Chem. 31 - 9/15 Lecture

Chem. 31 - 9/15 Lecture

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 Todays 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 Lets 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 = (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 values can be increased (use different solvents in HPLC or column with better selectivity) but effect on RS is

more complicated 1 1 kB RS N 4 1 kB Note: above equation is best used when deciding how to improve RS, not for calculating RS from chromatograms Chromatographic Theory Optimization Resolution Equation RS 1 1 kB N 4 1 kB

Dont 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 Resolution

2.00 1.80 Maximum RS 1.60 1.40 1.20 1.00 0.80 0.60 0.40 Baseline Resolved 0.20 0.00 0 2 Start Point

4 6 8 Time (of 2nd peak) (min) 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 = 56C) elutes at 7.82 min while diethyl ether (KOW =7.76, bp = 34.6C) 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 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 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 12 11 1 H 2 10 3

5 4 9 H 13 H 7 O 16 14 8 H 17

OH 15 H 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 nonvolatile 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 dont interact Liquid at each plate is mixture of distillates (only X

and Y) X 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 molecules 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 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 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 compounds 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 L, 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)

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