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 TheoryOptimization - 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 TheoryOptimization – 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 TheoryOptimization – 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 TheoryOptimization – 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 tR (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 = (16tR

2/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 TheoryOptimization – 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

B

BS k

kNR

11

41

Note: above equation is best used when deciding how to improve RS, not for calculating RS from chromatograms

Chromatographic TheoryOptimization – Resolution Equation

• 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)

B

BS k

kNR

11

41

Chromatographic TheoryGraphical Representation

Initial Separation Smaller H (narrower peaks)

Larger k - separation increases more than width

Increased alpha (more retention of 2nd compound)

Chromatographic TheoryOptimization – 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 TheoryOptimization – 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

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

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1.80

2.00

0 2 4 6 8 10 12

Time (of 2nd peak) (min)

Re

so

luti

on

Start Point

Maximum RS

Baseline Resolved

Chromatographic TheoryOptimization – 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 TheoryOptimization – 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 ret/T)

– 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 TheoryOptimization – 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

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149810

171211

15

16

756

H

H1

4

2

3H

H

H

compound A

O

O

compound B

Chromatographic TheoryOptimization – 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 TheoryOptimization – Some Questions

• Indicate how the chromatograms could be improved?

Chromatogram 1

1.5

1.7

1.9

2.1

2.3

2.5

2.7

2.9

3.1

0.0 0.5 1.0 1.5 2.0 2.5

time (min.)

resp

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Chromatogram 2

0

0.5

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Chromatogram 3

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Chromatographic TheoryReview Questions

1. What is the most common way to increase retention of analytes in gas chromatography? a) decrease flow rateb) decrease temperaturec) increase flow rated) use carrier gas with larger molecular weight

2. Increasing the flow rate in chromatography will increase which term in the van Deemter equation. (Give name or term).

3. What type of intermolecular force is typically the most important for analyte – stationary phase in reversed phase HPLC?

4. 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 TheoryOptimization – Some Questions

1. Why is it usually more difficult to improve the separation factor () when there are a larger number of analytes/contaminants?

2. Both using a longer column or using a column of smaller H will improve resolutions. Which method will lead to a better chromatogram? Why?

3. RS = 0.93 and kB = 2.7. What is the maximum RS value just by changing kB?

4. 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 ChromatographyOverview 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 ChromatographyComparison 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 ChromatographyCompressibility 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 ChromatographyAdvantages 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 ChromatographyDevelopment 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

Liquid at each plate is mixture of distillates (only X and Y)

X

X

Y

Y

Liquid (or solid) stationary phase interacts with x and y

Gas ChromatographyDevelopment 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 ChromatographyDevelopment 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 ChromatographyDevelopment 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

non-volatile 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 ChromatographyDevelopment 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 non-polar 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 ChromatographyStationary 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 InstrumentationMobile 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 InstrumentationSome Questions

1. If a set of compounds in a sample could be analyzed by GC or HPLC what would be two reasons for picking GC?

2. What is a concern in analyzing a liquid sample that has numerous highly volatile compounds?

3. 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?

4. What is one way in which low volatility samples can be analyzed by GC?

5. 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?

6. How is the retention of polar compounds affected by switching from He to H2 as a carrier gas?

GC InstrumentationFlow 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

Soap film

GC InstrumentationSample 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 InstrumentationSample Injection – Gas Samples

• 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

Gas sample in

Waste

6 port valve

He in

To GC column

LOAD POSITIONINJECT POSITION

GC InstrumentationSample 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)