Skip to main content

High performance liquid chromatography (HPLC)

High performance liquid chromatography (HPLC)

High performance liquid chromatography
Image Source-Google | Image by -QC/QA Methodology

Principal of HPLC:-

HPLC is separation technique based on a solid stationary phase and a liquid mobile phase.Separation is achieved by adsorption partition or ion exchange process.

Principal:
  • Adsorption
  • Partition
  • Ion exchange.
Parted:
  • Injector/Auto injector
  • Pump
  • Detector
  • Column

Advantage of HPLC:-

  • Analysed compound is dissolved in a suitable solvent.
  • Most of analysis of room temperature.
  • Most drugs being non volatile thermally unstable compound.
  • Chromatography always used for hydrocarbon soluble compound of molecular weight less than 1000.
Detector work:
  • Compound elutes from the column they passes through cell and absorb the radiation resulting in measurable energy level change.
Type of detector:
  • U.V. Visible (Multi-Wavelength detector)
  • P.D.A. (Photo-diode array detector)
  • R.I. (Refractor index detector)
  • Fluorometric detector
  • Electrochemical detector
Type of mobile phase:

Normal phase:
Polar stationary phase and non polar mobile phase are describe as normal phase.
Revers phase:
Non polar stationary phase and polar mobile phase are describe as revers phase.

Column work:
Separation is achieved by partition of compound in the test solution between the mobile phase and stationary phase.

Pump work:
Pump system deliver metered amount of mobile phase from the solvent reservoirs to the column through high presser.

Check the column performance or Qualification:

  • Number of theoretical plates 
  • Tailing factor
  • Capacity factor
  • Resolution
Number of Theoretical plates: (Not less than =3000)
Number of theoretical plates(N) is a measure of column efficiency for Gaussian peak.

N=16(T/W)2 
N=5.54(T/W h/2)2
Where,
          T=Retention time of peak.
          W=Width of the peak.

Tailing factor:
Tailing factor(T) a measure of peak symmetry is unit of perfectly symmetrical peak.

T=W 0.05/2f
0.05=Width of peak at 5% height
F=Half distance peak maximum to the loading edge of peak distance being measured at a point 5% of the peak height from the baseline.

Capacity factor:

Time spent of substance in stationary phase
K= ------------------------------------------------------
Time spent of substance in mobile phase 

Resolution: (Grater than=1.5)
The separation of two component in a mixture.

2(t2 - t1)
R=---------------------
W2 + W1
t2 and t1 are the retention time of two component. 
W2 and W1 are the corresponding width at the base of the peak.


Comments

Popular posts from this blog

To determine loss on drying (LOD)

To determine loss on drying (LOD) Definition:   Loss on drying compares the weight of product sample before and after drying. The result is the percentage of moisture in a product. % Moisture = (Begging weight-Ending weight) X 100                         -----------------------------------------------                                    Begging weight  Loss on drying determine by two method which is given below: Method (1):   Weigh accurately a dry empty glass Petri dish. Put the sample (about 0.5 to 5 gm as par requirement, Its mean if sample having less wet take 5 g weight. If sample having more wet than take 0.5 g weight ) in dish and weigh. Note down the reading. Distribute the sample in Petri dish by gentle shaking. Place the loaded dish (without cover) in the drying chamber for two hours. Maintain the oven temperature 105 +/- 5 0 C.  After drying is completed, open the drying chamber, cover the dish and allow it to cool at room temp.  Keep it in the desiccator for 15

IR स्पेक्ट्रोस्कोपी और इसका सिद्धांत

IR स्पेक्ट्रोस्कोपी और इसका सिद्धांत  IR स्पेक्ट्रोस्कोपी क्या है और इसका सिद्धांत क्या है? एक अणु या एक रासायनिक यौगिक में यह पता लगाने के लिए कि कौन से कार्यात्मक समूह या समूह मौजूद हैं, हम IR स्पेक्ट्रोस्कोपी तकनीक का उपयोग करते हैं। आईआर स्पेक्ट्रोस्कोपी तकनीक का प्रदर्शन करने के लिए हम विद्युत चुम्बकीय स्पेक्ट्रम की एक विशिष्ट श्रेणी का उपयोग करते हैं जिसे हम IR (इन्फ्रारेड) विकिरण कहते हैं आईआर विकिरण तरंग दैर्ध्य की विभिन्न श्रेणियों से बना है और IR विकिरण के इस अनुप्रयोग को IR स्पेक्ट्रोस्कोपी के रूप में जाना जाता है क्योंकि यह पहचानने के लिए कि सभी कार्यात्मक समूह मौजूद हो सकते हैं हम IR विकिरण का उपयोग करते हैं इसलिए जब हम किसी नमूने पर आईआर विकिरण लागू करते हैं, तो हम इस तकनीक को आईआर स्पेक्ट्रोस्कोपी कहते हैं  अब आइए नजर डालते हैं आईआर स्पेक्ट्रोस्कोपी के सिद्धांत पर   जिस किसी भी नमूना का हम विश्लेषण करना चाहते हैं (नमूना किसी भी रूप में हो सकता है - ठोस, तरल या गैस) हम उसका नमूना तैयार करते हैं और इसे आईआर स्पेक्ट्रोफोटोमीटर में डालते हैं IR स्पेक्ट्रोफोटोमीटर वह उपकरण है

Analytical Method Validation

Analytical Method Validation   The validation is performance, demonstration of any procedure, process, equipment, material, activity or any system performance as expected under given set of condition. In the most basis form, validation is proving that the performance is as intended. When extended to an analytical procedure,depending upon the application, it means that the method works reproducibaly, when carry out by same or different persons, in same or different laboratories, using different reagents, different equipment, etc. The analyst normally tends to assume that if an analytical method or process performs reproducibility in an limited number of experiment, it will continue doing so forever, which is too optimistic to assume. The Benefits:   The biggest   advantage  of method validation is that it builds a degree of confidence, not only for the developer but also to the user. Although the validation exercise may appears costly and time consuming. It is results in expens