Chem. 31 - 9/15 Lecture

Chem. 31 - 9/15 Lecture

Chem. 133 5/9 Lecture Announcements I Homework Set 3 due today Quiz 5 (Ave on Q5 was 1.75) Final Exam Thursday, May 18th 12:45-2:45 About 50% Review/50% New Material Allowed 1 8.5 x 11 sheet of notes (no equations provided) Will review new material on Thursday (5/11) Final topic covered will be GC Announcements II Todays Lecture Chromatography (general) Band broadening Resolution Gas Chromatography Columns Injectors Chromatography Measurement of Efficiency

Measuring N and H is valid under isocratic/isothermal conditions Later eluting peaks normally used to avoid effects from extra-column broadening (from injector, detector, etc.) Example: N = 16(14.6/0.9)2 = 4200 (vs. ~3000 for pk 3) H = L/N = 250 mm/4200 = 0.06 mm W ~ 0.9 min Chromatography Causes of Band Broadening There are three major causes of band broadening (according to theory) These depend on the linear velocity (u = L/tm) Given by van B Deemter Equation: H A u Cu

where H = Plate Height, and A, B, and C are constants Chromatography Band Broadening Most efficient velocity H C term B term A term u Chromatography Band Broadening Constant Terms A term: This is due to eddy diffusion or multiple paths Independent of u Smaller A term for: a)

small particles, or b) no particles (best) X X X dispersion Chromatography Band Broadening B Term Molecular Diffusion Molecular diffusion is caused by random motions of molecules Larger for smaller molecules Much larger for gases Dispersion increases with time spent in mobile phase Slower flow means more time in mobile phase X X X Band broadening Chromatography Band Broadening

C term Mass transfer to and within the stationary phase Analyte molecules in stationary phase are not moving and get left behind The greater u, the more dispersion occurs Less dispersion for smaller particles and thinner films of stationary phase X X dispersion Column particle Chromatography Some Questions 1. 2. 3. 4. Column A is 100 mm long with H = 0.024 mm. Column B is 250 mm long with H = 0.090 mm. Which column will give more efficient separations (under conditions for determining H)?

Which van Deemter term is negligible in open tubular GC? How can columns in HPLC be designed to decrease H? In open tubular GC? Both using a longer column or using a column of smaller H will improve resolutions. Which method will generally lead to a better chromatogram? Why? Chromatography Resolution Resolution = measure of how well separated two peaks are Resolution = ttr/wav (where wav = average peak width) (use this equation for calculating resolution) RS < 1, means significant overlap RS = 1.5, means about minimum for baseline resolution (at least for two peaks of equal height) Chromatography Resolution Example HO HO

H H H HO HH H mannosan 8.20 min. O O OH H H galactosan 9.09 min. ADC1A, ADC1CHANNELA(MATT\042709C2009-04-2715-58-52\042709000005.D) 10.879

Norm. 34 30 28 11.896 32 A9re.090 a: 57. 569 4 tR(1st pk) = 8.20 min., w (integrator) = w = 0.316 min, so w = 0.316(4/2.5) = 0.505 min. tR(2nd pk) = 9.09 min., w = 0.536 min Resolution = 0.89/0.521 = 1.70 (neglecting nonGaussian peak shape) Resolution not baseline

due to peak tailing OH O O OH H A8r.2 ea04 : 14 5.0 52 1st two retained peaks: H 1.834 RS calculation example: main difference: axial equatorial/ axial switch of 2 vs. 4 C OH groups

26 24 22 2 4 6 8 10 12 min Chromatography Optimization Resolution Equation 1 a 1 k2 RS N 4 a 1 k2

not in version of text we are using 2 for 2nd component to qualitatively eluteto figure out how Will use equation to improve chromatograms How to improve resolution Increase N (increase column length, use more efficient column) Increase a (use more selective column or mobile phase) Increase k values (increase retention) Which way works best? Increase in k is easiest (but only if k is initially small) Increase in a is best, but often hardest Often, changes in k lead to small, but unpredictable, changes in a Chromatography Graphical Representation Initial Separation

Increased alpha (more retention of 2nd peak) Smaller H (narrower peaks) Larger k or L - separation increases more than width Chromatography Resolution/Optimization Questions 1. Why is it usually more difficult to improve the separation factor (a) when there are a larger number of analytes/contaminants? 2. Why is it effective to increase k to improve resolution ONLY if k is small to begin with? Chromatography Optimization Some Questions Indicate how the chromatograms could be improved?

Chromatogram 1 3.1 2.9 response 2.7 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 Gas Chromatography (GC) Introduction Overview of Topics Applications Most common for volatile compounds

More common for non-polar to moderately polar compounds Columns (packed vs. open tubular) Sample Injection Detectors GC Columns Two Common Formats Packed columns (older style) Open tubular (typically long columns with small diameters) Advantages of open tubular columns Greater Efficiency Better sensitivity with most detectors (due to less band broadening vs. lower mass through

column) Advantage of packed Open Tubular (end on, cross section view) Column Wall (fused silica) Mobile phase Stationary phase GC Stationary Phase Selection of stationary phase affects k and a values Main concerns of stationary phase are: polarity, functional groups, maximum operating temperature, and column bleed (loss of stationary phase) 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 Injection Liquid Samples Most Common Overload (solvent or sample) is a common problem split/splitless injector minimizes this (next page) Gas Samples Syringe Injection (standard injector) Fixed Loop Injectors (common for HPLC) Solid Phase Microextraction (SPME) GC Sample Injection Split/Splitless Split/Splitless Injectors Injectors capable of running in two modes: split and splitless Split injections used to avoid overloading columns Septum Syringe port

outside Split vent Injection Process Syringe pierces septum and depressing plunger deposits liquid Analyte volatilizes Part injected (usually smaller fraction) Part passed to vent Fraction vented depends on split valve liner He in Split valve To Column GC Injection Split injection is used for:

Higher concentrations Smaller diameter (OT) columns Greater need for high resolution than high accuracy In split injection, solvent overload is less problematic Splitless injection is used for trace analysis (~50% of injected sample put on column)

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