HPLC Sample

In Hplc, sample refers to the solution that contains the analyte that is to be analyst, for this a proper sample preparation is important for accurate, reproducible, and reliable results.
In this there is mainly three components
1.The analyte (compounds to be analyzed)
2.A suitable solvent ( diluent)
3.Buffer or additives, depending on the analyte’s properties

HPLC Sample Types

Pharmaceuticals: Tablets, capsules, injections
Biological samples: Blood, plasma, urine
Environmental samples: Water, soil extracts
Food and beverages: Additives, preservatives, vitamins
Chemicals and polymers: Industrial raw materials

Sample Preparation Steps

Sample preparation must be done with utmost care as even a small deviation from the sample amount or change from the procedure will cause huge deviation in the final result. For a Hplc compatible and free from interference there are certain step that must be considered while preparing the sample which includes

Dissolution– the sample is dissolved in the solvent, here the solvent used must be compatible with mobile phase and also good enough to dissolve the sample in it. For proper dissolution of sample various methods like magnetic stirring, vortex shaker, rotary shaker or sonication can be used
Filtration– filtering the prepared sample is an important part in the for this normally syringe filter are used which uses 0.22 µm or 0.45 µm of member filter.
Dilution– for proper detection of compound it is important that the analyte concentration is within the linear range of the detector
pH Adjustment– Adjust the sample pH to improve solubility or stability

HPLC Sample Vials

They are small, cylindrical containers, typically made of glass and designed to be placed in an autosample tray from where the autosample injector takes the required amount of sample.

Sample Vials holds the sample for a required period of time until the completed analysis is not over. They are used in autosampler Hplc, Using the right vial ensures sample integrity, prevents contamination, and avoids instrument issues.

Types of HPLC Sample Vials

Based on Material

Glass Vials: Chemically resistant and ideal for organic solvents.
Plastic Vials: Suitable for aqueous samples or when breakage is a concern.

Based on Color

Clear Vials: For standard use when light sensitivity is not an issue.

Protect light-sensitive compounds (e.g., vitamins, some pharmaceuticals) from UV degradation.

Based on Neck Style

Crimp-Top Vials: Require a crimping tool; provide a tight seal.
Screw-Top Vials: Easy to use and reseal; most popular for routine analysis.
Snap-Top Vials: Convenient but may not seal as tightly; used for short-term analysis.

Parts of HPLC Vials

Caps: Usually made of aluminum or plastic.
Septa: Typically made of silicone or PTFE (Teflon) and prevent sample evaporation and contamination.

What is a Septa

It is a form of seal between the outside and inside vial, made of soft and rubber-like disc. The septum is typically pierced by the autosampler needle during injection and after piercing, the septa reseals to maintain sample integrity

Vial Sizes

Although there are many sizes available for vail, the most commonly used is 2ml. Microvials (0.3 mL–1 mL) are used for small-volume or precious samples. Larger vials (4 mL or more) are used in preparative or semi-preparative HPLC.

HPLC Sample Analysis

The sample analysis using Hplc happens in four steps,

Injecting a prepared sample into the HPLC system
Separating the analytes based on their chemical properties
Detecting them using a suitable detector (e.g., UV, PDA, MS)
Analyzing the data to identify or quantify the compounds.

Types of Sample Analysis in HPLC

Qualitative Analysis: Identifies what compounds are present, based on retention time.
Quantitative Analysis: Measures how much of each compound is present using peak area and calibration curves.
Purity Testing: Assesses the purity of a compound by checking for additional peaks.
Stability Studies: Checks how a compound degrades over time under different conditions.
Impurity Profiling: To detect and quantify trace impurities in drugs or formulations.
Dissolution Testing: To evaluate the rate at which a drug dissolves in a medium, simulating the human body.
Environmental Sample Analysis: To detect and quantify pollutants, pesticides, or toxins in water, soil, or air samples.
Natural Product/Plant Extract Analysis: To separate and quantify bioactive compounds in herbal formulations or plant extracts.
Enantiomeric or Chiral Analysis: To separate and measure chiral (enantiomeric) compounds using chiral HPLC columns.

HPLC sample loop

In Hplc the sample loop refers to the small, fixed-length piece of stainless steel that holds a defined volume of sample, this is found in both manual injector or autosampler mechanism and Helps in defining how much sample should be injected into the column for analysis.

Common Loop Volumes

Fixed Volume LoopsSample loops are available in various sizes, depending on theanalysis type:
Analytical HPLC -5 µL – 20 µL
Preparative HPLC–50 µL – 100 µL
Ultra-low volume analysis –< 2 µL

Need for sample loop to

Accurate Injection Volume-Delivers a precise, repeatable amount of sample every Time.
Consistent Peak Areas-Essential for quantitative analysis where reproducibility Matters.
Minimizes Sample Wastage-Especially useful when working with small or valuable Samples.

HPLC Sample Injector

Sample injector in Hplc helps in introduction of sample into the flow of the mobile phase, which in turn takes sample to column for chromatographic analysis. The injector must be able to withstand high pressure and deliver precise sample volume without contamination

Types of HPLC Injectors

Manual Injector
This type of injector used a basic model and for educational setups. The operator has to manually inject the sample every time for a new sample run. For this sample is loaded into a sample loop using a syringe and the valve it rotates to switch from “load” to “inject” position.

Autosampler (Automatic Injector)
Compared to manual injector in automatic injector for autosampler the sample is automatically drawn and injected into the port to be passed into the column. For this different samples are placed in multiple vials in sample tray which is temperature controlled, Because of this setup hundreds of samples can be tested without the presence of the user. Also the chance of user handling error is greatly reduced by this setup.