High performance Liquid chromatography(HPLC)

1.     Introduction

High-Performance Liquid Chromatography(HPLC: formerly referrd to ashigh-pressour liquid Chrometography), is a techique in analytical chemistry used to separate, identify, and quantify each component in a mixture.

The principle of chrometography, in chromatography a liquid is pumped through a bed of particles. The liquid is called the mobile phase and the particle the staionary phase . High performance liquid chrometography high performance liquid chrometography (HPLC) is basicallly a highy improved from liquid chrometography. Instead of solvent being chrometograhy allowed todrip though a column under gravity, it is forced through under high pressur of up to 400 atmospheres.That makes it much faster. All chrometograhy separation , includinHPLC operate under the sane basic principle; sepration of a samll into it’s  constituent parts because of the difference in the relative affinities of defferent molecules for the mobile phase and the stationary phase used in the sepration.

2.     Instrumentation of  HPLC

3.     HPLC Tuoubleshooting

A.   Peak Tailing

Possible Cause	Solution
1.     Blocked frit	1.       a.     Reverse flush column ( if allowed) b.     Replace inlet frit c.      Replace Column
2.     Column Void	2.     Fill void
3.  Interfering peak	3.                 a.  Use longer column                b. Change mobile phase                 and / or column/ selectivity
4.Wrong mobile phase pH	4.               a. Adjust pH               b. For basic compounds,  lower pH usually provide more symmetric peak.
4.     Sample reacting with active site	5.       a.     Add ion pair reagent or volatile basic modifier b.     Change column

B.   Peak Pronting

Possible Cause	Solution
1.     Low temperature	1.Increase cloumn temerature
2.     Wrong sample solvent	2.Use mobile phase for injection solvent
3.     Sample overload	3.Decrase sample concentration
4.     Bad Column	4.See A.1 and A.2

C.   Split Peaks

Possible Cause	Solution
1.     Contamination on guard or analytical column inlet           Fig. Split Peaks	1.               a.Remove guard column and attempt analysis.               b.Replace guard if necessary               c.If analytical column is obstructed, reverse and flush              d.If problam persists, column may be fouled with strongly reatined contaminats              e.Use appropriate restration procedure              f.If problam persists, inlet is probably plugged             g.Change frit or replace column
2.     Sample solvent incompatible with mobile phase	2.Change solvent; whenever possible, inject samples in mobile phase

D.   Distortion of Larger peaks

Possible Cause	Solution
1.     Sample overload	1.Reduce sample size

E.   Distoration of Early Peaks

Possible Cause	Solution
1.     Wrong injection	1. a. Reduce injection volume b. Use weaker injection solvent

F.    Extra peaks

Possible Cause	Solution
1.     Other components in sample	1.Normal
2.     Late- eluting peak from previous injection	2. a. Increase run time or gradient slope b. Increase flow rate
3.     Vacancy or ghost peaks	3. a. cheak purity of mobile phase b. Use mobile phase as injection solvent c. Reduce injection volume

G.  Retention Time Drifts

Possible Cause	Solution
1.     Poor temperature control	1.Thermotat column
2.     Mobile phase changing	2.Prevent change (evaporation, reaction.)

H.  Abrupt Retention Time Change

Possible Cause	Solution
1.     Flow rate change	1.Reset flow rate
2.     Air bubble in pump	2.Bleed air from pump
3.     Improper mobile phase	3.       a. Replace with proper mobile phase           b.Set proper mobile phase mixture on
4.     Weak detectour lamp	4.Replace lamp
5.     Column leaking silica or packing material	5.Replace Column
6.     Mobile phase mixture inadequate or malfunctioning	7.     Repair or replace the mix offline if isocratic

I.      Broad Peaks

Possible Cause	Solution
1.     Mobile phase composition changed	1.Prepare new mobile phase
2.     Mobile-phase flow rate too low	2.Adjust flow rate
3.     Leaks	3.          a. See Section 3          b. Cheak for loose fittings          c. Cheak pump for leaks, salt build- up , and unsual noises          d. Change seals if necessary
4.     Detector setting incorrect	4.Adjust setting
5.     Extra-column effect: a.     Column overloaded b.     Detectore response time oe call volume too large c.      Tubing between column and dectector too long or ID too larg d.     Recorder response time too high	5. a.     Inject smaller column (e.g. 10µl vs. 100 µl) or 1:100 and 1:100 dilution of sample b.     Reduce response time oe use smaller call c.      Use as short a piece of 0.007-0..10. inch ID tubing as practical d.     Reduce respose time
6.     Buffer concentration too low	6.Increase concentration
7.     Guard column contaminated / worn out	7.Replace guard colunm
8.     Column contaminateds / worn out: low plate number
9.     Void at column inlet

Comming Soon............................

Drx. Atul Jaiswal , Sourabh Kushwah

 and Vishal Kushwah

Cell Division-Mitosis and Meiosis

Cells divide for many reasons. For example, when you skin your knee, cells divide to replace old, dead, or damaged cells. Cells also divide so living things can grow. When organisms grow, it isn't because cells are getting larger. Organisms grow because cells are dividing to produce more and more cells. In human bodies, nearly two trillion cells divide every day.

Mitosis Cell Division

Mitosis is how somatic—or non-reproductive cells—divide. Somatic cells make up most of your body's tissues and organs, including skin, muscles, lungs, gut, and hair cells. Reproductive cells (like eggs) are not somatic cells.

In mitosis, the important thing to remember is that the daughter cells each have the same chromosomes and DNA as the parent cell. The daughter cells from mitosis are called diploid cells. Diploid cells have two complete sets of chromosomes.  Since the daughter cells have exact copies of their parent cell's DNA, no genetic diversity is created through mitosis in normal healthy cells. 

Mitosis cell division creates two genetically identical daughter diploid cells. The major steps of mitosis are shown here. (Image by Mysid from Science Primer and National Center for Biotechnology Information)

The Mitosis Cell Cycle

Before a cell starts dividing, it is in the "Interphase." It seems that cells must be constantly dividing (remember there are 2 trillion cell divisions in your body every day), but each cell actually spends most of its time in the interphase. Interphase is the period when a cell is getting ready to divide and start the cell cycle. During this time, cells are gathering nutrients and energy. The parent cell is also making a copy of its DNA to share equally between the two daughter cells.

Table- The mitosis cell cycle includes several phases that result in two new diploid daughter cells. Each phase is highlighted here and shown by light microscopy with fluorescence. Click on the image to learn more about each phase. (Image from OpenStax College with modified work by Mariana Ruiz Villareal, Roy van Heesheen, and the Wadsworth Center.)

The mitosis division process has several steps or phases of the cell cycle—interphase, prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis—to successfully make the new diploid cells

.

When a cell divides during mitosis, some organelles are divided between the two daughter cells. For example, mitochondria are capable of growing and dividing during the interphase, so the daughter cells each have enough mitochondria.  The Golgi apparatus, however, breaks down before mitosis and reassembles in each of the new daughter cells. Many of the specifics about what happens to organelles before, during and after cell division are currently being researched. (You can read more about cell parts and organelles by .)

Meiosis Cell Division 

Meiosis is the other main way cells divide. Meiosis is cell division that creates sex cells, like female egg cells or male sperm cells.  What is important to remember about meiosis? In meiosis, each new cell contains a unique set of genetic information. After meiosis, the sperm and egg cells can join to create a new organism.    

Meiosis is why we have genetic diversity in all sexually reproducing organisms. During meiosis, a small portion of each chromosome breaks off and reattaches to another chromosome. This process is called "crossing over" or "genetic recombination." Genetic recombination is the reason full siblings made from egg and sperm cells from the same two parents can look very different from one another. 

The Meiosis Cell Cycle

Meiosis has two cycles of cell division, conveniently called Meiosis I and Meiosis II. Meiosis I halves the number of chromosomes and is also when crossing over happens. Meiosis II halves the amount of genetic information in each chromosome of each cell. The end result is four daughter cells called haploid cells. Haploid cells only have one set of chromosomes - half the number of chromosomes as the parent cell.

Before meiosis I starts, the cell goes through interphase. Just like in mitosis, the parent cell uses this time to prepare for cell division by gathering nutrients and energy and making a copy of its DNA. During the next stages of meiosis, this DNA will be switched around during genetic recombination and then divided between four haploid cells. 

By Atul Jaiswal