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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

Methylparaben

Molecular Weight - 152.1

Ethylparaben

Ethylparaben

Molecular Weight 166.2

Propylparaben

Propylparaben

Molecular Weight 180.2

n-Butylparaben

n-Butylparaben

Molecular Weight 194.2

Benzoic Acid

Benzoic Acid

Molecular Weight 122.1

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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, n- butylparaben 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 µg 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 µg 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 C 8 ) C 8 , 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 µL

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 15µL 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 µL 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 µL 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

Ws

1

10

10

% Ethylparaben

=

-----

x

-----

x

-----

x

-----

x

-----

x

100

 

As

10

10

Wu

1

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)

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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 µg per mL of methylparaben, ethylparaben, propylparaben, n-butylparaben and benzoic acid).

Prepare a set of three vials for three users in Group 2.

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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 µg 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 C 18 , 150 x 4.6 mm

5.2. Column Temperature: Room Temperature

5.3. Injection Volume: 15 µL

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)

 

Flow rate

   

Time

(mL/min)

A (%)

B (%)

Initial - 0

1.0

90

10

25.0

1.0

30

70

28.0

1.0

30

70

31.0

1.0

90

10

40.0

1.0

90

10

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Instructor: Vikas Pande

Experimental Practice 1

Seneca College

HPLC METHOD DEVELOPMENT Course PFT869

6. Chromatographic Procedure:

6.1. Inject 15µL 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 µL 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 µL 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

Ws

1

10

10

% Ethylparaben

=

-----

x

-----

x

-----

x

-----

x

-----

x

100

 

As

10

10

Wu

1

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)

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Instructor: Vikas Pande