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How to organize your ebook collection with calibre.

Stop organizing your ebooks like they're just a tired pile of documents and start organizing them in style with Calibre---an open source ebook organizer that supports metadata, cover flow, format conversion, and more.

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Why calibre, what do i need to get started, installing and configuring calibre, adding books to calibre, editing metadata in calibre, adding books to your device and converting ebooks in calibre, further reading.

Calibre is an absolutely fantastic application for organizing your ebook collection. It supports 22 ebook formats including MOBI, EPUB, LIT, PDF, and more. It can accept those 22 formats and convert them to 16 formats, with customized output for dozens of specific ebook readers including the Kindle , Nook, Sony Readers, and more.

The interface is polished, easy to use, and makes managing your ebook collection like managing a media collection through popular media management applications such as iTunes. You'll be able to easily download covers, metadata, and tags from online databases and browse your collection using that information.

On top of all that Calibre is open source and available for Windows, Mac, and Linux operating systems.

You don't need much to get started with Calibre, to follow along with this guide you'll need the following:

  • A copy of Calibre for your operating system
  • A folder or drive to serve as a new home for your collection
  • Some books to add into your Calibre database

Once you have a copy of Calibre installed and you've grabbed a few books to use as test subjects as you follow along with the tutorial, you're ready to go.


For this tutorial we're going to be using the portable Windows version of Calibre. Once you unpack the portable version or install the traditional version there is no difference in operation. The primary reason we've opted to go with the portable version is because it allows us to pack Calibre right along with our ebook collection using the following directory structure:

/Ebook Library/ /Calibre Portable/ /Ebooks/ /temp/

Thus we can easily backup the entire library, management app and all, at once. If you're not using the portable version don't stress about not putting everything together. Calibre stores all information (meta data and covers) in each unique book directory.

When you run Calibre for the first time it will prompt you to select the language you want to use and the location of your library. Make sure to create a new folder for your new Calibre library. We're going to import your old ebooks into Calibre and let Calibre manage the directory structure. In our case we're pointing Calibre at /Ebook Library/Ebooks/.


In the second step you tell Calibre what ebook reader you use. If you don't use one go ahead and leave it as Generic/Generic e-ink device. If you do use one select the appropriate device such as Amazon/Kindle.


In the third step you can configure Calibre to email documents to your ebook reader, such as to your Kindle's email address. If you have a device that supports such a service we recommend you configure it now. After configuring your email service, click finish.


At this point Calibre should launch and you should see a screen like the one above with an empty library---save for the Calibre Quick-Start Guide. The setup wizard should have all the major settings already configured for you, if you need to tweak anything in the future click on the Preferences icon or press CTRL+P to access the configuration menu.

Open the Calibre, click "Add Books," and choose how you'd like to add books

Now it's time to start adding books to Calibre. Grab some free books online or some books you've already collected. For the purposes of this tutorial we downloaded some free ebooks from around the web and placed them, temporarily, in the /Ebook Library/temp/ folder.

You can add books in one of several ways. Choose carefully. If you're going to make a mess anywhere in the process of using Calibre it's right here---we speak from experience. Here's a quick primer:

Add books from a single directory: Opens a dialog box. You pick the books you want to add manually from a directory of files. Best for selecting single books or a few books in a single format.

Add books from directories, including sub-directories (One book per directory, assumes every ebook file is the same book in a different format): If you already have a batch of books sorted into folders (the titles and organization of the folders doesn't matter as long as each folder only has different-format copies of the same book) this is the one you want.

Add books from directories, including sub-directories (Multiple books per directory, assumes every ebook file is a different book): If your books are in multiple directories but every book is different this is the one you want. This command assumes that every directory in the directory structure you point it at has books in it and those books are all different. It's worth it to do a little manual sorting and use the prior one-book-per-directory command as this one can sometimes make a mess you'll need to spend a few minutes tidying up.

For a more detailed look at the add command, refer to the Calibre manual here .

We downloaded our free ebooks into the /temp/ directory with no special folder organization. It's simply one directory with a bunch of ebooks in it. In this instance we can use the first option, Add books from a single directory:


Depending on how many books you're adding this process can take anywhere from a few seconds to a half hour or more as Calibre extracts information from the incoming books. If you're planning on adding a large library we'd recommend starting with a small chunk, maybe 5-10 books to practice with, and then move up to importing larger portions.

After Calibre finishes adding your books you should see them listed in the main pane. Our five books, downloaded from Project Gutenberg, look good---the author and title are all properly placed. Unfortunately Project Gutenberg doesn't supply covers with their books, they all have a generic look ebook reader icon in place of cover data. In the next step we'll tidy things up.


Let's take a look at how we can tidy up the entries and get some new covers. There are two ways you can go about getting additional meta data and covers, you can individually edit items or you can bulk scan them. If your library look pretty clean (as our does) you can likely get away with bulk scanning. If you've got a bunch of jumbled titles and authors, however, you'll want to go with the manual one-by-one updating to make sure you can spot check each entry.


First let's look at what a manual edit looks like. Right click on any book in your library you want to manually edit. We'll start right at the top with Ulysses. Select Edit metadata and then Edit metadata individually . Note: you can highlight more than one book and still select Edit metadata individually and you'll be presented with the metadata panel for each book one by one.

In the Edit Metadata menu you'll see all the data Calibre has for the selected book including title, author, series/number (if applicable), which formats the library contains, and the cover. We're going to leave the author and title alone as they are already properly sorted. The cover is rather lackluster though. Let's click download cover and see what the databases turn up.


The Project Gutenberg edition has no official cover so we can pick from two covers discovered through Google and If neither is satisfactory we can hit cancel and go manually find an image online and then drag and drop the file into the Edit Metadata pane onto the Cover spot to manually add it. The provided cover looks good to us, we'll take that one.

Back in the Edit Metadata window we have a fresh cover and a decent amount of metadata. There's no rating and it might be nice to update the tags. Press the download metadata button to do so. Calibre will poll Google and Amazon again and present multiple versions of the book. You can check the rating, reviews, and tags for each source and select from them based on your preference (if the book is of the generic variety like a Project Gutenberg release) or for the exact Publisher/Edition if applicable.


If everything looks OK when you're done with your selection go ahead and click OK to return to the main book list. Now lets take a look at bulk downloading of metadata and covers. Go ahead and select as many of your books as you wish to bulk edit. Right click on an entry, as you did above, only this time select download metadata and covers or hit CTRL+D to start the process. Calibre will prompt you to ensure you want to bulk edit. Here you can opt to download just the metadata, just the covers, or both. In the beginning you can go wild and download everything. As time progresses and you've invested time in picking your favorite covers out and such you may opt to only update one category of metadata.


In the lower left hand of the Calibre window you should see a little rotating icon and a " Jobs: 1 " notation. Anytime Calibre is working on anything (downloading metadata, converting books, etc.) the lower right portion of the window will show you. You can click on the Jobs:1 text in order to see what is in the job queue.

When Calibre is done updating the metadata a Download Complete dialog box will pop up and ask you if you want to apply the updated metadata to your library. Your options are yes, no, and view log. If you're feeling particularly cautious you can check out the log but, be forewarned, it's quite lengthy. If you like what you see, click Yes.


Calibre will apply the metadata. When it is complete, click on any of your previously un-updated books to check out the new covers and metadata.

Start slowly to get a feel for editing metadata in Calibre. Once you've got the hang of it you can start pouring in your entire ebook collection. Although most of the menus are straight forward, you can read a detailed list of functions and some tips and tricks in the Calibre manual here .

Now that we've tidied things up, take a moment to browse through your fledgling collection. You can stick with the main pane interface and simply scroll up or you can click the small arrow in the lower right corner (or press SHIFT+ALT+B) to enter the cover flow mode to enjoy a swankier iTunes style look at your collection.


Ideally, you have books already in the format you need them in. Books natively in the file format you need have the most reliable text formatting. That said, Calibre is a whiz at converting between formats. First let's look at simply adding a book.

Calibre auto-detects dozens of ebook readers. Simply plug in your ebook read and wait. Within a moment or two an extra column, On Device, as seen in the screenshot above, will appear. If your device does not appear, check out this trouble shooting guide here .

If the ebook is already in a format supported by your device (such as MOBI for the Kindle) you can simply right click on the book entry, select Send to Device and Main Memory and Calibre will choose the most appropriate format and shuttle it over.

If your book is not in a compatible format, such as the book we selected The Importance of Being Earnest by Oscar Wilde, you have two options. You can take the simple route and follow the steps we outlined in the last paragraph---Calibre will ask you if you want to automatically convert the book to the right format for the device---or you can manually configure the conversion process. Most of the time Calibre does a fine job with the automatic conversions between document formats. If you have a moment to check the results on your ebook reader before heading out the door, it's a pretty safe gamble to let Calibre convert on its own with no supervision.


Alternatively you can right click on a book entry and select Convert Books and then Convert Individually . We recommend you only go with this option of the automatic conversion process has failed you in some way. Mucking about in the settings in the manual conversion menu when you're unfamiliar with them is a sure way to end up with less than pleasing results. One of the first steps you can take, before diving into editing every sub-menu in the conversion tool, is to try out Heuristic Processing . You can turn it on by selecting it in the sidebar of the conversion tool and checking Enable Heuristic Processing . It's like, for lack of a better simple analogy, error checking for books. It will scan your book for common conversion problems and attempt to correct them. If the heuristic process doesn't fix your issues, you'll need to roll up your sleeves and go through each sub-menu and tweak the specific issues with your conversion. Make sure to check out this conversion guide to ensure you're clear on the function of each tool.

Once your book is converted and transferred to your device, it should look like so on the main screen:


Simply rinse and repeat for all your books and you'll have a well organized library and a well stocked ebook reader.

Prior to this introductory guide, we've shared multiple guides to taking advantage of Calibre's cool features and other ebook-related tips and tricks. To increase your Calibre-fu and ebook reader enjoyment check out the following guides:

  • Calibre User Manual
  • Convert a PDF eBook to ePub Format for Your iPad, iPhone, or eReader
  • Convert Word Documents to ePub Format for Your eReader or iBooks
  • Edit ePub eBooks with Your Favorite HTML Editor
  • How to Convert PDF Files for Easy Ebook Reading
  • Access Your Ebook Collection Anywhere in the World
  • How to Find Thousands of Free Ebooks Online
  • Optimize Your Manga and Comic Collection for the Kindle
  • Jailbreak Your Kindle for Dead Simple Screensaver Customization
  • How to Read PDF Files on Your Amazon Kindle (Version 3)
  • Get More From Your Kindle: Tips, Tricks, Hacks, and Free Books

Have a tip to share? Experience converting books? Let's hear about it in the comments.

7 Hidden Calibre Features That'll Help You Manage Your Ebooks Better

Calibre is brilliant, but many of its features fly under the radar. Here are the best Calibre features to help you manage your ebook collection.

Calibre might not be the most polished app in the world, but it's definitely the best software for managing your ebook collection .

It ticks all the right boxes: it's free, there aren't any ads, and it boasts a vast number of powerful features. Sadly, many of those features fly under the radar, which is a shame because they can elevate your ebook management to the next level.

So, without further ado, let's take a look at some of the best hidden Calibre features.

Download: Calibre on Windows, Mac, and Linux

1. Merge and Split EPUB Ebooks

Some books come in multiple installments. Perhaps it's a series of books like the Lord of the Rings trilogy, or maybe it's a reference guide such as an encyclopedia.

Similarly, some books are exceptionally long; encyclopedias again spring to mind. A long book means a large file size, and that can be problematic when you're waiting for a book to fully load in your e-reader.

The solution is to use two Calibre plugins called EpubSplit and EpubMerge . Combined, they let you collate multiple books or divide single books at your choosing.

To install the plugins, follow the instructions below:

  • Open the Calibre app.
  • Right-click on the Preferences icon in the upper right-hand corner.
  • Select Get plugins to enhance Calibre from the drop-down menu.
  • Locate EpubSplit and highlight the plugin.
  • Click on Install in the lower right-hand corner.
  • Choose the toolbars and menus where you want the plugin option to appear.
  • Locate EpubMerge and highlight the plugin.
  • Again, click on Install in the lower right-hand corner.
  • And again, choose the toolbars and menus where you want the plugin option to appear.

epubmerge epubsplit

To use the plugins, click on the book you want to merge or split in your library, select the correct plugin in the menu bar, and choose the sections you either want to split or merge.

calibre split book

Note: The plugins only work with ebooks in the EPUB format. We'll discuss how to convert books into different formats shortly.

Interested in more helpful tools? Take a look at these awesome Calibre plugins for ebook lovers .

2. Get Your Favorite Magazines on Your E-Reader

Online magazine subscriptions can be confusing. Some offer an online-only digital version, some offer a Kindle version, and some are only available as Android apps .

Wouldn't it be great if you could cut through the fluff and download your favorite magazines onto your e-reader automatically?

Well, Calibre makes this possible.

To get started, click on the Fetch News tab at the top of the app's home screen. In the left-hand panel, you'll see a list of languages. Click on your dialect of choice to see what's available.

calibre library news

Find the title you want to read and---if required---enter your paywall login credentials. Next, in the right-hand panel, mark the checkbox next to Schedule for download and hit Download Now .

Lastly, you need to force Calibre to send your newly downloaded magazine issues to your e-reader. Go to Preferences > Behavior and mark the checkbox next to Automatically send downloaded news to ebook reader .

Note: For automatic downloading to work, Calibre needs to be running on your computer.

For more, check out how to get news updates on your Kindle with Calibre .

3. Turn Calibre Into a Sharing Server

If several members of your household have a Kindle, or if you own multiple Kindles, continually syncing your data manually quickly becomes tedious.

Instead, why not turn your Calibre app into a content server? By doing so, you can make your entire Calibre library available on all your devices. You can even upload new content to your Calibre library from those devices.

The process is remarkably straightforward. In Calibre's menu bar, go to Connect/share > Start content server . Your computer might prompt you to allow the app through its firewall.

Next, click on the Connect/share tab for a second time. You'll see your computer's local IP address followed by the port number. Make a note of them.

calibre library server ip

Now head to your Kindle's browser (or any other browser) and type [IP address]:[port number] in the address bar. You should see all your Calibre books appear on your screen.

Click on a book's title to open it or click on the + icon in the upper right-hand corner to add more content to your library.

calibre library server

Note: If you want to access your library when you're away from your home network, you'll need to allow port forwarding to Calibre's port number on your router.

4. Convert Ebooks Into Different Formats

Sadly, the world of ebook file formats is a bit of a mess.

In theory, the standard format is the open-source EPUB. However, Amazon's Kindle devices---which are the world's most popular e-readers---can't read it. Instead, they rely on the proprietary AZW format. And let's not start on MOBI, LIT, AZW3, or the other dozen different formats you see out there.

Suffice to say, you need a way to convert ebooks into different formats .

Thankfully, Calibre delivers. To convert an ebook into a different file format, use the following instructions:

  • Open Calibre.
  • Right-click on the book you want to convert and go to Convert Books > Convert Individually .
  • In the upper right-hand corner of the new window, choose your desired new format.
  • Click on OK and give the conversion a minute or two to complete.

calibre convert

You can verify if the conversion has been successful by clicking on Jobs in the bottom right-hand corner of the app's home screen. You can also check whether the new format is listed in the book's information panel on the right-hand side of the screen.

calibre convert success

Note: You can also automatically convert new ebooks when you import them into your library for the first time.

5. Remove DRM From Ebooks

We now live in an age in which true ownership of digital media is becoming less common. Luckily for bookworms, Calibre lets your wrestle back control of your ebooks by offering a way to remove the DRM from titles you've bought from Amazon and other online stores.

We covered the process in detail when we explained how to remove the DRM on every ebook you own . So we recommend reading that article for the full scoop.

6. Automatically Download Ebook Metadata

Like any media library, you need to keep the metadata organized. Doing so will lead to a much smoother and more enjoyable experience, especially if you have a vast number of books.

But who has the time to enter all that information manually?

Calibre offers a little-used feature that lets you scan for your books' metadata automatically. It can even find the correct book covers.

To scan for metadata, right-click on the book in question and go to Edit Metadata > Download metadata and covers . A new box will pop up on your screen. You need to select Download both .

metadata download

When the scan is finished, you'll see a notification appear. Click on Review downloaded metadata and make any tweaks as appropriate.

metadata download

Calibre can pull metadata from Amazon, Google Images, Overdrive, Open Library, Edelweiss, Douban Books, and more.

7. Put Your Ebook Library in the Cloud

If using the content server method that we described earlier sounds too finicky, you can instead use Dropbox as an alternative.

By keeping your books in Dropbox rather than on your local machine, you'll be able to access them from anywhere. However, if you set up the Calibre app correctly, you'll also be able to use the app to manage your library locally.

Firstly, you need to download and install Dropbox on your computer.

Next, head to Calibre and move your library to the shared Dropbox folder on your machine. The fastest way of achieving this is to go to Preferences > Run Setup Wizard and point the Calibre app at the Dropbox folder.

calibre library wizard

Lastly, drag-and-drop your ebooks from their current location into the Dropbox folder. Et voila, you now have a cloud-based ebook library that you can manage locally using Calibre.

Do You Need More Books to Read?

The seven features we have covered in this article are all great, but you won't be able to enjoy them if you don't have a library of ebooks to begin with.

But building a library doesn't necessarily involve spending money. There are lots of sites to get free ebooks , and you can even download free books directly from Amazon .

Technical How-tos, Tips, and Tricks

How to download calibre ebooks to hard drive or usb.

January 7, 2020 Matt Mills How to 0

What a few years ago for many was an accumulation of paper books on the shelves of his house, now it is reduced to a few files on the PC . But of course, like conventional copies, these files also need an order and concert. For this, instead of shelves or simply boxes, we use applications that we install on the computer, as is the case of the aforementioned Calibre and that you can download from here . And it is necessary to bear in mind that in many occasions we do not speak of some tens of electronic books , but of thousands of titles.

Manage your ebook library from the Calibre program

In fact, with this program, we are referring to the most used and extended solution of this type at this time and for some time. In this way, its developers have not stopped improving and growing this software through its constant updates. Therefore we will have the opportunity to create and manage a personal library of ebooks thanks to the functions presented here.

Calibre Guardar en disco

There are many features that over time have come to this software that we mentioned and that we have been talking about. Well, now we are going to show you how to download the titles stored in that personal library , to your hard drive or USB stick , for example.

As you can imagine, this will allow us to save or transport these digital contents as we want, for example to share them with others. It is possible that someone has lent us your Calibre  library , so after importing it, we need to download certain titles.

Download the eBooks you want from your library to the hard drive or USB memory

Well, how could it be otherwise, the program we mentioned, Calibre, also allows us to download these digital books locally. Therefore, in these same lines we will show you the way to do it in a few steps. Thus, the first thing will be to open the program as such so that all electronic books that make up our library appear on the screen.

Calibre descargar libros

Once we see them, we can place ourselves on any of them and click on the date to the right of the Save to disk button. Then we will see a drop-down list of options with which we can store those contents in local mode. So, as we see, we will have the possibility to choose Save to disk. This will download all the books we have marked, in a certain folder previously chosen.

But that’s not all, but we also have the possibility to store those books only in MOBI format, store everything in the same directory , or use only one format. This allows us to manage the contents of the library directly from the Explorer , or create a new one with the titles that we want to add.

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How to Use Calibre for Kindle – Step By Step Guide

Calibre e-book manager

I never owned a Kindle but recently I borrowed one from a friend . A few things that came to my mind were, how do  I transfer books? Will it let me transfer PDFs? Do I need to convert my files to Mobi format? All these are genuine questions if you’re starting out with using an e-book reader like Kindle .

Also Read: Kindle Paperwhite Tips and Tricks You Should Know in 2019

So I started searching for ways I could transfer, organize, create and convert e-books easily. An e-book management system that would let me do all that in one complete package. And I found Calibre .

What is Calibre?

Calibre is an open-source software that lets you manage your e-book reader. You can create digital libraries, manage your existing device, modify the metadata and much more. In addition to that, you can easily sync it with other devices . The software is cross-platform which means, it’s available for Windows , macOS ,  and Linux . There’s also a portable version that can straightway be loaded on a disk or a drive and simply put to use by inserting or plugging in. It is immensely convenient for someone who travels often or doesn’t own a PC.

There are plenty of features I need to share with you right away, so let’s start with the basics. After you install Calibre on your computer , connect your e-book device with a USB cable, in my case Kindle . This is how the toolbar will change after the software auto-detects the device.

Add, Convert and Transfer Books

These are the primary operations you’ll use if you’re using an e-book reader. You’ll add books to the library, convert books that are not in the native format and transfer it to your device . This is the bare minimum anyone would do. In Calibre after you’re connected, you see your device, which on clicking shows you the entire library of books . The ones with a green tick are ones in the Calibre Library but not in Kindle. So you can keep all your books in Calibre library and keep selected in your Kindle device.

Adding a book to your Kindle is easy if you’ve saved it on your PC. There is an ‘Add books’ on the to right, clicking on which will open the explorer, from where you can add books to your Calibre library. You can also add books that are not natively supported by the device as you’d be working with the Calibre library rather than the device. There is also an option to drag and drop books in case you want an easy way out.

As I said, the format of your book doesn’t matter when you’re adding it to the Calibre library but when you want to send it to your device you need to convert it to a format supported by the device. In the case of Kindle, it’s Mobi. In case you forget to convert and straightaway send it to your device, it’ll prompt you to convert and then transfer the book. It automatically detects the current format of the document and you can choose the format you want to convert it to through the drop-down menu on the top right corner.

Read: How to Send Articles to Kindle From Android

While you’re converting a book, you also get an option to tweak the Look & Feel of it.  A tool for the nerds among you, it lets you choose the font, size and even tweak space between two paragraphs.

Edit Metadata

I often fetch a book from the internet or a friend shares it with me, and it’s so frustrating to find the book cover, author’s name or publisher details missing. Thankfully, Calibre can download the missing metadata for all your books from various sources. You can even update specific parts of it like the book cover, title etc. When you search for a metadata, it searches through all possible platforms where the information can be and you can select the most appropriate. For example, when we search for a book cover it scrapes through Google Images, Amazon, Open Book etc to fetch you the details resulting in several options to choose from.

Library and Tags Browser

It has an inbuilt way to segregating subjects simply by tagging them. So you can easily select them and put them in various available categories. You can search and divide on the basis of authors, languages, publishing house or a custom tag of your own. I often tag my books in terms of genre, like an autobiography, music, fiction, etc.

Calibre Tags

This helps in not only managing the e-book reader but my entire library. When dealing with multiple book readers this feature can be really helpful in finding and sorting out books.

Read News and Magazines

The amount of things Caibre is capable of is outstanding. At least for me, it has all the necessary features that I could think of while writing this article. I read books on my Kindle but besides that, I also tend to use it for reading magazines and newspapers . With Calibre I don’t have to worry much.

Read: Best Places to Get Kindle Books (Free/ Cheap)

Calibre News

It has downloadable magazines from a huge global database. You can choose either by country or by language and select the publication of your choice. With the scheduling feature it’s possible to automate Calibre to download newspapers and magazine daily so you eliminate manually doing it.

Search For Books

With an integrated search option, Get books , you can search for the title you want. There are quick searches in case you want to see all available options for a specific author, title or if you want to search with a specific keyword (umbrella search). It skims through public as well as commercial sources to deliver what you’re looking for.  You can select specific stores like Amazon, Barnes & Noble, Mills and Boon etc.

Calibre Search For Books

The only downside is it’s a bit slow but provided the search is so vast, I can really manage along.

Forum Support

It originally started with the developer, Kovind Goyal not being able to use his e-reader with Linux, resulting in reverse-engineering the USB protocol to make it work. Then came the issue of a non-supportive format and the lack of an effective e-book manager, giving rise to Calibre , as we know it.

Ca(libre, freedom) hence is an free open source product with a large community of testers and volunteers . Therefore if there is a suggestion, an issue you can’t resolve or simply you being overwhelmed by the features, you can write on Calibre Forum or  reach out on Facebook or Twitter . You can also use the Bug Tracker to report bugs or suggest new features.

Closing Words

If you’re a Kindle user or as a matter of fact uses any e-book reader, Calibre is a capable software to handle all your reading and managing woes. From fetching the metadata, in-built reader, easy drag and drop transfers to converting a document it can do almost everything you throw at it. I talked about all the basic yet important features that you’ll use on a daily basis because I use them extensively. Calibre has never disappointed me and I’m sure you’ll be happy too. So take your e-book devices out and enjoy reading!

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Vaibhav is a broadcast journalist with a keen interest in tech. He doesn't believe in fanboying a specific product. He writes about things he believes are actually helpful in some way to the user.

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Analytical Methods

Separation and purification of short-, middle-, and long-stranded rnas by rp-hplc using different mobile phases and c18 columns with various pore sizes.

Nucleic acids, which have been employed in medicines for various diseases, are attracting attention as a new pharmaceutical model. Depending on the target substances, nucleic acid medicines with various nucleic acid chain lengths (several ten nucleotide [nt] to several thousand nt) exist. The purification of synthesized nucleic acids is crucial as various impurities remain in the crude product after synthesis. Presently, reversed-phase high-performance liquid chromatography (RP-HPLC) represents an effective purification method for nucleic acids. However, the information regarding the HPLC conditions for separating and purifying nucleic acids of various chain lengths is insufficient. Thus, this technical note describes the separation and purification of short-, middle-, and long-stranded nucleic acids (several ten nt to thousand nt) by RP-HPLC with various mobile phases and octadecyl-based columns with various pore sizes, such as normal (9–12 nm), wide (30 nm), and super wide (>30 nm) pores.

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M. Ozaki, T. Kuwayama, M. Shimotsuma and T. Hirose, Anal. Methods , 2024, Accepted Manuscript , DOI: 10.1039/D4AY00114A

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