Chromatography 4

Question 1

List the three(3) methods for quantitative in GC and HPLC

Answer 1

internal normalisation

• use of external standards
• use of internal standards

Question 2

Explain Internal normalization in Gas Chromatography

Answer 2

In an ideal case, an equal amount of any compound will give the same peak area on a chromatograph.
In such cases internal normalisation can be used to find the percentage of each component in the mixture.

This ideal case only occurs when the analytes are very similar and the detector response is linear. Usually this is not the case (i.e. the same number of moles of different components give different detector responses). In such cases the detector response must be calculated from a set of standards (external calibration). Or an internal standard must be used

Question 3

What is the formula used in calculating the % of each component in a mixture in an internal normalization of GC?

Answer 3

% of component x = (area of component x peak / total area of all peaks) x 100

Question 4

Facts about External standards in GC quantitative analysis

Answer 4

In this approach, a series of samples each containing various known amounts of the test substance is subjected to GC under identical conditions. A calibration curve of response area vs. concentration is then constructed and from this, the concentration of the test sample may be read from the GC response

Question 5

An analyst is particularly interested in the exact concentration of aspirin within a tablet extract. They therefore prepared a range of known concentrations of aspirin in methanol (0-1% v/v), injected them into the GC and recorded the area of the aspirin peak in each resulting chromatogram. Using this data a calibration graph of aspirin concentration vs. peak area was plotted

Answer 5

See chromatography 4 lecture, slide 5 for graph and other parameters

answer: 0.70%

Question 6

An aspirin tablet was dissolved in 10 mL of water (pH7) (Solution A). 1 mL of this solution was further diluted by placing it in a 10 mL flask and diluting to the mark with water (to make Solution B). Solution B was analysed by HPLC, and the peak area of the chromatographic peak for aspirin was found to be 0.25 cm2. Calibration standards had been prepared previously with concentrations ranging from 0.0 %w/v to 2.0 %w/v. The calibration curve of the standards had a linier fit of y = 0.955x + 0.01. What is the concentration of aspirin in Solution A?

Answer 6

1.251%w/v

Question 7

State the weakness of external standards in quantitative analysis of GC?

Answer 7

GC must be performed under exactly the same conditions which is not possible (changes in temperature column age etc).

Accurate microlitre volumes must be injected. Very difficult in GC – manual injections particularly poor. Much human error.

Question 8

How to overcome the problems associated with injection of samples into GC not being repeatable

Answer 8

an internal standard (something added to the thing you want to analysis) is added before injection onto the GC. This is known as “spiking”

Question 9

Facts about Internal standard

Answer 9

An internal standard must be;
closely related to the assayed component
be completely resolved from both the assay component and all other components of the mixture
ideally have a retention time close to that of the compound under analysis
ideally be used at a concentration similar to that you expect to find in the sample under analysis

Question 10

State the BP format for assays requiring internal standards

Answer 10

1. A calibration standard containing approximately equal amounts of the pure standard (of the component/s to be measured in the unknown) and an internal standard (SOLUTION 1)
2. An extract from the sample containing no internal standard (to check for interference from the formulation matrix) (SOLUTION 2) (validation)
3. An Extract from the sample containing the same amount of internal standard as solution 1 (SOLUTION 3) (unknown).

Question 11

State how detectors respond in a standard solution

Answer 11

Detectors do not always respond equally to the same amounts of different compounds

Therefore we have to work out a response factor (Rf) from the standard solution to tell us how much more/less the detector responds to the calibration Standard (analyte).

Question 12

state the equation for calculating the Rf for standard solution (Rfs)

Answer 12

Rf for standard solution (Rfs) =Area of peak due to analyte/Area of peak due to Internal Standard

Question 13

What is the formula for calculating the Rf for unknown mixture (Rfu)

Answer 13

Rf for unknown mixture (Rfu) =Area of peak due to analyte/Area of peak due to Internal Standard

Question 14

Facts about Solution 3: Unknown solution (Sample solution spiked with IS)

Answer 14

A known concentration of the internal standard (the same concentration used in solution 1) is added to the unknown mixture. The ratio between the area of the sample peak compared to the internal standard is known as the response factor (Rfu) for the unknown solution

Question 15

State the equation used to calculate the concentration of an analyte in an unknown mixture spiked with an internal standard

Answer 15

conc of analyte in sample= Rfu x conc analyte in standard solution/Rfs

Question 16

State the advantage of using an internal standard of quantitative analysis in GC

Answer 16

since all Responses are treated as ratios to the internal standard, this technique;

1 . Compensates for the changes in chromatographic conditions (providing these changes effect the sample and internal standard equally

2. Is unaffected by exact volume injected.

Question 17

The analysis of methyl testosterone in a tablet formulation using testosterone as an internal standard - Data

Answer 17

SEE slide 15 and onwards as it is a worked example from data sets

Question 18

Facts about Quantitative analysis in HPLC

Answer 18

If you can get quite reproducible results in between runs (often in HPLC) you do not need an internal standard and can use external calibrations:

1. Weigh out the analytical standard for the analyte and dissolve it in a precise volume of solvent  stock solution.
2. Prepare suitable dilutions (hopefully to cover the expected range of concentration of your unknown solution). Run them on the HPLC and plot a calibration graph.
3. Make up standard solution of your tablet/formulation and inject. Use calibration graph to determine the concentration of analyte in the mixture from its peak area.

Question 19

Analysis of paracetomal tablets using a calibration curve

Answer 19

see slide 20 and onwards for data and calculations

Question 20

facts about Assay using calibration against an internal standard

Answer 20

Not often required since HPLC is quite reproducible if you run an external calibration curve on the same day as your sample to analyse.

However, sometimes it is needed if you are concerned that your extraction procedure may not be complete. This is especially true in ointments and creams. Here an internal standard is added to the sample before it is extracted so any losses will effect both the internal standard and the analyte.

Question 21

state the applications of GC in Biomedical and Pharmaceutical analysis

Answer 21

1. Measurement of drugs and their metabolites
   in biological fluids.
2. The characterisation of some unformulated
   drugs, particularly with regard to detection
   of process impurities.
3. Limit tests for solvent residues and other
   volatile impurities in drug substances
4. Sometimes used for quantification of drugs
   in formulations, particularly if the drug lacks
   a chromophore
5. Characterisation of some raw materials used
   in the synthesis of drug molecules.
6. Characterisation of volatile oils, proprietary
   cough mixtures and tonics, and fatty acids in
   fixed oils.

Question 22

Strengths OF GC

Answer 22

Capable of the same quantitative accuracy and precision as HPLC, particularly when used in conjunction with an internal standard.
Much greater separating power than HPLC when used with capillary columns
Readily automated
Can be used to determine compounds which lack chromophores
The mobile phase does not vary and does not require disposal. Helium is relatively cheap compared to organic solvents used in HPLC.

Question 23

weaknesess of GC

Answer 23

Only thermally stable and volatile compounds can be analysed.
The sample may require derivatisation to convert it to a volatile form
Quantitative sample injection is more difficult than HPLC because of small volumes injected
Aqueous solutions and salts cannot be injected into the instrument.

Question 24

Applications of HPLC in Biomedical and Pharmaceutical analysis

Answer 24

Measurement of drugs and their metabolites in biological fluids
Provides an accurate, precise and robust method for quantitative analysis of pharmaceutical products and is the industry standard method for this purpose.
Monitoring the stability of pure drug substances in formulations with quantitation of any degradation products
Determination of partition coefficients and pKa values of drugs and of drug protein binding

Question 25

Strengths of using HPLC

Answer 25

Easily controlled and precise sample introduction ensures quantitative precision
Good technology – advancing rapidly
Variety of columns detectors, stationary phases and mobile phases – great diversity and applications
Less risk of sample degradation as in GC – and can be used for non-volatile compounds which can not be run with GC.
Can be readily automated

Question 26

Weaknesses of using HPLC

Answer 26

Still require a reliable and inexpensive detector which can monitor compounds lacking a chromaphore
Large amount of organic solvent waste
Drugs often have to be extracted from their formulations prior to analysis.