HPLC-15

Column efficiency

Column efficiency is the ability of the column to produce the good resolution and narrow peaks.

Factors affecting Column efficiency:

• Length of the column
• Linear velocity of flow in the column
• Extra column volume
• Retention factor
• Particle size of the stationary phase
• Packing strength

HPLC-14

Extra-Column band broadening

Extra-Column band broadening occurs due to more Extra-column volume or Dead volume (refer post HPLC-08). Tubing connections between column and injection system, column and detector region can cause band or peak broadening. Therefore larger the radius of connecting tube, more the extra-column peak broadening and smaller the radius of connecting tube, less the extra-column broadening.

HPLC-13

How CAD detector works?

The steps involved in CAD-Charged Aerosol Detector :

• The mobile phase from the column enters into CAD
• Pneumatic nebulization of mobile phase creates aerosol droplets
• Conditioning of aerosol occurs to remove the large droplets
• Evaporation of solvent from the formed aerosol leads to dried particles
• An Ion jet induces charge to the dried particles
• An Ion trap is used to select the particles and remove the excess ions from them
• The charge of those selected particles can be measured and detected by using Electromter

HPLC-12

Causes for Pressure Fluctuations

In HPLC, Pressure fluctuations mainly occur due to the following:

• Leaks in Pump seal
• Leaks in tubings
• Inline bubble traps in pump, column and solvent tubings

Solutions:

Bubble traps can be minimized by Degassing

Pump seal can be replaced to avoid leaks

HPLC-11

Causes for the the change of Retention time

 The molecule of analyte in a sample has to elute at 8th minute. But instead, If it gets eluted either before or after that 8th minute there is a change in retention time. It happens due to some factors like variation of column temperature, evaporation of mobile phase, Improper preparation of mobile phase. So it is necessary to maintain optimum temperature in column and prepare the mobile phase accurately.

HPLC -10

Relative Retention Time (RRT)

RRT is the ratio of retention time of peaks corresponding to the compounds other than analyte and the main peak retention time. For example, If the retention time of main peak is 8 minutes and the retention time of other impurity peak is 5 minutes. The RRT of impurity peak is 5/8=0.625 and the mainpeak RRT is 8/8=1.
RRT is mainly used to control the elution of impurity peaks from the sample.

HPLC-09

Peak Fronting and Tailing

Peak Fronting

It occurs mainly due to overloading of sample..i.e., Injection volume is too high. Even mobile phases with more viscosity can lead to fronting of peaks.

Peak Tailing
It occurs due to retention of some compounds inside the column. It is the beginning of peak doubling.

HPLC-08

Know about Void volume and Dead volume

Void volume
The volume of mobile phase in the column is Void volume. If the stationary phase occupies 45% of total column volume, the void volume would be 55% of total column volume.

Dead volume
The volume between the injection point and detection point excluding the stationary phase in the column. It is also known as Extra-column volume.

HPLC-07

S/N Ratio & Baseline Noise 

S/N- Signal to Noise Ratio
The signal for analyte peak and baseline noise both contribute to S/N ratio.

Baseline Noise: It is the variation of baseline from the straight one caused by many factors like electrical signal fluctuations, lamp instability, temperature fluctuations, etc..

General info-10

Difference between Deamidation and Deamination

Deamidation
It is the removal or convertion of amide functional group in the side chain of amino acids like asparagine or glutamine to aspartic acid or glumatic acid.

Deamination
It is the process in which amine group is removed and converted to ammonia.

HPLC-06

Charged Aerosol Detector (CAD)

CAD is the universal detector used to measure the chemical compounds by producing charged aerosols which are detected by electrometer. Here the particles are measured based on the size of aerosols.

HPLC-05

Know the difference between UV/VIS, PDA and DAD detectors

 Don't think all these three detectors work on different principle. (refer HPLC-03 post for abbreviations) PDA and DAD are actually UV/VIS detectors and they have an array of photodiodes. Photodiode is used to convert light into electrical signal.The UV/Vis detector can detect  single wavelength at one time while the DAD/PDA can scan a wavelength range (200-800 nm). If you want to scan a wavelength range DAD/PDA are better or If you know the particular absorbance wavelength, go with UV/VIS dtector. So UV/VIS (PDA or DAD) are the most commonly used detectors in HPLC analysis because it is also very easy to analyse/interpret the peaks in the chromatogram.

HPLC-04

Principle of UV Visible detector

UV VIS detector works on the principle of Beer Lambert's law. It means the Absorbance is directly proportional to the concentration of the analyte. 

HPLC-03

Detectors used mostly in HPLC

The most commonly used detectors in High Performance Liquid Chromatography:

• UV Visible detector
• Diode Array Detector (DAD)
• Photo Diode Array detector (PDA)
• Charged Aerosol Detector (CAD)

HPLC-02

Peak Broadening

The tendency of a chromatographic peak to broaden as it passes through the column. It is also  known as peak spreading or peak dispersion. The peak width or the number of theoretical plates in the column (N) is measure of peak broadening.

General info-09

UV cutoff wavelength

Every solvent has a UV-vis absorbance cutoff wavelength. UV cutoff means the wavelength at which the solvent has an absorbance of 1 AU for 1 cm path length by using water in the reference cell.

Example: UV cutoff for acetonitrile and water is 190 nm.

HPLC-01

Why Column Temperature is the important factor in HPLC?

The maintenance of optimum temperature plays a vital role in the separation of analyte of interest from a sample. Temperature of column directly influences with the viscosity of the mobile phase. Increase in temperature of column decreases the viscosity of the mobile phase. Therefore the viscosity gets decreased, the flow rate becomes fast and it also reduces longitudinal diffusion. So, Optimum temperature (It shouldn't be very high or low) should be maintained for the better separation of peaks.

General info-08

Guard columns

In HPLC, Guard columns are mostly used to extend the life of the analytical columns. Guard column is a small sized column packed with the same packing material as like analytical column. Since the frits on guard column also remains same as those on analytical column, the particles blocking the analytical column will block the guard column first. So the analytical column will be safeguarded by the use of guard columns.

(Analytical columns- Columns like IEX, RPC, SEC, HIC normally used for the purification and analysis of samples)

RPC-04

Use of Ion Pairing reagents in RPC

Ion pairing reagents are mainly used in RPC to make the analytes to interact with stationary phase (refer previous RPC posts). Ion pairing reagents will bind to the ionic molecules in the analyte via ionic interactions which make the hydrophobic groups present in the analyte to be exposed out for the affinity of binding with stationary phase. Trifluoroacetic acid is mostly used as an ion pairing reagent for the reversed phase separation of peptides (Glajch J L et al, 1987) (Shibue M et al, 2005). Here, the negatively charged trifluoroacetate interacts with the positively charged amino acid residues and makes the analyte less hydrophilic. (Geraldine Thevenon, 1989) 

IEX-03

Effect of Isoelectric point on IEX

Every protein has an Isoelectric point (pI) (refer General info-01). When a protein is at its pI the protein will not even bind to the ion-exchange resin. Below this pH the protein will have a net positive charge and will bind to a cation exchanger, and above this pH it will have a net negative charge and bind to an anion exchanger. 

IEX-02

Stationary phase in IEX

The cation exchanger has sulfomethyl, carboxymethyl groups and the anion exchanger has Quarternary ammonium, Diethylaminoethyl (DEAE) groups attached to the stationary phase resin. 

IEX-01

Ion Exchange Chromatography (IEX) 

Ion exchange chromatography (IEX) separates proteins based on their net surface charge, through electrostatic interactions that occur between proteins and a charged stationary phase.

Types of IEX 

Two types of IEX exist:

1) Anion exchange (Negatively charged proteins bind to positively charged stationary phase)

2) Cation exchange (Positively charged proteins bind to negatively charged stationary phase)

Image designed by Tutkia biotech

General info-07

Know about Ghost peaks 

One of the major problems in chromatography is the occurrence of ghost peaks in chromatogram. These peaks are mainly due to the contamination of column from previous run samples. So to avoid those ghost peaks, the column should be washed or flushed  properly with strong solvents at the end of every runs.

RPC-03

Endcapping

During manufacture of RP columns, some of the silanol groups remain on the surface of the silica after immobilization of non polar functional groups. This is because of the steric hindrance from C8 or C18 chains. Basically the stationary phase of  RPC should be non polar but the silanol groups are polar. This leads to the chance of interaction of unwanted analytes with stationary phase resulting in poor peak resolution and peak tailing. End capping is used to mask this interaction by replacing silanol groups with trimethylsilyl group.The reagents like trimethylchlorosilane and dichlorodimethylsilane are commonly used for end capping the reverse phase columns. 

General info-06

Modes of elution

Isocratic elution - The mobile phase composition remains the same throughout the run time. 

Example: Don't think only 100%  of  mobile phase A or B is Isocratic mode. If the run time is 8 minutes, either the mobile phase of 100% or  composition of A-80% + B-20%  runs same for 8 minutes in this mode.

Gradient elution - The mobile phase composition changes during the run time. 

Example: If the run time is same 8 minutes, the mobile phase composition of A-80% + B-20% runs for first 4 minutes and it changes to A-60% + B-40% for next 4 minutes in gradient mode.

RPC-02

Stationary phase in RPC

The stationary phase of RPC is non polar/hydrophobic (refer previous post RPC-01).The hydrophobic nature is achieved by the alkyl chains (C18, C8 and C4), amide, phenyl or cyano groups bonded to the stationary support silica. Order of increase in Hydrophobicity is C4<C8<C18. This statement shows C18 is more hydrophobic than C8 and C4 because 18 (CH3) alkyl groups are bounded to the silica support.

General info-05

Know about Transposons

Transposons are the genes having the ability to jump either from one piece of DNA to another or other position of the same DNA. These start to emerge when a gene is bounded by insertion sequences on both sides. This causes mutations when they jump to the middle of the gene. These are more resistant to antibiotics. (Bioprocess engineering Michael L. Shuler)

RPC-01

Reverse Phase Chromatography (RPC)

Reverse Phase chromatography separates molecules based on the differences in hydrophobicity of molecules. RPC uses hydrophobic stationary phase. The hydrophobic nature is achieved by the non polar functional groups bonded to the stationary phase. The mobile phase in RPC is polar. The non-polar compounds bind to the stationary phase by hydrophobic interaction. Then the bonded analytes are eluted from the column by using non-polar solvents.(Mant C T, 1996)(Jason Olbrich, 2013)

Principle of Reverse Phase Chromatography
Image designed by tutkiabiotech

Link-01

Reference books for Chromatographic techniques

Students interested in purification and chromatography can use the below links to gain more knowledge in different chromatographic techniques.

Introduction to Modern Liquid Chromatography - Important book

///D:/hplc%20study/Introduction%20to%20Modern%20Liquid%20Chromatography%202nd%20Edition%20(L.R.Snyder%20-%20J.J.Kirkland).pdf

Reverse Phase & Hydrophobic Interaction Chromatography

wolfson.huji.ac.il/purification/PDF/HCIC/AMERSHAM_HIC_RPC.pdf

Ion Exchange Chromatography

sevierlab.vet.cornell.edu/resources/GE_Ion-Exchange-Chromatography.pdf

Size Exclusion Chromatography

sevierlab.vet.cornell.edu/resources/GE_Size-Exclusion.pdf

Higher studies-01

Competitive exams after B.Tech BT

 Students who are more passionate about doing masters degree after B.Tech Biotechnology have the dream to enter into good institutes. Most of them know only about GATE. The following are the few competitive exams after B.Tech:

 

                   1. GATE 

                   2. TIFR

                   3. DBT  

                   4. CSIR UGC NET

                   5. JNU

                   6. TANCET

                   7. GRE  (for abroad)

                   

                   8. IELTS  (for abroad)

                   

                   9. TOFEL (for abroad)

Tb info-02

Resume Building

The students stepped into final year are now at the stage of making decisions about their career. Some plan for higher studies and some would like to be a part of  any core companies. The first step for that is to build a good resume. In Interview, most of the questions will be raised from the resume. So be aware of the contents what you are adding in the resume. Even your resume will sometimes impress the recruiters among other candidates.

 For this week, Tb comes to help you in resume building. Kindly make use of this and feel free to drop your doubts in our contact form. 

Biotech Companies in Bangalore

Don't think there is no scope in Biotechnology. There are ocean full of companies as like IT  to make your dreams come true. See the list of biotech companies only in Bangalore area. Visit those websites for the career opportunities and make your life to shine in Biotech... 

Anthem Biosciences
www.anthembio.com

Accelrys Inc

www.accelrys.com

Advanta India Ltd

www.advantaindia.com

Azyme Biosciences Private Limited

 www.azymebio.com

Avestha Gengraine Technologies Pvt Ltd

www.avesthagen.com

Alfa Laval India Ltd

www.alfalaval.com

Alltech

www.alltech.com

Aurigene Discovery Technologies Limited

www.aurigene.com

AstraZeneca India Pvt Ltd

www.astrazenecaindia.com

Bangalore Genei Pvt Ltd

www.bangaloregenei.com

Bigtec Labs  Pvt Ltd

www.bigteclabs.com

Bhat Biotech India Pvt Ltd

www.bhatbiotech.com

Biocon Limited

www.biocon.com

Biozeen

www.biozeen.com

Clinigene International Pvt Ltd

www.clinigeneintl.com

Creative Biolabs 

www.creative-biolabs.com

Clintec International 

www.clintec.com

CytoGenomics (P) Ltd

www.cytogenomic.com

EID Parry (India) Ltd

www.eidparry.com

GE Healthcare Life Sciences

www.gelifesciences.com

Genotypic Technology Pvt Ltd

www.genotypictech.com

Glaxo SmithKline 

www.gsk-india.com

Greenearth Biotechnologies Ltd

www.greenearthbiotech.com

Gangagen Biotechnologies Pvt Ltd

www.gangagen.com

Indo-American Hybrid Seeds (India) Pvt Ltd

www.indamseeds.com

Jubilant Biosys (P) Ltd

www.jubilantbiosys.com

Icon Clinical Research

www.iconclinical.com

Khoday Biotek-Palm Grove Nurseries

www.khodayindia.com

Kewaunee scientific corporation

www.kewaunee.com

Keygene

www.keygene.com

Lifeguard Laboratories

www.lifeguardlabs.com

Lotus Labs

www.lotuslabs.com

Millipore India Ltd

www.millipore.com

Mylan Pharmaceuticals Pvt Ltd

 www.mylan.in

Metahelix Life Sciences Pvt Ltd

www.meta-helix.com

Micro Labs Ltd
 www.microlabsltd.com

Monsanto India Ltd

www.monsantoindia.com

Multiplex Bio-Tech Pvt Ltd

www.multiplexbiotech.com

Molecular connections

www.molecularconnections.com

Merck Life Science Pvt Ltd

www.merck.co.in
     

Natural Remedies Pvt Ltd

www.naturalremedy.com

Novonordisk  India Limited

www.novonordisk.co.in

Novozymes 

www.novozymes.com

Namdhari Seeds

www.namdhariseeds.com 

Ontop pharmaceuticals Pvt Ltd
www.ontoppharma.com

Parry Neutraceuticals Ltd

www.parryneutraceuticals.com

Phalada Agro Research Foundations Pvt Ltd

www.phaladaagro.com

     

Rallis Research Centre

www.rallis.co.in

Richcore Lifesciences

www.richcoreindia.com

Sartorius India Pvt Ltd

www.sartorius.com

Sun Pharmaceutical Industries Ltd

 www.sunpharma.com

Sami Labs Ltd

www.samilabs.com

Stelis Biopharma Pvt Ltd

 www.stelis.com

Strand Life Sciences

www.strandls.com 

Syngene International (P) Ltd

www.syngeneintl.com

Vittal Mallya Scientific Research Foundation

www.vmsrf.com

Wexford laboratories

www.wexfordindia.com

Wipro BioMed

www.wiproindia.com

XCyton Diagnostics

www.xcyton.com

SEC-03

Advantages of SEC

Gel filtration can be used to remove salts from the sample, due to its ability to separate small components from the larger ones.

Mechanism: Low molecular weight salts and other compounds are removed from non-ionic and high molecular weight compounds.

General Info-04

Why do proteins detect at 280 nm?

The main reason is because of the presence of amino acids with aromatic ring structure. Example of aromatic amino acids are Tyrosine, Tryptophan and Phenylalanine.

General Info-03

Why caffeine is mostly used for calibration of UV detectors in HPLC?

Caffeine gives response at two maximas: one at 203 nm, second at 273 nm & one minima at 245nm.

Since Methyl, Ethyl & Propyl Paraben  absorbs at two maximas and one minima, It is also used as a standard for calibration of UV detectors.

SEC-02

Exclusion Limit

The upper limit of molecular weight or size beyond which molecules will elute at the same retention volume.

Retention Volume

    Retention Volume is directly proportional to the retention time at constant flow rate

  Example:  If the retention is 8 minutes and flow rate is 1 ml/min, then the retention volume is 8 ml. 

General Info-02

Know about Unit of Pressure and Use of BET method

Unit of Pressure

• Bar- A unit of pressure equal to one atmosphere. 
• It is equivalent to 14.5 pounds per square inch (psi) or 0.1 Megapascal.

BET Method

• A method developed by Bruner, Emmett and Teller for measuring surface area.
• The level of liquid nitrogen adsorption within the pores of the phase is measured at very low temperatures. 
• Pore volume and pore size distribution methods can also be obtained by this method.

SEC-01

Size Exclusion Chromatography (SEC)

• Used mainly to separate high molecular weight samples and to determine their molecular weight distribution by virtue of their size in solution. 
• Also known as gel permeation, gel filtration or steric exclusion chromatography.
• Stationary phase is made of beads with pores of defined diameter. 
• Very large molecules can’t enter into the pores and so elute first, Medium sized molecule enter with difficulties and so elute second. Small molecules enter all pores  and elute as last.

Principle of SEC
Image designed by tutkiabiotech

General Info-01

Know about Isoelectric Point or Isoionic point (pI)

One of the  most probable Interview questions in many pharma industries

•  The pH point at which protein no longer has a net charge. This doesn't mean that the molecule has no charge. It has charge  i.e., all the charges are neutralized.
•  we can also say pI is the pH at which the amino acid does not migrate in an electric field

• Ion exchange chromatography works mostly based on pI of the protein to be separated.

Tb info-01