Setting Up Chromatography Consumables & Solutions Equipment in Your Lab

Chromatography is an important analytical technique used in many laboratories for separating, identifying, and quantifying components in a mixture. Setting up and maintaining chromatography equipment properly is crucial for generating accurate and reliable results. This guide will walk you through the key steps for setting up consumables and solutions for common chromatography techniques like HPLC, GC, IC, and LC-MS.

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Introduction to Chromatography Consumables & Solutions

Chromatography techniques rely on the differential partitioning of analytes between a mobile phase and a stationary phase to achieve separation. Key consumables and solutions required for setting up chromatography systems include:

Stationary Phases

The column contains the chromatographic stationary phase and is the heart of the separation. Common stationary phases include:

• Silica particles, alumina, or polymer beads functionalized with C18, C8, phenyl, cyano or other chemically bonded phases for reverse phase LC.
• Porous graphitic carbon, ZIC-HILIC or other polar stationary phases for normal phase LC.
• Ion exchange resins for ion chromatography.
• Waxes, polysiloxanes and other coatings on capillary columns for gas chromatography.
• Immunoaffinity media, protein A resins or other ligands for affinity chromatography.

The specific stationary phase is chosen based on analytes of interest and separation mode (reverse phase, normal phase, ion exchange etc). Key specifications like particle size, pore size, column length and inner diameter are optimized for the separation.

Mobile Phases

Caption: Mobile phase solvents

Alt text: Bottles of various organic solvents used as mobile phases in chromatography
Source: Welch Materials

Mobile phases can be gases, liquids or supercritical fluids. Typical mobile phases include:

• Organic solvents like acetonitrile and methanol for reverse phase LC.
• Buffered aqueous solutions for ion chromatography.
• Gases like helium, nitrogen and hydrogen for gas chromatography.

Additives like acids, bases, ion pairing agents and buffers are used to control mobile phase pH, improve analyte stability and adjust selectivity. Graded solvent mixtures are used for gradient elution. Degassing is critical for liquid mobile phases.

Sample Handling

• Autosampler vials hold samples at controlled temperatures before injection. Volumetric glass vials with leakproof caps are required.
• Precision syringes and needles inject reproducible sample volumes. Optimized tip designs improve injections.
• Septa caps placed over vials and injection ports seal and re-seal after injections. Advanced polymer/silicone septa resist coring.
• Vial trays hold vials in precisely spaced autosampler locations for reliable sampling.

Reference Materials

• Certified reference standards are required for retention time markers, calibration curves, and quality control. Proper weighing, dilution, and documentation is critical.
• Gases must be ultra high purity with inline filters to remove contaminants. Cylinder regulators control inlet gas pressures.

With careful selection and preparation of these core consumables, laboratories can optimize chromatography workflows and data quality.

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Step-by-Step Guide to Setting Up Consumables & Solutions

Properly setting up consumables and solutions is absolutely vital for successful chromatography analyses and reliable data generation. Follow these comprehensive best practices:

Select Optimal Columns

The column is the heart of any chromatography system and choosing the right stationary phase is critical. Carefully select columns based on:

• Analytes of interest – Different column chemistries like C18, C8, phenyl, cyano, Hilic etc. separate various compounds differently. Match the stationary phase to your target analytes.
• Separation mode – Reverse phase, normal phase, ion exchange, size exclusion etc. require specific column chemistries optimized for the mode.
• Required resolution – Longer columns with smaller particles provide more theoretical plates, improving peak resolution. Shorter columns with larger particles reduce analysis time but sacrifice resolution.
• Analysis time – When faster analysis is needed, use short columns and larger particles. For more resolved peaks, use long columns and small particles. Balance time vs. resolution.
• Sample complexity – Highly complex samples require very high efficiency columns with more theoretical plates to resolve more peaks. Simpler samples may not need ultra-high resolution columns.
• Sample loading capacity – Maximum sample load capacity relates to column length and inner diameter. For high sample loads, choose bigger ID and longer columns.

Standard columns like C18 work well for many small molecule pharmaceuticals in reverse phase LC. HILIC phases suit polar compounds. Ion exchange handles ionizable analytes. Affinity media separates proteins, peptides, oligos.

Carefully optimize key column dimensions like length, inner diameter (ID), and particle size based on analysis goals around resolution, run time, and load capacity.

Install Columns Correctly

Follow best practices around column installation and handling:

• Keep columns sealed with end plugs when not in use to prevent stationary phase drying or contamination.
• Avoid damage by finger tightening column nuts gently without tools. Overtightening can crack fittings and cause leaks.
• Flush brand new columns with initial conditioning solvents to prepare the stationary phase surface chemistry prior to first use.
• Position the column inlet frit properly so the tubing reaches fully to the bottom of the column bed for smooth and even mobile phase flow.
• Secure the column tightly to avoid leaks. Check for leaks at all connections periodically to prevent loss of precious samples or mobile phase.

Prepare High Quality Mobile Phases

Mobile phase quality is absolutely critical for good separation and system performance. Recommendations:

• When possible, use fresh HPLC grade solvents, triple filtered through 0.2 μm membranes and unopened from sealed containers. Older solvents risk more contaminants.
• Make buffers using 18-Megohm ultrapure water, carefully weighing out components on analytical balances. Use high purity buffer salts and reagents.
• Mix mobile phase components thoroughly. Label clearly indicating contents, date of preparation, pH etc. Record details in lab notebooks.
• Degas via vacuum filtration, helium sparging or ultrasonic baths for at least 15-30 minutes minimum to remove dissolved gases thoroughly.
• Always filter buffers again through 0.2 μm membranes after complete mixing and pH adjustment.
• Adjust pH carefully using calibrated digital meters. Check and readjust pH periodically if needed.
• Store mobile phases in tightly sealed, clean glass containers away from light and ambient humidity. Discard older solution.

Rinse Flow Path Extremely Thoroughly

When changing between mobile phases, flush the entire system flow path meticulously to avoid cross-contamination:

• Rinse pump heads, all tubing, injectors, column, and detector flow cells exhaustively. Residual salts or immiscible solvents causes issues.
• Use an intermediate solvent like isopropanol or methanol that is mutually miscible with your mobile phases to bridge between them and assist rinsing.
• Flush at least 10-20 column volumes of rinse solvents, allowing time for columns to fully equilibrate, especially for gradient methods.
• Soak the injection system in rinse solvent for ~30 minutes between analyses if needed to prevent carryover.

Prepare Standards Precisely

High quality standards ensure accurate calibration and reliable analyte quantification:

• Use certified reference material from reputable vendors when available, with documentation of purity assay and traceability.
• Carefully weigh out solid materials on recently calibrated analytical balances, recording weights and calculations in lab notebooks.
• Make high concentration stock solutions volumetrically using Class A grade volumetric flasks and graduated cylinders.
• Store reference materials and stocks properly in conditions like cold, dark, inert headspace that prevent degradation.
• Make working standards fresh daily by diluting stocks. Refrigerate and re-analyze working standards later if needed.
• Prepare calibration curves spanning 80-120% of expected analyte concentrations using ~6-8 levels plus blank.
• Include Quality Control (QC) standards from separate stock to assess intra-run precision and system suitability.

Fill Autosampler Vials Precisely

Follow stringent guidelines for sample vials to avoid problems:

• Use recommended vials matching your autosampler dimensions and tray design. Standard clear 2 mL glass vials with screw caps work for most HPLC systems.
• Fill vials about half to three-quarters full to provide sufficient headspace for the autosampler syringe needle.
• Cap extremely tightly using septa made specifically for each vial size - 11 mm, 13 mm etc. Best are pre-slit silicone/PTFE septa.
• Load vials firmly into trays in specified numbered positions. Press down completely into the bottom of the tray to prevent dislodgement.
• Inspect samples and standards for bubbles which can cause issues. Centrifuge samples briefly before injection if needed to consolidate.

Setup Ultra High Purity Gas Supplies for GC

For GC analyses, ultra high purity carrier gases are essential:

• Use gas generators or cylinders with certified 5.0-7.0 grade purity gases specifically for chromatography.
• Employ high capacity purification scrubbers, hydrocarbon/oxygen traps and 0.5 micron in-line filters to remove contaminants.
• Secure cylinders tightly. Check for leaks periodically with soapy water at connections.
• Attach regulators properly using clamps, nuts and gaskets designed for the cylinder. Set inlet pressure based on method requirements.

Maintain Autosamplers

Autosamplers improve precision but require regular careful maintenance:

• Visually inspect and replace injection port septa frequently before coring or leaks occur to maintain integrity.
• Check autosampler syringes closely for scratches causing carryover. Lubricate plungers and replace needles periodically.
• Follow manufacturer directions for fully cleaning injector ports, sleeves, sample trays etc. to avoid salt/sample buildup over time.
• Update autosampler firmware and software when available to take advantage of improvements.

By fastidiously selecting quality columns, preparing impeccable mobile phases, rinsing thoroughly between solvents, handling standards stringently, filling vials precisely, installing ultra pure gases securely and maintaining autosamplers properly, laboratories can consistently achieve excellent chromatography day after day. Partnering with a premium consumables provider like IT Tech assists labs in sustained success.

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Troubleshooting Common Issues

While proper setup of consumables and solutions prevents many problems, issues can still arise in chromatography systems if best practices are not precisely followed:

Ghost Peaks

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• Cause: Ghost peaks appear when sample material builds up and carries over between runs. They elute close to normal retention times.
• Solution: Thoroughly rinse the entire flow path including column, tubing, injector etc with strong solvents to remove buildup. Replace septa and autosampler needles regularly. Analyze a blank after high concentration samples. Use a different column if needed.

Irreproducible Retention Times

• Cause: Retention time drifting or poor reproducibility is often caused by contaminated mobile phases, buffer issues, pH drift or column degradation.
• Solutions:

1. Prepare fresh mobile phase solutions using highest grade solvents and buffer components. Degas thoroughly. Filter buffers.
2. Check and manually adjust mobile phase pH again right before use. Confirm meter calibration.
3. Test column efficiency periodically with a standardurity compound. Replace column if failing.
4. Verify vacuum degasser, in-line filters, and solvent mixing are functioning properly.

Low Efficiency/Resolution

• Causes: Low plate counts and poor resolution stem from consumable problems like:

1. Blocked inlet filters causing uneven flow and peak broadening.
2. Old degraded columns with contaminated stationary phase or altered surface chemistry.
3. Dirty autosampler injectors introducing extra band broadening.
4. Tiny bubbles in mobile phases interfering with uniform flow.

• Solutions:

1. Replace inlet filters and degas solutions.
2. Install fresh efficient columns matched to analysis needs.
3. Clean or replace autosampler injector ports and sample loops.
4. Filter and degas mobile phases thoroughly right before use.

Pressure Fluctuations

• Causes: Unstable pressures arise from:

1. Tiny bubbles in solvent lines or pumps causing flow interruptions.
2. Leaks at tubing connections allowing pumping of air.
3. Blockage in flow path increasing back pressure.
4. Worn pump piston seals allowing erratic flow.

• Solutions:

1. Degas solvents thoroughly and check for bubbles throughout flow path.
2. Inspect connections and tighten fittings to eliminate leaks.
3. Rinse to remove any column or filter blockages.
4. Test and replace pump seals if worn according to maintenance schedule.

Baseline Noise/Drift

• Causes: Rising, falling, noisy baselines come from:

1. Contaminated mobile phases, buffer issues, or column degradation.
2. Bubbles passing through the detector flow cell.
3. Clogged detector cell allowing buildup.
4. Loose or worn detector lamp/electrical connections.

• Solutions:

1. Replace mobile phases and column. Verify pH. Degas thoroughly.
2. Eliminate bubbles through degassing, inline filters and rinsing.
3. Clean detector flow cell and replace if scratched.
4. Check detector lamp, cell and electrical connections. Tighten or replace parts.

By carefully preparing chromatography consumables, precisely following best practices, and methodically troubleshooting issues, laboratories can achieve excellent system performance and reliable results. Partnering with a premium consumables provider like IT Tech assists labs in success.

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Partnering With IT Tech for Chromatography Success

As a full-service provider of chromatography consumables, equipment, and services, IT Tech helps laboratories optimize their analytical workflows. With over 40 years of experience assisting pharma, biotech, food, environmental, and petrochemical labs, IT Tech offers:

• The widest selection of columns, vials, and accessories from leading vendors
• Mobile phase solutions including HPLC solvents, deionized water, and buffer concentrates
• Autosampler vials rigorously tested for leak proof caps and precise dimensions
• Certified standard reference materials for calibration and method development
• Expert technical support for method development and troubleshooting
• IQ/OQ services for seamless chromatography equipment commissioning

By partnering with IT Tech for their chromatography consumables and services, laboratories can setup efficient, high-performance analytical systems generating the reliable data needed to drive confident decisions.

Ready to tackle your chromatography challenges? Contact our experts at IT Tech today to discuss your laboratory's needs.

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