace the electrode.

The glass can wear out. This gives slow response times as well as
a lower slope of the mV versus pH curve. The first remedy possible is
to put the electrode in a 3 Molar KCl solution at 55 degrees celsius for
5 hours. This should revitalise to some extend the electrode. If this
does not help, one can refurbish the electrode by removing a layer of the
glass. This is done by putting the electrode for two minutes in a (plastic!)
container containing a mixture of HCl and KF (be careful, do not breath
the fumes; wear gloves). Afterwards the electrode is put two more minutes
in HCl, and rinsed thoroughly. As a part of the glass in physically removed,
the new surface will be about as good as the original new surface. However
because the glass is now less thick, this shortens the life of the electrode.
After this remedy the first days, a very frequent recalibration is
required.

The glass can be dirty. If a film of some product lays on the glass, the
glass still measures correctly but does not measure the pH in the solution
to be measured but the pH in the layer of surrounding product instead.
This is seen normally by very slow response times and obviously wrong pH
values. Also the pH may vary according to the buffer capacity and/or the
stirring rate in the solution to be measured.
If one knows exactly what product it is, one should dissolve the product
using an adequate solvent. In the general case one should normally first
dip the affected electrode a few minutes in a strongly alcaline solution,
followed by immersing it in a strong acid (HCl) solution. If this does
not help, one should try pepsin in HCl. If still unsuccessful, one can
use the HCl/KF method described in the previous paragraph.

Errors of the reference

The diaphragm of the reference can be blocked. This is seen as unstable
or wrong pH measurements. If one measures the electrical resistivity
over the diaphragm, one find high values. (Most multimeters will give
an overrange). The most common reason is that AgS did form a precipitate
in the diaphragm. The diaphragm will be black in this case. The electrode
should be immersed in a solution of acidic thiourea until the diaphragm
is white again. Afterwards the internal filling liquid of the reference
electrode should be replaced.

There is no contact over the diaphragm, due to some air bubbles. This is
seen exactly as if the diagram were blocked, except that the diaphragm
has its normal color. In this case one should make sure that the liquid
is at all times (slowly) flowing from the reference electrode towards
the liquid to measure.

A polluting substance did enter the reference electrode. This is seen as
unstable or wrong pH measurements. Often the pH at which the output of the
system is 0 mV differs considerably from pH 7. The diaphragm has its normal
color and the electrical resistivity is normal. However, quite often this
case is combined with the previous case, which invalidates the previous
statement. The remedy is to replace, eventually several times the reference
liquid. In many cases, however, the electrode will be permanently damaged.
One can prevent this to happen by choosing for gel filled reference
electrodes, double junction electrodes or by making sure that there is at
all times an net outflow of reference liquid towards the solution to be
measured.

The electrode was filled with a wrong reference solution. This is seen
as pH measurements which are shifted. Replace the reference liquid.

Errors in the electrical part

The input stage of the meter is broken. This gives random measurements.
Shorting both input wires does not make any difference. Remedy : one
should repair the meter.

The input stage seems broken. Shorting both input wires gives a stable
pH measurement of about 7. The meter can in fact be broken, but most
probably the problem is elsewhere.

The input stage of the meter is contaminated with some liquid. This gives
an almost constant measurement of about pH 7, even with the pH electrode
disconnected. Sometimes this is also seen as a pH which seems to vary only
to some proportion of what it should, when tested with two standard 
solutions. In this case one should clean the contaminated part, first with 
distilled water, afterwards with ethanol and dry thoroughly.

Water did enter into the connecting cable. This appear exactly as the
previous case, except that if one disconnects the cable the pH will
start to drift. The remedy is the same as in previous case, only the
contaminated part is different.

There is a short circuit in the cable. This gives similar results as
the previous case. Sometimes one does not know that in most pH cables
between the two copper conductors there are two layers which seem to
be insulators. However the inner layer is in fact an isulator whereas
the outer layer is a conductor to avoid trace electrical effects. If
this outer layer does make a connection to the inner conductor, there is
a short circuit. Remedy : make sure that there are no such contacts.

Externally generated errors

If there is a marked flux of liquid around the electrode, then there can
be a trace electric effect. This generated some potential on the glass
membrane, which is superposed on the actual pH measurement. This effect
becomes negligible for good conducting liquids. It is seldom observed.
In case the trace electric effect does influence pH measurements, one can
add a little salt to increase the conductivity or one can try to change
the flux of liquid around the electrode.

In case of ground loops or spurious currents, there are electrical currents
flowing on places where one should not expect them. Such currents can
strongly influence pH measurements. It is not unlikely to observe a pH
in the range of -15 to +20 even if the real pH is 7, just due of such
electrical phenomena. One can remove those ground loops by correctly
grounding the setup. One should also check the insulation. Often
those problems can be extremely difficult to detect and remedy.

26.3  What are ion-selective electrodes? 

Ion selective electrodes are electrochemical sensors whose potential varies
with the logarithm of the activity of an ion in solution. Available types:
The membrane can be a single compound or a homogeneous mixture of compounds
The support, containing an ionic species, or uncharged species, forms the
    membrane. The support can be solid or porous.
The membrane can be thin glass whose chemical composition determines the
    response to specific ions. 
Popular texts on applications of ion-selective electrodes include 
"Ion-Selective Electrodes in Analytical Chemistry" [3], and "Ion-selective
Electrode Methodology" [4].

26.4  Who supplies pH and ion-selective electrodes?

The best known manufacturer of ion-selective electrodes is Orion Research. 
There are several pH electrode manufacturers, including Radiometer and
Metrohm.

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