Download to read offline
QUANTIFICATION METHODS OF HPLC
The document is a seminar presentation on quantification methods in High-Performance Liquid Chromatography (HPLC) aimed at first-year M.Pharm students. It covers the steps involved in quantitative analysis, the principles and requirements of chromatographic techniques, and various quantification methods such as external standard analysis and internal standard analysis. It also highlights the importance of calibration curves and the standard addition method for accurate quantification in complex samples.
More Related Content
Similar to QUANTIFICATION METHODS OF HPLC
More from Raghavendra institute of pharmaceutical education and research .
QUANTIFICATION METHODS OF HPLC
- 1. QUANTIFICATION METHODS OFHPLC A Seminar as a part of curricular requirement for 1st year M.Pharm I semester Presented by G.HIMA BINDU Regd no.(20L81S0703) RIPER AUTONOMOUS NAAC & NBA (UG) SIRO-DSIR Under the Guidance of/Mentorship of Dr. Ramalingam, M.Pharm., Ph.D., R&D Director Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 1
- 2. Contents Introduction Stepsinvolved in quantitative analysis Chromatographic requirements Principles of quantitative analysis Different Quantification methods References RIPER AUTONOMOUS NAAC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 2
- 3. Quantitative Analysis After thepeaks have been integrated and identified, the next step in the analysis is quantification. “Chemical analysis designed to determine the amounts or proportions of the components of a substance”. Quantification uses peak areas or heights to determine the concentration of a compound in the sample. RIPER AUTONOMOUS NAAC & NBC(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 3
- 4. RIPER AUTONOMOUS NAAC & NBC(UG) SIRO-DSIR Raghavendra Instituteof Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 4
- 5. Steps Involved InQuantitative Analysis Known the compound you are analysing. Establish a method for analysing samples containing this compound. Analyse a sample containing a known concentration (the Standard) of the compound to obtain the response due to that concentration. RIPER AUTONOMOUS NAAC & NBA (UG( SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 5
- 6. Analyse the samplecontaining an unknown concentration of the compound to obtain the response due to the unknown concentration. Compare the response of the unknown concentration to the response of the known (standard) concentration to determine how much of the compound is present. Cntd. RIPER AUTONOMOUS NAAC & NBC(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 6
- 7. Chromatographic Requirements Good Chromatographicresolution. Response of the compound is within the linear range of detector. Good peak performance. No peak shoulders or tailing. Peak symmetry. RIPER AUTONOMOUS NAAC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 7
- 8. Principles of QuantitativeAnalysis Each detector will produce a response that depends upon the amount of analyte to which it is responding. Some general requirements that must be met before a quantitative analysis can be taken include: 1)Identity of the component to be analysed should be known. 2)Best possible separation of the component to be achieved. 3)Standards of known purity should be available(otherwise accuracy may be compromised). 4)If the internal standards are not used, then mobile phase flow rate and injection volume should be reproducible. RIPER AUTONOMOUS NACC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 8
- 9. Primary methodologies forquantitative analysis RIPER AUTONOMOUS NACC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 9
- 10. Area % orHeight % (Normalization) The area % calculation procedure reports the area of each peak in the chromatogram as a percentage of the total area of all peaks. Height % calculation procedure reports the height of each peak in the run as a percentage of the total height of all peaks in the run. RIPER AUTONOMOUS NACC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 10
- 11. Cntd. RIPER AUTONOMOUS NAAC & NBC(UG) SIRO-DSIR Raghavendra Instituteof Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 11
- 12. Peak area isespecially useful because HPLC peaks may be tailed. In this case, because peak heights may vary, area values are more repeatable. For trace analysis, when the peak of interest is very small, use peak height for calculations, this reduces the error sustained in small changes in peak start and end time variation. Areas of a chromatographic peak will change, if the flow rate changes. Be aware that poorly maintained pump systems will have an unstable flow rate resulting in loss of peak area precision. Peak Height or Peak Area RIPER AUTONOMOUS NAAC & NBC(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 12
- 13. For this reason,always perform routine maintenance on the pump seals, check valves and filters. Make certain that the pump has been primed and dissolved gasses removed. Both peak height and area measurements will be irreproducible if injection volume varies. Cntd. RIPER AUTONOMOUS NAAC & NBC(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 13
- 14. External Standard Quantification Theexternal standard (ESTD) is the basic quantification procedure in which both calibration and unknown samples are analysed under the same conditions. The external standard method is the technique used most frequently to gather quantitative information from a chromatogram. In this case, a pure reference substance is injected in increasing concentrations. The peak areas or peak heights obtained are plotted versus the concentration. RIPER AUTONOMOUS NAAC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 14
- 15. These calibrationcurves should show a constant slope (linear curves) and the intercept should be as close to zero as possible. A calibration curve must be recorded for each component of interest. The peak areas heights of the sample chromatogram are then compared with those in the calibration curve. CONC unknown = area unknown/ area known × conc known RIPER AUTONOMOUS NAAC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 Cntd. 15
- 16. Cntd. External Standard Quantification RIPER AUTONOMOUS NAAC& NBC(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 16
- 17. Internal Standard Analysis Adefined amount of a chosen compound (internal standard) is added to the sample, so that two peaks(that of the internal standard and that of the compound of interest) must be measured to obtain one result. Requirements of Internal Standard: a.It must not already be present in the sample. b.It should elute close to the peak of interest and the two peak sizes should not differ too much. RIPER AUTONOMOUS NAAC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 17
- 18. RIPER AUTONOMOUS NAAC& NBA(UG) SIRO-DSIR Raghavendra Institute ofPharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 c. If possible, peaks should not overlap, and no chemical interaction the internal standard and other sample components should occur. d. The internal standard must also have good storage, stability and be available in high purity. Cntd. 18
- 19. CONC UNKNOWN =Area of internal STD known/ Area of internal std unknown × Area of unknown/ Area of known RIPER AUTONOMOUS NAAC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 Cntd. 19
- 20. RIPER AUTONOMOUS NACC& NBA(UG) SIRO-DSIR Raghavendra Institute ofPharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 Standard Addition Methods 1.Most convenient when a small number of samples are to be analysed. 2.Useful when the analysis is present in a complicated matrix and no ideal blank is available. 20
- 21. Standard Addition MethodProcedure 1.Add one or more increments of a standard solution to sample aliquots of the same size. 2.Each mixture is then diluted to the same volume. 3.Prepare a plot of Analytical Signal versus: a) volume of standard solution added or b)concentration of analyte added. RIPER AUTONOMOUS NACC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 21
- 22. 4. The x-interceptof the standard addition plot corresponds to the amount of analyte that must have been present in the sample (after accounting for dilution). 5. The standard addition method assumes: a) the curve is linear over the concentration range. b) the y-intercept of a calibration curve would be 0. RIPER AUTONOMOUS NACC& NBA(UG) SIRO-DSIR Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 Cntd. 22
- 23. RIPER AUTONOMOUS NACC& NBA(UG) SIRO-DSIR Raghavendra Institute ofPharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 Cntd. 23
- 24. References RIPER AUTONOMOUS NACC& NBA(UG) SIRO-DSIR 1.Quality- Control AnalyticalMethods: High Performance Liquid Chromatography “ International Journal of Pharmaceutical compounding, Vol. 8 No. 3 May/June 2004. 2.Sharma Y. Elementary organic spectroscopy: principles and chemical applications. Chand and company Ltd, New Delhi, India. 2009:23. 3.A Text Book of Principles of Instrumental Analysis: Skoog Raghavendra Institute of Pharmaceutical Education and Research- Autonomous K.R.Palli Cross, Chiyyedu, Ananthapuramu, A.P. - 515721 24
- 25.
Editor's Notes
- #3 INTRODUCTION STEPS INVLOVED IN QUANTITATIVE ANALYSIS CHROMATOGRAPHIC REQUIREMENTS PRINCIPLES OF QUANTITATIVE ANALYSIS Area % Normalization External Standard Quantification Internal Standard Analysis CALIBRATION CURVE
- #4 After the peaks have been integrated and identified, the next step in the analysis is quantification. “Chemical analysis designed to determine the amounts or proportions of the components of a substance”. Quantification uses peak areas or heights to determine the concentration of a compound in the sample.
- #6 Known the compound you are analysing. Establish a method for analysing samples containing this compound. Analyse a sample containing a known concentration (the Standard) of the compound to obtain the response due to that concentration. Analyse the sample containing an unknown concentration of the compound to obtain the response due to the unknown concentration. Compare the response of the unknown concentration to the response of the known (standard) concentration to determine how much of the compound is present.
- #8 Good Chromatographic resolution. Response of the compound is within the linear range of detector. Good peak performance. No peak shoulders or tailing. Peak symmetry.
- #9 Each detector will produce a response that depends upon the amount of analyte to which it is responding. Some general requirements that must be met before a quantitative analysis can be taken include: Identity of the component to be analysed should be known. Best possible separation of the component to be achieved. Standards of known purity should be available(otherwise accuracy may be compromised). If the internal standards are not used, then mobile phase flow rate and injection volume should be reproducible.
- #11 The area % calculation procedure reports the area of each peak in the chromatogram as a percentage of the total area of all peaks. Height % calculation procedure reports the height of each peak in the run as a percentage of the total height of all peaks in the run.
- #15 The external standard (ESTD) is the basic quantification procedure in which both calibration and unknown samples are analysed under the same conditions. The external standard method is the technique used most frequently to gather quantitative information from a chromatogram. In this case, a pure reference substance(ideally the same compound as the one to be determined in the sample) is injected in increasing concentrations and the peak areas or peak heights obtained are plotted versus the concentration(calibration curve). These calibration curves should show a constant slope (linear curves) and the intercept should be as close to zero as possible.
- #16 A calibration curve must be recorded for each component of interest. The peak areas heights of the sample chromatogram are then compared with those in the calibration curve. CONC unknown = area unknown/ area known × conc known
- #18 A defined amount of a chosen compound (internal standard) is added to the sample, so that two peaks(that of the internal standard and that of the compound of interest) must be measured to obtain one result. Requirements of Internal Standard: It must not already be present in the sample. It should elute close to the peak of interest and the two peak sizes should not differ too much. If possible, peaks should not overlap, and no chemical interaction the internal standard and other sample components should occur. The internal standard must also have good storage, stability and be available in high purity.
- #21 Most convenient when a small number of samples are to be analysed. Useful when the analysis is present in a complicated matrix and no ideal blank is available.
- #22 Add one or more increments of a standard solution to sample aliquots of the same size. Each mixture is then diluted to the same volume. Prepare a plot of Analytical Signal versus: a) volume of standard solution added or b)concentration of analyte added.
- #23 The x-intercept of the standard addition plot corresponds to the amount of analyte that must have been present in the sample (after accounting for dilution). The standard addition method assumes: a) the curve is linear over the concentration range. b) the y-intercept of a calibration curve would be 0.
- #25 Quality- Control Analytical Methods: High Performance Liquid Chromatography “ International Journal of Pharmaceutical compounding, Vol. 8 No. 3 May/June 2004. Sharma Y. Elementary organic spectroscopy: principles and chemical applications. Chand and company Ltd, New Delhi, India. 2009:23.