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: 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 p-

nitrophenyl 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.

! $!

EXPERIMENTAL PROCEDURE:

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

! 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 buffer-

acetonitrile 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

! %!

(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.

 ! &!

DATA AND RESULTS:

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

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

peak
peak width
peak # ret. time (min.) width area height area %
(half-height)
(proper)

phthalic acid

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

salicylic acid 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

benzoic acid 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

p-nitrophenyl
2 1.038 0.094 0.159 10.715 1.507 0.568
ethanoic acid
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
! LC 1: Q1 – Q2
width
buffer:aceton
peak # tR (min.) (half- width (proper) area % k' Rs
-itrile
height)

1 0.912 0.061 0.103 1.929

2 2.171 0.077 0.132 29.718 1.381

90:10 3 6.654 0.161 0.274 2.634 6.299

4 12.186 0.298 0.507 9.389 12.366

4.863
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

80:20 3 2.276 0.095 0.162 4.132 1.574

4 3.829 0.126 0.215 10.524 3.330

2.960
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

70:30
4 1.313 0.071 0.120 4.283 1.074

5 1.929 0.069 0.117 8.633 2.047

1.324
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

60:40 3 0.840 0.064 0.108 17.475 0.383

1.496
4 0.991 0.054 0.093 3.905 0.631

5 1.354 0.062 0.105 51.181 1.228

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

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

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

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

! LC 1: Q3
longest tR Rs :longest tR
buffer:acetonitrile Rs
(min.) 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.

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

! **!

! 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

! 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

 ! *"!

! LC 1: Q7: isocratic vs gradient elution

peak
sample buffer:aceto time tR area Rs/longest tR
peak # width Rs
run -nitrile (min.) (min.) (%) ratio
(proper)

65:35 0 1 0.697 0.062 1.877

80:20 0.6 2 0.866 0.101 26.007

2.369 2.152
O 90:10 0.8 3 1.101 0.097 5.023

4 1.589 0.109 8.585

0.944 0.543
5 1.737 0.205 58.509

65:35 0 1 0.714 0.073 2.342

80:20 0.7 2 0.847 0.069 26.199

2.809 1.626
P 90:10 0.9 3 1.105 0.115 4.803

4 1.592 0.103 7.648

0.908 0.525
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

2.661 1.534
S 80:20 0.8 3 1.101 0.108 5.199

90:10 1 4 1.590 0.108 7.926

0.933 0.538
5 1.735 0.203 55.862

!
! the higher resolutions are of reduced significance since an instrument is as good

as the lowest provided resolution

! only the four best of ten runs are shown

! Theoretically, a gradient elution allows for a quick separation of components

combined with equal or better resolution since the mobile phase composition…
! *#!

… 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).
! *$!

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

! LC 2: Q1

[analyte] peak tR width area

(ppm) # (min.) (proper) (%)

1 0.679 0.070 3.392 water

2 0.827 0.088 24.631 phthalic

0
3 1.354 0.111 65.177 unresolved

4 3.300 0.263 6.800 -

1 0.678 0.066 2.361 water

10 2 0.827 0.089 18.407 phthalic

3 1.351 0.104 79.232 unresolved

1 0.679 0.070 1.958 water

20 2 0.821 0.090 13.164 phthalic

3 1.345 0.102 84.878 unresolved

1 0.679 0.069 1.631 water

30 2 0.823 0.089 10.680 phthalic

3 1.345 0.099 87.690 unresolved

1 0.679 0.069 1.326 water

40 2 0.823 0.090 9.162 phthalic

3 1.343 0.097 89.511 unresolved

! *%!

! Chromatogram of unknown + 0 ppm analyte mixture under 60:40

buffer:acetonitrile

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

! *&!

! LC 2: Q2

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conc of std. (ppm) 100

vol. of unknown (mL) 50

vol. of std. added

area (%)
(mL)

0 65.177

1 79.232

2 84.878

3 87.69

4 89.511

best-fit line characteristics

gradient 5.713

y-intercept 69.872

vol. intercept -12.231

conc. in unknown 24.463

! *'!
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

! concentration of p-nitrophenyl ethanoic acid in the unknown:

(24.46 ± 7.018) ppm

24.46 ppm ± 28.7 %

! 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.
! *)!

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 %.