Transcript Chem. 230 – 10/07 Lecture Announcements I • Second Homework Set Due • Exam 2 – Next Week – You can bring a 3”

Chem. 230 – 10/07 Lecture
Announcements I
• Second Homework Set Due
• Exam 2
– Next Week
– You can bring a 3” x 5” note card with notes (front
and back) to the exam
– I will provide constants but no equations
– Topics Covered:
• Simple Separations vs. Chromatography
• Chromatographic Theory (Basic definitions of parameters,
meaning of parameters, how to read chromatograms, rate
theory)
• Intermolecular Forces + Their Effects
• Optimization
Announcements II
• Should Sign Up for Presentation Topic Today
• Today’s Topics
– Optimization (last topic on Exam I)
– Gas Chromatography
•
•
•
•
•
Comparison of methods
Historical Development
Column types
Analytes and Samples
Instrumentation (mobile and stationary phases, flow control,
injection?)
Chromatographic Theory
Optimization - Overview
• How does “method development” work?
– Goal of method development is to select and improve a
chromatographic method to meet the purposes of the
application
– Specific samples and analytes/solutes will dictate many of the
requirements (e.g. how many solutes are being separated and in
what concentration? what is the purpose of the separation?,
what other compounds will be present?)
– Coarse method selection (e.g. GC vs HPLC and selection of
column type and detectors) is often based on past work or can
be based on initial assessment showing problems (e.g. 20
compounds all with k between 0.2 and 2.0 with no easy way to
increase k)
– Optimization then involves making equipment work as well as
possible (or limiting equipment changes)
Chromatographic Theory
Optimization – What are we optimizing?
• Ideally, we want sufficient resolution (Rs of 1.5
or greater for analyte/solute of interest peaks)
• We also want the separation performed in a
minimum amount of time
• Other parameters may also be of importance:
– sufficient quantity if performing “prep” scale
separation
– sufficient sensitivity for detection (covered more with
instrumentation and quantitation)
– ability to identify unknowns (e.g. with MS detection)
Chromatographic Theory
Optimization – Some trade offs
• Flow rate at minimum H vs. higher flow rates (covered
with van Deemter Equation) – low flow rate not always
desired because of time required and sometimes smaller
S/N
• Maximum flow rate often based on column/instrument
damage – this can set flow rate
• Trade-offs in reducing H
– In packed columns, going to small particle sizes results in
greater back-pressure (harder to keep high flow)
– In GC, small column and film diameters means less capacity and
greater likelihood of column overloading
• Trade-offs in lengthening column (N = L/H)
– Longer times due to more column (can be considerably longer
for HPLC due to pressure limits)
Chromatographic Theory
Optimization – Improved Resolution Through Increased Column Length
Example:
Compounds X and Y are separated on a 100 mm column. tM = 2 min, tX = 8
min, tY = 9 min, wX = 1 min, wY = 1.13 min, so RS = 0.94. Also, N = 1024
and H = 100 mm/1024 = 0.097 mm
Let’s increase L to 200 mm. Now, all times are doubled:
tM = 4 min, tX = 16 min, tY = 18 min. So DtR (or d) now = 2 min. Before
considering widths, we must realize that N = L/H (where H is a constant for
given packing material).
N200 mm = 2*N100 mm. Now, N = 16(tR/w)2 so w = (16tR2/N)0.5
w200 mm/w100 mm = (tR200 mm/tR100 mm)*(N100 mm/N200 mm)0.5
w200 mm/w100 mm = (2)*(0.5)0.5 = 21-0.5 = (2)0.5
w200 mm = 1.41w100 mm
RS = d/ave(w) = 2/1.5 = 1.33
Or RS 200/RS 100 = d/wave = (d200/d100)*(w100/w200)= (L200/L100)*(L100/L200)0.5
So RS is proportional to (L)0.5
Chromatographic Theory
Optimization – Resolution Equation
• Increasing column length is not usually the most desired
way to improve resolution (because required time
increases and signal to noise ratio decreases)
• Alternatively, k values can be increased (use lower T in
GC or weaker solvents in HPLC); or α values can be
increased (use different solvents in HPLC or column with
better selectivity) but effect on RS is more complicated
1
   1  k B
RS 
N

4
   1  k B



Note: above equation is best used when deciding how to improve RS,
not for calculating RS from chromatograms
Chromatographic Theory
Optimization – Resolution Equation
1
   1  k B
RS 
N

4
   1  k B



• Don’t use above equation for calculating Rs
• How to improve resolution
– Increase N (increase column length, use more efficient column)
– Increase  (use more selective column or mobile phase)
– Increase k values (increase retention)
• Which way works best?
– Increase in k is easiest (but best if k is initially small)
– Increase in  is best, but often hardest
– Often, changes in k lead to small, but unpredictable, changes
in  also (for problems in this class we will assume no change
in  with change in T or solvent composition)
Chromatographic Theory
Graphical Representation
Initial Separation
Increased alpha (more retention of 2nd
compound)
Smaller H (narrower peaks)
Larger k - separation increases
more than width
Chromatographic Theory
Optimization – Back to 1st Example
Compounds X and Y are separated on a 100
mm column. tM = 2 min, tX = 8 min, tY =
9 min, wX = 1 min, wY = 1.13 min, so RS
= 0.94. Also, N = 1024, kY = 4.5 and  =
1.13.
What change is needed in N, k, and  to get
RS = 1.5?
Chromatographic Theory
Optimization – 2nd Example
•
•
tM = 1 min, tX = 2 min, wX = 0.1 min, tY = 2.1 min, wY = 0.105 so: RS =
0.98,  = 1.1, kY = 1.1
With small initial k values, increasing k helps more
After k > 5, only minor increases in resolution possible
2.00
Maximum
RS
1.80
Resolution
•
1.60
1.40
1.20
1.00
0.80
Baseline Resolved
0.60
0.40
0.20
0.00
0
2
4
6
8
Time (of 2nd peak) (min)
Start Point
10
12
Chromatographic Theory
Optimization – Changes in  - I
In GC analysis on a DB-1 (non-polar) GC column,
the compounds acetone (KOW = 0.58, bp =
56°C) elutes at 7.82 min while diethyl ether (KOW
=7.76, bp = 34.6°C) elutes at 7.97 min. Peak
widths are around 0.2 min. If the unretained
time is 1.00 min., this is a difficult separation
with this column.
Occasionally, changing T to change k will also
increase  (more on this on next slide)
Suggest a column switch (aimed at increasing  to
improve the separation).
Chromatographic Theory
Optimization – Changes in  - II
• Changes in  with T:
– Example: alkanes and toluene
– In Plot, most alkanes show
similar temp. – retention
behavior (similar slopes – no
overlap)
– If two alkanes overlap (e.g. two
branched alkanes), there is not
much chance in increasing 
(since both have same Dret/DT)
– If a separation of octane and
toluene had been performed at
150, coeluting peaks would be
observed
– Decreasing T would lead to
improvement because different
slopes lead to a change in 
note: if chromatogram started at 200C, one
would be disappointed by initial change
Chromatographic Theory
Optimization – Changes in  - III
In HPLC, it is possible to change the mobile phase to affect
solute – solvent interactions and retention.
For example, if molecules A and B are separated by normal
phase HPLC using 15% 2-propanol/85% hexane and are
found to co-elute, solvent changes may resolve.
One might expect that changing solvent to 25% toluene
75% hexane will increase affinity of compound B for
mobile phase relative to compound A (due to compound
B being aromatic) leading to increase retention of B
H
12
11
9
2
10
3
5
O
16
14
8
H
4
17
H
H
1
13
OH
15
H
7
6
compound A
O
compound B
Chromatographic Theory
Optimization – Changes in  - IV
The two compounds below are found to give
retention times of 8.91 and 9.02 min. (aniline
and benzaldehyde, respectively) when separated
using HPLC on a C18 column using 60%
methanol/40% water vs. an unretained time of
1.62 min.
There is an easy way to increase  for this
separation. How can the mobile phase be
changed to increase ?
NH2
O
Chromatographic Theory
Optimization – Some Questions
Chromatogram 1
3.1
2.9
2.7
response
• Indicate how the
chromatograms could
be improved?
2.5
2.3
2.1
1.9
1.7
1.5
0.0
0.5
1.0
1.5
2.0
2.5
time (min.)
Chromatogram 3
Chromatogram 2
1.4
1.2
2
response
response
2.5
1.5
1
0.5
1
0.8
0.6
0.4
0.2
0
0.0
1.0
2.0
3.0
time (min.)
4.0
5.0
6.0
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
time (min.)
7.0
8.0
9.0
10.0
Chromatographic Theory
Review Questions
1.
2.
3.
4.
What is the most common way to increase retention of analytes
in gas chromatography?
a) decrease flow rate
b) decrease temperature
c) increase flow rate
d) use carrier gas with larger molecular weight
Increasing the flow rate in chromatography will increase which
term in the van Deemter equation. (Give name or term).
What type of intermolecular force is typically the most important
for analyte – stationary phase in reversed phase HPLC?
An obviously tailing peak is observed in a chromatogram. The
concentration of the standard is decreased by a factor of 10 and
the sample is re-injected. The tailing looks about the same.
What can be concluded about the source of tailing? List one
other possible source of the tailing. (added later)
Chromatographic Theory
Optimization – Some Questions
1.
2.
3.
4.
Why is it usually more difficult to improve the
separation factor () when there are a larger number
of analytes/contaminants?
Both using a longer column or using a column of
smaller H will improve resolutions. Which method will
lead to a better chromatogram? Why?
RS = 0.93 and kB = 2.7. What is the maximum RS
value just by changing kB?
An initial run of two standards at moderate
concentrations results in RS = 1.9, kA = 3.3 and kB =
4.0. Why might an analytical chemist and a prep
chemist change k in opposite directions?
Gas Chromatography
Overview of Topics
• Comparison of mobile phases (Chapter 6)
• History, analyte – stationary phase
interaction (Section 7.1)
• Instrumentation (Section 7.2, 7.3)
• Stationary phase (Section 7.4)
• Temperature issues (Section 7.6)
Gas Chromatography
Comparison of Mobile Phases
• Two key differences between GC and LC:
– No analyte – mobile phase interaction in GC
– Temperature is routinely changed (and always
controlled) in GC
• Effects of gases (vs. liquids)
– Much higher diffusivity (larger B term of van Deemter
equation but very small CM term)
– Lower viscosity of gases (backpressure is not as big
an issue)
– Much lower density (capacity of column is a big issue
with liquid samples)
– Gases are compressible
Gas Chromatography
Compressibility of Gases
• The volume flow rate will not be a
constant along a column because as the
pressure drops, the volume increases
• There are various ways to calculate
average flow rates which we will not go
into
Gas Chromatography
Advantages vs. HPLC
• Main practical advantage comes from high N
values (although H is usually larger) achieved
with open tubular columns.
• Another advantage comes from being able to
use quite long columns (60 m vs. 250 mm for
HPLC) because backpressure is not a major
issue
• Other advantages have to do with instrument
cost and better detectors
• Main disadvantage is for analysis of non-volatile
compounds
Gas Chromatography
Development and Theory
• Initially, GC was developed to
improve upon fractional
distillations
• In fractional distillations, the
liquid at each plate is a
mixture of analytes
• In gas chromatography
analytes are present, but
stationary phase interactions
are dominant and analyte X
and Y generally don’t interact
X Y
Liquid at each plate is
mixture of distillates
(only X and Y)
XY
Liquid (or solid)
stationary phase
interacts with x and y
Gas Chromatography
Development and Theory
• Types of Columns
– Packed Columns
• Older type of column
• Both solid and liquid stationary phase
• Best column for preparatory GC and for use with thermal
conductivity detectors
• Sometimes used for very specific applications (low production
volume less of an issue)
– Open Tubular Columns
•
•
•
•
More modern columns
Much better analytical performance (large N values)
Most common in wall coated format (WCOT)
Variety of diameters (0.25 to 0.53 mm most common) allow high
resolution vs. easier injection
• Stationary phases are mainly bonded of varying amounts of polarity
• Good reliability
• Disadvantages: harder to make and less capacity
Gas Chromatography
Development and Theory
• Retention of Compounds
– KC value depends on:
• Volatility
• Polarity of analyte vs. polarity of stationary phase
– Measure of volatility
• Best measure is vapor pressure at temperature
• Boiling point temperature is used more frequently
• Depends on molecule’s size and polarity
– Polarity in separations
• Compounds of similar polarity as stationary phase will be
more retained than similar compounds of different polarity if
their boiling points are the same (ether vs. acetone example)
Gas Chromatography
Development and Theory
• Application of GC
– Gas samples
• Somewhat different equipment (injector and oven range) is
needed vs. liquid samples
– Liquid samples
• Compounds must be volatile (plus small amounts of nonvolatile interferences)
• Compounds must be stable at GC temperatures
• Separations are better for less polar compounds
• Issues occur for very volatile and low volatility samples (due
to min and max temperatures)
Gas Chromatography
Development and Theory
• Application of GC
– Extension to non-volatile, thermally labile
compounds
• Derivatization: example – fatty acids are highly
polar and do not produce narrow peak with nonpolar columns, but they can be reacted to produce
fatty acid methyl ester (same reaction used to
produce biodiesel) that are volatile and stable
• Pyrolysis GC: non-volatile samples are heated and
breakdown products are measured by GC. This
give information about compound’s “building
blocks”
Gas Chromatography
Stationary Phase
• Selection of stationary phase affects k and  values
• Main concerns of stationary phase are: polarity, functional groups,
maximum operating temperature, and column bleed (loss of
stationary phase due to decomposition)
• More polar columns suffer from lower maximum temperatures and
greater column bleed
Type
Functional Groups
Polarity
OV-1
methyl
Non-polar
OV-17
50% methyl/50%
phenyl
Somewhat polar
OV-225
Cyanopropyl,
methyl, and phenyl
More polar
carbowax
Ether groups
polar
GC Instrumentation
Mobile Phase
•
Since there is no mobile phase –
analyte interaction in GC, why
does the mobile phase matter?
– Affects diffusion
• Smallest MW gases diffuse faster
• van Deemter B term at low flow
rates (fast is worse) and C term at
higher flow rates (fast is better)
• Hmin not affected much, but umin
affected by gas chosen
• Smallest MW allows fastest runs
at min. H
– Detector requirements
– He is most common (inert, safe
gas with high diffusivity for better
efficiency at high flow rate)
– H2 also can be used with even
better efficiency, but is less safe
CO2 min
H2 min
GC Instrumentation
Some Questions
1.
2.
3.
4.
5.
6.
If a set of compounds in a sample could be analyzed by GC or
HPLC what would be two reasons for picking GC?
What is a concern in analyzing a liquid sample that has numerous
highly volatile compounds?
In the case of the situation in question 2, would you want a
column with the stationary similar to or different from the polarity
of the analytes?
What is one way in which low volatility samples can be analyzed
by GC?
In response to high He prices, a lab director says that no more He
can be purchased. Would you want to use Ne or N2? (assuming
reasonable prices for both of those gases)? What other change
would be needed to get reasonable separations with Ne or N2
carrier gases?
How is the retention of polar compounds affected by switching
from He to H2 as a carrier gas?
GC Instrumentation
Flow Control
• Flow can be controlled by
regulating inlet pressure (either
constant pressure or
compensation for constant linear
velocity).
• Equipment consists of valves for
regulating pressure (constant
pressure) in older instruments or
electronic pressure control
(solenoid valve opens or closes
in response to pressure).
• Flow rate is typically checked at
detector using bubble meter.
Pressure
Transducer
Solenoid valve
Soap film
soap
GC Instrumentation
Sample Injection
• Several types of injectors are available and choice of
injector depends on sample phase, analyte
concentration, and other sample properties
• The most common injectors are designed for liquids (but
can be used for gases)
• Injectors for gases only can be used for gases
• Liquids require much smaller volumes (1 μL, a typical
liquid injection volume, is equivalent to ~ 1 mL after
evaporation) and column overloading is common
• Column overloading is most common with narrow
diameter OT columns and least common with packed
columns
• Most injectors are heated (except on-column)
GC Instrumentation
Sample Injection – Gas Samples
6 port valve
• Fixed Loop Injectors
– A loop of fixed volume is
filled with a gas
– The injection valve is
twisted so that the mobile
phase pushes the gases in
the loop into the column
– Very similar to most
common injections in
HPLC (Covered later)
– Very reproducible injection
He in
To GC column
Waste
Gas
sample in
INJECT
POSITION
LOAD POSITION
GC Instrumentation
Sample Injection – Gas Samples
• Specialized Injectors (Fixed loop injectors with trapping capability)
– Best for trace analysis
– In place of loop is a trap (adsorbant or cold trap) so that all gas sent into
loop gets trapped, then injected
– These allow injection of greater volumes but may require removal of
interferents (oxygen, water) and require better quantitative control of
gases (careful volume or pressure monitoring)
– Thermal trapping (cool to trap, then hot to desorb) can increase
efficiency
• Other ways to inject gas samples (using injectors designed for
liquids)
– Direct syringe injection (samples at higher concentrations)
– Solid phase microextraction (SPME with fibers exposed to gas samples)