Chem. 231 – 2/11 Lecture

Announcements I

• Return Homework Set 1• Quiz 1 Today (15 min.)• New Homework Set (Set 2)• Website Update

– Homework 1 Solutions– Adding demonstration for HW2

Announcements II

• Today’s Topics:– Finish Extractions

• Quantitative calculations• How to determine if method is working and how to

improve methods

– Low Performance Chromatography• Lower pressure chromatography• Thin layer chromatography

– Quantitative Chromatography • Starting early• Focus today on integrating chromatograms

Simple ExtractionsCalculations for Liquid – Liquid

Extraction• For liquid-liquid extractions, partitioning between

two layers can be calculated:

– for unreactive compounds, Kp = [X]raffinate/[X]extractant

– k = nraf/nextr = Kp(Vraf/Vext)

– Q = fraction transferred to extractant phase– Q = 1/(1+k)– For weak acids/bases, we need to consider

that X can exist in two forms in aqueous layer (HA or A- for weak acids and B or BH+ for weak bases)

raffinate = original sample layer


Extraction• Sample Calculation for Butyric acid CH3(CH2)2CO2H with

KOW = 5.75 and Ka = 4.82.• Assuming an octanol raffinate phase, lets calculate

fraction extracted to an aqueous phase as a function of pH assuming 20 mL aqueous phase and 10 mL octanol

• KD = [HA]total aq/[HA]Octanol and Kp = 5.75• Ka = [H+][A-]/[HA] = 10-4.82 = 1.51 x 10-5

• Since Kp = [HA]Oct/[HA]aq and KD = [HA]Oct/([A-] + [HA])aq,KD/Kp = [HA]aq/([A-] + [HA]) = = nonionized fraction

= [HA]/([A-] + [HA]) = [HA]/(Ka[HA]/[H+]+ [HA]) = [H+]/(Ka+ [H+]) = f(pKa, pH) [note: different equation for weak bases]

• KD = Kp• k = Kp(Voct/Vaq)• Q = 1/(1 + k)• Go to Excel Demonstration


Extraction• For best separation, want high Q value for one

compound and low Q value for other compound• Go to 3-Mepyridine, 2-naphthaleneamine

separation

Simple ExtractionsCalculations for other methods (SPE)

• Not Quantitative (too many variables)• Can Make Predictions about Relative

Retention• Example: want to know if 2-

chlorophenol will be retained on SPE cartridge– If phenol has smaller KOW and has near

100% retention, 2-chlorophenol should also be retained

Simple ExtractionsTests

• Numerous losses are possible:– inefficient retention– inefficient sample transfers– inefficient removal of trapped

compounds

• Best strategy is to test recovery (and for each step if recovery is low)

• Small sample sizes and concentrations are challenging

Low Performance Chromatography

Lower Pressure Chromatography• Purpose of Separation

– Typically used for preparative chromatography– Commonly used when simple extractions don’t have sufficient

resolution (e.g. removal of desired product from reactants and distinctly different side reaction products)

• Equipment– packed columns (usually normal phase)– injection system or manual placement of sample– flow provided by low pressure pump, gravity or gas pressure

(Flash)– means for fraction collection more common than means for

detection

• Advantages/Disadvantages– better separation than simple methods and lower cost than HPLC– limited resolution is main disadvantage


Thin Layer Chromatography• Purpose of Separation

– Typically used for fast qualitative analysis

– Can be used for initial method optimization or preparative separations

• Equipment– plate (typically normal

phase)– method to “spot” sample– solvent/container– visual detection or

fluorescent detection common

Chamber (solvent in base)

spots placed at base


Thin Layer Chromatography• Advantages

– relatively fast (especially considering start up time)– low cost– simple detection– can run multiple samples in parallel– whole sample seen (whether doesn’t migrate or moves

with solvent)

• Disadvantages– not very quantitative– limited sample size– limited resolution– not as reproducible

Quantitative ChromatographyIntegration Concerns

• Goals of Integration:– pick up peaks, but

avoid noise– pick up small peaks that

overlap with big peaks– give realistic

separations of overlapping peaks

– avoid bias in peak integrations

min5 10 15 20 25

mAU

75

100

125

150

175

200

225

250

ADC1 A, ADC1 CHANNEL A (TOM\APR11A000003.D)

0.91

0 1.

041

1.12

3 1.

410

1.49

0 1.

662

1.78

9 1.

951

2.27

3 2.

393

2.56

0 2.

752

3.48

3 3.

683

3.79

4 3.

881

3.95

5 4.

091

4.29

4 4.

457

4.59

6 4.

866

4.98

0 5.

302

6.12

6

7.11

1

8.20

9

9.33

1

10.35

6

11.33

6 12

.137

12.50

8 12

.816

13.20

7 13

.427

14.02

0 14

.183

14.48

9 14

.746

15.13

1 15

.315

15.55

8 16

.007

16.25

5 16

.619

16.97

8 17

.428

17.49

6 17

.968

18.12

7 18

.375

18.52

3 18

.654

18.82

2 19

.050

19.49

1 19

.889

20.13

7 20

.412

21.15

9 21

.604

21.93

0 22

.481

Both Peaks and Noise Picked Up

min5 10 15 20

mV

58

59

60

61

62

63

64

65

ADC1 A, ADC1 CHANNEL A (DIXON\011408000002.D)

5.3

24

15.

247

No Noise Picked Up, but peaks missed


• How to process collected chromatograms

• Shown for Agilent 1100 (Chemstation)

• Use Correct WindowView → Data Analysis

• Select your file– File → Load Signal

use “offline” version


• Picking up peaks vs. noise– Choose channel of interest and then go to Integration

Events to set “Slope Sensitivity” (may need to change peak width, peak area reject or peak height reject)

• Slope Sensitivity Set to 1• Slope Sensitivity Set to 15

min5 10 15 20 25

mAU

75

100

125

150

175

200

225

250


0.91

0 1.

041

1.12

3 1.

410

1.49

0 1.

662

1.78

9 1.

951

2.27

3 2.

393

2.56

0 2.

752

3.48

3 3.

683

3.79

4 3.

881

3.95

5 4.

091

4.29

4 4.

457

4.59

6 4.

866

4.98

0 5.

302

6.12

6

7.11

1

8.20

9

9.33

1

10.35

6

11.33

6 12

.137

12.50

8 12

.816

13.20

7 13

.427

14.02

0 14

.183

14.48

9 14

.746

15.13

1 15

.315

15.55

8 16

.007

16.25

5 16

.619

16.97

8 17

.428

17.49

6 17

.968

18.12

7 18

.375

18.52

3 18

.654

18.82

2 19

.050

19.49

1 19

.889

20.13

7 20

.412

21.15

9 21

.604

21.93

0 22

.481

min5 10 15 20 25

mAU

75

100

125

150

175

200

225

250


2.56

0

5.30

2 6.

126

7.11

1

8.20

9

9.33

1

10.35

6

11.33

6

Quantitative Chromatography

Integration Concerns• Picking up peaks vs. noise - more– May need to zoom in to “see” small peaks

• Slope Sensitivity too low• Improved Settings

– Last peak can be picked up, but at the expense of poor integration; better to just use manual integration

min5 10 15 20

mV

58

59

60

61

62

63

64

65


5.3

24

15.

247

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mV

59

60

61

62

63

64

65


5.32

4

9.76

4

15.24

7

17.09

2

min2.5 5 7.5 10 12.5 15 17.5 20 22.5

mV

59

60

61

62

63

64

65

66


1.69

4 2.

150

2.35

4 2.

881

3.06

3 3.

287

3.83

0 4.

328

5.32

2

6.54

9 6.

702

6.94

9 7.

217

7.64

3

8.49

3 9.

034

9.77

0 10

.441

11.40

1 11

.613

11.82

0 12.36

4

13.23

6

15.24

4

16.41

8 17

.092

21.94

5

min12 14 16 18 20 22 24

mV

60

61

62

63

64


15.24

7

17.08

6

Area:

33.14

71 21

.942

Area on diagonal indicates manual integration

Quantitative Chromatography

Integration Concerns• Other problems

– large solvent (especially GC) or unretained peaks– overlapping peaks

min2 4 6 8 10 12 14 16 18

mAU

200

250

300

350

400

ADC1 A, ADC1 CHANNEL A (TOM\AUG23A000001.D)

0.84

8 1.

046

1.50

0

2.22

4

3.33

4 3.

777

4.04

5 4.

329

4.69

1 5.

132

5.72

8 6.

185

6.80

0

7.46

2

8.21

8

9.06

6

10.02

6

11.09

6

12.36

0

13.58

5

18.71

8

Relatively good “auto” integration settings

however, some small peaks may have area overestimated

“Best” baseline between pink and black lines

Chem. 231 – 2/11 Lecture

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Transcript of Chem. 231 – 2/11 Lecture