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HPLC

This document contains a report submitted by Prashant Kumar to Dr. Parveen Kumar Sappati analyzing caffeine concentration using HPLC. It includes caffeine extraction data from two standard curves which are used to determine that a soft drink sample contains approximately 9.6-10.58 ppm of caffeine. Potential sources of error in the HPLC method are discussed. Key components of HPLC including the pump, injector, column, and detector are explained along with their functions.

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prashant kumar
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28 views

HPLC

This document contains a report submitted by Prashant Kumar to Dr. Parveen Kumar Sappati analyzing caffeine concentration using HPLC. It includes caffeine extraction data from two standard curves which are used to determine that a soft drink sample contains approximately 9.6-10.58 ppm of caffeine. Potential sources of error in the HPLC method are discussed. Key components of HPLC including the pump, injector, column, and detector are explained along with their functions.

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prashant kumar
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© © All Rights Reserved
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Download as PDF, TXT or read online on Scribd
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FOOD ANALYSIS

LAB(AG69010)

HPLC

Submitted By: PRASHANT KUMAR


(21AG63R26)

Submitted To: Dr. Parveen Kumar Sappati

Agricultural and Food Engineering Department Indian


Institute of Technology Kharagpur
1. Caffeine Extraction HPLC Data

caffeine concentration (ppm) caffeine peak area 1 caffeine peak area 2


2 4261294 4299851
10 7784362 7848574
15 9840841 9821236
20 14406202 14392666
40 27459422 27628968

After the analysis, if the measured soft drink sample gives an area of 8019998
and 8678858 for two replicates, what will be the concentration of the caffeine
in the sample? What is your coefficient of variation in your sample? List
possible sources of error.
Standard curve 1

caffeine peak area 1


30000000
y = 627077x + 2E+06
25000000

20000000

15000000

10000000

5000000

0
0 10 20 30 40 50

Area= 8019998
Let Y= 8019998
Put the y in equation, y = 627077x + 2E+06
X= 9.6
Concentration of caffeine is 9.6 ppm.
Coefficient = 627077
Errors may be occurred due to
• Not constant speed of the mobile phase during all the time.
• May be due to injection of rapidly introduces the sample into the system
with minimal disruption of the solvent flow.
• Contaminants in the mobile phase are especially troublesome in
gradient elution.
• Leaks at pump fittings or seals will result in poor chromatography.

Standard Curve 2

caffeine peak area 2


30000000
y = 630480x + 2E+06
25000000

20000000

15000000

10000000

5000000

0
0 10 20 30 40 50

y = 630480x + 2E+06
Area= 8678858
Let Y= 8678858
Put the y in equation, y = 630480x + 2E+06
X= 10.58
Concentration of caffeine is 10.58 ppm
Coefficient = 630480
2. Name 5 parts/ component of a high-performance liquid chromatography
(HPLC) and give their functions.
Answer-
1) Degassing device -Bubbles are often seen in the mobile phase solution
due to dissolved oxygen or air mixed in. Degassing device is used to
remove these bubbles from the HPLC.
2) Pump -The role of the pump is to propel (force) a liquid (the mobile
phase) through the chromatograph at a specific flow rate.
3) Injector – it serves to introduce the liquid sample into the flow stream of
the mobile phase.
4) column – column is considered the heart of the chromatograph. The
success or failure of a particular analysis depends on the choice
of column. The column’s stationary phase separates the sample
components using various physical and chemical parameters.
5) Detector - The detector is a device that is used to continuously monitor
the composition and content changes of the effluent separated by the
chromatographic column

3) explain the concept that governs the HPLC method


Answer-
• High Performance Liquid Chromatography (HPLC) is a process of
separating components in a liquid mixture.
• A liquid sample is injected into a stream of solvent (mobile
phase) flowing through a column packed with a separation
medium (stationary phase).
• Sample components separate from one another by a process
of differential migration as they flow through the column.
• As bands emerge from the column, flow carries them to one or
more detectors which deliver a voltage response as a function
of time. This is called a chromatogram.
• For each peak, the time at which it emerges identifies the
sample constituent with respect to a standard. The peak’s area
represents the quantity.
4) What are the different types of HPLC detectors, and suggest possible
detector applicable to phytochemicals.
Answer-
1) UV/Vis Detectors

2) Photodiode Array (PDA) Detectors

3) Fluorescence Detectors

4) Refractive Index Detectors

5) Evaporative Light Scattering Detectors

I think PDA (photodiode Array Detector) is applicable for detection


of Phytochemicals substance.

1) Its response to chromophoric analytes in the range 190 –


800nm.

2) PDA can be used for Multi-wavelength monitoring

3) Complete spectral profiles of chromatographic peaks

4) Good for the majority of organic analytes

5) Gradient elution compatible

5)
n- butanol

2-methyl-1-propanol

2-butanol

Tert-butyl alcohol

Chloroform is more in solvent, as solvent is chloroform: ethanol (4:1), This will


make the mobile phase slightly non- polar.
More non-polar substance will have less residence time because solvent
(mobile phase) is non- polar.
There must be polar stationary phase.
Stationary phase will show more affinity towards polar substance.
This will lead to more residence time for less non polar component.
The order of non-polarity is n-butanol, 2-methyl-1-propanol, n-propanol
and at last Tert-butyl alcohol.

So the peak representation are

a) n-butanol

b) 2-methyl-1-propanol

c) n-propanol

d) Tert-butyl alcohol.

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