Selectivity and/or Specificity

Chromatography

Presented by :
Indarto Adikusumo

112210101036

Zulviyati

112210101038

Yuniar Wahyu Rahmawati

112210101042

Nikmatur Rohmah

112210101044

What is Chromatography?

Chromatography is the ability to separate molecules using partitioning

characteristics of molecule to remain in a stationary phase versus a mobile
phase. Once a molecule is separated from the mixture, it can be isolated and
quantified.

Chromatographic separation

Mobile phase flows through column, carries analyte.

1. Gas = Gas Chromatography (GC)

2. Liquid = Liquid Chromatography (LC), Thin Layer Chromatography (TLC)
3. Supercritical fluid = Supercritical Fluid Chromatography (SFC)

Stationary phase stays in a place, does not move.

1. GC, LC placed inside of the column

2. TLC layer of a sorbent on the plate

The SEPARATION is based on the partitioning between the mobile and

stationary phase.

Steps in the Chromatographic Methods Validation

Process

The process of validating chromatographic methods can be broken down into

four steps. These steps include:

1. Method evaluation and further method development,

2. Final method development and trial methods validation,
3. Formal methods validation, and
4. Formal data review and report issuance.

The following are typical analytical performance characteristics

which maybe tested during methods validation:

Accuracy

Precision

Repeatability

Intermediate precision

Specificity

Detection limit

Quantitation limit

Linearity

Range

Robustness

System suitability determination

Forced degradation studies

Solution stability studies

Filter retention studies

Extraction efficiency studies

Additional methods validation information

Representative instrument output

Representative calculations

Listing and characterization of known impurities

Degradation pathways (if known)

Description of Specificity and/or Selectivity

Selectivity: the selectivity of an assay is a measure of the extent to which the

method can determine a particular compound in the analysed matrices without
interference from matrix components. A method that is perfectly selective for
an analyte or group of analytes is specific.

Specificity: the validation procedure should confirm the ability of the method to
unequivocally assess the analyte in the presence of other components that may
be present (for example, impurities, degradation products and matrix
components).

Experimental Determination of Specificity and/or Selectivity

Make an injection of a blank/diluent during each chromatographic run to ensure

there are no interferences.

Prepare a placebo for both the 5-mg and 10-mg tablet strength.

Inject each placebo preparation twice.

Confirm that no peaks can be attributed to the blank/diluent or placebo.

Define any peaks observed by RRT indexed to the active component.

Prepare and inject twice, samples of related A and related B at the

impurity/degradant (e.g., related compound) specification limit of 0.50%.

Prepare two separate spiked solutions containing the active at 100% and each
impurity (from two separate impurity preparations) at the limit.

Inject the spiked samples twice to confirm specificity.

Acceptance Criteria

Confirm that no interference in the elution zone of the active occurred

from the blank/diluent, internal standard (if applicable), or the placebo.

Confirm that the impurities/degradants do not elute in the elution zone of

the active.

The Determination Acetaminophen by High Performance

Liquid Chromatography

Preparation of Eluent :
Methanol is mixed with water at a ratio of 75: 25 to 500 mL volume and then filtered
Preparation of standard solution:
6.25 mg of acetaminophen dissolved in 25 mL of eluent

6.25 mg / 25 mL x 1000 = 250 ppm

Of the solution in the pipette 1,2,3,4 and 5 mL and then diluted to 10 mL

Standart 1 = 1 mL/ 10 mL x 250 ppm = 25 ppm

Standart 2 = 2 mL/ 10 mL x 250 ppm = 50 ppm

Standart 3 = 3 mL/ 10 mL x 250 ppm = 75 ppm

Standart 4 = 4 mL/ 10 mL x 250 ppm = 100 ppm

Standart 5 = 5 mL/ 10 mL x 250 ppm = 125 ppm

results
concentrati
on (ppm)

area

Retention
time

25

2488335

1,51

50

4126411

1,50

75

6744285

1,51

100

8901360

1,51

125

10667748

1,51

Regression equation:
y= 84535x + 24549

Preparation of sample

6 tablets crushed, then weighed 27 mg of powder (27 mg powder contains 25

mg of acetaminophen). Dissolved into 25 mL of eluent.

Of the solution is then taken 1 mL and then diluted to 10 mL

Result

Peak
1
2

1st injection

Retention
time

Area

1,51

7762424

2,31

13143

Peak

Retention
time

Area

0,06

7100

0,26

57665

1,51

7777816

2,30

6844

2nd injection

Calculation

Sample 1 ( y = 7762424)
y= 84535x + 24549
7762424 = 84535x + 24549
x

= 91,534 ppm

91,534 ppm x 10 x 25 mL / 1000 = 22,883 mg

Sample 2 ( y = 7777816 )
y= 84535x + 24549
7777816 = 84535x + 24549
x

= 91,717 ppm

91,717 ppm x 10 x 25 mL / 1000 = 22,929

Average = 22,883 + 22,929 /2 =22,906 mg
% concentration in sample = 22,906 mg / 25 mg x 100% = 91,624 %

Thank you