HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Flashcards
• Technique used in analytical chemistry to separate, identify, and quantify specific components in a mixture
• Have high resolution and separation capacity
• Used in quantitative and qualitative analysis
High Performance Liquid Chromatography (HPLC)
High performance liquid Chromatography (HPLC) can only analyze mixtures dissolve in ___
Solvents
(basic principles)
High performance liquid Chromatography (HPLC) depends on the nature of stationary phase whether if it is an ____ chromatography or ____ chromatography
Absorption
Partition
2 commonly used type of HPLC
Absorption and Partition chromatography
Type of chromatography where separation is based on absorption
Absorption chromatography
Type of chromatography where separation is based on partition
Partition chromatography
(basic principle)
The separation principle of HPLC is based on the distribution of sample (analyte) between the ____ and ____
Mobile phase (eluent)
Stationary phase (packing material in column)
The time which is required for the compounds to be transported from the injection site to the detector unit is called ___
Retention time
Retention time the data for ____ and ___ of the compounds in the mixture
identification
quantification
(basic principle)
HPLC can separate compounds in the mixture through their interaction with ____, wherein the higher polarity, the slower it will move through the column
Polar stationary phase
Higher polarity = ?
Slower movement through column
(Basic principle)
Compounds are identified through the difference in their peak’s ___ time, with prior injected ____ standards
retention
reference
____ and related data from the detector helps calculate the concentration of each compound (or quantify specific compounds
Chromatogram
The more concentrated, the ?
stronger the signal
General components of HPLC
Solvent
Pump
Injector
HPLC column
Detector
PC (for data acquisition)
Waste container
Are a group of organic liquid used in HPLC
Solvents
Generates a flow of eluent from the solvent reservoir into the system
Pump
• liquid that dissolves the target compound
• polar and non polar mixture of solvent can be used
Mobile phase
Remove gases from a liquid such as the mobile phase in HPLC
Degasser
The main cause of noise and cycling in the baseline, as well as poor compressibility of the eluent
Gases
It allows the introduction of a precise sample volume onto the HPLC column
Injector
Device used in chromatography for the separation of chemical compounds
HPLC column
• It is made up of an absorbent material that has a very small particle size and the particle size are kept uniform to obtain better performance
• part of a column that interacts with the target compound
Stationary phase
Stationery phase is made up of an ____ material that has a very small particle size
Adsorbent
HPLC are largely influenced by _____. In order to obtain repeatable results it is important to be consistent in this condition
Temperature
• Designed to allow to keep columns at a constant temperature or (isothermal)
• utilize a series of temperature increases and constant hold temperatures ( gradient elution)
Column heater
• Used to monitor the compounds themselves in order to quantify and identify the compound separated in the column
• called “eyes” for hplc
Detector
Detector could be
UV detector
IR detector
refractive index detector
mass spectrometer
Translate the signal generated by the detector into a graph called a chromatogram
Recorder
Recorder translates the signal generated by the detector into a graph called
Chromatogram
safely collects all the mobile phase and sample components after they pass through the HPLC system
Waste reservoir
The composition of the eluent is consistent without the presence of
Analyte
Two common types of hplc
Normal phase hplc
Reverse phase hplc
• Utilizes a polar stationary phase (usually silica) and less polar (non-aqueous) diluting solvents
•used to separate hydrophobic compounds and matrices that are retained too strongly by reversed phase and have minimal solubility in aqueous mobile phase
Normal phase hplc
Normal phase hplc utilizes a ___ stationary phase and less ___ eluting solvents
Polar
Less polar
• Technique can be used to separate identify and or quantitative components in mixtures of soluble organic components based on their hydrophobicity
• the stationary phase is nonpolar (C-18 silica beads) and the mobile phase is polar (methanol)
Reverse phase hplc
Reverse phase hplc separate, identify or quantitate components in mixtures of soluble organic components based on their ___
Hydrophobicity
The stationary phase is ___ and the mobile phase is __
Nonpolar (C-18 silica beads
Polar (methanol)
In normal phase hplc nonpolar moves ___
Quickly
In reverse phase hplc nonpolar moves___
Slowly
Journal article
Applications of hplc combined with laser induced fluorescence for protein profile analysis of tissue homogenates in cervical cancer
A total of 44 samples including normal cervical biopsy samples from ______ patients or patients whose uterus was removed and the patient suffering from different stages of ___ ___ were recorded using the hplcif
hysterectomy
cervical cancer
The collection of samples from normal cervical tissue and those with cervical cancer were obtained:
15 samples were collected from normal tissues
7 samples were collected from stage 2B cancer
19 samples were collected from stage 3b cancer
1 in stage 4 cancer
1 in CIN1(or the cells that line the cervix have abnormal changes wherein it is not yet considered as cancerous but if left and treated it will develop into one)
1 in CIS (or pre cancerous cells which still rest on the surface of the cervix and have not yet invaded to the basement membrane into the deeper tissues)
the high performance liquid chromatography with laser induced fluorescence or hplc-lif technique gives very good diagnostic results for ____ ___
cervical tissues
specificity and sensitivity of the analysis are found to be very high nearly ____%
100 %
this technique can provide objective diagnosis of cervical cancer if compared to histopathology wherein the heterogeneity of the tissue samples and the subjectivity of the operator in histopathology may lead to __
Possible errors
