Experimental Practice 1

Seneca College
HPLC METHOD DEVELOPMENT
Course PFT869
_____________________________________________________________________________________

Student Name :_____________________ Date :___________________

Objective:
1. Learn how to select column and buffer solution for acidic and neutral compounds
during HPLC method development
2. Understand the relevance of stationary phase and pH selection on retention time
and resolution

Page 1

Instructor: Vikas Pande

Experimental Practice 1

Seneca College
HPLC METHOD DEVELOPMENT
Course PFT869
_____________________________________________________________________________________

Material Information:
Methylparaben

Molecular Weight - 152.1

Ethylparaben
Molecular Weight 166.2

Propylparaben
Molecular Weight 180.2

n-Butylparaben
Molecular Weight 194.2

Benzoic Acid
Molecular Weight 122.1

Page 2

Instructor: Vikas Pande

Experimental Practice 1

Seneca College
HPLC METHOD DEVELOPMENT
Course PFT869
_____________________________________________________________________________________

HPLC Group 1 (Three users)

1. Solutions
1.1. Solution A: Mix 2 mL of phosphoric acid with 2000 mL of purified water. Filter.
1.2. Solution B: Acetonitrile
2. Mobile Phase Preparation
Mix 1200 mL of Solution A with 800 mL of Solution B, degas. (Total Volume 2 L)
3. Sample Solvent Preparation
Mix purified water and Solution B (acetonitrile) in volume ratio of 40: 60.
4. Preparation of Identification and Standard Solutions
4.1. Accurately weigh about 10 mg each of methylparaben, ethylparaben,
propylparaben, n-butylparaben and benzoic acid and transfer quantitatively into
separate five 10 mL volumetric flasks. To each flask, add about 5 mL of
acetonitrile, shake until dissolved and then dilute to volume each flask with water
to give solutions 1, 2, 3, 4 and 5 respectively.
(Concentration: 1 mg per mL of methylparaben, ethylparaben, propylparaben, nbutylparaben and benzoic acid)
4.2. Further dilute 1.0 mL of each of the above solution to 10.0 mL into separate
volumetric flasks with Sample Solvent (water /acetonitrile : 40/60). Filter a
portion of each solution through the membrane filter using filtration kit and collect
the filtrates separately in to HPLC vials. Name the vials as a, b, c, d and e
containing methylparaben, ethylparaben, propylparaben, n-butylparaben and
benzoic acid solutions each.
(Concentration: 100 g per mL of methylparaben, ethylparaben, propylparaben,
n-butylparaben and benzoic acid).
Prepare a set of three vials for three users in Group 1.

Page 3

Instructor: Vikas Pande

Experimental Practice 1

Seneca College
HPLC METHOD DEVELOPMENT
Course PFT869
_____________________________________________________________________________________

5. Sample Preparation
Pipette1.0 mL each of of solutions 1, 2, 3, 4 and 5 and quantitatively transfer to
the same 10 mL volumetric flask, dilute to volume with sample solvent. Filter a
portion through membrane filter using sample filtration kit and collect the filtrate
into HPLC vial.
(Concentration: This solution contains 100 g per mL of methylparaben,
ethylparaben, propylparaben, n-butylparaben and benzoic acid)
Note: Stability of Sample Preparation: The sample preparation is stable for up to
48 hours at room temperature).
Prepare a set of three vials for three users in Group 1.
Chromatographic Conditions
5.1. Column: Any brand (e.g. Waters C8) C8, 150 x 4.6 mm
5.2. Column Temperature: Room Temperature
5.3. Flow Rate: 1.0 mL per min.
5.4. Injection Volume: 15 L
5.5. Detection wavelength: 225 nm
5.6. Sample cooler compartment: Room Temperature
5.7. Run Time: 25 minutes (The run time may be adjusted depending upon the
elution time)
6. Chromatographic Procedure:
6.1. Inject 15L of Sample Solvent; check baseline drift or any other signs of system
instability. Repeat injections until the stable baseline is obtained. Note the
retention time of any solvent related peaks. All HPLC users in group 1 will inject
this solution.

Page 4

Instructor: Vikas Pande

Experimental Practice 1

Seneca College
HPLC METHOD DEVELOPMENT
Course PFT869
_____________________________________________________________________________________

6.2. Inject 15 L of Identification / Standard solution. Each HPLC users in group will
inject the solutions as per plan given below:

HPLC User 1

Solution a

Solution b

Solution c

HPLC User 2

Solution c

Solution b

Solution d

HPLC User 3

Solution d

Solution b

Solution e

1.1. Inject 15 L of sample preparation. The typical chromatogram is given in Figure

1. All HPLC users in group 1 will inject this solution.
2. Column Storage Conditions
After completion of the sequence, flush the column with acetonitrile : water
mixture in volume ratio of 60:40 for at least 10 minutes.
3. Calculations
Calculate the content of ethylparaben in the Sample Preparation using the
following formula:
Au
% Ethylparaben

----As

Ws
x

----10

1
x

----10

10
x

----Wu

10
x

-----

100

Where:
Au: Area of Sample
As: Area of Standard
Ws: Weight of Standard ethylparaben
Wu: Weight of ethylparaben (in this case, it is same as the weight of standard
ethylparaben)

Page 5

Instructor: Vikas Pande

Experimental Practice 1

Seneca College
HPLC METHOD DEVELOPMENT
Course PFT869
_____________________________________________________________________________________

HPLC Group 2 (Three users):

1. Solutions
1.1. Mobile Phase A (Buffer solution): Add approximately 400 - 600 mL of deionized
water to a 2 L volumetric flask. Add 30 mL acetic acid to the flask and mix.
Weigh 30 g of ammonium acetate into a beaker, and dissolve in a 200 mL of
deionized water. Transfer aqueous ammonium acetate solution to the 2L flask
containing the aqueous acetic acid. Mix and bring to volume with deionized
water. Mix well and filter.
1.2. Solution B: Methanol
2. Sample Solvent Preparation
Mix purified water and Solution B (acetonitrile) in volume ratio of 40: 60.
3. Preparation of Identification and Standard Solutions
Accurately weigh about 10 mg each of methylparaben, ethylparaben,
propylparaben, n-butylparaben and benzoic acid and transfer quantitatively into
five 10 mL volumetric flasks. To each flask, add about 5 mL of acetonitrile,
shake until dissolved and then dilute to volume each flask with water to give
solutions 1, 2, 3, 4 and 5.
(Concentration: Each of the solution contains 1 mg per mL of methylparaben,
ethylparaben, propylparaben, n-butylparaben and benzoic acid)
3.1. Further dilute 1.0 mL of each of the above solution to 10.0 mL separately in
volumetric flask with Sample Solvent (water /acetonitrile : 40/60). Filter a portion
of each solution through the membrane filter using filtration kit and collect the
filtrates separately in to HPLC vials. Name the vials as a, b, c, d and e
containing methylparaben, ethylparaben, propylparaben, n-butylparaben and
benzoic acid solutions respectively.
(Concentration: 100 g per mL of methylparaben, ethylparaben, propylparaben,
n-butylparaben and benzoic acid).
Prepare a set of three vials for three users in Group 2.
Page 6

Instructor: Vikas Pande

Experimental Practice 1

Seneca College
HPLC METHOD DEVELOPMENT
Course PFT869
_____________________________________________________________________________________

4. Sample Preparation
Pipette1.0 mL each of of solutions 1, 2, 3, 4 and 5 and quantitatively transfer to
the same 10 mL volumetric flask, dilute to volume with sample solvent. Filter a
portion through membrane filter using filtration kit and collect the filtrate into
HPLC vial.
(Concentration: This solution contains 100 g per mL of methylparaben,
ethylparaben, propylparaben, n-butylparaben and benzoic acid)
Note: Stability of Sample Preparation: The sample preparation is stable for up to
48 hours at room temperature).
Prepare a set of three vials for three users in Group 2.
5. Chromatographic Conditions
5.1. Column: Any brand C18, 150 x 4.6 mm
5.2. Column Temperature: Room Temperature
5.3. Injection Volume: 15 L
5.4. Detection wavelength: 254 nm
5.5. Sample cooler compartment: Room Temperature
5.6. Run Time: The run time may be adjusted depending upon the elution time)
Time
Initial - 0
25.0
28.0
31.0
40.0

Page 7

Flow rate
(mL/min)
1.0
1.0
1.0
1.0
1.0

A (%)

B (%)

90
30
30
90
90

10
70
70
10
10

Instructor: Vikas Pande

Experimental Practice 1

Seneca College
HPLC METHOD DEVELOPMENT
Course PFT869
_____________________________________________________________________________________

6. Chromatographic Procedure:
6.1. Inject 15L of Sample Solvent; check baseline drift or any other signs of system
instability. Repeat injections until the stable baseline is obtained. Note the
retention time of any solvent related peaks. All HPLC users in group 2 will inject
this solution.
6.2. Inject 15 L of Identification / Standard solution. Each HPLC users in group will
inject the solutions as per plan given below:
HPLC User 1

Solution a

Solution b

Solution c

HPLC User 2

Solution c

Solution b

Solution d

HPLC User 3

Solution d

Solution b

Solution e

1.1. Inject 15 L of sample preparation. All HPLC users in group 2 will inject this
solution.
7. Column Storage Conditions
After completion of the sequence, flush the column with acetonitrile : water or
methanol : water mixture in volume ratio of 50:50 for at least 10 minutes.
8. Calculations
Calculate the content of ethylparaben in the Sample Preparation using the
following formula:
Au
% Ethylparaben

----As

Ws
x

----10

1
x

----10

10
x

----Wu

10
x

-----

100

Where:
Au: Area of Sample
As: Area of Standard
Ws: Weight of Standard ethylparaben
Wu: Weight of ethylparaben (in this case, it is same as the weight of standard
ethylparaben)
Page 8

Instructor: Vikas Pande