Lc Report

Name:

NAUMAN MITHANI

Student no.:

301016320; group C

Course:

CHEM 316

Object:

Expt. IV lab report: LIQUID CHROMATOGRAPHY: SEPARATION AND IDENTIFICATION OF FOUR ORGANIC ACIDS

Due date:

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27-3-2008!

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ABSTRACT: In this reversed phase (H)igh (P)erformace/(P)ressure (L)iquid (C)hromatography experiment, samples of a mixture comprised of phthalic, salicylic, benzoic and pnitrophenyl ethanoic acid were analysed. The organic acids are cited in ascending order of retention times, thus descending order of polarity. It was determined that an 80:20 ratio of the potassium hydrogen phosphate – phosphoric acid buffer and acetonitrile (as mobile phase) yielded the best balance between desired low retention times and high resolutions. Its polarity index was 9.32; it was also calculated that a 83:17 ratio of the buffer:methanol would be required to produce the same polarity index. The experiment also involved comparison of isocratic versus gradient elution; results show that isocratic elution was the better option, though this may be at odds with normalcy. A less than optimum choice of composition of the buffer:acetonitrile and durations could account for this. By combining the methods of standard addition and linear regression analysis, the concentration of p-nitrophenyl ethanoic acid was confirmed to be (~25 ppm) 24.46 ppm ± 28.7 % in a prepared unknown sample.

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INTRODUCTION: The experiment involved the separation and identification of a mixture of four organic acids: phtalic acid, salicylic acid, benzoic acid and p-nitrophenyl ethanoic acid; by the technique of high performance liquid chromatography (combined with UV-visible absorption for identification and quantification). The technique involved running the analyte sample through a column packed with siloxane-coated silica particles; this was the stationary phase. The eluent-mixture (mobile phase) was a mixture of aqueous buffer and acetonitrile (methyl cyanide). The column was sealed under high pressure and the carrier fluid/eluent was liquid, hence the name high performance/pressure liquid chromatography. The use of the polar mobile phase and the relatively non-polar stationary phase lends the term reversed-phase as sub-type of the HPLC. The components of the analyte sample mixture separate out by their different and characteristic affinities for the polar mobile phase and non-polar stationary phase. The more polar components exit the column before the less polar ones, thus having a lesser retention time, due to a greater affinity for the polar mobile phase than the stationary phase. The arrival (and potential quantification) of a component through the column is detected by an attached UV-visible spectrometer.

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EXPERIMENTAL PROCEDURE: !

Composition of buffer: H3PO4, K2HPO4 and water at pH of 3.10

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Characteristics of column: length of 5 cm; packing of siloxane-coated octadecylsilyl (particles of 5 µm diameter) A 50 mL test mixture was prepared from 12.5 mL each of phthalic, salicylic,

benzoic and p-nitrophenyl ethanoic acid of 100 ppm; thereby reducing the concentration of each component to 25 ppm. This test mixture was then run through the column at a rate of 1.5 mL/min (constant throughout the experiment) in a 80:20 mixture of the buffer and acetonitrile as mobile phase; the test mixture was then run at the same speed but under different ratios of buffer and acetonitrile, 90:10, 70:30 and 60:40. Each run was terminated once all the components had eluted. Then, the 100 ppm standard of each substance was run through the column. The next series of experimental runs were conducted under a gradient of bufferacetonitrile ratios. One such run is cited here (refer to pg ?? for the data): 65:35 from 0 to 0.6 min., 80:20 from 0.6 min. to 0.8 min. and 90:10 from 0.8 min. to 3 min. The second section of the experiment was commenced with the preparation of 50 mL of an unknown solution containing p-nitrophenyl ethanoic acid, benzoic acid and phthalic acid at a concentration of 25 ppm each. This was done by adding 12.5 mL of 100 ppm standard solutions of each substance along with 12.5 mL of water. Using the 100 ppm p-nitrophenyl ethanoic acid standard as the new analyte, five solutions (10 mL) were prepared of varying concentrations, 0, 10, 20, 30 and 40 ppm. These solutions were prepared by adding 5 mL of the unknown in each case and 0, 1, 2, 3 and 4 mL

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(corresponding to 0, 10, 20, 30 and 40 ppm) of the analyte; the total volume was brought to 10 mL using water. Each of the five solutions was then run through the column under a 60:40 ratio of buffer:acetonitrile.

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DATA AND RESULTS:

---------------- Section 1 ----------------

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Table and graph of retention times (ret. time or tR) of 100 ppm standards

peak #

ret. time (min.)

peak width (half-height)

peak width (proper)

area

height

area %

phthalic acid

salicylic acid

benzoic acid

p-nitrophenyl ethanoic acid

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1

0.879

0.056

0.095

19.772

4.888

2.064

2

1.035

0.041

0.069

938.174

347.153

97.936

1

0.875

0.057

0.097

14.215

3.365

9.212

2

1.028

0.061

0.103

17.688

4.093

11.463

3

2.252

0.076

0.130

122.403

23.770

79.325

1

0.880

0.034

0.058

8.385

3.736

2.445

2

1.032

0.065

0.111

15.875

3.352

4.628

3

3.779

0.118

0.201

318.766

40.519

92.927

1

0.883

0.057

0.097

20.282

4.888

1.075

2

1.038

0.094

0.159

10.715

1.507

0.568

3

4.509

0.146

0.249

1,855.364

190.601

98.357

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(! Sample chromatogram of test mixture under 80:20 buffer:acetonitrile!

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)! Isochratic separation and

! buffer:aceton -itrile

90:10

LC 1: Q1 – Q2 peak #

tR (min.)

width (halfheight)

width (proper)

area %

k'

1

0.912

0.061

0.103

1.929

2

2.171

0.077

0.132

29.718

1.381

3

6.654

0.161

0.274

2.634

6.299

4

12.186

0.298

0.507

9.389

12.366

5

15.454

0.492

0.837

56.330

15.951

1

0.884

0.060

0.103

2.697

2

1.036

0.043

0.074

29.069

0.172

3

2.276

0.095

0.162

4.132

1.574

4

3.829

0.126

0.215

10.524

3.330

5

4.566

0.166

0.283

53.578

4.164

1

0.633

0.277

0.471

5.772

2

0.769

0.073

0.124

2.396

3

0.879

0.040

0.068

25.363

0.389

4

1.313

0.071

0.120

4.283

1.074

5

1.929

0.069

0.117

8.633

2.047

6

2.105

0.088

0.149

53.554

2.325

1

0.608

0.415

0.707

3.582

2

0.670

0.035

0.060

2.470

3

0.840

0.064

0.108

17.475

0.383

4

0.991

0.054

0.093

3.905

0.631

5

1.354

0.062

0.105

51.181

1.228

Rs

4.863

80:20

2.960

70:30

1.324

60:40

1.496

details provided on following page

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! The first peaks are those of water !

width (proper) pertain to peak-widths; equal to peak-widths at half height ÷ 2.35 ! 4

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retention factor, k’ = [(tR)B - (tR)A] ÷ (tR)A

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resolution, Rs = 2•[(tR)B - (tR)A] ÷ [wB + wA]; where w? is peak-width (proper)

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the order of elution of the components does not change and so is not shown

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LC 1: Q3 buffer:acetonitrile

longest tR (min.)

Rs

Rs :longest tR ratio

90:10

15.454

4.863

0.315

80:20

4.566

2.96

0.648

70:30

2.105

1.324

0.629

60:40

2.539

1.496

0.589

the 80:20 mixture yields the best compromise between resolution and ret. time.

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LC 1: Q4 {refer to pg 18: Discussion section}

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**! LC 1: Q5: polarity index of the optimal “solvent blend”

= (0.8 ! 10.2) + (0.2 ! 5.8) = 9.32 where

": volume fraction P’: polarity index of particular substance A: aqueous buffer B: acetonitrile

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LC 1: Q6: composition of the hypothetical buffer:methanol for same P’AB

9.32 = (x ! 10.2) + (y ! 5.1) and x + y = 1 "

x:y = buffer:methanol = 83:17

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! sample run

O

P

S

*"! LC 1: Q7: isocratic vs gradient elution

time (min.)

65:35

0

1

0.697

0.062

1.877

80:20

0.6

2

0.866

0.101

26.007

90:10

0.8

3

1.101

0.097

5.023

4

1.589

0.109

8.585

5

1.737

0.205

58.509

peak #

tR (min.)

peak width (proper)

buffer:aceto -nitrile

area (%)

65:35

0

1

0.714

0.073

2.342

80:20

0.7

2

0.847

0.069

26.199

90:10

0.9

3

1.105

0.115

4.803

4

1.592

0.103

7.648

5

1.728

0.195

59.008

65:35

0

1

0.708

0.079

2.448

80:20

0.6

2

0.853

0.078

28.566

80:20

0.8

3

1.101

0.108

5.199

90:10

1

4

1.590

0.108

7.926

5

1.735

0.203

55.862

Rs

Rs/longest tR ratio

2.369

2.152

0.944

0.543

2.809

1.626

0.908

0.525

2.661

1.534

0.933

0.538

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the higher resolutions are of reduced significance since an instrument is as good as the lowest provided resolution

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only the four best of ten runs are shown

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Theoretically, a gradient elution allows for a quick separation of components combined with equal or better resolution since the mobile phase composition…

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*#! … can be changed in real-time during the experiment run. However, as the processed data suggests, isocratic elution was more efficient with a higher Rs / longest tR ratio compared to the highest provided by gradient elution (0.648 vs. 0.543).

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---------------- Section 2 ----------------

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LC 2: Q1 [analyte] (ppm)

0

10

20

30

40

peak #

tR (min.)

width (proper)

area (%)

1

0.679

0.070

3.392

water

2

0.827

0.088

24.631

phthalic

3

1.354

0.111

65.177

unresolved

4

3.300

0.263

6.800

1

0.678

0.066

2.361

water

2

0.827

0.089

18.407

phthalic

3

1.351

0.104

79.232

unresolved

1

0.679

0.070

1.958

water

2

0.821

0.090

13.164

phthalic

3

1.345

0.102

84.878

unresolved

1

0.679

0.069

1.631

water

2

0.823

0.089

10.680

phthalic

3

1.345

0.099

87.690

unresolved

1

0.679

0.069

1.326

water

2

0.823

0.090

9.162

phthalic

3

1.343

0.097

89.511

unresolved

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*%! Chromatogram of unknown + 0 ppm analyte mixture under 60:40 buffer:acetonitrile

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the furthest peak (peak # 4) is ignored since it is an erroneous signal

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LC 2: Q2

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conc of std. (ppm)

100

vol. of unknown (mL)

vol. of std. added (mL)

50

area (%)

0

65.177

1

79.232

2

84.878

3

87.69

4

89.511

best-fit line characteristics gradient y-intercept vol. intercept conc. in unknown

5.713 69.872 -12.231 24.463

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*'! uncertainty parameters std. error in y

4.433

N

5

Sxx

10

y bar

81.298

std. dev. in vol.

3.509

std. dev. in conc.

7.018

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concentration of p-nitrophenyl ethanoic acid in the unknown: (24.46 ± 7.018) ppm 24.46 ppm ± 28.7 %

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LC 2: Q3:

{refer to pg 18 Discussion section}

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DISCUSSION ---------------- Section 1 ---------------!

LC 1: Q4: change in order of elution There is to be no change in the order in which the different components of the

sample elute as that would require alteration of the polarity of the components’ molecules. Since there is no physical nor chemical alteration to the molecules of the components, there is no alteration in their polarities. Only the retention times are affected.

---------------- Section 2 ---------------!

LC 2: Q3: comments on standard addition

Advantages:

allows for analysis of complex samples with potentially significant matrix effects.

Downside:

assumption of a linear relationship between the dependant and independent variables; in this case, detector signal vs. concentration of a standard.

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CONCLUSION: The buffer:acetonitrile composition ratio of 80:10 was found to offer the best compromise of resolution and retention times. Its polarity index was calculated to be 9.32, and that a 83:17 composition of the buffer:methanol would be required to produce the same polarity index. Secondly, it is concluded that isocratic elution provided better results than gradient elution; this may be attributed to less than optimum choice of buffer:acetonitrile compositions and durations. The concentration of the p-nitrophenyl ethanoic acid was, by the methods of linear regression analysis and standard additions, calculated to be 24.46 ppm ± 28.7 %.